RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/394,849 filed on August 3, 2022 and U.S. Provisional Application No. 63/471,167 filed on June 5, 2023; the entire contents of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 2, 2023, is named V2071-3006PCT_SL.xml and is 4,777,611 bytes in size.

FIELD OF THE DISCLOSURE

[0002] The disclosure relates to compositions and methods for the preparation, use, and/or formulation of active agents conjugated to ligands for increased crossing of the blood brain barrier.

BACKGROUND

[0003] Administering active agents such as therapeutic agents and diagnostic agents to the adult central nervous system (CNS) remains a significant challenge. Engineered compositions comprising said active agents fused or coupled with ligands capable of binding receptors on cells present in the blood brain barrier represent an attractive solution to the limitations of CNS delivery.

[0004] Attempts at providing compositions with improved ability to cross the blood brain barrier, have met with limited success. As such, there is a need for improved methods of producing and delivering active agents of interest to a target cell or tissue, e.g., a CNS cell or tissue.

SUMMARY OF THE DISCLOSURE

[0005] The present disclosure pertains at least in part, to compositions and methods for the production and use of a composition comprising a ligand capable of binding a receptor present on a cell in the blood brain barrier. In some embodiments, the ligand is fused or coupled, e.g., covalently or non-covalently, to an active agent, e.g., a therapeutic agent or a diagnostic agent. Said compositions can be useful for delivery of an active agent, e.g., a therapeutic agent or a diagnostic agent described herein, to a cell or tissue, e.g., a CNS cell or tissue, for the treatment of a disorder, e.g., a neurological or a neurodegenerative disorder, a muscular or a neuromuscular disorder, or a neuro-oncological disorder.

[0006] Accordingly, in one aspect, the present disclosure provides a composition e.g., a fusion molecule or a conjugate molecule, comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, e.g., a therapeutic agent or a diagnostic agent, wherein the ligand is fused or coupled, e.g., covalently or non-covalently, to the active agent.

[0007] In another aspect, the present disclosure provides multispecific antibody molecule comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

[0008] In yet another aspect, the present disclosure provides a method of making a composition described herein, the method comprises (i) providing the ligand that binds to the GPI anchored protein, e.g., ALPL, and the active agent; and (ii) incubating the ligand and active agent under conditions suitable to fuse or couple the ligand to the active agent, thereby generating the composition.

[0009] In yet another aspect, the present disclosure provides a method of delivering an active agent, e.g., a therapeutic agent or a diagnostic agent, to a cell or tissue (e.g., a CNS cell or a CNS tissue). The method comprising administering to the subject an effective amount of a composition comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, described herein.

[0010] In yet another aspect, the present disclosure provides a method of increasing central nervous system transduction (e.g., increased crossing of the blood brain barrier) in a subject. The method comprising administering to the subject an effective amount of a composition comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, described herein.

[0011] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with having a genetic disorder, e.g., a monogenic disorder or a polygenic disorder. The method comprising administering to the subject an effective amount of a composition comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, described herein.

[0012] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with having neurological, e.g., a neurodegenerative, disorder. The method comprising administering an effective amount of a composition comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, described herein.

[0013] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with having a neuro-oncological disorder. The method comprising administering an effective amount of a composition comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, described herein. [0014] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.

Enumerated Embodiments

1. A composition, e.g., a fusion molecule or a conjugate molecule, comprising:

(i) a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and

(ii) an active agent, e.g., a therapeutic agent or a diagnostic agent, wherein the ligand is fused or coupled, e.g., covalently or non-covalently, to the active agent; optionally wherein the ligand is capable of binding the GPI anchored protein, e.g., ALPL, at a KD of at least about 10-250 nM, 10-150 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 175 nM, 200 nM, 215 nM, or 250 nM), e.g., when measured by an SPR assay, e.g., as described in Example 8.

2. The composition of claim 1, wherein the ligand is capable of binding the GPI anchored protein, e.g., ALPL, at a KD of:

(a) at least about 10-250 nM;

(b) at least about 10-150 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM), e.g., wherein the ligand is a viral particle or a peptide;

(c) at least about 10-55 nM, 15-30 nM, 20-30 nM, 25-50 nM, or 30-50 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, or 55 nM), e.g., wherein the ligand is a viral particle (e.g., an AAV viral particle) or a peptide; or

(c) at least about 150-250 nM, 150-225 nM, 175-250 nM, 175-225 nM, 200-225 nM, 200-250 nM (e.g., 150 nM, 175 nM, 200 nM, 215 nM, or 250 nM), e.g., wherein the ligand is an antibody molecule; optionally, when (a), (b), (c), and (d) are measured by an SPR assay, e.g., as described in Example 8 or 13.

3. The composition of claim 1 or 2, wherein the ligand is capable of binding the GPI anchored protein e.g., ALPL, in a pH dependent manner, optionally wherein the ligand binds to ALPL at physiological pH (e.g., at a pH of at least about 6.5-8.0, 7.0-8.0, 6.5-7.5, 7.0-7.5, 7.0, 7.1, 7.2, 7.3, or 7.4) and/or does not substantially bind ALPL at an acidic pH (e.g., at a pH of at least about 1.0-5.7, 1.0-5.5, 2.0- 5.7, 2.5-5.5, 2.5-5.7, 3.0-5.7, 3.0-5.5, 3.5-5.7, 3.5-5.5, 4.0-5.7, 4.0-5.5, 4.5-5.7, 4.5-5.5, 5.0-5.7, 5.5- 5.7, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5), e.g., as measured by an assay e.g., an SPR or Biacore assay, e.g., as described in Example 8 or 13. 4. The composition of any one of embodiments 1-3, wherein the ligand is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule.

5. The composition of any one of embodiments 1-3, wherein the ligand comprises a linear peptide or a circular peptide.

6. The composition of any one of embodiments 1-5, wherein the active agent is or comprises a therapeutic agent chosen from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

7. The composition of any one of embodiments 1-5, wherein the active agent is or comprises a ribonucleic acid complex (e.g., a Cas9/gRNA complex), a plasmid, a closed-end DNA, a circ-RNA, or an mRNA.

8. The composition of any one of embodiments 1-5, wherein the active agent is a diagnostic agent is or comprises an imaging agent (e.g., a protein or small molecule compound coupled to a detectable moiety).

9. The composition of any one of embodiments 1-8, wherein the ligand is covalently linked to the active agent.

10. The composition of any one of embodiments 1-9, wherein the ligand is conjugated to the active agent.

11. The composition of any one of embodiments 1-8, wherein the ligand is fused to the active agent, e.g., as part of a fusion peptide or protein.

12. The composition of any one of embodiments 1-11, wherein the ligand is not a component of a viral particle, e.g., an adeno-associated viral (AAV) particle.

13. The composition of any one of embodiments 1-12, wherein the ligand is not a component of a capsid protein, e.g., an AAV capsid protein.

14. The composition of embodiment 13, wherein the ligand is not a component of an AAV9 capsid or variant thereof. 15. The composition of any one of embodiments 1-14, wherein the GPI anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mice, NHPs (e.g., Macaca fascicularis), and/or humans).

16. The composition of embodiment of embodiment 15, wherein the at least two GPI anchored proteins are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to each other.

17. The composition of any one of embodiments 1-16, wherein the GPI anchored protein is present on the surface of a cell in the blood brain barrier.

18. The composition of any one of embodiments 1-17, wherein the GPI anchored protein is ALPL, CD59, LY6E, CA4, GPC5, NTM, HYAL2, LSAMP, BST2, EMP2, ALPL, CPM, NCAM1, EFNA1, PIBF1, SEC24B, PRNP, TFPI, OPCML, CD109, DPM3, CNTN4, PIGN, HBP1, CNTN2, CD55, NEGRI, EFNA5, RECK, NRN1, CNTN1, GPAA1, PGAP1, PIGF, PIGK, MDGA2, DPMI, SVIP, NTNG1, CNTN5, GPC6, PIGG, TMEM8A, THY1, GPIHBP1, PIGT, PIGL, ZFAND2B, PLAUR, DPM2, or GPC1.

19. The composition of any one of embodiments 1-18, wherein the GPI anchored protein is ALPL.

20. The composition of any one of embodiments 1-19, wherein the ligand binds human, cynomolgus, or murine ALPL.

21. The composition of any one of embodiments 1-20, wherein ligand is fused or coupled to a therapeutic agent or diagnostic agent.

22. The composition of any one of embodiments 1-21, wherein the ligand is covalently linked, e.g., directly or indirectly via a linker, to the active agent.

23. The composition of embodiment 22, wherein the ligand is covalently linked to the active agent via a linker.

24. The composition of any one of embodiments 1-23, wherein the ligand is conjugated, e.g., directly or indirectly via linker, to the active agent.

25. The composition of 24, wherein the ligand is conjugated to the active agent via a linker. 26. The composition of any one of embodiments 22-25, wherein the linker is a cleavable linker or a non-cleavable linker.

27. The composition of embodiment 26, wherein the cleavable linker is a pH sensitive linker or an enzyme sensitive linker.

28. The composition of embodiment 27, wherein the pH sensitive linker comprises a hydrazine/hydrazone linker or a disulfide linker.

29. The composition of embodiment 28, wherein the enzyme sensitive linker comprises a peptide based linker, e.g., a peptide linker sensitive to a protease (e.g., a lysosomal protease); or a betaglucuronide linker.

30. The composition of embodiment 26, wherein the non-cleavable linker is a linker comprising a thioether group or a maleimidocaproyl group.

31. The composition of any one of embodiments 1-23, wherein the ligand is fused, e.g., directly or indirectly via linker, to the active agent, e.g., as part of a fusion peptide or protein.

32. The composition of any one of embodiments 1-31, wherein the ligand and the active agent are fused or coupled post-translationally, e.g., using click chemistry.

33. The composition of any one of embodiments 1-32, wherein the ligand and the active agent are fused or couple via chemically induced dimerization.

34. The composition of any one of embodiments 1-33, wherein the ligand is present N-terminal relative to the active agent.

35. The composition of any one of embodiments 1-33, wherein the ligand is present C-terminal relative to the active agent.

36. The composition of any one of embodiments 1-33, wherein the ligand is fused or coupled at or near the C-terminus of the active agent, wherein the active agent is a therapeutic protein, enzyme, or antibody molecule.

37. The composition of embodiment 36, wherein the ligand is fused or coupled within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids from the C-terminus of the therapeutic protein, enzyme, or antibody molecule. 38. The composition of any one of embodiments 1-36, wherein the ligand is or comprises a protein or a peptide comprising an amino acid sequence having the following formula: [N1]-[N2]-[N3], wherein:

(i) optionally [Nl] comprises XI, X2, and X3, wherein at least one of XI, X2, or X3 is G;

(ii) [N2] comprises the amino acid sequence of SPH, optionally wherein S comprises a modification, e.g., comprises a phosphate group;

(ii) [N3] comprises X4, X5, and X6, wherein at least one of X4, X5, or X6 is a basic amino acid, e.g., a K or R.

39. The composition of embodiment 38, wherein X4, X5, or both of [N3] is a K.

40. The composition of embodiment 38 or 39, wherein X4, X5, or X6 of [N3] is an R.

41. The composition of any one of embodiments 38-40, wherein:

(a) position X4 of [N3] is independently chosen from: K, S, A, V, T, G, F, W, V, N, or R;

(b) position X5 of [N3] is independently chosen from: S, K, T, F, I, L, Y, H, M, or R; and/or

(c) position X6 of [N3] is independently chosen from: G, A, R, M, I, N, T, Y, D, P, V, L, E,

W, N, Q, K, or S; optionally, wherein the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).

42. The composition of any one of embodiments 38-41, wherein [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG.

43. The composition of any one of embodiments 38-42, wherein [N3] is SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG.

44. The composition of any one of embodiments 38-43, wherein [N2]-[N3] comprises SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKR (SEQ ID NO: 4717), SPHGA (SEQ ID NO: 4718), SPHSR (SEQ ID NO: 4719), SPHSL (SEQ ID NO: 4720), SPHSS (SEQ ID NO: 4721), SPHFL (SEQ ID NO: 4722), SPHWK (SEQ ID NO: 4723), SPHGK (SEQ ID NO: 4724), SPHTK (SEQ ID NO: 4725), SPHNK (SEQ ID NO: 4726), SPHAK (SEQ ID NO: 4727), SPHKH (SEQ ID NO: 4728), SPHKM (SEQ ID NO: 4729), or SPHRS (SEQ ID NO: 4730).

45. The composition of any one of embodiments 38-44, wherein [N2]-[N3] is or comprises:

(i) SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKTY (SEQ ID NO: 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPHSSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

46. The composition of any one of embodiments 38-45, wherein [Nl] comprises XI, X2, and X3, wherein at least one of XI, X2, or X3 is G.

47. The composition of any one of embodiments 38-46, wherein:

(a) position XI of [Nl] is independently chosen from: G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C; (b) position X2 of [Nl] is independently chosen from: S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q; and/or

(c) position X3 of [Nl] is independently chosen from: G, C, L, D, E, Y, H, V, A, N, P, or S; optionally wherein the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).

48. The ligand of any one of embodiments 38-47, wherein [Nl] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, Al, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS.

49. The composition of any one of embodiments 38-48, wherein [Nl] is or comprises GSG, GHD, GQD, VSG, CSG, GRG, CSH, GQS, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS.

50. The composition of any one of embodiments 38-49, wherein [N1]-[N2] comprises:

(i) SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758), NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i). 51. The composition of any one of embodiments 38-50, wherein [N1]-[N2] is or comprises:

(i) GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802), ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GSASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIGSPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

52. The composition of any one of embodiments 38-51, wherein [N1]-[N2]-[N3] comprises:

(i) SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHAS (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSPHSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SGSPHNK (SEQ ID NO: 4879), LGSPHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892), PDSPHKI (SEQ ID NO: 4893), PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

53. The composition of any one of embodiments 38-52, wherein [N1]-[N2]-[N3] is or comprises:

(i) GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CSGSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPHKFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSKD (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939), GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW (SEQ ID NO: 4952), GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA (SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA (SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989), GNGSPHSKS (SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKSL (SEQ ID NO: 5001), GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 5012), GHDSPHKHG (SEQ ID NO: 5013), GQVSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSPHKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

54. The composition of any one of embodiments 38-53, wherein [N3] comprises SK, KA, KS, or SG.

55. The composition of any one of embodiments 38-54, wherein [N3] is or comprises SKA, KSG, or KYG.

56. The composition of any one of embodiments 38-55, wherein [N2]-[N3] comprises SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), or SPHKY (SEQ ID NO: 4715).

57. The composition of any one of embodiments 38-56, wherein [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941).

58. The composition of any one of embodiments 38-56, wherein [N2]-[N3] is or comprises SPHKSG (SEQ ID NO: 946).

59. The composition of any one of embodiments 38-56, wherein [N2]-[N3] is or comprises SPHKYG (SEQ ID NO: 966).

60. The composition of any one of embodiments 38-59, wherein [Nl] comprises GS, SG, GH, or HD.

61. The composition of any one of embodiments 38-60, wherein [Nl] is or comprises GSG.

62. The composition of any one of embodiments 38-60, wherein [Nl] is or comprises GHD.

63. The composition of any one of embodiments 38-57, 60, or 61, wherein [N1]-[N2]-[N3] comprises SGSPHSK (SEQ ID NO: 4839).

64. The composition of any one of embodiments 38-56, 58, 60, or 62, wherein [N1]-[N2]-[N3] comprises HDSPHKS (SEQ ID NO: 4840).

65. The composition of any one of embodiments 38-56 or 59-61, wherein [N1]-[N2]-[N3] comprises SGSPHKYG (SEQ ID NO: 5027). 66. The composition of any one of embodiments 38-57, 60, 61, or 63, wherein [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697).

67. The composition of any one of embodiments 38-56, 58, 60, 62, or 54, wherein [N1]-[N2]-[N3] is or comprises GHDSPHKSG (SEQ ID NO: 4698).

68. The composition of any one of embodiments 38-56, 59-61, or 65, wherein [N1]-[N2]-[N3] is or comprises GSGSPHKYG (SEQ ID NO: 4927).

69. The composition of any one of embodiments 38-68, which further comprises [N4], wherein [N4] comprises X7 X8 X9 X10, and wherein:

(a) position X7 is independently chosen from Q, W, K, R, G, L, V, S, P, H, K, I, M, A, E, or F;

(b) position X8 is independently chosen from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L;

(c) position X9 is independently chosen from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y; and

(d) position X10 is independently chosen from Q, H, L, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V; optionally wherein the protein comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(d).

70. The composition of embodiment 69, wherein:

(a) position X7 of [N4] is Q or R;

(b) position X8 of [N4] is N or R;

(c) position X9 of [N4] is Q or R; and

(d) position X10 of [N4] is Q, L, or R.

71. The composition of embodiment 69 or 70, wherein [N4] is or comprises:

(i) QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR (SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), SKRQ (SEQ ID NO: 5048), QLYR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066), PNLQ (SEQ ID NO: 5067), HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), QRTM (SEQ ID NO: 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093), QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), QTQL (SEQ ID NO: 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 5133), QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), QNQM (SEQ ID NO: 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159), QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), QYRA (SEQ ID NO: 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198), HTQQ (SEQ ID NO: 5199), QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), RTQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), QIIV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225), QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQL (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, or 3 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

72. The composition of any one of embodiments 69-71, wherein [N1]-[N2]-[N3]-[N4] is or comprises:

(i) the amino acid sequence of any of SEQ ID NOs: 1800-2241;

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i). 73. The composition of any one of embodiments 69-72, wherein [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (SEQ ID NO: 1801).

74. The composition of any one of embodiments 69-72, wherein [N1 ]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (SEQ ID NO: 1800).

75. The composition of any one of embodiments 69-72, wherein [N1 ]-[N2]-[N3]-[N4] is or comprises GSGSPHKYGQNQQT (SEQ ID NO: 910).

76. The composition of any one of embodiments 38-75, which further comprises [NO], wherein [NO] comprises XA XB and XC, and wherein:

(a) position XA is independently chosen from T, S, Y, M, A, C, I, R, L, D, F, V, Q, N, H, E, or G;

(b) position XB is independently chosen from I, M, P, E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L; and

(c) position XC is independently chosen from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S; and optionally wherein the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).

77. The composition of embodiment 76, wherein [NO] is or comprises TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, IET, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM, AEV, MDA, VEW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, Til, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS, TID, TCN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TTN, TED, TNN, EVH, TIS, TVR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TIR, NAL, VIN, TIQ, TEF, TRE, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof.

78. The composition of embodiment 76 or 77, wherein [NO] -[N1]-[N2]-[N3]-[N4] is or comprises:

(i) the amino acid sequence of any one of SEQ ID NOs: 2242-2886;

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).

79. The composition of any one of embodiments 76-78, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (SEQ ID NO: 2242).

80. The composition of any one of embodiments 76-78, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TINGHDSPHKSGQNQQ (SEQ ID NO: 2243).

81. The composition of any one of embodiments 76-78, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHKYGQNQQT (SEQ ID NO: 5246).

82. The composition of any one of embodiments 38-81, wherein [N3] is present immediately subsequent to [N2].

83. The composition of any one of embodiments 38-82, which comprises from N-terminus to C- terminus, [N2]-[N3].

84. The composition of any one of embodiments 38-83, which comprises from N-terminus to C- terminus, [N1]-[N2]-[N3].

85. The composition of any one of embodiments 76-84, which comprises from N-terminus to C- terminus, [NO]-[N1]-[N2]-[N3].

86. The composition of any one of embodiments 69-85, which comprises from N-terminus to C- terminus, [N1]-[N2]-[N3]-[N4],

87. The composition of any one of embodiments 76-86, which comprises from N-terminus to C- terminus, [NO]-[N1]-[N2]-[N3]-[N4],

88. The composition of any one of embodiments 1-87, wherein the ligand comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35-84. 89. The composition of embodiment 88, wherein the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides comprise the same amino acid sequence.

90. The composition of embodiment 88, wherein the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides comprise different amino acid sequences.

91. The composition of any one of embodiments 88-90, wherein the at least 1-5, e.g., at least 1, 2, 3,

4, or 5, proteins or peptides are present in tandem (e.g., connected directly or indirectly via a linker) or in a multimeric configuration.

92. The composition of any one of embodiments 38-91, wherein the protein or peptide comprises an amino acid sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or 35 amino acids in length.

93. The composition of embodiment of 92, wherein the protein or peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of the amino acids TLKFSVAGPSNMAVQG (SEQ ID NO: 4694), optionally wherein the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of the amino acids LKFSVAGPSNMAVQG (SEQ ID NO: 21) is present C-terminal relative to [N4].

94. The composition of any one of embodiments 5-93, wherein the peptide comprises the amino acid sequence of SPH, wherein the S comprises a modification, e.g., comprises a phosphate group.

95. The composition of any one of embodiments 5-94, wherein the peptide comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), optionally wherein the S at position 1, numbered according to SEQ ID NO: 941, comprises a modification, e.g., comprises a phosphate group.

96. The composition of any one of embodiments 2-94, wherein the peptide comprises the amino acid sequence of SPHK (SEQ ID NO: 6398), optionally wherein S comprises a modification, e.g., comprises a phosphate group.

97. The composition of any one of embodiments 2-94 or 96, wherein the peptide comprises the amino acid sequence of HDSPHK (SEQ ID NO: 2), optionally wherein S comprises a modification, e.g., comprises a phosphate group.

98. The composition of any one of embodiments 38-97, wherein the modification comprises a phosphate group. 99. The composition of any one of embodiments 38-98, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids present N-terminal relative the amino acid sequence of SPH.

100. The composition of any one of embodiments 96-99, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids present N-terminal relative the amino acid sequence of HDSPHK (SEQ ID NO: 2).

101. The composition of any one of embodiments 96-100, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids present C-terminal relative the amino acid sequence of HDSPHK (SEQ ID NO: 2).

102. The composition of any one of embodiments 94, 95, or 98, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids present N- terminal relative the amino acid sequence of SPHSKA (SEQ ID NO: 941).

103. The composition of any one of embodiments 94, 95, 98, or 102, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids present C-terminal relative the amino acid sequence of SPHSKA (SEQ ID NO: 941).

104. The composition of any one of embodiments 76-94, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids present N-terminal relative the amino acid sequence of [N0]-[N2]-[N3]-[N4].

105. The composition of any one of embodiments 76-94 or 104, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids present C-terminal relative the amino acid sequence of [NO] -[N2]-[N3]-[N4].

106. The composition of any one of embodiments 5-105, wherein the peptide comprises the amino acid sequence of:

(i) GHDSPHKS (SEQ ID NO: 4487), optionally wherein the S at position 4 of SEQ ID NO: 4487, comprises a modification, e.g., comprises a phosphate group;

(ii) NGHDSPHKSG (SEQ ID NO: 4489), optionally wherein the S at position 5 of SEQ ID NO: 4489, comprises a modification, e.g., comprises a phosphate group;

(iii) INGHDSPHKSGQ (SEQ ID NO: 4490), optionally wherein the S at position 6 of SEQ ID NO: 4490, comprises a modification, e.g., comprises a phosphate group; (iv) TINGHDSPHKSGQN (SEQ ID NO: 4491), optionally wherein the S at position 7 of SEQ ID NO: 4491, comprises a modification, e.g., comprises a phosphate group;

(v) KTINGHDSPHKSGQNQ (SEQ ID NO: 4492), optionally wherein the S at position 8 of SEQ ID NO: 4492, comprises a modification, e.g., comprises a phosphate group;

(vi) LYYLSKTINGHDSPHKSGQNQQTLKF (SEQ ID NO: 4518), optionally wherein the S at position 13 of SEQ ID NO: 4518, comprises a modification, e.g., comprises a phosphate group;

(vii) RLMNPLIDQYLYYLSKTINGHDSPHKSGQNQQTLKFSVAGPSNMAV (SEQ ID NO: 4519), optionally wherein the S at position 23 of SEQ ID NO: 4519, comprises a modification, e.g., comprises a phosphate group;

(viii) GSPHSKAQ (SEQ ID NO: 4493), optionally wherein the S at position 2 of SEQ ID NO: 4493, comprises a modification, e.g., comprises a phosphate group;

(ix) SGSPHSKAQN (SEQ ID NO: 4494), optionally wherein the S at position 3 of SEQ ID NO: 4494, comprises a modification, e.g., comprises a phosphate group;

(x) GSGSPHSKAQNQ (SEQ ID NO: 4495), optionally wherein the S at position 4 of SEQ ID NO: 4495, comprises a modification, e.g., comprises a phosphate group;

(xi) NGSGSPHSKAQNQQ (SEQ ID NO: 4496), optionally wherein the S at position 5 of SEQ ID NO: 4496, comprises a modification, e.g., comprises a phosphate group; or

(xii) INGSGSPHSKAQNQQT (SEQ ID NO: 4497), optionally wherein the S at position 6 of SEQ ID NO: 4497, comprises a modification, e.g., comprises a phosphate group.

107. The composition of any one of embodiments 5-94 or 96-106, wherein the peptide comprises the amino acid sequence of NGHDpSPHKSG (SEQ ID NO: 4515).

108. The composition of any one of embodiments 5-94 or 96-107, wherein the peptide comprises the amino acid sequence of KTINGHDpSPHKSGQNQ (SEQ ID NO: 4516).

109. The composition of any one of embodiments 5-94 or 96-107, wherein the peptide comprises the amino acid sequence of YLSKTINGHDpSPHKSGQNQQTLKFS (SEQ ID NO: 4517).

110. The composition of embodiment 1-109, wherein the ligand is a conjugate comprising at least 2-5, e.g., at least 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 38-109, wherein the conjugate comprises a chemical linkage, e.g., succinimidyl ester or biotin.

111. The composition of any one of embodiments 1-110, wherein the ligand is a fusion protein comprising at least 2-5, e.g., at least 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 3-108, wherein each protein or peptide of the fusion protein is connected directly or via a linker. 112. The composition of any one of embodiments 1-111, wherein the peptide or protein is identified using phage-display.

113. The composition of any one of embodiments 1-37, wherein the ligand is or comprises an aptamer.

114. The composition of embodiment 113, wherein the aptamer binds to human, murine, or NHP ALPL.

115. The composition of embodiment 113 or 114, wherein the aptamer is or comprises DNA, RNA, modified DNA, modified RNA, or a combination thereof.

116. The composition of any one of embodiment 114-115, wherein the aptamer is fused or coupled to a therapeutic agent chosen from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

117. The composition of any one of embodiments 1-37, wherein the ligand is or comprises an antibody molecule that binds to the GPI anchored protein, e.g., ALPL.

118. The composition of embodiment 117, wherein the antibody molecule comprises a full antibody or an antigen binding fragment.

119. The composition of embodiment 117 or 118, wherein the antigen binding fragment is a Fab or a Fab fragment, a F(ab)2 fragment, an Fv fragment, dAb fragment, a single chain antibody (scFv) or a scFv fragment, an antibody variable region, a diabody, a VHH, a camelid antibody, a single domain antibody or a nanobody.

120. The composition of any one of embodiments 117-119, wherein the antibody molecule is a monospecific antibody, a multispecific antibody, e.g., a bispecific or biparatopic antibody.

121. The composition of any one of embodiments 117-120, wherein the antibody molecule is a human antibody, a humanized antibody, a chimeric antibody, a phage display antibody, a recombinant antibody, a murine antibody.

122. The composition of any one of embodiments 117-121, wherein the antibody molecule comprises a half-life extender. 123. The composition of any one of embodiments 117-122, wherein the variable domain of the antibody molecule binds to ALPL, e.g., human ALPL.

124. The composition of any one of embodiments 117-123, wherein the antibody molecule is an antibody as provided in Table 40 (e.g., Ab 9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, 2F4, or a variant thereof.

125. The composition of any of any one of embodiments 117-124, wherein the antibody molecule binds the same or substantially the same epitope as any one of an antibody as provided in Table 40 (e.g., Ab 9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, or 2F4.

126. The composition of any of any one of embodiments 117-125, wherein the antibody molecule competes for binding with any one of an antibody as provided in Table 40 (e.g., Ab 9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, or 2F4.

127. The composition of any one of 117-126, which further comprises a therapeutic antibody molecule, e.g., a multispecific antibody comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

128. A multispecific antibody molecule comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

129. The multispecific antibody molecule of embodiment 128, wherein the first and/or second binding domain is a full-length antibody, or an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv, a single chain Fv (scFv), a single domain antibody, a half-arm antibody, a diabody (dAb), a bivalent antibody, a bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody).

130. The multispecific antibody molecule of embodiment 128 or 129, wherein:

(i) the anti-ALPL binding domain is an Fab and the second binding domain is an scFv;

(ii) the anti-ALPL binding domain is an Fab and the second binding domain is an Fab;

(iii) the anti-ALPL binding domain is an scFv and the second binding domain is an scFv; or

(iv) the anti-ALPL binding domain is an scFv and the second binding domain is an Fab.

131. The multispecific antibody molecule of any one of embodiments 128-130, wherein the multispecific antibody molecule comprises an immunoglobulin constant region (e.g., an Fc region). 132. The multispecific antibody molecule of embodiment 131, wherein the immunoglobulin constant region (e.g., an Fc region) is linked (e.g., covalently linked) to the first and/or the second binding domain.

133. The multispecific antibody molecule of any one of embodiments 128-132, wherein the first and/or second binding domain comprises a light chain constant region chosen from the light chain constant region of kappa or lambda, or a fragment thereof.

134. The multispecific antibody molecule of any one of embodiments 128-133, wherein the first binding domain and the second binding domain comprise a common light chain variable region.

135. The multispecific antibody molecule of any one of embodiments 128-134, comprising a dimerization domain, e.g., an interface of a first and second immunoglobulin chain constant regions (e.g., Fc regions).

136. The multispecific antibody molecule of embodiment 135, wherein the dimerization domain is engineered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface.

137. The multispecific antibody molecule of embodiment 136, wherein the dimerization of the immunoglobulin chain constant regions (e.g., Fc regions) is enhanced by providing an Fc interface of a first and a second Fc regions with one or more of: a paired cavity-protuberance ("knob-in-a hole"), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer :homomultimer forms, e.g., relative to a non-engineered interface.

138. The multispecific antibody molecule of any one of embodiments 135-137, wherein the immunoglobulin chain constant region (e.g., Fc region) comprises an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgGl.

139. The multispecific antibody molecule of any one of embodiments 135-138, wherein the immunoglobulin chain constant region (e.g., Fc region) comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protuberance or knob), or a combination thereof. 140. The multispecific antibody molecule of any one of embodiments 128-139, wherein: the anti- ALPL binding domain comprises a first polypeptide and a second polypeptide, and the second binding domain comprises a third polypeptide and a fourth polypeptide, wherein:

(i) the first polypeptide comprises, e.g., from N-terminal to C-terminal: a first heavy chain variable region (VH), a first heavy chain constant region 1 (CHI), and a first Fc region that promotes association between the first and third polypeptides, wherein the first Fc region comprises a first heavy chain constant region 2 (CH2) and a first heavy chain constant region 3 (CH3);

(ii) the second polypeptide comprises, e.g., from N-terminal to C-terminal: a first light chain variable region (VL) and a first light chain constant region (CL);

(iii) the third polypeptide comprises, e.g., from N-terminal to C-terminal: a second heavy chain variable region (VH), a second heavy chain constant region 1 (CHI), and a second Fc region that promotes association between the first and third polypeptides, wherein the second Fc region comprises a second heavy chain constant region 2 (CH2) and a second heavy chain constant region 3 (CH3); and

(iv) the fourth polypeptide comprises, e.g., from N-terminal to C-terminal: a second light chain variable region (VL) and a second light chain constant region (CL).

141. The multispecific antibody molecule of any one of embodiments 128-139, wherein:

(i) the anti-ALPL binding domain (e.g., an anti-ALPL Fab or scFv), is situated N-terminal relative to the second binding domain that binds to a therapeutic target (e.g., a Fab or scFv); or

(ii) the second binding domain that binds to a therapeutic target (e.g., a Fab or scFv) is situated N-terminal relative to the anti-ALPL binding domain (e.g., an anti-ALPL Fab or scFv), optionally wherein an Fc region is situated between the anti-ALPL binding domain binding domain and the second binding domain that binds to a therapeutic target.

142. The multispecific antibody molecule of any one of embodiments 128-141, wherein the Fc region of the first and/or second binding domain:

(i) has reduced affinity, e.g., ablated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor;

(ii) comprises a mutation at one, two, or all of positions 1253 (e.g., I253A), H310 (e.g., H310A or H310Q), and/or H435 (e.g., H435A or H435Q), numbered according to the EU index as in Kabat;

(iii) has reduced effector function (e.g., reduced ADCC), compared to a reference wherein the reference is a wild-type Fc receptor;

(iv) comprises a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index as in Kabat. 143. The multispecific antibody molecule of any one of embodiments 128-142, wherein the therapeutic target comprises:

(i) a CNS related target, e.g., an antigen associated with a neurological or neurodegenerative disorder, e.g., P-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and/or synuclein;

(ii) a muscular or neuromuscular related target, e.g., an antigen associated with a muscular or neuromuscular disorder; or

(iii) a neuro-oncology related target, e.g., an antigen associated with a neuro-oncological disorder, e.g., HER2, or EGFR (e.g., EGFRvIII).

144. The composition of any one of embodiments 1-37, wherein ligand is or comprises a first Fc polypeptide.

145. The composition of embodiment 144, wherein the first Fc polypeptide is fused or coupled to an active agent comprising a second Fc polypeptide.

146. The composition of embodiment 145, wherein the first Fc polypeptide and the second Fc polypeptide form a dimer.

147. The composition of embodiment 145 or 146, wherein the second Fc polypeptide is fused or coupled (e.g., directly or indirectly via a linker) to a therapeutic protein or variant thereof (e.g., an enzyme).

148. The composition of any one of embodiments 145-147, wherein the second Fc polypeptide is covalently linked to the therapeutic protein or variant thereof.

149. The composition of any one of embodiments 145-148, wherein the second Fc polypeptide is connected to the therapeutic protein or variant thereof indirectly via a linker.

150. The composition of embodiment 149, wherein the linker is a peptide linker (e.g., a flexible peptide linker (e.g., a glycine-serine linker) or a peptide linker sensitive to a protease), a cleavable linker (e.g., a pH sensitive linker or an enzyme sensitive linker), or a non-cleavable linker (e.g., a linker comprising a thioether group or a maleimidocaproyl group).

151. The composition of embodiment 149 or 150, wherein the linker is a glycine-serine linker, e.g., a G4S linker (SEQ ID NO: 6407) or a (G4S)2 linker (SEQ ID NO: 6408). 152. The composition of any one of embodiments 147-151, wherein the therapeutic protein is present at the N-terminus of the second Fc polypeptide.

153. The composition of any one of embodiments 147-151, wherein the therapeutic protein is present at the C-terminus of the second Fc polypeptide.

154. The composition of any one of embodiments 147-153, wherein the therapeutic protein or functional variant thereof is associated with (e.g., aberrantly expressed in) a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder.

155. The composition of any one of embodiments 147-154, wherein the therapeutic protein or functional variant thereof is chosen from apolipoprotein E (APOE) (e.g., ApoE2, ApoE3 and/or ApoE4); human survival of motor neuron (SMN) 1 or SMN2; glucocerebrosidase (GBA1); aromatic L-amino acid decarboxylase (AADC); aspartoacylase (ASPA); tripeptidyl peptidase I (CLN2); betagalactosidase (GLB1); N-sulphoglucosamine sulphohydrolase (SGSH); N-acetyl-alpha- glucosaminidase (NAGLU); iduronate 2-sulfatase (IDS); intracellular cholesterol transporter (NPC1); or gigaxonin (GAN).

156. The composition of any one of embodiments 144-155, wherein the first Fc polypeptide is fused or coupled to a second therapeutic protein or variant thereof, e.g., an enzyme, optionally wherein the therapeutic protein or variant thereof is fused or coupled to the N-terminus or the C-terminus of the first Fc polypeptide.

157. The composition of any one of embodiments 145-156, wherein the first Fc polypeptide and the second Fc polypeptide comprise a dimerization domain, e.g., an interface of a first and second Fc polypeptides.

158. The composition of embodiment 157, wherein the dimerization domain is engineered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface.

159. The composition of embodiment 158, wherein the dimerization of the first Fc polypeptide and the second Fc polypeptide is enhanced by providing an Fc interface of the first and a second Fc polypeptides with one or more of: a paired cavity-protuberance ("knob-in-a hole"), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer:homomultimer forms, e.g., relative to a non-engineered interface. 160. The composition of any one of embodiments 145-159, wherein the first Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof).

161. The composition of any one of embodiments 145-160, wherein the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob).

162. The composition of any one of embodiment 145-161, wherein the first Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof); and the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob).

163. The composition of any one of embodiments 145-162, wherein the second Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof).

164. The composition of any one of embodiments 145-159 or 163, wherein the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob).

165. The composition of any one of embodiment 145-159, 163, or 164, wherein the second Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof); and the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob).

166. The composition of any one of embodiments 145-165, wherein the first Fc polypeptide, the second Fc polypeptide, or both:

(i) has reduced affinity, e.g., ablated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor;

(ii) comprises a mutation at one, two, or all of positions 1253 (e.g., I253A), H310 (e.g., H310A or H310Q), and/or H435 (e.g., H435A or H435Q), numbered according to the EU index as in Kabat;

(iii) has reduced effector function (e.g., reduced ADCC), compared to a reference wherein the reference is a wild-type Fc receptor;

(iv) comprises a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index as in Kabat. 167. The composition of any one of embodiments 145-166, wherein the first Fc polypeptide, the second Fc polypeptide, or both comprises a half-life extender or an amino acid modification that increases serum half-life (e.g., (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to EU numbering).

168. The composition of any one of embodiments 144-167, wherein the first Fc polypeptide comprises the protein or peptide according to any one of embodiments 35-84.

169. The composition of any one of embodiments 168, wherein the protein or peptide is present in the CH3 domain of the first Fc polypeptide.

170. The composition of embodiment 169, wherein the CH3 domain is modified from a human IgGl, IgG2, IgG3, or IgG4 CH3 domain.

171. The composition of embodiment 169 or 170, wherein the CH3 domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions in a set of amino acid positions comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, according to EU numbering.

172. The composition of any one of embodiments 168-171, wherein the protein or peptide is present at or near the C-terminus of the first Fc polypeptide (e.g., within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids from the C-terminus of the therapeutic protein, enzyme, or antibody molecule).

173. The composition of any one of embodiments 145-172, wherein the first Fc polypeptide, the second Fc polypeptide or both the first Fc polypeptide and the second Fc polypeptide does not comprise an immunoglobulin heavy and/or light chain variable region sequence or an antigen-binding portion thereof.

174. The composition of embodiment 1-11 or 15-37, wherein the ligand is a component of a viral particle, e.g., an AAV particle or a lentivirus.

175. The composition of any one of embodiments 1-11, 15-37, or 174, wherein the ligand is a component of a capsid protein, e.g., an AAV capsid protein.

176. The composition of any one of embodiments 1-11, 15-37, 174, or 175, wherein the ligand is a component of an AAV9 capsid or variant thereof. 177. The composition of any one of embodiments 1-11, 15-37, or 174-176, wherein the ligand is an AAV9 capsid variant comprising a modification, e.g., a substitution, insertion, and/or deletion, in loop IV of AAV9.

178. The composition of any one of embodiments 1-11, 15-37, or 174-176, wherein ligand is an AAV9 capsid variant comprising the amino acid sequence of any one of embodiments 35-84.

179. The composition of any one of embodiments 1-11, 15-37, or 174, wherein the ligand is a lentiviral particle, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the lentiviral particle comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides, e.g., ALPL -binding peptides, or a peptide or protein according to any one of embodiments 38-109.

180. The composition of embodiment 1-37, wherein the ligand is a small molecule.

181. The composition of embodiment 180, wherein the small molecule is an inhibitor of ALPL, e.g., a small molecule that interferes with ALPL dimerization.

182. The composition of embodiment 180 or 181, wherein the small molecule is an aryl sulfonamide, a phosphonate derivative, a pyrazole, a triazole, or an imidazole, optionally wherein the small molecule is 2,5-Dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (Tissue -Nonspecific Alkaline Phosphatase Inhibitor (TNAPi)) or 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI- 425).

183. The composition of any one of the preceding embodiments, wherein binding to ALPL results in increased cellular transduction, e.g., as compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by a transduction assay or binding/internalization assay as described (e.g., as described in Example 8).

184. The composition of any one of the preceding embodiments, wherein binding to ALPL results in increased crossing of the blood brain barrier, e.g., as compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by a transduction assay or binding/internalization assay as described (e.g., as described in Example 8).

185. The composition of any one of embodiments 1-127 or 144-184, wherein the therapeutic agent or diagnostic agent is an antibody molecule or an Fc polypeptide. 186. The composition of embodiment 185, wherein the antibody molecule comprises a full antibody or an antigen binding fragment.

187. The composition of embodiment 186, wherein the antigen binding fragment is a Fab or a Fab fragment, a F(ab)2 fragment, an Fv fragment, dAb fragment, a single chain antibody (scFv) or a scFv fragment, an antibody variable region, a diabody, a VHH, a camelid antibody, a single domain antibody or a nanobody.

188. The composition of any one of embodiments 185-187, wherein the antibody molecule is a monospecific antibody, a multispecific antibody, e.g., a bispecific or biparatopic antibody.

189. The composition of any one of embodiments 185-188, wherein the antibody molecule is a human antibody, a humanized antibody, a chimeric antibody, a phage display antibody, a recombinant antibody, a murine antibody.

190. The composition of any one of embodiments 185-189, wherein the antibody molecule is an antibody-drug conjugate.

191. The composition of embodiment 190, wherein the antibody molecule is conjugated to a cytotoxic or cytostatic agent, e.g., a chemotherapeutic agent or an anti-neoplastic drug.

192. The composition of any one of embodiments 185-189, wherein the antibody molecule is coupled to a radioactive isotope, e.g., a-, P-, or y-emitter, or P-and y-emitter.

193. The composition of any one of embodiments 185-192, wherein the antibody molecule comprises an Fc region, which comprises amino acid modifications that increase serum half-life.

194. The composition of embodiment 193, wherein the amino acid modifications that increase serum half-life comprise (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to EU numbering.

195. The composition of embodiment 193 or 194, wherein the Fc region of the antibody molecule:

(i) has reduced affinity, e.g., ablated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor;

(ii) comprises a mutation at one, two, or all of positions 1253 (e.g., I253A), H310 (e.g., H310A or H310Q), and/or H435 (e.g., H435A or H435Q), numbered according to the EU index as in Kabat; (iii) has reduced effector function (e.g., reduced ADCC), compared to a reference wherein the reference is a wild-type Fc receptor;

(iv) comprises a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index as in Kabat.

196. The composition of any one of embodiments 185-195, wherein the antibody molecule binds:

(i) a CNS related target, e.g., an antigen associated with a neurological or neurodegenerative disorder, e.g., P-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and/or synuclein;

(ii) a muscular or neuromuscular related target, e.g., an antigen associated with a muscular or neuromuscular disorder; or

(iii) a neuro-oncology related target, e.g., an antigen associated with a neuro-oncological disorder, e.g., HER2, or EGFR (e.g., EGFRvIII).

197. The composition of any one of embodiments 1-127 or 144-118565, wherein the ligand is present or coupled to a carrier, e.g., an exosome, a microvesicle, or a lipid nanoparticle (LNP).

198. The composition of embodiment 197, wherein the carrier is an exosome or a LNP.

199. The composition of embodiment 197 or 198, wherein the ligand is present on the surface of the carrier.

200. The composition of any one of embodiments 197-199, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the carrier comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35-84.

201. The composition of any one of embodiments 197-200, wherein the carrier comprises a therapeutic agent.

202. The composition of any one of embodiments 197-201, wherein the carrier comprises an RNAi agent, an mRNA, a ribonucleoprotein complex (e.g., a Cas9/gRNA complex), or a circRNA.

203. The composition of any one of embodiments 197-202, wherein the ligand is conjugated to the surface of the carrier by post-insertion. 204. The composition of any one of embodiments 197-202, wherein the ligand is conjugated to the surface of the carrier via a covalent bond (e.g., using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry or thiol-maleimide linkage reactions).

205. The composition of any one of embodiments 1-127 or 144-184, wherein the ligand is coupled to an RNAi agent directly or via a linker.

206. The composition of embodiment 205, wherein the RNAi agent is a dsRNA, a siRNA, a shRNA, a pre-miRNA, a pri-miRNA, a miRNA, a stRNA, a IncRNA, a piRNA, an antisense oligonucleotide agent (ASO), or a snoRNA.

207. The composition of embodiment 205 or 206, wherein the RNAi agent is a siRNA or an ASO.

208. The composition of embodiment 206 or 207, wherein the siRNA or the ASO comprises at least one modified nucleotide.

209. The composition of any one of embodiment 206-208, wherein not more than five of the sense strand nucleotides of the siRNA and not more than five of the nucleotides of the antisense strand of the siRNA are unmodified nucleotides.

210. The composition of any one of embodiment 206-209, wherein all of the nucleotides of the sense strand of the siRNA and all of the nucleotides of the antisense strand of the siRNA are modified.

211. The composition of any one of embodiment 206-208, wherein not more than five of the nucleotides of ASO are unmodified nucleotides.

212. The composition of any one of embodiment 206-208 or 211, wherein all of the nucleotides of the ASO are modified.

213. The composition of any one of embodiment 208-312, wherein the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3 ’-terminal deoxythimidine (dT) nucleotide, a 2’-O-methyl modified nucleotide, a 2’-fluoro modified nucleotide, a 2’-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl- modified nucleotide, 2’-C-alkyl-modified nucleotide, a 2 ’-methoxy ethyl modified nucleotide, a 2’-O- alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5 ’-phosphate, a nucleotide comprising a 5 ’-phosphate mimic, a glycol modified nucleotide, and a 2-0-(N- methylacetamide) modified nucleotide; and combinations thereof.

214. The composition of any one of embodiments 205-213, wherein the RNAi agent modulates, e.g., inhibits, expression of, a CNS related gene, mRNA, and/or protein.

215. The composition of embodiment 214, wherein the CNS gene is chosen from SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, SCN8A-SCN11A, SMN, or a combination thereof.

216. The composition of any one of embodiments 205-215, wherein the ligand comprises the protein or peptide according to any one of embodiments 35-84.

217. The composition of embodiments 205-216, wherein the ligand comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35-84.

218. The composition of embodiment 216 or 217, wherein the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides are present in tandem (e.g., connected directly or indirectly via a linker) or in a multimeric configuration.

219. The composition of any one of embodiments 216-218, wherein the protein or peptide comprises an amino acid sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or 35 amino acids in length.

220. The composition of embodiment of 219, wherein the protein or peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of the amino acids LKFSVAGPSNMAVQG (SEQ ID NO: 21).

221. The composition of any one of embodiments 205-221, wherein the ligand is covalently linked, e.g., directly or indirectly via a linker to the RNAi agent.

222. The composition of any one of embodiments 205-221, wherein the ligand is conjugated, e.g., directly or indirectly via a linker to the RNAi agent. 223. The composition of any one of embodiments 205-222, wherein the ligand is conjugated to the RNAi agent via a linker, e.g., a crosslinker.

224. The composition of embodiment 223, wherein the crosslinker comprises succinimidyl-4-(N- maleimidomethyl) and/or a saturated or unsaturated hydrocarbon chain (e.g., cyclohexane- 1- carboxylate).

225. The composition of embodiment 223 or 224, wherein the crosslinker comprises succinimidyl-4- (N-maleimidomethyl) cyclohexane- 1 -carboxylate.

226. The composition of any one of embodiments 205-224, wherein the ligand is conjugated to the RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

227. The composition of any one of embodiments 205-226, wherein the ligand is conjugated, e.g., directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent.

228. The composition of any one of embodiments 205-226, wherein the ligand is conjugated, e.g., directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent.

229. The composition of any one of embodiments 205-226, wherein the ligand is conjugated, e.g., directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent.

230. The composition of any one of embodiments 227-229, wherein the at least one strand of the RNAi agent is the sense strand.

231. The composition of any one of embodiments 205-230, wherein the composition further comprises a lipophilic moiety.

232. The composition of embodiment 231, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

233. The composition of embodiment 231 or 232, wherein the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. 234. The composition of any one of embodiments 231-233, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

235. The composition of embodiment 234, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain, e.g., a saturated or unsaturated C16 hydrocarbon chain.

236. The composition of any one of embodiments 231-235, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) of the iRNA agent, e.g., the siRNA or ASO.

237. The composition of embodiment 236, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

238. The composition of any one of embodiments 231-237, wherein the lipophilic moiety is conjugated to the RNAi agent, e.g., the siRNA or ASO, via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

239. The composition of any one of embodiments 231-238, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

240. The composition of any one of embodiments 231-239, wherein the lipophilic moiety is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

241. The composition of any one of embodiments 231-240, wherein the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent. 242. The composition of any one of embodiments 231-241, wherein the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent.

243. The composition of any one of embodiments 231-242, wherein the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent.

244. The composition of any one of embodiments 241-243, wherein the at least one strand of the RNAi agent is the sense strand.

245. The composition of any one of embodiments 231-244, wherein the ligand and the lipophilic moiety are present on the same strand, e.g., the sense strand.

246. The composition of any one of embodiments 231-244, wherein the ligand and the lipophilic moiety are present on different strands.

247. The composition of any one of embodiments 206-246, wherein a 3’ end of a sense strand of a siRNA agent is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

248. The composition of any one of embodiments 205-247, wherein the composition further comprises an N-acetylgalactosamine (GalNAc) conjugate.

249. The composition of embodiment 248, wherein the GalNAc conjugate is attached through a monovalent linker; or a bivalent, trivalent, or tetravalent branched linker.

250. The composition of any one of embodiments 1-127 or 144-184, wherein the active agent is a diagnostic agent.

251. The composition of embodiment 250, wherein the diagnostic agent is or comprises an imaging agent (e.g., a protein or small molecule compound coupled to a detectable moiety).

252. The composition of embodiment 251, wherein the imaging agent comprises a PET or MRI ligand, or an antibody molecule coupled to a detectable moiety. 253. The composition of embodiment 252, wherein the detectable moiety is or comprises a radiolabel, a fluorophore, a chromophore, or an affinity tag.

254. The composition of embodiment 253, wherein the radiolabel is or comprises tc99m, iodine-123, a spin label, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen- 15, oxygen-17, gadolinium, manganese, or iron.

255. A vector comprising a polynucleotide encoding the ligand of any one of embodiments 1-127 or 144-184.

256. A cell comprising the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the vector of embodiment 255, optionally wherein the cell is a mammalian cell, a cell of the central nervous system, or and/or a cell present in the blood brain barrier.

257. A method of making the composition of any one of embodiments 1-127 or 144-254, comprising:

(i) providing the ligand that binds to the GPI anchored protein, e.g., ALPL, and the active agent; and

(ii) incubating the ligand and active agent under conditions suitable to fuse or couple the ligand to the active agent, thereby generating the composition.

258. A pharmaceutical composition comprising the composition of any one of embodiments 1-127 or 144-254 or the multispecific antibody molecule of any one of embodiments 128-143, and a pharmaceutically acceptable excipient.

259. A method of delivering an active agent, e.g., a therapeutic agent or a diagnostic agent, to a cell or tissue (e.g., a CNS cell or a CNS tissue), comprising administering the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128- 143, or the pharmaceutical composition of embodiment 258.

260. The method of embodiment 259, wherein the cell is a cell of a brain region or a spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate, cerebellar cortex, cerebral cortex, brain stem, hippocampus, or thalamus.

261. The method of embodiment 259 or 260, wherein the cell or tissue is in a subject. 262. A method of increasing central nervous system transduction (e.g., increased crossing of the blood brain barrier) in a subject comprising administering the composition of any one of embodiments 1- 127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258.

263. The method of embodiment 261 or 262, wherein the subject has, has been diagnosed with having, or is at risk of having a genetic disorder, e.g., a monogenic disorder or a polygenic disorder.

264. The method of any one of embodiments 261-263, wherein the subject has, has been diagnosed with having, or is at risk of having a neurological, e.g., a neurodegenerative disorder.

265. The method of any one of embodiments 261-264, wherein the subject has, has been diagnosed with having, or is at risk of having a neuro-oncological disorder.

266. The method of any one of embodiments 261-265, wherein the subject has, has been diagnosed with having, or is at risk of having a muscular disorder or a neuromuscular disorder.

267. A method of treating a subject having or diagnosed with having a genetic disorder, e.g., a monogenic disorder or a polygenic disorder, comprising administering to the subject the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258.

268. A method of treating a subject having or diagnosed with having a neurological disorder, e.g., a neurodegenerative disorder, comprising administering to the subject an effective amount of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258.

269. A method of treating a subject having or diagnosed with having a muscular disorder or a neuromuscular disorder, comprising administering to the subject an effective amount of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258.

270. A method of treating a subject having or diagnosed with having a neuro-oncological disorder, comprising administering to the subject an effective amount of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128- 143, or the pharmaceutical composition of embodiment 258. 271. The method of any one of embodiments 263-270, wherein the genetic disorder, neurological disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, or neuro- oncological disorder is Huntington’s Disease, Amyotrophic Lateral Sclerosis (ALS), Gaucher Disease, Dementia with Lewy Bodies, Parkinson’s disease, Spinal Muscular Atrophy, Alzheimer’s Disease, a leukodystrophy (e.g., Alexander disease, autosomal dominant leukodystrophy with autonomic diseases (ADLD), Canavan disease, cerebrotendinous xanthomatosis (CTX), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease, or Refsum disease), or a cancer (e.g., a HER2/neu positive cancer or a glioblastoma).

272. The method of any one of embodiments 267-271, where treating comprises prevention of progression of the disease or disorder in the subject.

273. The method of embodiment 261-272, wherein the subject is a human.

274. The method of any one of embodiments 261-273, wherein the composition is administered to the subject intravenously, via intra-cisterna magna injection (ICM), intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, intraarterially, or intramuscularly.

275. The method of any one of embodiments 261-274, wherein the composition is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

276. The method of any one of embodiments 261-275, wherein the composition is administered to the subject intravenously.

277. The method of any one of embodiments 261-276, wherein the composition is administered to the subject via intra-cisterna magna injection (ICM).

278. The method of any one of embodiments 261-277, wherein the composition is administered to the subject intraarterially.

279. The method of any one of embodiments 274-278, wherein administration of the composition results in a decreased presence, level, and/or activity of a gene, mRNA, protein, or combination thereof. 280. The method of any one of embodiments 274-278, wherein administration of the composition results in an increased presence, level, and/or activity of a gene, mRNA, protein, or a combination thereof.

281. The composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, for use in a method of delivering a payload to a cell or tissue.

282. The composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, for use in a method of treating a genetic disorder, a neurological disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, or a neuro-oncological disorder.

283. The composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, for use in the manufacture of a medicament.

284. The composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, for use in a method of increasing central nervous system transduction (e.g., increased crossing of the blood brain barrier) in a subject.

285. Use of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, in the manufacture of a medicament.

286. Use of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, in the manufacture of a medicament for treating a genetic disorder, a neurological disorder, a neurodegenerative disorder, a muscular disorder, a neuromuscular disorder, or a neuro-oncological disorder.

287. Use of the composition of any one of embodiments 1-127 or 144-254, the multispecific antibody molecule of any one of embodiments 128-143, or the pharmaceutical composition of embodiment 258, in the manufacture of a medicament for increasing central nervous system transduction (e.g., increased crossing of the blood brain barrier) in a subject. [0015] The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Certain terms are defined in the Definition section and throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] FIG. 1A is a violin plot showing expression level of the payload on the Y-axis in various cell types as shown on the X-axis, which includes from left to right, microglia, astrocytes, endothelial cells subset 1, vascular smooth cells, pericytes, endothelial cells subset 2, committed oligodendrocytes, macrophages, vascular and leptomeningeal cells, committed oligodendrocytes subset 2, and mature oligodendrocytes. FIG. 1A relates to data shown in Example 7 and Table 22. FIG. IB is a violin plot showing expression level of ALPL on the Y-axis in various cell types as shown on the X-axis, which includes from left to right, microglia, astrocytes, endothelial cells subset 1, vascular smooth cells, pericytes, endothelial cells subset 2, committed oligodendrocytes, macrophages, vascular and leptomeningeal cells, committed oligodendrocytes subset 2, and mature oligodendrocytes .

[0018] FIG. 2A is a graph showing TTM-002 binding to ALPL at increasing concentrations of AAV by surface plasmon resonance (SPR) over time. FIG. 2B is a graph showing AAV9 binding to ALPL at increasing concentrations of AAV by SPR over time. FIG. 2C is a graph showing ALPL binding to TTM-002 at increasing concentrations of ALPL by surface plasmon resonance (SPR) over time. FIG. 2D is a graph showing ALPL binding to AAV9 at increasing concentrations of ALPL by SPR over time.

[0019] FIG. 3A is a graph showing ALPL binding to TTM-002 at increasing concentrations of ALPL by surface plasmon resonance (SPR) over time at a pH of 7.4, where the left half of the graph shows the association and the right half of the graph shows the dissociation. FIG. 3B is a graph showing ALPL binding to TTM-002 at increasing concentrations of ALPL by surface plasmon resonance (SPR) over time at a pH of 5.5, where the left half of the graph shows the association and the right half of the graph shows the dissociation.

[0020] FIG. 4 is a graph showing the luciferase activity (RLU) as a measure of TTM-002 (right side of graph) or AAV9 (left side of graph) at 24-hours post-transduction and 48-hours posttransfection with siRNA 1 , 2 or both siRNA 1 and 2 targeting ALPL or a non-ALPL control siRNA that did not knockdown ALPL.

[0021] FIGs. 5A-5C are a series of graphs demonstrating the effects of the small molecule inhibitor, TNAPi, of the ALPL receptor on TTM-002 transduction in vitro in HeLa cells expressing ALPL. FIG. 5A is a graph showing luciferase activity as a measure of transduction of the TTM-002 capsid variant or the AAV9 capsid variant in the present of increasing concentrations of the TNAPi inhibitor. The concentrations tested include, from left to right on the X-axis, 0 nM (no inhibitor control), 24 nM, 48 nM, 95 nM, 190 nM, and 380 nM. FIG. 5B a graph showing luciferase activity as a measure of transduction of the TTM-002 capsid variant in the presence of increasing concentration of the TNAPi inhibitor or the DMSO vehicle control. The concentrations tested include, from left to right on the X-axis, 0 nM, 0.019 nM, 0.19 nM, 1.9 nM, 19 nM, and 190 nM. FIG. 5C is a graph showing the IC50 of the TNAPi inhibitor compared to the vehicle control for the TTM-002 capsid variant.

[0022] FIGs. 6A-6C are a series of graphs demonstrating the effects of the small molecule inhibitor, SBI-425, of the ALPL receptor on TTM-002 transduction in vitro in HeLa cells expressing ALPL.FIG. 6A is a graph showing luciferase activity as a measure of transduction of the TTM-002 capsid variant in the presence of increasing concentration of the SBI-425 inhibitor or the DMSO vehicle control. The concentrations tested include, from left to right on the X-axis, 0 nM, 0.00019 nM, 0.0019 nM, 0.019 nM, 0.19 nM, 1.9 nM, or 19.0 nM. FIG. 6B is a graph showing luciferase activity as a measure of transduction of the AAV9 capsid control in the presence of increasing concentration of the SBI-425 inhibitor or the DMSO vehicle control. The concentrations tested include, from left to right on the X-axis, 0 nM, 0.00019 nM, 0.0019 nM, 0.019 nM, 0.19 nM, 1.9 nM, or 19.0 nM. FIG. 6C is a graph showing the IC50 of the SNBI-425 inhibitor compared to the vehicle control for the TTM-002 capsid variant.

[0023] FIG. 7A is a series of graphs demonstrating ALPL binding to GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or GSGSKTINGHDpSPHKSGQNQ (SEQ ID NO: 4513) (right graph) at increasing concentrations of ALPL by SPR over time (seconds). FIG. 7B is a series of graphs demonstrating ALPL binding to GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or GSGSNGHDpSPHKSG (SEQ ID NO: 4512) (right graph) at increasing concentrations of ALPL by SPR over time (seconds).

[0024] FIG. 8A is a series of graphs demonstrating the binding of GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or GSGSKTINGHDpSPHKSGQNQ (SEQ ID NO: 4513) (right graph) to ALPL at increasing concentrations of said peptides by SPR over time (seconds). FIG. 8B is a series of graphs demonstrating binding of GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or GSGSNGHDpSPHKSG (SEQ ID NO: 4512) (right graph) to ALPL at increasing concentrations of said peptides by SPR over time (seconds).

[0025] FIG. 9A is a series of graphs showing binding of GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or GSGSKTINGHDpSPHKSGQNQ (SEQ ID NO: 4513) (right graph) to ALPL over time by Bio Layer Interferometry (BLI)/Octet. FIG. 9B is a series of graphs showing binding of GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or GSGSNGHDpSPHKSG (SEQ ID NO: 4512) (right graph) to ALPL over time by Bio Layer Interferometry (BLI)/Octet.

[0026] FIG. 10A is a graph showing the binding (OD450) to ALPL to increasing concentrations of GSGSNGHDSPHKSG (SEQ ID NO: 4500) or GSGSNGHDpSPHKSG (SEQ ID NO: 4512) (pg/mL) by ELISA. FIG. 10B is a graph showing the binding (OD450) to ALPL to increasing concentrations of GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) or GSGSKTINGHDpSPHKSGQNQ (SEQ ID NO: 4513) (pg/mL) by ELISA.

[0027] FIG. 11A is a graph depicting the antibody concentration in the top of the chamber prior to the transcytosis assay measured in pg/ml. The antibodies from left to right on the X-axis include: PT3 (non- ALPL binding control), MOPC (isotype control), Ab 9 (ALPL binding antibody), and Ab 22 (ALPL binding antibody). FIG. 1 IB is a graph showing the antibody concentration in the bottom chamber (pg/ml) for the antibodies indicated on the X-axis. FIG. 11C is a graph showing the percentage of the antibody detected in the bottom chamber relative to the load for the antibodies indicated on the X-axis. The left portion of the graph (labeled “MDCK ALPL Single Clone”) depicts the percentage in the single clone MDCK ALPL expressing cells generated in Example 8 and the right portion of the graph (labeled “MDCK”) shows the percentage in MDCK cells that do not express ALPL.

[0028] FIG. 12 is a graph showing the luciferase activity (RLU) in cells pre-incubated with the antibody to ALPL as listed on the X-axis and described in Table 40, and subsequently transduced with an AAV particle comprising a TTM-002 capsid variant and encoding a GFP luciferase transgene. Low luciferase activity measured indicates that the antibody was able to compete for binding to ALPL with the TTM-002 capsid variant.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0029] Described herein, inter alia, are compositions comprising e.g., a fusion molecule or a conjugate molecule, comprising a ligand that binds to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL); and an active agent, e.g., a therapeutic agent or a diagnostic agent. In some embodiments, the ligand is fused or coupled, e.g., covalently or non- covalently to the active agent. In some embodiments, the GPI anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mice, NHPs (e.g., Macacafascicularis), and/or humans). In some embodiments, the GPI anchored protein is present on the surface of a cell in the blood brain barrier. In some embodiments, the GPI anchored protein is ALPL, e.g., human or murine ALPL.

[0030] In some embodiments, a ligand to be used in a composition described herein is a ligand capable of binding ALPL. In some embodiments a ligand of the present disclosure is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule. In some embodiments, an active agent described herein is a therapeutic agent (e.g., a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule). In some embodiments the active agent described herein is a diagnostic agent.

[0031] Without wishing to be bound by theory, it is believed in some embodiments, fusing or coupling, e.g., covalently (e.g., directly or via a linker) or non-covalently, a ligand that can bind ALPL to an active agent increases crossing of the blood brain barrier by the active agent relative to a active agent that is not fused or coupled to a ligand that can bind ALPL. Without wishing to be bound by theory, it is believed in some embodiments, that peptides comprising the amino acid sequences provided herein, e.g., in Tables 1, 2A, 2B, 2C, 13-19 (e.g., SEQ ID NOs: 2, 941, or 943), when fused or coupled, e.g., covalently (e.g., directly or via a linker) or non-covalently to an active agent, e.g., a therapeutic agent or a diagnostic agent, can enhance blood brain barrier crossing and biodistribution in the CNS of the active agent relative to the active agent alone.

Ligands

[0032] Disclosed herein are ligands that are capable of binding a protein present on a cell, e.g., a cell present in the blood brain barrier. In some embodiments, the ligand binds a GPI anchored protein. In some embodiments, the GPI anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mice, NHPs (e.g., Macaca fascicularis), and/or humans). In some embodiments, the GPI anchored protein is alkaline phosphatase issue-nonspecific isozyme (NM_000478.4, which is incorporated by reference herein) (ALPL).

[0033] ALPL is part of a family of membrane-bound glycoproteins that hydrolyze monophosphate esters at a high pH (see, e.g., Weiss et al., Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc. Nat. Acad. Sci., 83: 7182-7186 (1986), the contents of which are hereby incorporated by reference in their entirety). ALPL is highly conserved across humans, mice, and cynomolgus macaques (Macaca fascicularis) when compared by sequence alignment (e.g., as shown in Table 24). Additionally, in humans ALPL is expressed on endothelial cells and neurons, and at a low level on astrocytes. The highest level of ALPL expression in human is on endothelial cells. In mice, ALPL is more highly expressed on astrocytes, oligodendrocyte progenitor cells (OPCs), and to a lesser extent on endothelial cells. Without wishing to be bound by theory, it is believed in some embodiments that highly conserved nature of the ALPL receptor protein across species is predictive of cross-species compatibility of the AAV capsid variants described herein.

[0034] In some embodiments, the ligand binds an ALPL protein comprising an amino acid sequence or encoded by a nucleotide sequence provided in Table 32, or a sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical thereto. In some embodiments, the ligand binds a human ALPL protein, e.g., a human ALPL protein comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical thereto. In some embodiments, the ALPL is a murine ALPL, e.g., a murine ALPL comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical thereto.

Table 32: Exemplary ALPL Sequences

[0035] In some embodiments, the GPI anchor protein described herein is CD59, LY6E, CA4, GPC5, NTM, HYAL2, LSAMP, BST2, EMP2, ALPL, CPM, NCAM1, EFNA1, PIBF1, SEC24B, PRNP, TFPI, OPCML, CD109, DPM3, CNTN4, PIGN, HBP1, CNTN2, CD55, NEGRI, EFNA5, RECK, NRN1, CNTN1, GPAA1, PGAP1, PIGF, PIGK, MDGA2, DPMI, SVIP, NTNG1, CNTN5, GPC6, PIGG, TMEM8A, THY1, GPIHBP1, PIGT, PIGL, ZFAND2B, PLAUR, DPM2, or GPC1.

[0036] In some embodiments, the ligand is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule.

[0037] In some embodiments, the ligand is not a component of a viral particle, e.g., an AAV viral particle. In some embodiments, the ligand is not a component of a capsid protein, e.g., an AAV capsid protein.

[0038] In some embodiments, the ligand is covalently attached, e.g., directly or indirectly via a linker, to an active agent described herein (e.g., a therapeutic agent or a diagnostic agent). In some embodiments, the ligand is conjugated, e.g., directly or indirectly via a linker, to an active agent described herein (e.g., a therapeutic agent or a diagnostic agent). In some embodiments, the ligand is fused to the active agent, e.g., as part of a fusion peptide or protein.

[0039] In some embodiments, the ligand is conjugated directly to an active agent described herein. In some embodiments, direct conjugation includes but is not limited to formation of a covalent bond between a reactive group on the ligand and a corresponding group or acceptor on the active agent; modification (e.g., genetic modification) of the ligand or active agent to be conjugated to a reactive group (e.g., a sulfhydryl group or a carboxyl group) that forms a covalent attachment to the other molecule to be conjugated under appropriate conditions. For example, a desired active group may be introduced to the ligand, active agent, or both and a disulfide bond may be formed.

[0040] In some embodiments, the ligand is coupled or fused, e.g., conjugated, to the active agent non-covalently, e.g., by a hydrophobic bond, an electrostatic interaction, and/or an ionic bond.

[0041] In some embodiments, the ligand is conjugated to the ligand by a linker. In some embodiments, the linker is a cleavable linker (e.g., an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide -containing linker). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is an enzyme sensitive linker or a pH sensitive linker. In some embodiments, the pH sensitive linker comprises a hydrazine/hydr azone linker or a disulfide linker. In some embodiments, the enzyme sensitive linker comprises a peptide based linker, e.g., a peptide linker sensitive to a protease (e.g., a lysosomal protease); or a beta- glucuronide linker. In some embodiments, the non-cleavable linker is a linker comprising a thioether group or a maleimidocaproyl group. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker, e.g., a flexible polypeptide. In some embodiments, the linker is a glycine serine linker. In some embodiments, the linker is a cross-linker, e.g., a cross-linker selected from BMPS, EMCS, GMBS, HBVS, LC-SM CC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfoKMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, or SVSB (succinimidyl(4-vinylsulfone)benzoate).

[0042] In some embodiments, a ligand may be conjugated to an active agent described herein using a bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidom ethyl) cyclohexane -1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate H ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene).

[0043] In some embodiments, the ligand and the active agent are fused or coupled post- translationally, e.g., using click chemistry. In some embodiments, the ligand and the active agent are fused or couple via chemically induced dimerization.

[0044] In some embodiments, a ligand may be conjugated to an active agent described herein using a method described in Shadish JA and DeForest CA, Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials. Matter 2020 2:50-70; Fu et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy 2022 7:93; and Drago et al. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol 2021 18:327-344; Eyford et al. A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood Brain Barrier to Attenuate Ischemic Stroke. Front Mol Biosci 2021 8:611367; A microfluidic method for synthesis of transferrin-lipid nanoparticle loaded with siRNA EOR-1284 for therapy of acute myeloid leukemia. Nanoscale 2014 6(16):9742-9751; or US20220125823A1; which are all hereby incorporated by reference in their entirety.

[0045] In some embodiments, the ligand is present N-terminal relative to the active agent. In some embodiments the ligand is present C-terminal relative to the active agent. In some embodiments, the ligand is fused or coupled at or near the C-terminus of the active agent, wherein the active agent is a therapeutic protein, enzyme, or antibody molecule. In some embodiments, the ligand is fused or coupled within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids from the C- terminus of the therapeutic protein, enzyme, or antibody molecule. [0046] In some embodiments, binding to ALPL results in one or both of increased cell signaling and/or transcytosis. In some embodiments, binding to ALPL results in increased crossing of the blood brain barrier, e.g., as compared to a reference sequence of SEQ ID NO: 138.

Peptides

[0047] Disclosed herein are ligands comprising peptides or proteins, for binding a protein on cell, e.g., a cell present in the blood brain barrier. In some embodiments, the protein is a GPI anchored protein. In some embodiments, the protein is ALPL, e.g., human or murine ALPL. In some, embodiments, the peptide is an isolated, e.g., recombinant, peptide. In some embodiments, the nucleic acid encoding the peptide, is an isolated, e.g., recombinant nucleic acid.

[0048] The present disclosure also provides peptides and associated AAV particles comprising an AAV capsid variant and a peptide for enhanced or improved transduction of a target cell or tissue (e.g., a cell or tissue of the CNS). In some embodiments, the peptide may increase distribution of an AAV particle to a cell, region, or tissue of the CNS. The cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or supporting cells of the brain such as immune cells (e.g., T cells). The tissue of the CNS may be, but is not limited to, the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei. In some embodiments, the peptide may increase distribution of an AAV particle to the CNS (e.g., the cortex) after intravenous administration.

[0049] In some embodiments, a peptide of a ligand described herein may vary in length. In some embodiments, the peptide is about 3 to about 20 amino acids in length. As non-limiting examples, the peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3- 15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length. In some embodiments, a peptide comprises about 6 to 12 amino acids in length, e.g., about 9 amino acids in length. In some embodiments, a peptide comprises about 5 to 10 amino acids in length, e.g., about 7 amino acids in length. In some embodiments, a peptide comprises about 7 to 11 amino acids in length, e.g., about 8 amino acids in length. In some embodiments, a peptide comprises about 4 to 9 amino acids in length, e.g., about 6 amino acids in length.

[0050] In some embodiments, a ligand described herein comprises a protein or a peptide, which comprises a sequence as set forth in Table 1 (e.g., comprising the amino acid sequence of any one of SEQ ID NOs: 200-940, 1800-2241, 2242-2886, or 2887-3076). In some embodiments, the peptide may comprise a sequence as set forth in Table 2A, 2B, or 2C. In some embodiments, the peptide may comprise a sequence set forth in Table 13 or 14. In some embodiments, the peptide may comprise a sequence as set forth in Table 15. In some embodiments, the peptide may comprise a sequence as set forth in Table 16. In some embodiments, the peptide may comprise a sequence as set forth in Table 17. In some embodiments, the peptide may comprise a sequence as set forth in Table 18. In some embodiments, the peptide may comprise a sequence as set forth in Table 19. In some embodiments, the peptide is isolated, e.g., recombinant.

Table 1. Exemplary Peptide Sequences

Table 2A. Exemplary Peptide Sequences

Table 2B. Exemplary Peptide Sequences

Table 2C. Exemplary Phosphorylated Peptide Sequences

[0051] In some embodiments, a ligand described herein comprises a protein or a peptide comprising an amino acid sequence having the formula [N1]-[N2]-[N3], wherein [N2] comprises the amino acid sequence of SPH and [N3] comprises X4, X5, and X6, wherein at least one of X4, X5, or X6 is a basic amino acid, e.g., a K or R. In some embodiments, position X4 of [N2] is K. In some embodiments, position X5 of [N2] is K.

[0052] In some embodiments, [Nl] comprises XI, X2, and X3, wherein at least one of XI, X2, or X3 is G. In some embodiments, position XI of [Nl] is independently chosen from G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C. In some embodiments, position X2 of [Nl] is independently chosen from: S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q. In some embodiments, position X3 of [Nl] is independently chosen from: G, C, L, D, E, Y, H, V, A, N, P, or S. In some embodiments, [Nl] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, Al, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS. In some embodiments, [Nl] comprises GS, SG, GH, or HD. In some embodiments [Nl] is or comprises GSG, GHD, GQD, VSG, CSG, GRG, CSH, GQS, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS. In some embodiments, [Nl] is or comprises GSG. In some embodiments, [Nl] is or comprises GHD. In some embodiments, [N1]-[N2] comprises SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758), NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [Nl]- [N2] is or comprises GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802), ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GSASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIGSPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2] is or comprises GSGSPH (SEQ ID NO: 4695). In some embodiments, [N1]-[N2] is or comprises GHDSPH (SEQ ID NO: 4784).

[0053] In some embodiments, X4, X5, or both of [N3] are K. In some embodiments, X4, X5, or X6 of [N3] is R. In some embodiments, position X4 of [N3] is independently chosen from: A, K, V, S, T, G, F, W, V, N, or R. In some embodiments, position X5 of [N3] is independently chosen from: S, K, T, F, I, L, Y, H, M, or R. In some embodiments, position X6 of [N3] is independently chosen from: G, R, A, M, I, N, T, Y, D, P, V, L, E, W, N, Q, K, or S. In some embodiments, [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG. In some embodiments, [N3] comprises SK, KA, KS, or SG. In some embodiments, [N3] is or comprises SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG. In some embodiments, [N3] is or comprises SKA. In some embodiments, [N3] is or comprises KSG. In some embodiments, [N2]-[N3] comprises SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKR (SEQ ID NO: 4717), SPHGA (SEQ ID NO: 4718), SPHSR (SEQ ID NO: 4719), SPHSL (SEQ ID NO: 4720), SPHSS (SEQ ID NO: 4721), SPHFL (SEQ ID NO: 4722), SPHWK (SEQ ID NO: 4723), SPHGK (SEQ ID NO: 4724), SPHTK (SEQ ID NO: 4725), SPHNK (SEQ ID NO: 4726), SPHAK (SEQ ID NO: 4727), SPHKH (SEQ ID NO: 4728), SPHKM (SEQ ID NO: 4729), or SPHRS (SEQ ID NO: 4730). In some embodiments [N2]-[N3] comprises (SEQ ID NO: 4701) or SPHKS (SEQ ID NO: 4704). In some embodiments, [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKTY (SEQ ID NO: 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPHSSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976). In some embodiments, [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941). In some embodiments, [N2]-[N3] is or comprises SPHKSG (SEQ ID NO: 946).

[0054] In some embodiments, [N1]-[N2]-[N3] comprises SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHAS (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSPHSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SGSPHNK (SEQ ID NO: 4879), LGSPHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892), PDSPHKI (SEQ ID NO: 4893), PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905). In some embodiments, [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CSGSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPHKFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSKD (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939), GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW (SEQ ID NO: 4952), GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA (SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA (SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989), GNGSPHSKS (SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKSL (SEQ ID NO: 5001), GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 5012), GHDSPHKHG (SEQ ID NO: 5013), GQVSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSPHKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697). In some embodiments, [N1]-[N2]-[N3] is or comprises GHDSPHKSG (SEQ ID NO: 4698). [0055] In some embodiments, ligand comprising the protein or the peptide comprising an amino acid sequence having the formula [N1]-[N2]-[N3], further comprises [N4] which comprises X7 X8 X9 X10. In some embodiments, position X7 of [N4] is independently chosen from W, Q, K, R, G, L, V, S, P, H, K, I, M, A, E, or F. In some embodiments, position X8 of [N4] is independently chosen from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L. In some embodiments, position X9 of [N4] is independently chosen from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y. In some embodiments, position X10 of [N4] is independently chosen from Q, H, E, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V. In some embodiments [N4] is or comprises QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), QNQQ (SEQ ID NO: 5028), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR (SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), SKRQ (SEQ ID NO: 5048), QLYR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066), PNLQ (SEQ ID NO: 5067), HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), QRTM (SEQ ID NO: 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093), QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), QTQL (SEQ ID NO: 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 5133), QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), QNQM (SEQ ID NO: 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159), QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), QYRA (SEQ ID NO: 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198), HTQQ (SEQ ID NO: 5199), QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), RTQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), QIIV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225), QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQL (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245), or any dipeptide or tripeptide thereof. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises: the amino acid sequence of any of SEQ ID NOs: 1800-2241; an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (SEQ ID NO: 1801). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (SEQ ID NO: 1800). [0056] In some embodiments, the ligand comprising the protein or peptide comprising an amino acid sequence having the formula [N1]-[N2]-[N3], further comprises [NO], which comprises XA XB and XC. In some embodiments, XA of [NO] is independently chosen from T, S, Y, M, A, C, I, R, L,

D, F, V, Q, N, H, E, or G. In some embodiments, XB of [NO] is independently chosen from I, M, P,

E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L. In some embodiments, XC of [NO] is independently chosen from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S. In some embodiments, [NO] is or comprises TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, IET, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM, AEV, MDA, VEW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, Til, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS, TID, TCN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TTN, TED, TNN, EVH, TIS, TVR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TIR, NAL, VIN, TIQ, TEF, TRE, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is or comprises the amino acid sequence of any one of SEQ ID NOs: 2242-2886; an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N0]- [N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (SEQ ID NO: 2242). In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TINGHDSPHKSGQNQQ (SEQ ID NO: 2243).

[0057] In some embodiments, [N3] is present immediately subsequent to [N2]. In some embodiments, the peptide comprises from N-terminus to C-terminus, [N2]-[N3]. In some embodiments, the peptide comprises from N-terminus to C-terminus, [N1]-[N2]-[N3]. In some embodiments, the peptide comprises from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4]. In some embodiments, the peptide comprises from N-terminus to C-terminus, [NO] -[Nl ]-[N2]-[N3] . In some embodiments, the peptide comprises from N-terminus to C-terminus, [NO]-[N1]-[N2]-[N3]-[N4].

[0058] In some embodiments, a ligand comprises a protein or a peptide comprising an amino acid sequence having the formula [A] [B] (SEQ ID NO: 6410), wherein [A] comprises the amino acid sequence of GSGSPH (SEQ ID NO: 4695) and [B] comprises XI X2 X3 X4 X5 X6 X7. In some embodiments, position XI of [B] is independently chosen from S, C, F, or V. In some embodiments, position X2 of [B] is independently chosen from K, L, R, I, E, Y, V, or S. In some embodiments, X3 of [B] is independently chosen from A, R, L, G, I, Y, S, F, or W. In some embodiments X4 of [B] is independently chosen from W, Q, R, G, L, V, S, or F. In some embodiments, position X5 of [B] is independently chosen from N, Y, R, C, K, or L. In some embodiments, position X6 of [B] is independently chosen from Q, G, K, R, T, L, or Y. In some embodiment, position X7 of [B] is independently chosen from Q, L, R, or V. In some embodiments, [B] comprises SLLWNQQ (SEQ ID NO: 5247), SKAQYYV (SEQ ID NO: 5248), SKLRRQQ (SEQ ID NO: 5249), SIWQNQQ (SEQ ID NO: 5250), SKAGCGQ (SEQ ID NO: 5251), SRAQNQQ (SEQ ID NO: 5252), SKRLRQQ (SEQ ID NO: 5253), SLRRNQQ (SEQ ID NO: 5254), SRGRNQQ (SEQ ID NO: 5255), SEIVNQQ (SEQ ID NO: 5256), SSRRNQQ (SEQ ID NO: 5257), CLLQNQQ (SEQ ID NO: 5258), SKAFRLQ (SEQ ID NO: 5259), CLAQNQQ (SEQ ID NO: 5260), FLRQNQQ (SEQ ID NO: 5261), SLRFNQQ (SEQ ID NO: 5262), SYLRNQQ (SEQ ID NO: 5263), CSLQNQQ (SEQ ID NO: 5264), VLWQNQQ (SEQ ID NO: 5265), SKWLLQQ (SEQ ID NO: 5266), SLWSNQQ (SEQ ID NO: 5267), SKRRLQQ (SEQ ID NO: 5268), SVYLNQQ (SEQ ID NO: 5269), SLWLNQQ (SEQ ID NO: 5270), SKAQRKL (SEQ ID NO: 5271), SKALRRQ (SEQ ID NO: 5272), SKAQRLR (SEQ ID NO: 5273), SKAQNQQ (SEQ ID NO: 5274), SKAQRRL (SEQ ID NO: 5275), SKARRQQ (SEQ ID NO: 5276), SKARRLQ (SEQ ID NO: 5277), SKSRRQQ (SEQ ID NO: 5278), SKARLRQ (SEQ ID NO: 5279), SKASKRQ (SEQ ID NO: 5280), VRRQNQQ (SEQ ID NO: 5281), SKAQLYR (SEQ ID NO: 5282), SLFRNQQ (SEQ ID NO: 5283), SKAQLTV (SEQ ID NO: 5284), or any dipeptide, tripeptide, tetrapeptide, pentapeptide, or hexapeptide thereof. In some embodiments, [A] [B] comprises GSGSPHSLLWNQQ (SEQ ID NO: 5285), GSGSPHSKAQYYV (SEQ ID NO: 2060), GSGSPHSKLRRQQ (SEQ ID NO: 2061), GSGSPHSIWQNQQ (SEQ ID NO: 5286), GSGSPHSKAGCGQ (SEQ ID NO: 2062), GSGSPHSRAQNQQ (SEQ ID NO: 2063), GSGSPHSKRLRQQ (SEQ ID NO: 2064), GSGSPHSLRRNQQ (SEQ ID NO: 2065), GSGSPHSRGRNQQ (SEQ ID NO: 2066), GSGSPHSEIVNQQ (SEQ ID NO: 5287), GSGSPHSSRRNQQ (SEQ ID NO: 2067), GSGSPHCLLQNQQ (SEQ ID NO: 5288), GSGSPHSKAFRLQ (SEQ ID NO: 2068), GSGSPHCLAQNQQ (SEQ ID NO: 5289), GSGSPHFLRQNQQ (SEQ ID NO: 2070), GSGSPHSLRFNQQ (SEQ ID NO: 2071), GSGSPHSYLRNQQ (SEQ ID NO: 5290), GSGSPHCSLQNQQ (SEQ ID NO: 5291), GSGSPHVLWQNQQ (SEQ ID NO: 5292), GSGSPHSKWLLQQ (SEQ ID NO: 2072), GSGSPHSLWSNQQ (SEQ ID NO: 5293), GSGSPHSKRRLQQ (SEQ ID NO: 2073), GSGSPHSVYLNQQ (SEQ ID NO: 5294), GSGSPHSLWLNQQ (SEQ ID NO: 5295), GSGSPHSKAQRKL (SEQ ID NO: 2074), GSGSPHSKALRRQ (SEQ ID NO: 2075), GSGSPHSKAQRLR (SEQ ID NO: 2076), GSGSPHSKAQNQQ (SEQ ID NO: 1801), GSGSPHSKAQRRL (SEQ ID NO: 2077), GSGSPHSKARRQQ (SEQ ID NO: 2078), GSGSPHSKARRLQ (SEQ ID NO: 2079), GSGSPHSKSRRQQ (SEQ ID NO: 2080), GSGSPHSKARLRQ (SEQ ID NO: 2082), GSGSPHSKASKRQ (SEQ ID NO: 2083), GSGSPHVRRQNQQ (SEQ ID NO: 2084), GSGSPHSKAQLYR (SEQ ID NO: 2085), GSGSPHSLFRNQQ (SEQ ID NO: 5296), GSGSPHSKAQLTV (SEQ ID NO: 2086), or any portion thereof, e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, thereof. In some embodiments, [B] is present immediately subsequent to [A]. In some embodiments, the peptide comprises from N-terminus to C- terminus, [A][B].

[0059] In some embodiments, the ligand comprises a protein or peptide comprising an amino acid sequence having the formula [A] [B] (SEQ ID NO: 6411), wherein [A] comprises XI X2 X3 X4 X5 X6 and [B] comprises SPHKSG (SEQ ID NO: 946). In some embodiments, position XI of [A] is independently chosen from T, M, A, C, I, R, L, D, F, V, Q, N, or H. In some embodiments, position X2 of [A] is independently chosen from I, P, E, N, D, S, A, T, M, or Q. In some embodiments, position X3 of [A] is independently chosen from N, E, G, Y, W, M, T, I, K, Q, F, S, V, A, or L. In some embodiments, position X4 of [A] is independently chosen from G, D, R, or E. In some embodiments, position X5 of [A] is independently chosen from H, Q, N, or D. In some embodiments, position X6 of [A] is independently chosen from D or R. In some embodiments, [A] comprises TINGHD (SEQ ID NO: 5297), MPEGHD (SEQ ID NO: 5298), MEGGHD (SEQ ID NO: 5299), MEYGHD (SEQ ID NO: 5300), AEWGHD (SEQ ID NO: 5301), CEWGHD (SEQ ID NO: 5302), ANNGQD (SEQ ID NO: 5303), IPEGHD (SEQ ID NO: 5304), ADMGHD (SEQ ID NO: 5305), IEYGHD (SEQ ID NO: 5306), ADYGHD (SEQ ID NO: 5307), IETGHD (SEQ ID NO: 5308), MEWGHD (SEQ ID NO: 5309), CEYGHD (SEQ ID NO: 5310), RINGHD (SEQ ID NO: 5311), MEIGHD (SEQ ID NO: 5312), LEYGHD (SEQ ID NO: 5313), ADWGHD (SEQ ID NO: 5314), IEIGHD (SEQ ID NO: 5315), TIKDND (SEQ ID NO: 5316), DIMGHD (SEQ ID NO: 5317), FEQGHD (SEQ ID NO: 5318), MEFGHD (SEQ ID NO: 5319), CDQGHD (SEQ ID NO: 5320), LPEGHD (SEQ ID NO: 5321), IENGHD (SEQ ID NO: 5322), MESGHD (SEQ ID NO: 5323), AEIGHD (SEQ ID NO: 5324), VEYGHD (SEQ ID NO: 5325), TSNGDD (SEQ ID NO: 5326), IEVGHD (SEQ ID NO: 5327), MEMGHD (SEQ ID NO: 5328), AEVGHD (SEQ ID NO: 5329), MDAGHD (SEQ ID NO: 5330), VEWGHD (SEQ ID NO: 5331), AEQGHD (SEQ ID NO: 5332), LEWGHD (SEQ ID NO: 5333), MELGHD (SEQ ID NO: 5334), METGHD (SEQ ID NO: 5335), MEAGHD (SEQ ID NO: 5336), TINRQR (SEQ ID NO: 5337), IESGHD (SEQ ID NO: 5338), TAKDHD (SEQ ID NO: 5339), MEVGHD (SEQ ID NO: 5340), CEIGHD (SEQ ID NO: 5341), ATNGHD (SEQ ID NO: 5342), MDGGHD (SEQ ID NO: 5343), QEVGHD (SEQ ID NO: 5344), ADQGHD (SEQ ID NO: 5345), NMNGHD (SEQ ID NO: 5346), TPWEHD (SEQ ID NO: 5347), IEMGHD (SEQ ID NO: 5348), TANEHD (SEQ ID NO: 5349), QQQGHD (SEQ ID NO: 5350), TPQDHD (SEQ ID NO: 5351), HDWGHD (SEQ ID NO: 5352), IEGGHD (SEQ ID NO: 5353), or any dipeptide, tripeptide, tetrapeptide, or pentapeptide thereof. In some embodiments, [A] [B] comprises TINGHDSPHKR (SEQ ID NO: 5354), MPEGHDSPHKS (SEQ ID NO: 5355), MEGGHDSPHKS (SEQ ID NO: 5356), MEYGHDSPHKS (SEQ ID NO: 5357), AEWGHDSPHKS (SEQ ID NO: 5358), CEWGHDSPHKS (SEQ ID NO: 5359), ANNGQDSPHKS (SEQ ID NO: 5360), IPEGHDSPHKS (SEQ ID NO: 5361), ADMGHDSPHKS (SEQ ID NO: 5362), IEYGHDSPHKS (SEQ ID NO: 5363), ADYGHDSPHKS (SEQ ID NO: 5364), IETGHDSPHKS (SEQ ID NO: 5365), MEWGHDSPHKS (SEQ ID NO: 5366), CEYGHDSPHKS (SEQ ID NO: 5367), RINGHDSPHKS (SEQ ID NO: 5368), MEIGHDSPHKS (SEQ ID NO: 5369), LEYGHDSPHKS (SEQ ID NO: 5370), ADWGHDSPHKS (SEQ ID NO: 5371), IEIGHDSPHKS (SEQ ID NO: 5372), TIKDNDSPHKS (SEQ ID NO: 5373), DIMGHDSPHKS (SEQ ID NO: 5374), FEQGHDSPHKS (SEQ ID NO: 5375), MEFGHDSPHKS (SEQ ID NO: 5376), CDQGHDSPHKS (SEQ ID NO: 5377), LPEGHDSPHKS (SEQ ID NO: 5378), IENGHDSPHKS (SEQ ID NO: 5379), MESGHDSPHKS (SEQ ID NO: 5380), AEIGHDSPHKS (SEQ ID NO: 5381), VEYGHDSPHKS (SEQ ID NO: 5382), TSNGDDSPHKS (SEQ ID NO: 5383), IEVGHDSPHKS (SEQ ID NO: 5384), MEMGHDSPHKS (SEQ ID NO: 5385), AEVGHDSPHKS (SEQ ID NO: 5386), MDAGHDSPHKS (SEQ ID NO: 5387), VEWGHDSPHKS (SEQ ID NO: 5388), AEQGHDSPHKS (SEQ ID NO: 5389), LEWGHDSPHKS (SEQ ID NO: 5390), MELGHDSPHKS (SEQ ID NO: 5391), METGHDSPHKS (SEQ ID NO: 5392), MEAGHDSPHKS (SEQ ID NO: 5393), TINRQRSPHKS (SEQ ID NO: 5394), IESGHDSPHKS (SEQ ID NO: 5395), TAKDHDSPHKS (SEQ ID NO: 5396), MEVGHDSPHKS (SEQ ID NO: 5397), CEIGHDSPHKS (SEQ ID NO: 5398), ATNGHDSPHKS (SEQ ID NO: 5399), MDGGHDSPHKS (SEQ ID NO: 5400), QEVGHDSPHKS (SEQ ID NO: 5401), ADQGHDSPHKS (SEQ ID NO: 5402), NMNGHDSPHKS (SEQ ID NO: 5403), TPWEHDSPHKS (SEQ ID NO: 5404), IEMGHDSPHKS (SEQ ID NO: 5405), TANEHDSPHKS (SEQ ID NO: 5406), TINGHDSPHKS (SEQ ID NO: 5407), QQQGHDSPHKS (SEQ ID NO: 5408), TPQDHDSPHKS (SEQ ID NO: 5409), HDWGHDSPHKS (SEQ ID NO: 5410), IEGGHDSPHKS (SEQ ID NO: 5411), or any portion thereof, e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, thereof. In some embodiments, [B] is present immediately subsequent to [A]. In some embodiments, the peptide comprises from N-terminus to C- terminus, [A][B].

[0060] In some embodiments, a ligand described herein comprises a protein or peptide comprising an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from any one of the sequences provided in Tables 1, 2A, 2B, 2C, 13-19. In some embodiments, the peptide comprises an amino acid sequence comprising at least 3, 4, or 5 consecutive amino acids from any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids from any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903- 909. In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue.

[0061] In some embodiments, the 3 consecutive amino acids comprise SPH. In some embodiments, the 4 consecutive amino acids comprise SPHS (SEQ ID NO: 4700). In some embodiments, the 5 consecutive amino acids comprise SPHSK (SEQ ID NO: 4701). In some embodiments, the 6 consecutive amino acids comprise SPHSKA (SEQ ID NO: 941). In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue present at position one, numbered according to SEQ ID NO: 941. [0062] In some embodiments, 3 consecutive amino acids comprise HDS. In some embodiments, the 4 consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, the 5 consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, the 6 consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, the 7 consecutive amino acids comprise HDSPHKS (SEQ ID NO: 4840). In some embodiments, the 8 consecutive amino acids comprise HDSPHKSG (SEQ ID NO: 943).

[0063] In some embodiments, 3 consecutive amino acids comprise HDS. In some embodiments, the 4 consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, the 5 consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, the 6 consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue present at position three, numbered according to SEQ ID NO: 2.

[0064] In some embodiments, the 3 consecutive amino acids comprise SPH. In some embodiments, the 4 consecutive amino acids comprise SPHK (SEQ ID NO: 6398). In some embodiments, the 5 consecutive amino acids comprise SPHKY (SEQ ID NO: 4715). In some embodiments, the 6 consecutive amino acids comprise SPHKYG (SEQ ID NO: 966).

[0065] In some embodiments, a ligand described herein comprises a protein or a peptide comprising an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of SEQ ID NO: 1754. [0066] In some embodiments, a ligand described herein comprises a protein or a peptide comprising an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0067] In some embodiments, a ligand described herein comprises a protein or a peptide comprising comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943). In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943). [0068] In some embodiments, a ligand described herein comprises a protein or a peptide comprising comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2). In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0069] In some embodiments, a ligand described herein comprises a protein or a peptide comprising comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHKYG (SEQ ID NO: 966). In some embodiments, the peptide comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids relative to the amino acid sequence of SPHKYG (SEQ ID NO: 966).

[0070] In some embodiments, a ligand described herein comprises a protein or a peptide comprising the amino acid sequence of any of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises the amino acid sequence of any of S EQ ID NOs: 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 941. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 943. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1754.

[0071] In some embodiments, a ligand described herein comprises a protein or a peptide comprising comprises an amino acid sequence encoded by a nucleotide sequence described herein, e.g., a nucleotide sequence of Table 2A. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the peptide comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the peptide comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.

[0072] In some embodiments, a ligand described herein comprises a protein or a peptide, which comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue present at position three, numbered according to SEQ ID NO: 2. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue present at position one, numbered according to SEQ ID NO: 941. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, on a serine residue present in the amino acid sequence of SPH. [0073] In some embodiments, the nucleotide sequence encoding a peptide of a ligand described herein comprises a nucleotide sequence described herein, e.g., as described in Table 2A. In some embodiments, the nucleotide sequence encoding a peptide described herein is codon optimized. In some embodiments, the nucleotide sequence encoding a peptide described herein is isolated, e.g., recombinant.

[0074] In some embodiments the nucleotide sequence encoding a peptide of a ligand described herein comprises the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the nucleotide sequence encoding a peptide described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments the nucleic acid sequence encoding a peptide described herein comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.

[0075] In some embodiments, the nucleic acid encoding a peptide of a ligand described herein comprises the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the nucleotide sequence encoding a peptide described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments the nucleic acid encoding a peptide described herein comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.

[0076] The present disclosure also provides a nucleic acid or polynucleotide encoding any of the peptides described herein and ligands, compositions, AAV capsid variants, AAV particles, vectors, and cells comprising the same.

Antibody Molecules

[0077] In some embodiments, a ligand described herein is or comprises an antibody molecule. In other embodiments, an active agent described herein, e.g., a therapeutic agent or a diagnostic agent, is or comprises an antibody molecule.

[0078] As used herein, the term "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “antibody molecule” includes, for example, a monoclonal antibody (including a full length antibody which has an immunoglobulin Fc region). In an embodiment, an antibody molecule comprises a full length antibody, or a full length immunoglobulin chain. In an embodiment, an antibody molecule comprises an antigen binding or functional fragment of a full length antibody, or a full length immunoglobulin chain.

[0079] In an embodiment, an antibody molecule is a monospecific antibody molecule and binds a single epitope, e.g., a monospecific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds the same epitope.

[0080] In an embodiment an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or tetraspecific antibody molecule.

[0081] In an embodiment a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.

[0082] In some embodiments, the antibody molecule comprises at least one immunoglobulin variable domain sequence. An antibody molecule may include, for example, full-length, mature antibodies and antigen-binding fragments of an antibody. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The antibody molecules can be monoclonal or polyclonal. The encoded antibody can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda.

[0083] Examples of antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); and (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. An antibody fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).

[0084] The term “antibody” includes intact molecules as well as functional fragments thereof. Constant regions of the antibodies can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

[0085] In some embodiments, the antibody molecule can be single domain antibody. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4- chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

[0086] In some embodiments, the VH and VL regions of the antibody molecule can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).

[0087] The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0088] “Complementarity determining region”, and “CDR”, as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

[0089] The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (Chothia numbering scheme). In some embodiments, the CDRs defined according the Chothia number scheme are also sometimes referred to as hypervariable loops.

[0090] For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

[0091] In some embodiments, the antigen binding domain of the antibody molecules of the present disclosure is the part of the antibody molecule that comprises determinants that form an interface that binds a therapeutic protein or an epitope thereof. With respect to proteins (or protein mimetics), the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid mimics) that form an interface that binds to the therapeutic protein. Typically, the antigen-binding site of an antibody molecule includes at least one or two CDRs and/or hypervariable loops, or more typically at least three, four, five or six CDRs and/or hypervariable loops.

[0092] The antibody molecule can be a monoclonal antibody molecule or a polyclonal antibody molecule. In some embodiments, a monoclonal antibody or a monoclonal antibody composition refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods). [0093] In some embodiments, the sequences of an antibody molecule to be included in an encoded payload described herein can be generated by recombinant libraries, e.g., generated by phage display or by combinatorial methods.

[0094] Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al.

International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0095] In some embodiments, the sequences of an antibody molecule to be included in an encoded payload described herein can be generated from an antibody molecule that is designed using the VERSITOPE™ Antibody Generation or BIOATLA®, e.g., in US20130303399, US20130281303, W02012009026, WO2016033331, WO2016036916, and US8859467, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the sequences of an antibody molecule to be included in an encoded payload described herein can be derived from an antibody molecule that is designed and/or produced using the methods described, e.g., in WO2017189959 and WO2020223276, the contents of which are herein incorporated by reference in their entirety.

[0096] In some embodiments, the antibody molecule comprises an amino acid sequence of a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.

[0097] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0098] In some embodiments, the antibody comprises an amino acid sequence of an antibody in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Antibody molecules comprising chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibody molecules comprising the sequences of antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention. [0099] An effectively human protein is a protein that does substantially not evoke a neutralizing antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA can be problematic in a number of circumstances, e.g., if the antibody molecule is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeated antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0100] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559). [0101] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the donor and the immunoglobulin providing the framework is called the acceptor. In some embodiments, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0102] In some embodiments, the consensus sequence refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. In some embodiments, the consensus framework refers to the framework region in the consensus immunoglobulin sequence.

[0103] An antibody can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference).

[0104] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incorporated by reference.

[0105] In some embodiments, the antibodies comprise the sequences of humanized antibodies in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, published on December 23, 1992.

[0106] In some embodiments, the antibody molecule can be a single chain antibody. A singlechain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.

[0107] In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function). In some embodiments the antibody has: effector function; and can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement. In other embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, it is an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0108] Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

[0109] An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0110] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0111] Useful detectable agents with which an antibody molecule of the invention may be derivatized (or labeled) to include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms, e.g., europium (Eu), and other anthanides, and radioactive materials (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5dimethylamine-l- napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, P-galactosidase, acetylcholinesterase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody molecule may also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin). For example, an antibody may be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin.

[0112] Labeled antibody molecule can be used, for example, diagnostically and/or experimentally in a number of contexts, including (i) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation; (ii) to detect a predetermined antigen (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein; (iii) to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.

[0113] An antibody molecules may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety. Radioactive isotopes can be used in diagnostic or therapeutic applications. Radioactive isotopes that can be coupled to the antibodies described herein include, but are not limited to a-, P-, or y-emitters, or P-and y-emitters. Such radioactive isotopes include, but are not limited to iodine ( ¹³¹ I or ¹²⁵ I), yttrium ( ⁹⁰ Y), lutetium ( ¹⁷⁷ Lu), actinium ( ²²⁵ Ac), praseodymium, astatine ( ²¹¹ At), rhenium ( ¹⁸⁶ Re), bismuth ( ²¹² Bi or ²¹³ Bi), indium ( ⁱⁿ In), technetium (" mTc), phosphorus ( ³² P), rhodium ( ¹⁸⁸ Rh), sulfur ( ³⁵ S) , carbon ( ¹⁴ C), tritium ( ³ H), chromium ( ⁵¹ Cr), chlorine ( ³⁶ C1), cobalt ( ⁵⁷ Co or ⁵⁸ Co), iron ( ⁵⁹ Fe), selenium ( ⁷⁵ Se), or gallium ( ⁶⁷ Ga). Radioisotopes useful as therapeutic agents include yttrium ( ⁹⁰ Y), lutetium ( ¹⁷⁷ Lu), actinium ( ²² 5 Ac), praseodymium, astatine ( ²¹¹ At), rhenium ( ¹⁸⁶ Re), bismuth ( ²¹² Bi or ²¹³ Bi), and rhodium ( ¹⁸⁸ Rh). Radioisotopes useful as labels, e.g., for use in diagnostics, include iodine ( ¹³¹ I or ¹²⁵ I), indium ( ⁱⁿ In), technetium ( ⁹⁹ mTc), phosphorus ( ³² P), carbon ( ¹⁴ C), and tritium ( ³ H), or one or more of the therapeutic isotopes listed above.

[0114] The invention provides radiolabeled antibody molecules and methods of labeling the same. In one embodiment, a method of labeling an antibody molecule is disclosed. The method includes contacting an antibody molecule, with a chelating agent, to thereby produce a conjugated antibody. The conjugated antibody is radiolabeled with a radioisotope, e.g., ^{1 1 1} Indium, "Yttrium and ¹⁷⁷ Lutetium, to thereby produce a labeled antibody molecule.

[0115] As is discussed above, the antibody molecule can be conjugated to a therapeutic agent. Therapeutically active radioisotopes have already been mentioned. Examples of other therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846, 545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclinies (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

[0116] In some embodiments, a ligand described herein is or comprises an antibody molecule that binds to a GPI-anchored protein. In some embodiments, the antibody molecule binds to ALPL, e.g., human or murine ALPL. In some embodiments, the antibody molecule is F2910-SP, AF2909, NBP2- 67295, LS-B3666, MA524845, 2F4, or a variant thereof. In some embodiments the antibody molecule is an antibody provided in Table 40 or a variant thereof, e.g., Ab 9 of Table 40.

Multispecific Antibody Molecules

[0117] In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or tetraspecific antibody molecule. In some embodiments, an antibody molecule described herein is a multispecific antibody molecule.

[0118] In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope. In some embodiment, an antibody molecule as described herein is a bispecific antibody molecule.

[0119] In some embodiments, the sequences of the antibody molecules can be generated from bispecific or heterodimeric antibody molecules produced using protocols known in the art; including but not limited to, for example, the “knob in a hole” approach described in, e.g., US5731168; the electrostatic steering Fc pairing as described in, e.g., WO 09/089004, WO 06/106905 and WO 2010/129304; Strand Exchange Engineered Domains (SEED) heterodimer formation as described in, e.g., WO 07/110205; Fab arm exchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO 2013/060867; double antibody conjugate, e.g., by antibody crossdinking to generate a bi-specific structure using a heterobifunctional reagent having an amine -reactive group and a sulfhydryl reactive group as described in, e.g., US4433059; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., US 4444878; trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhdryl reactive groups, as described in, e.g., US5273743; biosynthetic binding proteins, e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross-linking, as described in, e.g., US5534254; bifunctional antibodies, e.g., Fab fragments with different binding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., US5582996; bispecific and oligospecific mono-and oligovalent receptors, e.g., VH- CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CHI region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g., US5591828; bispecific DNA-antibody conjugates, e.g., crosslinking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g., US5635602; bispecific fusion proteins, e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., US5637481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also disclosed creating bispecific, trispecific, or tetraspecific molecules, as described in, e.g., US5837242; minibody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CH3 region, which can be dimerized to form bispecific/multivalent molecules, as described in, e.g., US5837821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described in, e.g., US5844094; String of VH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C- terminus further associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., US5864019; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFv or diabody type format, as described in, e.g., US5869620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found, for example, in US5910573, US5932448, US5959083, US5989830, US6005079, US6239259, US6294353, US6333396, US6476198, US6511663, US6670453, US6743896, US6809185, US6833441, US7129330, US7183076, US7521056, US7527787, US7534866, US7612181, US2002/004587A1,

US2002/076406A1, US2002/103345A1, US2003/207346A1, US2003/211078A1, US2004/219643 Al, US2004/220388A1, US2004/242847A1, US2005/003403A1, US2005/004352A1, US2005/069552A1, US2005/079170A1, US2005/100543A1, US2005/136049A1, US2005/136051A1, US2005/163782A1, US2005/266425A1, US2006/083747A1, US2006/120960A1, US2006/204493A1, US2006/263367A1, US2007/004909A1, US2007/087381A1, US2007/128150A1, US2007/141049A1, US2007/154901A1, US2007/274985A1, US2008/050370A1, US2008/069820A1, US2008/152645A1, US2008/171855A1, US2008/241884A1, US2008/254512A1, US2008/260738A1, US2009/130106A1, US2009/148905A1, US2009/155275A1, US2009/162359A1, US2009/162360A1, US2009/175851A1, US 2009/ 175867A1, US2009/232811A1, US2009/234105A1, US2009/263392A1, US2009/274649A1, EP346087A2, WQ00/06605A2, WO02/072635A2, W004/081051A1, W006/020258A2, W02007/044887A2, W02007/095338A2, W02007/137760A2, WO2008/119353A1, W02009/021754A2, W02009/068630A1, WO91/03493A1, WO93/23537A1, WO94/09131A1, WO94/12625A2, WO95/09917A1, WO96/37621A2, WO99/64460A1. The contents of the above -referenced applications are incorporated herein by reference in their entireties. [0120] In some embodiments, a ligand described herein comprises a multispecific, e.g., bispecific, antibody molecule comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

Fc Polypeptides

[0121] In some embodiments, a ligand described herein comprises an Fc polypeptide. In some embodiments, the ligand is or comprises a first Fc polypeptide. In some embodiments, the ligand is a first Fc polypeptide and the active agent is a second Fc polypeptide.

[0122] In some embodiments, the first Fc polypeptide and the second Fc polypeptide form a dimer. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise a dimerization domain, e.g., an interface of a first and second Fc polypeptides. In some embodiments, the dimerization domain is engineered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface. In some embodiments, the dimerization of the first Fc polypeptide and the second Fc polypeptide is enhanced by providing an Fc interface of the first and a second Fc polypeptides with one or more of: a paired cavity-protuberance ("knob-in-a hole"), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer :homomultimer forms, e.g., relative to a non-engineered interface. In some embodiments, the first Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof). In some embodiments, the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob). In some embodiments, the first Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof); and the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob). In some embodiments, the second Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof). In some embodiments, the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob). In some embodiments, the second Fc polypeptide comprises an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole) (or a combination thereof); and the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protuberance or knob).

[0123] In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both (i) has reduced affinity, e.g., ablated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor; (ii) comprises a mutation at one, two, or all of positions 1253 (e.g., I253A), H310 (e.g., H310A or H310Q), and/or H435 (e.g., H435A or H435Q), numbered according to the EU index as in Kabat; (iii) has reduced effector function (e.g., reduced ADCC), compared to a reference wherein the reference is a wild-type Fc receptor; (iv) comprises a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index as in Kabat. In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both comprises a half-life extender or an amino acid modification that increases serum half-life (e.g., (i) a Leu at position 428 and a Ser at position 434, or (ii) a Ser or Ala at position 434, according to EU numbering).

[0124] In some embodiments, the ligand comprises a first Fc polypeptide, wherein the first Fc polypeptide comprises a protein or peptide sequence provided herein, e.g., as set forth in any of Tables 1, 2A, 2B, 13-19. In some embodiments, the protein or peptide sequence is present in the CH3 domain of the first Fc polypeptide. In some embodiments, the CH3 domain is modified from a human IgGl, IgG2, IgG3, or IgG4 CH3 domain. In some embodiments, the CH3 domain comprises one, two, three, four, five, six, seven, eight, nine, ten, or eleven substitutions in a set of amino acid positions comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, according to EU numbering. In some embodiments, the protein or peptide is present at or near the C-terminus of the first Fc polypeptide (e.g., within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids from the C- terminus of the therapeutic protein, enzyme, or antibody molecule). In some embodiments, the first Fc polypeptide, the second Fc polypeptide or both the first Fc polypeptide and the second Fc polypeptide does not comprise an immunoglobulin heavy and/or light chain variable region sequence or an antigen-binding portion thereof.

[0125] In some embodiments, the second Fc polypeptide is fused or coupled (e.g., directly or indirectly via a linker) to a therapeutic protein or variant thereof (e.g., an enzyme).

Other Exemplary Ligands

[0126] In some embodiments, a ligand described herein comprises a nucleic acid molecule. In some embodiments a ligand described herein comprises an aptamer. In some embodiments the aptamer binds to a GPI anchored protein. In some embodiments, the aptamer binds to ALPL, e.g., human or murine ALPL. In some embodiments the aptamer is or comprises DNA, RNA, modified DNA, modified RNA, or a combination thereof. In some embodiments, the aptamer is fused or coupled to a therapeutic agent chosen from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

[0127] In some embodiments a ligand described herein is or comprises a small molecule. In some embodiments, the small molecule is an inhibitor of ALPL, e.g., a small molecule that interferes with ALPL dimerization. In some embodiments, the small molecule is an aryl sulfonamide, a phosphonate derivative, a pyrazole, a triazole, or an imidazole. In some embodiments, the small molecule is 5-((5- chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425). In some embodiments, the small molecule is 2,5-Dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (Tissue -Nonspecific Alkaline Phosphatase Inhibitor (TNAPi)). [0128] In some embodiments, a ligand described herein is present or coupled to a carrier, e.g., an exosome, a microvesicle, or a lipid nanoparticle (LNP). In some embodiments, the carrier is an exosome or LNP. In some embodiments, the ligand is present on the surface of the carrier. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the carrier comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides comprising an amino acid sequence provided herein, e.g., as set forth in any one of Tables 1, 2A, 2B, or 13-19. In some embodiments, the ligand is conjugated to the surface of the carrier by post-insertion. In some embodiments, the ligand is conjugated to the surface of the carrier via a covalent bond (e.g., using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry or thiol-maleimide linkage reactions). In some embodiments, the carrier is coupled to a therapeutic agent. In some embodiments, the carrier comprises an RNAi agent, an mRNA, a ribonucleoprotein complex (e.g., a Cas9/gRNA complex), or a circRNA.

AAV serotypes and capsids

[0129] In some embodiments, a ligand described herein is a component of a viral particle, e.g., an AAV particle or a lentivirus. In some embodiments, the ligand is a component of a capsid protein, e.g., and AAV capsid protein described herein.

[0130] In some embodiments, an AAV particle may comprise a capsid protein or variant thereof any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profiles. While not wishing to be bound by theory, it is believed in some embodiments, that the AAV capsid protein, e.g., an AAV capsid variant, can modulate, e.g., direct, AAV particle tropism to a particular tissue.

[0131] In some embodiments, an AAV comprises a small non-enveloped icosahedral capsid virus of the Parvoviridae family and is characterized by a single stranded DNA viral genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. The Parvoviridae family comprises the Dependovirus genus which includes AAV, capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

[0132] In some embodiments, AAV are used as a biological tool due to a relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. The genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload.

[0133] In some embodiments, the AAV, is a naturally occurring (e.g., wild-type) AAV or a recombinant AAV. In some embodiments, the wild-type AAV vector genome is a linear, singlestranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome. In some embodiments, an AAV viral genome typically comprises two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145 nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell. [0134] In some embodiments, the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are used for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid polypeptide, e.g., an AAV capsid variant. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV serotype, as a non-limiting example, for AAV9/hu.l4 (SEQ ID NO: 123 of US 7,906,111, the contents of which are herein incorporated by reference in their entirety) VP1 refers to amino acids 1- 736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, for any one of the amino acid sequences of SEQ ID NO: 981 or 982, VP1 comprises amino acids 1-742, VP2 comprises amino acids 138-742, and VP3 comprises amino acids 203-742. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region, are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1 : 1 : 10 of VP1 :VP2: VP3.

[0135] AAV vectors of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) reference sequences. In addition to single stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) viral genomes. scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell. In some embodiments, the AAV particle of the present disclosure is an scAAV. In some embodiments, the AAV particle of the present disclosure is an ssAAV.

[0136] Methods for producing and/or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos. W0200028004; W0200123001; W02004112727; W02005005610; and W02005072364, the content of each of which is incorporated herein by reference in its entirety).

[0137] As described herein, the AAV particles of the disclosure comprising an AAV capsid variant, and a viral genome, have enhanced tropism for a cell-type or a tissue, e.g., a CNS cell-type, region, or tissue.

[0138] In some embodiments, an AAV capsid variant described herein allows for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments the AAV capsid variant allows for increased distribution to a brain region. In some embodiments, the brain region comprises a frontal cortex, sensory cortex, motor cortex, caudate, dentate nucleus, cerebellar cortex, cerebral cortex, brain stem, hippocampus, thalamus, putamen, or a combination thereof. In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant allows for transduction in a non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof).

[0139] In some embodiments, an AAV capsid variant allows for increased distribution to a spinal cord region. In some embodiments, the spinal region comprises a cervical spinal cord region, thoracic spinal cord region, and/or lumbar spinal cord region.

[0140] In some embodiments, the AAV capsid variant, is suitable for intramuscular administration and/or transduction of muscle fibers. In some embodiments the AAV capsid variant, allows for increased distribution to a muscle region. In some embodiments, the muscle region comprises a heart muscle, quadriceps muscle, a diaphragm muscle region, or a combination thereof. In some embodiments, the muscle region comprises a heart muscle region, e.g., a heart atrium muscle region or a heart ventricle muscle region.

[0141] In some embodiments, the initiation codon for translation of the AAV VP1 capsid protein, e.g., a capsid variant, described herein may be CTG, TTG, or GTG as described in US Patent No. US8163543, the contents of which are herein incorporated by reference in its entirety.

[0142] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (e.g., capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met- aminopeptidases. This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.

[0143] Where the Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Met 1 /A Al amino acid (Met+/AA+) and some of which may lack a Met 1 /A Al amino acid as a result of Met/AA-clipping (Met-/AA-). For further discussion regarding Met/AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno- Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in its entirety.

[0144] According to the present disclosure, references to capsid proteins, e.g., AAV capsid variants, is not limited to either clipped (Met-/AA-) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a capsid protein or capsid polypeptide (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Metl/AAl amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Met/AA-clipping (Met-/AA-).

[0145] Further according to the present disclosure, a reference to a specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Metl/AAl amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Metl/AAl amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Metl/AAl).

[0146] As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length, and which includes a “Metl” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length, and which does not include the “Metl” amino acid (Met-) of the 736 amino acid Met-i- sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length, and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length, and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.

Il l [0147] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Metl/AAl amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Met/AAl -clipping (Met-/AA1-), and combinations thereof (Met+/AA1+ and Met-/AA1-).

[0148] As a non-limiting example, an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met-/AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met-/AA1-). An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+ZAAl) and VP2 (Met-/AA1-).

AAV Capsid Variant

[0149] In some embodiments, an AAV capsid variant disclosed herein comprises a modification in loop IV of AAV9, e.g., at positions between 449-460, e.g., at position 454 and/or 455, numbered relative to SEQ ID NO: 138, 981, or 982. In some embodiments, loop (e.g., loop IV) is used interchangeably herein with the term variable region (e.g., variable region IV), or VR (e.g., VR-IV). In some embodiments loop IV comprises positions 449-475 (e.g., amino acids KTINGSGQNQQTLKFSVAGPSNMAVQG (SEQ ID NO: 6404)), numbered according to SEQ ID NO: 138. In some embodiments loop IV comprises positions 449-460 (e.g., amino acids KTINGSGQNQQT (SEQ ID NO: 6405)), numbered according to SEQ ID NO: 138.

[0150] The AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles of the disclosure demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, an AAV particle of the disclosure may, in addition, or alternatively, have decreased tropism for a cell-type, tissue or organ.

[0151] As demonstrated in the Examples herein below, certain AAV capsid variants described herein show multiple advantages over wild- type AAV9, including (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, and/or (iii) elevated payload expression in multiple brain regions. Without wishing to be being bound by theory, it is believed that these advantages may be due, in part, to the dissemination of the AAV capsid variants through the brain vasculature. In some embodiments, the AAV capsids described herein enhance the delivery of a payload to multiple regions of the brain including for example, the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus.

[0152] In some embodiments, an AAV particle described herein comprises an AAV capsid variant, e.g., an AAV capsid variant described herein (e.g., an AAV capsid variant comprising a peptide described herein). In some embodiments, an AAV capsid variant comprises a peptide as set forth in any of Tables 1, 2A, 2B, 13-19.

[0153] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having the formula [N1]-[N2]-[N3], wherein [N2] comprises the amino acid sequence of SPH and [N3] comprises X4, X5, and X6, wherein at least one of X4, X5, or X6 is a basic amino acid, e.g., a K or R. In some embodiments, position X4 of [N2] is K. In some embodiments, position X5 of [N2] is K.

[0154] In some embodiments, [Nl] comprises XI, X2, and X3, wherein at least one of XI, X2, or X3 is G. In some embodiments, position XI of [Nl] is independently chosen from G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C. In some embodiments, position X2 of [Nl] is independently chosen from: S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q. In some embodiments, position X3 of [Nl] is independently chosen from: G, C, L, D, E, Y, H, V, A, N, P, or S. In some embodiments, [Nl] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, Al, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS. In some embodiments, [Nl] comprises GS, SG, GH, or HD. In some embodiments [Nl] is or comprises GSG, GHD, GQD, VSG, CSG, CSH, GQS, GRG, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS. In some embodiments, [Nl] is or comprises GSG. In some embodiments, [Nl] is or comprises GHD. In some embodiments, [N1]-[N2] comprises SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758), NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [Nl]- [N2] is or comprises GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802), ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GSASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIGSPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2] is or comprises GSGSPH (SEQ ID NO: 4695). In some embodiments, [N1]-[N2] is or comprises GHDSPH (SEQ ID NO: 4784).

[0155] In some embodiments, X4, X5, or both of [N3] are K. In some embodiments, X4, X5, or X6 of [N3] is R. In some embodiments, position X4 of [N3] is independently chosen from: A, K, V, S, T, G, F, W, V, N, or R. In some embodiments, position X5 of [N3] is independently chosen from: S, K, T, F, I, L, Y, H, M, or R. In some embodiments, position X6 of [N3] is independently chosen from: G, R, A, M, I, N, T, Y, D, P, V, L, E, W, N, Q, K, or S. In some embodiments, [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG. In some embodiments, [N3] comprises SK, KA, KS, or SG. In some embodiments, [N3] is or comprises SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG. In some embodiments, [N3] is or comprises SKA. In some embodiments, [N3] is or comprises KSG. In some embodiments, [N2]-[N3] comprises SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKR (SEQ ID NO: 4717), SPHGA (SEQ ID NO: 4718), SPHSR (SEQ ID NO: 4719), SPHSL (SEQ ID NO: 4720), SPHSS (SEQ ID NO: 4721), SPHFL (SEQ ID NO: 4722), SPHWK (SEQ ID NO: 4723), SPHGK (SEQ ID NO: 4724), SPHTK (SEQ ID NO: 4725), SPHNK (SEQ ID NO: 4726), SPHAK (SEQ ID NO: 4727), SPHKH (SEQ ID NO: 4728), SPHKM (SEQ ID NO: 4729), or SPHRS (SEQ ID NO: 4730). In some embodiments [N2]-[N3] comprises SPHSK (SEQ ID NO: 4701) or SPHKS (SEQ ID NO: 4704). In some embodiments, [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKTY (SEQ ID NO: 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPHSSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976). In some embodiments, [N2]-[N3] is SPHSKA (SEQ ID NO: 941). In some embodiments, [N2]-[N3] is or comprises SPHKSG (SEQ ID NO: 946).

[0156] In some embodiments, [N1]-[N2]-[N3] comprises SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHAS (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSPHSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SGSPHNK (SEQ ID NO: 4879), LGSPHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892), PDSPHKI (SEQ ID NO: 4893), PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905). In some embodiments, [N1]-[N2]-[N3] is GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CSGSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPHKFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSKD (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939), GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW (SEQ ID NO: 4952), GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA (SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA (SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989), GNGSPHSKS (SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKSL (SEQ ID NO: 5001), GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 5012), GHDSPHKHG (SEQ ID NO: 5013), GQVSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSPHKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697). In some embodiments, [N1]-[N2]-[N3] is or comprises GHDSPHKSG (SEQ ID NO: 4698). [0157] In some embodiments, the AAV capsid variant comprising an amino acid sequence having the formula [N1]-[N2]-[N3], further comprises [N4], wherein [N4] comprises X7 X8 X9 X10. In some embodiments, position X7 of [N4] is independently chosen from W, Q, K, R, G, L, V, S, P, H, K, I, M, A, E, or F. In some embodiments, position X8 of [N4] is independently chosen from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L. In some embodiments, position X9 of [N4] is independently chosen from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y. In some embodiments, position X10 of [N4] is independently chosen from Q, H, E, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V. In some embodiments [N4] comprises QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR (SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), SKRQ (SEQ ID NO: 5048), QLYR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066), PNLQ (SEQ ID NO: 5067), HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), QRTM (SEQ ID NO: 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093), QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), QTQL (SEQ ID NO: 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 5133), QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), QNQM (SEQ ID NO: 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159), QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), QYRA (SEQ ID NO: 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198), HTQQ (SEQ ID NO: 5199), QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), RTQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), QIIV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225), QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQL (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245), or any dipeptide or tripeptide thereof. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises: the amino acid sequence of any of SEQ ID NOs: 1800-2241; an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1 ]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (SEQ ID NO: 1801). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (SEQ ID NO: 1800).

[0158] In some embodiments, the AAV capsid variant comprising an amino acid sequence having the formula [N1]-[N2]-[N3], further comprises [NO], wherein [NO] comprises XA XB and XC. In some embodiments, XA of [NO] is independently chosen from T, S, Y, M, A, C, I, R, L, D, F, V, Q, N, H, E, or G. In some embodiments, XB of [NO] is independently chosen from I, M, P, E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L. In some embodiments, XC of [NO] is independently chosen from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S. In some embodiments, [NO] comprises TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, IET, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM, AEV, MDA, VEW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, Til, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS, TID, TCN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TTN, TED, TNN, EVH, TIS, TVR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TIR, NAL, VIN, TIQ, TEF, TRE, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof. In some embodiments, [NO] -[Nl ]-[N2]-[N3]-[N4] is or comprises the amino acid sequence of any one of SEQ ID NOs: 2242-2886; an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N0]- [N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (SEQ ID NO: 2242). In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TINGHDSPHKSGQNQQ (SEQ ID NO: 2243).

[0159] In some embodiments, [Nl ]-[N2]-[N3] is present in loop IV of the AAV capsid variant. In some embodiments [NO] and [N4] are present in loop IV of the AAV capsid variant. In some embodiments, [NO] -[Nl ]-[N2]-[N3]-[N4] is present in loop IV of the AAV capsid variant. In some embodiments, [NO] is present immediately subsequent to position 449, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, wherein [NO] is present immediately subsequent to position 449, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 981 or 982. In some embodiments, [NO] replaces positions 450, 451, and 452 (e.g., amino acids T450, 1451, and N452), relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. wherein [NO] is present immediately subsequent to position 449 and wherein [NO] replaces positions 450-452 (e.g., T450, 1451, and N452), relative to a reference sequence numbered according to SEQ ID NO: 138,

981, or 982. In some embodiments, [Nl] is present immediately subsequent to position 452, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981 or

982. In some embodiments, wherein [Nl] replaces positions 453- 455 (e.g., G453, S454, and G455), relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [Nl] is present immediately subsequent to position 452 and wherein [Nl] replaces positions 453-455 (e.g., G453, S454, and G455), relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N2] is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, [N2]-[N3] is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments [N1]-[N2]-[N3] is present immediately subsequent to position 452, numbered relative to SEQ ID NO: 138, 981, or 982. In some embodiments, [Nl]- [N2]-[N3] replaces positions 453-455 (e.g., G453, S454, and G455), relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [Nl] is present immediately subsequent to position 452 and wherein [Nl ]-[N2]-[N3] replaces positions 453-455 (e.g., G453, S454, and G455), relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N4] is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N4] replaces positions 456-459 (e.g., Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N4] is present immediately subsequent to position 455, and [N4] replaces positions 456-459 (e.g., Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] replaces positions 456-459 (e.g., Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] is present immediately subsequent to position 455, and wherein [N2]-[N3]-[N4] replaces positions 456- 459 (e.g., Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] replaces positions 453-459 (e.g., G453, S454, G455, Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 452, and wherein [N1]-[N2]-[N3]-[N4] replaces positions 453-459 (e.g., G453, S454, G455, Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] replaces positions 450-456 (e.g., T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N0]- [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 449, and wherein [NO]-[N1]- [N2]-[N3]-[N4] replaces positions 450-456 (e.g., T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, and Q459), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0160] In some embodiments, [N3] is present immediately subsequent to [N2].

[0161] In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus,

[N2]-[N3]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [N1]-[N2]-[N3]. In some embodiments, the AAV capsid variant comprises from N-terminus to C- terminus, [N1]-[N2]-[N3]-[N4]. In some embodiments, the AAV capsid variant comprises from N- terminus to C-terminus, [NO] -[Nl ] -[N2]-[N3] . In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [NO]-[N1]-[N2]-[N3]-[N4].

[0162] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, or 17 consecutive amino acids from any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, or 5 consecutive amino acids from any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids from any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903- 909. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is present immediately subsequent to position 448, 452, 453, 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, numbered according to SEQ ID NO: 982. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, numbered according to SEQ ID NO: 981. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., 1451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and 460 (e.g., T460), numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises one or more amino acid substitutions at positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., 1451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and/or 460 (e.g., T460), numbered according to SEQ ID NO: 138.

[0163] In some embodiments, the 3 consecutive amino acids comprise SPH. In some embodiments, the 4 consecutive amino acids comprise SPHS (SEQ ID NO: 4700). In some embodiments, the 5 consecutive amino acids comprise SPHSK (SEQ ID NO: 4701). In some embodiments, the 6 consecutive amino acids comprise SPHSKA (SEQ ID NO: 941).

[0164] In some embodiments, 3 consecutive amino acids comprise HDS. In some embodiments, the 4 consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, the 5 consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, the 6 consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, the 7 consecutive amino acids comprise HDSPHKS (SEQ ID NO: 4840). In some embodiments, the 8 consecutive amino acids comprise HDSPHKSG (SEQ ID NO: 943).

[0165] In some embodiments, 3 consecutive amino acids comprise HDS. In some embodiments, the 4 consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, the 5 consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, the 6 consecutive amino acids comprise HDSPHK (SEQ ID NO: 2).

[0166] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four different amino acids, from the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is present immediately subsequent to position 448, 452, 453, 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, numbered according to SEQ ID NO: 982. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, numbered according to SEQ ID NO: 981. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., 1451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and 460 (e.g., T460), numbered according to SEQ ID NO: 138.

[0167] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three, but no more than four different amino acids from the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0168] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three, but no more than four different amino acids that relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943).

[0169] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, two, or three, but no more than four different amino acids that relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0170] In some embodiments, the AAV capsid variant, comprises the amino acid sequence of any of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 941. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 943. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is present immediately subsequent to position 448, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 449-460 (e.g., K449, T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 448 and replaces positions 449-460 (e.g., K449, T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 449, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 450-460 (e.g., T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 449, and replaces positions 450-460 (e.g., T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 450, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 451-460 (e.g., 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 450 and replaces positions 451-460 (e.g., 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 451, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 452-460 (e.g., N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 451 and replaces positions 452-460 (e.g., N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 452, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 453-460 (e.g., G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 452, and replaces positions 453-460 (e.g., G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 454 and 455 (e.g., S454 and G455), numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, and replaces positions 454 and 455 (e.g., S454 and G455), numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 454-460 (e.g., S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 453, and replaces positions 454-460 (e.g., S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 454, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 454, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 981. In some embodiments, the amino acid sequence replaces positions 455-460 (e.g., positions G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to positions 454, and replaces positions 455-460 (e.g., positions G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982. In some embodiments, the amino acid sequence replaces positions 456-460 (e.g., Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 455, and replaces positions 456-460 (e.g., Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138.

[0171] In some embodiments, the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942 or 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the AAV capsid variant described herein, comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942 or 944, or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 942 or 944. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 942 or 944.

[0172] In some embodiments, the nucleotide sequence encoding the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleic acid sequence encoding the AAV capsid variant comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequences of SEQ ID NO: 942. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 942.

[0173] In some embodiments, the nucleotide sequence encoding the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleic acid sequence encoding the AAV capsid variant comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but no more than ten modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequences of SEQ ID NO: 944. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.

[0174] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence is present immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 981.

[0175] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of HDSPHKSG (SEQ ID NO: 943), wherein the amino acid sequence is present immediately subsequent to position 453, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of HDSPHKSG (SEQ ID NO: 943), wherein the amino acid sequence is present immediately subsequent to position 453, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.

[0176] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of HDSPHK (SEQ ID NO: 2), wherein the amino acid sequence is present immediately subsequent to position 453, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of HDSPHK (SEQ ID NO: 2), wherein the amino acid sequence is present immediately subsequent to position 453, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.

[0177] In some embodiments, an AAV capsid variant described herein comprises (i) the amino acid sequence of HDSPHKSG (SEQ ID NO: 943), which is present immediately subsequent to position 453; and (ii) a deletion of amino acids SG at position 454 and 455; wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0178] In some embodiments, an AAV capsid variant described herein comprises (i) the amino acid sequence of HDSPHSKA (SEQ ID NO: 4486), which is present immediately subsequent to position 453; and (ii) a deletion of amino acids SG at position 454 and 455; wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0179] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than S at position 454 and/or an amino acid other than G at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid H at position 454 and the amino acid D at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SPHKSG (SEQ ID NO: 946). In some embodiments, the AAV capsid variant comprises: (i) the amino acid H at position 454 and the amino acid D at position 455, and (ii) the amino acid sequence SPHKSG (SEQ ID NO: 946), wherein the amino acid sequence of SPHKSG (SEQ ID NO: 946) is present immediately subsequent to position 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0180] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than S at position 454 and/or an amino acid other than G at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid H at position 454 and the amino acid D at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises: (i) the amino acid H at position 454 and the amino acid D at position 455, and (ii) the amino acid sequence SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence of SPHSKA (SEQ ID NO: 941) is present immediately subsequent to position 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0181] In some embodiments, an AAV capsid variant described herein comprises a modification, e.g., substitution, relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at position S454 and/or G455, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a S454H substitution and/or G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a S454H substitution and a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SPHKSG (SEQ ID NO: 946). In some embodiments, the AAV capsid variant comprises: (i) a S454H substitution and a G455D substitution, and (ii) the amino acid sequence SPHKSG (SEQ ID NO: 946), wherein the amino acid sequence of SPHKSG (SEQ ID NO: 946) is present immediately subsequent to position 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0182] In some embodiments, an AAV capsid variant described herein comprises a modification, e.g., substitution, relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at position S454 and/or G455, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a S454H substitution and/or G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a S454H substitution and a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises: (i) a S454H substitution and a G455D substitution, and (ii) the amino acid sequence SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence of SPHSKA (SEQ ID NO: 941) is present immediately subsequent to position 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0183] In some embodiments, the AAV capsid variant further comprises one, two, or all of an amino acid other than T at position 450 (e.g., S, Y, or G), an amino acid other than I at position 451 (e.g., M or L), and/or an amino acid other than N at position 452 (e.g., S), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 450 and an M at position 451, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Y at position 450, an L at position 451, and an S at position 452, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a G at position 450, an L at position 451, and an S at position 452, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. [0184] In some embodiments, the AAV capsid variant further comprises one, two, three, four, or all of an amino acid other than Q at position 456 (e.g., R or L), N at position 457 (e.g., H, K, or R), Q at position 458 (e.g., R or T), Q at position 459 (H), and/or T at position 460 (N or S), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 456, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an L at position 456, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an H at position 457 and an R at position 458, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a K at position 457 and an N at position 460, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 458, an H at position 459, and an S at position 460, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 456, an R at position 457, and an R at position 458, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0185] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than I at position 451, an amino acid other than N at position 452, and an amino acid other than G at position 453, numbered according to SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises E at position 451, R at position 452, and V at position 453, numbered according to SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the substitutions I451E, N452R, and G453V, numbered according to SEQ ID NO: 138 or 981.

[0186] In some embodiments, the AAV capsid variant comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence is present immediately subsequent to position 455 and wherein the AAV capsid variant comprises the E at position 451, R at position 452, and V at position 453, numbered according to the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the substitutions 145 IE, N452R, and G453V, and further comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), wherein the amino acid sequence is present immediately subsequent to position 455, all numbered according to SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of ERVSGSPHSKA (SEQ ID NO: 6399), and wherein the amino acid sequence is present immediately subsequent to position 449 and replaces positions 450-455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence of KTERVSGSPHSKAQNQQT (SEQ ID NO: 3589), wherein the amino acid sequence is present immediately subsequent to position 448 and replaces positions 449-460, numbered according to SEQ ID NO: 138.

[0187] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than T at position 450, an amino acid other than I at position 451 , and an amino acid other than N at position 452, numbered according to SEQ ID NO: 138 or 982. In some embodiments, the AAV capsid variant comprises A at position 450, E at position 451, and I at position 452, numbered according to SEQ ID NO: 138 or 982. In some embodiments, the AAV capsid variant comprises the substitutions T450A, I451E, and N452I, numbered according to SEQ ID NO: 138 or 982.

[0188] In some embodiments, the AAV capsid variant comprises the amino acid sequence of SPHKSG (SEQ ID NO: 946), which is present immediately subsequent to positions 455, and further comprises A at position 450, E at position 451, 1 at position 452, H at position 454, and D at position 455, all numbered according to SEQ ID NO: 138 or 982. In some embodiments, the AAV capsid variant comprises the substitutions T450A, 145 IE, N452I, S454H, and G455D, and further comprises the amino acid sequence SPHKSG (SEQ ID NO: 946) present immediately subsequent to position 455, all numbered according to SEQ ID NO: 138 or 982. In some embodiments, the AAV capsid variant comprises the amino acid sequence of AEIGHDSPHKSG (SEQ ID NO: 6400), wherein the amino acid sequence is present immediately subsequent to position 449 and replaces positions 450- 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence of KAEIGHDSPHKSGQNQQT (SEQ ID NO: 1754), wherein the amino acid sequence is present immediately subsequent to position 448 and replaces positions 449- 460, numbered according to SEQ ID NO: 138.

[0189] In some embodiments, the AAV capsid variant, further comprises a substitution at position K449, e.g., a K449R substitution, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant, further comprises an amino acid other than K at position 449 (e.g., R), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an R at position 449, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification, e.g., an insertion, substitution, and/or deletion in loop I, II, VI, and/or VIII.

[0190] In some embodiments, the AAV capsid variant, further comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant, further comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 amino acids that differ from the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0191] In some embodiments, the AAV capsid variant further comprises (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 138, 981, or 982; (b) a VP2 protein comprising the amino acid sequence of positions 138-736 of SEQ ID NO: 138 or positions 138-742 of SEQ ID NO: 981 or 982; (c) a VP3 protein comprising the amino acid sequence of positions 203-736 of SEQ ID NO: 138 or positions 203-742 of SEQ ID NO: 981 or 982; or (d) an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity to any of the amino acid sequences in (a)-(c), an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids relative to any of the amino acid sequences in (a)-(c), or an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (a)-(c).

[0192] In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137.

[0193] In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137. [0194] In some embodiments, an AAV capsid variant of the present disclosure comprises an amino acid sequence as described herein, e.g., an amino acid sequence of an AAV capsid variant of TTM-001 or TTM-002, e.g., as described in Tables 3 and 4.

[0195] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, and/or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of an AAV capsid variant of TTM-001 or TTM-002, e.g., as described in Tables 3 and 4.

[0196] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTM-001 or TTM-002, e.g., as described in Tables 3 and 5.

[0197] In some embodiments, a polynucleotide or nucleic acid encoding an AAV capsid variant, of the present disclosure comprises a nucleotide sequence described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTM-001 or TTM-002, e.g., as described in Tables 3 and 5.

Table 3. Exemplary full length capsid sequences

Table 4. Exemplary full length capsid amino acid sequences

Table 5. Exemplary full length capsid nucleic acid sequences [0198] In some embodiments, the polynucleotide encoding an AAV capsid variant, described herein comprises the nucleotide sequence of SEQ ID NO: 983 or 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0199] In some embodiments, the polynucleotide encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 983. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.

[0200] In some embodiments, the polynucleotide encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 984. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 984. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.

[0201] In some embodiments, an AAV capsid variant described herein, comprises the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 981. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 981.

[0202] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 982. In some embodiments, the AAV capsid variant, comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 982.

[0203] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983 or 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 983 or 984. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 983 or 984.

[0204] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, VP3 protein, or a combination thereof. In some embodiments, an AAV capsid variant comprises the amino acid sequence corresponding to positions 138-742, e.g., a VP2, of SEQ ID NO: 981 or 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid protein comprises the amino acid sequence corresponding to positions 203-742, e.g., a VP3, of SEQ ID NO: 981 or 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence corresponding to positions 1-742, e.g., a VP1, of SEQ ID NO: 981 or 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0205] In some embodiments, an AAV capsid variant, described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138. [0206] In some embodiments, an AAV capsid variant described herein transduces a brain region, e.g., a midbrain region (e.g., the hippocampus, or thalamus) or the brain stem. In some embodiments, the level of transduction is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65-fold greater as compared to a reference sequence of SEQ ID NO: 138. In some embodiments, the level of transduction is at least 30, 35, 40, 45, 50, 55, 60, or 65-fold greater as compared to a reference sequence of SEQ ID NO: 138.

[0207] In some embodiments, an AAV capsid variant described herein is enriched at least about 3, 4, 5, 6, 7, 8, 9, or 10-fold in the brain compared to a reference sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85-fold in the brain compared to a reference sequence of SEQ ID NO: 138.

[0208] In some embodiments, an AAV capsid variant described herein is enriched in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse) species, compared to a reference sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse) species, compared to a reference sequence of SEQ ID NO: 138. In some embodiments, the at least two to three species are Macacafascicularis, Chlorocebus sabaeus, Callithrixjacchus, and/or mouse (e.g., BALB/c mice, C57B1/6 mice, and/or CD-I outbred mice).

[0209] In some embodiments, an AAV capsid variant described herein is enriched at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8-fold, in the brain compared to a reference sequence of SEQ ID NO: 981. In some embodiments, an AAV capsid variant described herein is enriched about 2, 2.5, 3, 3.5, 4, 4.5, 5, or 5.5-fold, in the brain compared to a reference sequence of SEQ ID NO: 982. [0210] In some embodiments, an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region. In some embodiments, the level of viral genomes is increased by at least 20, 25, 30, 35, 40, 45, or 50-fold, as compared to a reference sequence of SEQ ID NO: 138.

In some embodiments, the brain region comprises a midbrain region (e.g., the hippocampus or thalamus) and/or the brainstem.

[0211] In some embodiments, an AAV capsid variant described herein delivers an increased level of a payload to a brain region. In some embodiments, the level of the payload is increased by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70-fold, as compared to a reference sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a midbrain region (e.g., the hippocampus or thalamus) and/or the brainstem.

[0212] In some embodiments, an AAV capsid variant described herein is enriched at least about 5, 10, 15, 20, 25, 30, or 35-fold, in the spinal cord compared to a reference sequence of SEQ ID NO: 138. [0213] In some embodiments, an AAV capsid variant described herein shows preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the liver. In some embodiments, the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the liver and the DRG. In some embodiments, the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the heart. In some embodiments, the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the heart, and DRG. In some embodiments, the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the heart, DRG, and liver.

[0214] In some embodiments, an AAV capsid variant described herein is capable of transducing non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes). In some embodiments, the AAV capsid variant described herein is capable of transducing neuronal cells and non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes). In some embodiments, the non-neuronal cells are glial cells, oligodendrocytes (e.g., Olig2 positive oligodendrocytes), or astrocytes (e.g., Olig2 positive astrocytes). In some embodiments, the AAV capsid variant is capable of transducing Olig2 positive cells, e.g., Olig2 positive astrocytes or Olig2 positive oligodendrocytes.

[0215] In some embodiments, an AAV capsid variant described herein is capable of binding to a glycosylphosphatidylinositol (GPI) anchored protein, e.g., alkaline phosphatase (ALPL). In some embodiments, the GPI anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mice, NHPs (e.g., Macacafascicularis), and/or humans). In some embodiments, the GPI anchored protein is present on the surface of a cell in the blood brain barrier. In some embodiments, the GPI anchored protein is ALPL. In some embodiments, the AAV capsid variant is capable of binding N-linked galactose. In some embodiments, binding to ALPL results in increased cellular transduction, e.g., as compared to a reference sequence of SEQ ID NO: 138. In some embodiments, binding to ALPL results in increased crossing of the blood brain barrier, e.g., as compared to a reference sequence of SEQ ID NO: 138. Without wishing to be bound by theory, it is believed in some embodiments, that the binding of the AAV capsid variants described herein to ALPL is part of the mechanism leading to increased crossing of the blood brain barrier relative to the AAV9 control. Without wishing to be bound by theory, it is believed in some embodiments, that ALPL is upregulated in aging brain (e.g., as described in Yang et al. “Physiological blood-brain transport is impaired with age by a shift in transcytosis,” Nature. 2020 583:425-430, the contents of which are hereby incorporated by reference in its entirety).

[0216] In some embodiments, an AAV capsid variant of the present disclosure is isolated, e.g., recombinant. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure is isolated, e.g., recombinant. [0217] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.

Additional AAV Sequences

[0218] In some embodiments, the AAV capsid variant, comprises immediately subsequent to position 448, 452, 453, 455, numbered relative to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)), at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids of any of amino acid sequence provided in Tables 1, 2A, 2B, 2C, 13-19. In some embodiments, the amino acid sequence replaces at least one, two, three, four, five, six, seven, eight, nine or all of positions T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, and/or Q459, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the amino acid sequence replaces positions S454, G455, or both positions S454 and G455, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type, e.g., native, amino acid, at one, two, three, four, five, six, seven, eight, nine or all of positions T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, and/or Q459, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type, e.g., native, amino acid, at position S454, G455, or both positions S454 and G455, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at one, two, three, four, five, six, seven, eight, nine or all of positions T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, and/or Q459, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at position S454, G455, or both positions S454 and G455, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 1, SEQ ID NO: 11, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)).

[0219] In some embodiments, an AAV capsid polypeptide or AAV capsid variant described herein may comprise a VOY101 capsid polypeptide, an AAVPHP.B (PHP.B) capsid polypeptide, a AAVPHP.N (PHP.N) capsid polypeptide, an AAV 1 capsid polypeptide, an AAV2 capsid polypeptide, an AAV5 capsid polypeptide, an AAV9 capsid polypeptide, an AAV9 K449R capsid polypeptide, an AAVrhlO capsid polypeptide, or a functional variant thereof. In some embodiments, the AAV capsid polypeptide, e.g., AAV capsid variant, comprises an amino acid sequence of any of the AAV capsid polypeptides in Table 6, or an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid polypeptide comprises any one of the nucleotide sequences in Table 6, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.

[0220] In some embodiments, an AAV capsid polypeptide or an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid polypeptide or the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the AAV capsid polypeptide or the AAV capsid variant, comprises substitution at position K449, e.g., a K449R substitution, numbered according to SEQ ID NO: 138.

[0221] In some embodiments, the AAV capsid polypeptide or the AAV capsid variant, comprises a peptide comprising the amino acid sequence of TLAVPFK (SEQ ID NO: 4680). In some embodiments, the peptide is present immediately subsequent to position 588, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, the capsid polypeptide comprises the amino acid substitutions of A587D and Q588G, numbered according to SEQ ID NO: 138.

[0222] In some embodiments, the AAV capsid polypeptide or the AAV capsid variant comprises the amino acid substitution of K449R, numbered according to SEQ ID NO: 138; and a peptide comprising the amino acid sequence of TLAVPFK (SEQ ID NO: 4680), wherein the peptide is present immediately subsequent to position 588, relative to a reference sequence numbered according to SEQ ID NO: 138.

[0223] In some embodiments, the AAV capsid polypeptide or the AAV capsid variant comprises the amino acid substitution of K449R, numbered according to SEQ ID NO: 138; an peptide comprising the amino acid sequence of TLAVPFK (SEQ ID NO: 4680), wherein the insert is present immediately subsequent to position 588, relative to a reference sequence numbered according to SEQ ID NO: 138; and the amino acid substitutions of A587D and Q588G, numbered according to SEQ ID NO: 138.

[0224] In some embodiments, the AAV capsid polypeptide or the AAV capsid variant comprises a peptide comprising the amino acid sequence of TLAVPFK (SEQ ID NO: 4680), wherein the insert is present immediately subsequent to position 588, relative to a reference sequence numbered according to SEQ ID NO: 138; and the amino acid substitutions of A587D and Q588G, numbered according to SEQ ID NO: 138.

[0225] In some embodiments, the AAV capsid polypeptide or the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20, or 10 modifications, e.g., substitutions (conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 11, optionally wherein position 449 is not R.

[0226] In some embodiments, the AAV capsid polypeptide or AAV capsid variant, comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 1. Table 6. AAV Sequences

Viral Genome of the AAV particle

[0227] In some embodiments, an AAV particle as described herein comprising an AAV capsid variant described herein, may be used for the delivery of a viral genome to a tissue (e.g., CNS, DRG, and/or muscle). In some embodiments, an AAV particle comprising an AAV capsid variant described herein can be used for delivery of a viral genome to a tissue or cell, e.g., CNS, DRG, or muscle cell or tissue. In some embodiments, an AAV particle of the present disclosure is a recombinant AAV particle. In some embodiments, an AAV particle of the present disclosure is an isolated AAV particle.

[0228] The viral genome may encode any payload, such as but not limited to a polypeptide (e.g., a therapeutic polypeptide), an antibody, an enzyme, an RNAi agent and/or components of a gene editing system. In one embodiment, the AAV particles described herein are used to deliver a payload to cells of the CNS, after intravenous delivery. In another embodiment, the AAV particles described herein are used to deliver a payload to cells of the DRG, after intravenous delivery. In some embodiments, the AAV particles described herein are used to deliver a payload to cells of a muscle, e.g., a heart muscle, after intravenous delivery.

[0229] In some embodiments, a viral genome of an AAV particle comprising an AAV capsid variant, as described herein, comprises a nucleotide sequence comprising a transgene encoding a payload. In some embodiments, the viral genome comprises an inverted terminal repeat sequence (ITR). In some embodiments, the viral genome comprises two ITR sequences, one at the 5’ end of the viral genome (e.g., 5’ relative to the encoded payload) and one at the 3’ end of the viral genome (e.g., 3’ relative to the encoded payload). In some embodiments, a viral genome of an AAV particle, e.g., an AAV particle comprising an AAV capsid variant described herein, may comprise a regulatory element (e.g., promoter), untranslated regions (UTR), a miR binding site, a polyadenylation sequence (poly A), a filler or stuffer sequence, an intron, and/or a linker sequence, e.g., for enhancing transgene expression.

[0230] In some embodiments, the viral genome components are selected and/or engineered for expression of the payload in a target tissue (e.g., CNS, muscle, or DRG).

Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0231] In some embodiments, the AAV particle comprising an AAV capsid variant described herein comprises a viral genome comprising an ITR and a transgene encoding a payload. In some embodiments, the viral genome comprises two ITRs. In some embodiments, the two ITRs flank the nucleotide sequence encoding the pay load at the 5’ and 3’ ends. In some embodiments, the ITRs function as origins of replication comprising recognition sites for replication. In some embodiments, the ITRs comprise sequence regions which can be complementary and symmetrically arranged. In some embodiments, the ITRs incorporated into viral genomes as described herein may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences. [0232] In some embodiments, the ITR may be from the same serotype as the capsid polypeptide, e.g., capsid variant, selected from any of the known serotypes, or a variant thereof. In some embodiments, the ITR may be of a different serotype than the capsid. In some embodiments, the viral genome comprises two ITR sequence regions, wherein the ITRs are of the same serotype as one another. In some embodiments, the viral genome comprises two ITR sequence regions, wherein the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid. In one embodiment both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

Viral Genome Component: Promoters

[0233] In some embodiments, viral genome of an AAV particle described herein comprises at least one element to enhance the payload target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety). Nonlimiting examples of elements to enhance payload target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.

[0234] In some embodiments, an AAV particle comprising an AAV capsid variant described herein comprises a viral genome comprising a nucleic acid comprising a transgene encoding a payload, wherein the transgene is operably linked to a promoter. In some embodiments, the promoter is a species specific promoter, an inducible promoter, a tissue-specific promoter, or a cell cyclespecific promoter (e.g., a promoter as described in Parr et al., Nat. Med.3: 1145-9 (1997); the contents of which are herein incorporated by reference in their entirety). [0235] In some embodiments, the Promoter may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include those derived from viruses, plants, mammals, or humans. In some embodiments, the promoters may be those derived from human cells or systems. In some embodiments, the promoter may be truncated or mutated, e.g., a promoter variant.

[0236] In some embodiments, the promoter is a ubiquitous promoter, e.g., capable of expression in multiple tissues. In some embodiments the promoter is an human elongation factor la-subunit (EFla) promoter, the cytomegalovirus (CMV) immediate -early enhancer and/or promoter, the chicken P-actin (CBA) promoter and its derivative CAG, glucuronidase (GUSB) promoter, or ubiquitin C (UBC) promoter. In some embodiments, the promoter is a cell or tissue specific promoter, e.g., capable of expression in tissues or cells of the central or peripheral nervous systems, targeted regions within (e.g., frontal cortex), and/or sub-sets of cells therein (e.g., excitatory neurons). In some embodiments, the promoter is a cell-type specific promoters capable of expression of a payload in excitatory neurons (e.g., glutamatergic), inhibitory neurons (e.g., GABA-ergic), neurons of the sympathetic or parasympathetic nervous system, sensory neurons, neurons of the dorsal root ganglia, motor neurons, or supportive cells of the nervous systems such as microglia, glial cells, astrocytes, oligodendrocytes, and/or Schwann cells.

[0237] In some embodiments, the promoter is a liver specific promoter (e.g., hAAT, TBG), skeletal muscle specific promoter (e.g., desmin, MCK, C512), B cell promoter, monocyte promoter, leukocyte promoter, macrophage promoter, pancreatic acinar cell promoter, endothelial cell promoter, lung tissue promoter, and/or cardiac or cardiovascular promoter (e.g., aMHC, cTnT, and CMV- MLC2k).

[0238] In some embodiments, the promoter is a tissue-specific promoter for payload expression in a tissue or cell of the central nervous system. In some embodiments, the promoter is a synapsin (Syn) promoter, glutamate vesicular transporter (VGLUT) promoter, vesicular GABA transporter (VGAT) promoter, parvalbumin (PV) promoter, sodium channel Na _v 1.8 promoter, tyrosine hydroxylase (TH) promoter, choline acetyltransferase (ChaT) promoter, methyl -CpG binding protein 2 (MeCP2) promoter, Ca ²⁺ /calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light (NFL) or heavy (NFH) promoter, neuron-specific enolase (NSE) promoter, P-globin minigene np2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, and excitatory amino acid transporter 2 (EAAT2) promoter, or a fragment thereof. In some embodiments, the promoter is a cell-type specific promoter capable of expression in an astrocyte, e.g., a glial fibrillary acidic protein (GFAP) promoter and a EAAT2 promoter, or a fragment thereof. In some embodiments, the promoter is a cell-type specific promoter capable of expression in an oligodendrocyte, e.g., a myelin basic protein (MBP) promoter or a fragment thereof. [0239] In some embodiments, the promoter is a GFAP promoter. In some embodiments, the promoter is a synapsin (syn or synl) promoter, or a fragment thereof. [0240] In some embodiments, the promoter comprises an insulin promoter or a fragment thereof.

[0241] In some embodiments, the promoter of the viral genome described herein (e.g., comprised within an AAV particle comprising an AAV capsid variant described herein) comprises an EF-la promoter or variant thereof, e.g., as provided in Table 8. In some embodiments, the EF-la promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 987, 988, 990, 991, 995, 996, 998- 1007 or any one of the sequences provided in Table 8, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NOs: 987, 988, 990, 991, 995, 996, 998-1007 or any one of the sequences provided in Table 8, or a nucleotide sequence with at least 70% (e.g., 80, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 987, 988, 990, 991, 995, 996, 998-1007 or any one of the sequences provided in Table 8.

Table 8. Exemplary Promoter Variants

Viral Genome Component: Untranslated Regions (UTRs)

[0242] In some embodiments, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0243] Features typically found in abundantly expressed genes of specific target organs (e.g., CNS tissue, muscle, or DRG) may be engineered into UTRs to enhance stability and protein production. As a non-limiting example, a 5’ UTR from mRNA normally expressed in the brain (e.g., huntingtin) may be used in the viral genomes of the AAV particles described herein to enhance expression in neuronal cells or other cells of the central nervous system.

[0244] While not wishing to be bound by theory, wild- type 5' untranslated regions (UTRs) include features which play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another ’G.

[0245] In one embodiment, the 5 ’UTR in the viral genome includes a Kozak sequence.

[0246] In one embodiment, the 5 ’UTR in the viral genome does not include a Kozak sequence.

[0247] While not wishing to be bound by theory, wild-type 3' UTRs are known to have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in its entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class III ARES, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0248] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of a polynucleotide. When engineering specific polynucleotides, e.g., payload regions of viral genomes, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0249] In one embodiment, the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

[0250] In one embodiment, the viral genome may include at least one miRNA seed, binding site or full sequence. microRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. In some embodiments, a microRNA sequence comprises a seed region, e.g., a sequence in the region of positions 2-8 of the mature microRNA, which has Watson-Crick sequence fully or partially complementarity to the miRNA target sequence of the nucleic acid.

[0251] In one embodiment, the viral genome may be engineered to include, alter or remove at least one miRNA binding site, full sequence or seed region.

[0252] Any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location. In one embodiment, the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered” as it relates to a UTR, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.

[0253] In one embodiment, the viral genome of the AAV particle comprises at least one artificial UTR which is not a variant of a wild type UTR.

[0254] In one embodiment, the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature or property.

Viral Genome Component: Poly adenylation Sequence

[0255] The viral genome of the AAV particle described herein (e.g., an AAV particle comprising an AAV capsid variant, described herein) may comprise a polyadenylation sequence. In some embodiments, the viral genome of the AAV particle (e.g., an AAV particle comprising an AAV capsid variant, described herein) comprises a polyadenylation sequence between the 3’ end of the nucleotide sequence encoding the payload and the 5’ end of the 3’ITR. Viral Genome Component: Introns

[0256] In some embodiments, the viral genome of the AAV particle as described herein (e.g., an AAV particle comprising an AAV capsid variant), comprises an element to enhance the payload target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, Discov. Med, 2015, 19(102): 49-57; the contents of which are herein incorporated by reference in their entirety), such as an intron. Non-limiting examples of introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), P- globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/immunoglobin splice acceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S) (180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).

Viral Genome Component: Staffer sequences

[0257] In some embodiments, the viral genome of an AAV particle described herein (e.g., an AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid variant), comprises an element to improve packaging efficiency and expression, such as a stuffer or filler sequence. Nonlimiting examples of stuffer sequences include albumin and/or alpha- 1 antitrypsin. Any known viral, mammalian, or plant sequence may be manipulated for use as a stuffer sequence.

[0258] In one embodiment, the stuffer or filler sequence may be from about 100-3500 nucleotides in length. The stuffer sequence may have a length of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 nucleotides.

Viral Genome Component: miRNA

[0259] In one embodiment, the viral genome comprises a sequence encoding a miRNA to reduce the expression of the payload in a tissue or cell, e.g., the DRG (dorsal root ganglion), or neurons of other ganglia, such as those of the sympathetic or parasympathetic nervous system. In some embodiments, a miRNA, e.g., a miR183, a miR182, and/or miR96, may be encoded in the viral genome to modulate, e.g., reduce the expression, of the viral genome in a DRG neuron. As another non-limiting example, a miR-122 miRNA may be encoded in the viral genome to modulate, e.g., reduce, the expression of the viral genome in the liver. In some embodiments, a miRNA, e.g., a miR- 142-3p, may be encoded in the viral genome to modulate, e.g., reduce, the expression, of the viral genome in a cell or tissue of the hematopoietic lineage, including for example immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B -lymphocytes). In some embodiments, a miRNA, e.g., a miR-1, may be encoded in the viral genome to modulate, e.g., reduce, the expression, of the viral genome in a cell or tissue of the heart. Viral Genome Component: miR Binding Site

[0260] Tissue- or cell-specific expression of the AAV viral particles disclosed herein can be enhanced by introducing tissue- or cell-specific regulatory sequences, e.g., promoters, enhancers, microRNA binding sites, e.g., a detargeting site. Without wishing to be bound by theory, it is believed that an encoded miR binding site can modulate, e.g., prevent, suppress, or otherwise inhibit, the expression of a gene of interest on the viral genome disclosed herein, based on the expression of the corresponding endogenous microRNA (miRNA) or a corresponding controlled exogenous miRNA in a tissue or cell, e.g., a non-targeting cell or tissue. In some embodiments, a miR binding site modulates, e.g., reduces, expression of the payload encoded by a viral genome of an AAV particle described herein in a cell or tissue where the corresponding mRNA is expressed.

[0261] In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a microRNA binding site, e.g., a detargeting site. In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a miR binding site, a microRNA binding site series (miR BSs), or a reverse complement thereof.

[0262] In some embodiments, the nucleotide sequence encoding the miR binding site series or the miR binding site is located in the 3’-UTR region of the viral genome (e.g., 3’ relative to the nucleotide sequence encoding a payload), e.g., before the polyA sequence, 5’-UTR region of the viral genome (e.g., 5’ relative to the nucleotide sequence encoding a payload), or both.

[0263] In some embodiments, the encoded miR binding site series comprise at least 1-5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS). In some embodiments, all copies are identical, e.g., comprise the same miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g., a non-coding sequence. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides, nucleotides in length. In some embodiments, the spacer coding sequence or reverse complement thereof comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)- (iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions relative to the nucleotide sequence of GATAGTTA.

[0264] In some embodiments, the encoded miR binding site series comprise at least 1-5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS). In some embodiments, at least 1, 2, 3, 4, 5, or all of the copies are different, e.g., comprise a different miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g., a non-coding sequence. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides, in length. In some embodiments, the spacer comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, but no more than four modifications, e.g., substitutions, insertions, or deletions relative to the nucleotide sequence of GATAGTTA.

[0265] In some embodiments, the encoded miR binding site is substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), to the miR in the host cell. In some embodiments, the encoded miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches to a miR in the host cell. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous. In some embodiments, the mismatched nucleotides occur outside the seed region-binding sequence of the miR binding site, such as at one or both ends of the miR binding site. In some embodiments, the miR binding site is 100% identical to the miR in the host cell.

[0266] In some embodiments, the nucleotide sequence encoding the miR binding site is substantially complementary (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementary), to the miR in the host cell. In some embodiments, to complementary sequence of the nucleotide sequence encoding the miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches to a miR in the host cell. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are noncontiguous. In some embodiments, the mismatched nucleotides occur outside the seed region-binding sequence of the miR binding site, such as at one or both ends of the miR binding site. In some embodiments, the encoded miR binding site is 100% complementary to the miR in the host cell.

[0267] In some embodiments, an encoded miR binding site or sequence region is at least about 10 to about 125 nucleotides in length, e.g., at least about 10 to 50 nucleotides, 10 to 100 nucleotides, 50 to 100 nucleotides, 50 to 125 nucleotides, or 100 to 125 nucleotides in length. In some embodiments, an encoded miR binding site or sequence region is at least about 7 to about 28 nucleotides in length, e.g., at least about 8-28 nucleotides, 7-28 nucleotides, 8-18 nucleotides, 12-28 nucleotides, 20-26 nucleotides, 22 nucleotides, 24 nucleotides, or 26 nucleotides in length, and optionally comprises at least one consecutive region (e.g., 7 or 8 nucleotides) complementary (e.g., fully or partially complementary) to the seed sequence of a miRNA (e.g., a miR122, a miR142, a miR183, or a miRl).

[0268] In some embodiments, the encoded miR binding site is complementary (e.g., fully or partially complementary) to a miR expressed in liver or hepatocytes, such as miR122. In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR 122 binding site sequence. In some embodiments, the encoded miR122 binding site comprises the nucleotide sequence of ACAAACACCATTGTCACACTCCA (SEQ ID NO: 4673), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., insertions, deletions, or substitutions, relative to the nucleotide sequence of SEQ ID NO: 4673, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR122 binding site, e.g., an encoded miR122 binding site series, optionally wherein the encoded miR 122 binding site series comprises the nucleotide sequence of: ACAAACACCATTGTCACACTCCACACAAACACCATTGTCACACTCCACACAAACACCATT GTCACACT CCA (SEQ ID NO: 4674), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4674, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, at least two of the encoded miR122 binding sites are connected directly, e.g., without a spacer. In other embodiments, at least two of the encoded miR122 binding sites are separated by a spacer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive encoded miR122 binding site sequences. In embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8, in length. In some embodiments, the spacer coding sequence or reverse complement thereof comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, an encoded miR binding site series comprises at least 3-5 copies (e.g., 4 copies) of a miR 122 binding site, with or without a spacer, wherein the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions relative to the nucleotide sequence of GATAGTTA.

[0269] In some embodiments, the encoded miR binding site is complementary (e.g., fully or partially complementary) to a miR expressed in the heart. In embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR-1 binding site. In some embodiments, the encoded miR-1 binding site comprises the nucleotide sequence of ATACATACTTCTTTACATTCCA (SEQ ID NO: 4679), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4679, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR-1 binding site, e.g., an encoded miR-1 binding site series. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR-1 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA.

[0270] In some embodiments, the encoded miR binding site is complementary (e.g., fully or partially complementary) to a miR expressed in hematopoietic lineage, including immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B -lymphocytes). In some embodiments, the encoded miR binding site complementary to a miR expressed in hematopoietic lineage comprises a nucleotide sequence disclosed, e.g., in US 2018/0066279, the contents of which are incorporated by reference herein in its entirety.

[0271] In embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR-142-3p binding site sequence. In some embodiments, the encoded miR-142-3p binding site comprises the nucleotide sequence of TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 4675), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4675, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR-142-3p binding site, e.g., an encoded miR-142-3p binding site series. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR-142-3p binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA.

[0272] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, a miR182, and/or miR96 binding site. In some embodiments, the encoded miR binding site is complementary to a miR expressed in expressed in a DRG neuron comprises a nucleotide sequence disclosed, e.g., in WO2020/132455, the contents of which are incorporated by reference herein in its entirety.

[0273] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR183 binding site sequence. In some embodiments, the encoded miR183 binding site comprises the nucleotide sequence of AGTGAATTCTACCAGTGCCATA (SEQ ID NO: 4676), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4676, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the sequence complementary to the seed sequence corresponds to the double underlined of the encoded miR- 183 binding site sequence. In some embodiments, the viral genome comprises at least comprises at least 2, 3, 4, or 5 copies (e.g., at least 2 or 3 copies) of the encoded miR183 binding site, e.g., an encoded miR183 binding site. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR183 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).

[0274] In some embodiments, the encoded miR binding site or the encoded miR binding site series comprises a miR182 binding site sequence. In some embodiments, the encoded miR182 binding site comprises, the nucleotide sequence of AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 4677), a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4677, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR182 binding site, e.g., an encoded miR182 binding site series. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR182 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).

[0275] In certain embodiments, the encoded miR binding site or the encoded miR binding site series comprises a miR96 binding site sequence. In some embodiments, the encoded miR96 binding site comprises the nucleotide sequence of AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 4678), a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 4678, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR96 binding site, e.g., an encoded miR96 binding site series. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR96 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).

[0276] In some embodiments, the encoded miR binding site series comprises a miR122 binding site, a miR-1, a miR142 binding site, a miR183 binding site, a miR182 binding site, a miR 96 binding site, or a combination thereof. In some embodiments, the encoded miR binding site series comprises at least 2, 3, 4, or 5 copies of a miR122 binding site, a miR142 binding site, a miR183 binding site, a miR182 binding site, a miR 96 binding site, or a combination thereof. In some embodiments, at least two of the encoded miR binding sites are connected directly, e.g., without a spacer. In other embodiments, at least two of the encoded miR binding sites are separated by a spacer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive encoded miR binding site sequences. In embodiments, the spacer is at least about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer coding sequence or reverse complement thereof comprises one or more of (i) GGAT ; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA.

[0277] In some embodiments, an encoded miR binding site series comprises at least 2-5 copies (e.g., 2 or 3 copies) of a combination of at least two, three, four, five, or all of a miR-1, miR 122 binding site, a miR142 binding site, a miR183 binding site, a miR182 binding site, a miR96 binding site, wherein each of the miR binding sites within the series are continuous (e.g., not separated by a spacer) or are separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA.

[0278] In some embodiments, an encoded miR binding site series comprises at least 2-5 copies (e.g., 2 or 3 copies) of a combination of a miR-122 binding site and a miR-1 binding site, wherein each of the miR binding sites within the series are continuous (e.g., not separated by a spacer) or are separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, insertions, or deletions, but no more than four modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of GATAGTTA.

Genome Size

[0279] In one embodiment, the AAV particle described herein (e.g., an AAV particle comprising an AAV capsid variant), may comprise a single-stranded or double-stranded viral genome. The size of the viral genome may be small, medium, large or the maximum size. As described above, the viral genome may comprise a promoter and a polyA tail.

[0280] In one embodiment, the viral genome may be a small single stranded viral genome. A small single stranded viral genome may be 2.1 to 3.5 kb in size such as, but not limited to, about 2.1,

2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size.

[0281] In one embodiment, the viral genome may be a small double stranded viral genome. A small double stranded viral genome may be 1.3 to 1.7 kb in size such as, but not limited to, about 1.3, 1.4, 1.5, 1.6, and 1.7 kb in size.

[0282] In one embodiment, the viral genome may be a medium single stranded viral genome. A medium single stranded viral genome may be 3.6 to 4.3 kb in size such as, but not limited to, about

3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size.

[0283] In one embodiment, the viral genome may be a medium double stranded viral genome. A medium double stranded viral genome may be 1.8 to 2.1 kb in size such as, but not limited to, about 1.8, 1.9, 2.0, and 2.1 kb in size.

[0284] In one embodiment, the viral genome may be a large single stranded viral genome. A large single stranded viral genome may be 4.4 to 6.0 kb in size such as, but not limited to, about 4.4, 4.5,

4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb in size.

[0285] In one embodiment, the viral genome may be a large double stranded viral genome. A large double stranded viral genome may be 2.2 to 3.0 kb in size such as, but not limited to, about 2.2,

2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size.

Payloads and Active Agents

[0286] In some embodiments, a ligand described herein is fused to an active agent. In some embodiments the active agent is a therapeutic agent or a diagnostic agent. In some embodiments, the ligand is a component of an AAV particle, wherein the AAV particle comprises a viral genome encoding a payload. In some embodiments, the encoded payload comprises a therapeutic agent.

[0287] In some embodiments, the encoded payload or active agent comprises a therapeutic protein, an antibody molecule, an enzyme, one or more components of a genome editing system, an Fc polypeptide fused or coupled (e.g., covalently or non covalently) to a therapeutic agent, and/or an RNAi agent (e.g., a dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, IncRNA, piRNA, or snoRNA). In some embodiments, the encoded payload or active agent modulates, e.g., increases or decreases, the presence, level, and/or activity of a gene, mRNA, protein, or a combination thereof, e.g., in a cell or a tissue.

Polypeptides

[0288] In some embodiments, the encoded payload or the active agent comprises a polypeptide, protein, or peptide, e.g., a polypeptide, protein, or peptide described herein. The nucleic acid encoding the payload, may encode a product of any known gene and/or a recombinant version thereof. The active agent can be any known protein or a recombinant version thereof. In some embodiments, the encoded payload or the active agent is an apolipoprotein E (APOE) protein such as, but not limited to ApoE2, ApoE3 and/or ApoE4 protein. In one embodiment, the encoded payload or the active agent is ApoE2 (cysl 12, cysl58) protein or a fragment or variant thereof. In one embodiment, the encoded payload or the active agent is an ApoE3 (cysl 12, argl58) protein or fragment or variant thereof. In one embodiment, the encoded payload or the active agent is an ApoE4 (argl 12, argl58) protein or fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises an aromatic L-amin acid decarboxylase (AADC) protein. As another non-limiting example, the encoded payload or the active agent comprises an antibody, or a fragment thereof. As another non-limiting example, the encoded payload or the active agent comprises a human survival of motor neuron (SMN) 1 or SMN2 protein, or fragments or variants thereof. As another non-limiting example, the encoded payload or the active agent comprises a glucocerebrosidase (GBA1) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises a granulin precursor or progranulin (GRN) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises an aspartoacylase (ASPA) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises a tripeptidyl peptidase I (CLN2) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises a beta-galactosidase (GLB1) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises a N-sulphoglucosamine sulphohydrolase (SGSH) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises an N-acetyl-alpha-glucosaminidase (NAGLU) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises an iduronate 2-sulfatase (IDS) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises an intracellular cholesterol transporter (NPC1) protein, or a fragment or variant thereof. As another non-limiting example, the encoded payload or the active agent comprises a gigaxonin (GAN) protein, or a fragment or variant thereof.

[0289] In some embodiments, the encoded payload or the active agent comprises an Fc polypeptide. In some embodiments, the Fc polypeptide is fused or coupled to a therapeutic agent, e.g., a therapeutic protein or enzyme.

Antibody Molecules and Antibody Binding Fragments

[0290] In some embodiments, the encoded payload or the active agent is an antibody molecule. In some embodiments, the antibody molecule binds a CNS related target, e.g., an antigen associated with a neurological or neurodegenerative disorder. In some embodiments, the antibody molecule binds a muscular or neuromuscular related target, e.g., an antigen associated with a muscular or neuromuscular disorder. In some embodiments, the antibody molecule binds a neuro-oncology related target, e.g., an antigen associated with a neuro-oncological disorder.

[0291] In some embodiments, the antibody molecule binds to P-amyloid, APOE, tau, SOD1, TDP-43, huntingtin, and/or synuclein. In some embodiments, the encoded payload comprises an antibody or antibody fragment that binds to a neuro-oncology related target, e.g., HER2, EGFR (e.g., EGFRvIII). In some embodiments, the antibody molecule binds to HER2/neu. In some embodiments, the antibody molecule binds to P-amyloid. In some embodiments, the antibody molecule binds to tau. [0292] In some embodiments, the active agent comprises an antibody-drug conjugate. In some embodiments, the antibody molecule is conjugated to a cytotoxic or cytostatic agent, e.g., a chemotherapeutic agent or an anti-neoplastic drug. In some embodiments, the antibody is conjugated to a radioactive isotope, e.g., a-, P-, or y-emitter, or P-and y-emitter.

Gene Editing System

[0293] In some embodiments, the encoded payload or the encoded active agent comprises a gene editing system or one or more components thereof. In some embodiments, the gene editing system comprises nucleic acid sequences that encode proteins having enzymatic activity to (i) selectively induce double or single stranded breaks in a DNA or RNA sequence, or (ii) substitute, insert or delete a particular base or set of bases of a DNA or RNA sequence in the absence of a double or single stranded break in the DNA or RNA. In some embodiments, the gene editing system includes, but is not limited to a CRISPR-Cas system (including different Cas or Cas-related nucleases), a Zinc finger nuclease, a meganuclease, a TALEN or a base editors. In some embodiments, the gene editing system comprises a chromosomal integration of a transgene, e.g., introduced by a parvovirus vector in the absence of an exogenous nuclease or an enzymatic entity.

RNAi agents

[0294] In some embodiments, the encoded payload or the active agent comprises an RNAi agent, e.g., an RNAi agent described herein. In some embodiments, the encoded payload or the active agent comprises a dsRNA, a siRNA, a shRNA, a pre-miRNA, a pri-miRNA, a miRNA, a stRNA, a IncRNA, a piRNA, an antisense oligonucleotide (ASO), or a snoRNA. In some embodiments, the encoded payload or the active agent comprises an RNAi agent for inhibiting expression of a SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A- SCN5A, or SCN8A-SCN11A gene, protein, and/or mRNA. In some embodiments, the RNAi agent described herein inhibits SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, or SCN8A-SCN11A.

[0295] In some embodiment, the encoded payload or the active agent comprises an RNAi agent which targets the mRNA of a gene to modulate, e.g., interfere with gene expression and/or protein production. In some embodiments, the RNAi agent may target a gene at the location of a singlenucleotide polymorphism (SNP) or variant within the nucleotide sequence of the gene. In some embodiments, the RNAi agent is a siRNA. In some embodiments, the RNAi agent is an ASO.

[0296] The RNAi agent may be an siRNA duplex, wherein the siRNA duplex contains an antisense strand (guide strand) and a sense strand (passenger strand) hybridized together forming a duplex structure, wherein the antisense strand is complementary to the nucleic acid sequence of the targeted gene, and wherein the sense strand is homologous to the nucleic acid sequence of the targeted gene. In some aspects, the 5’end of the antisense strand has a 5’ phosphate group and the 3’end of the sense strand contains a 3 ’hydroxyl group. In other aspects, there are none, one or 2 nucleotide overhangs at the 3’end of each strand.

[0297] Each strand of a siRNA duplex targeting a gene of interest may be about 19 to 25, 19 to 24 or 19 to 21 nucleotides in length, preferably about 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.

[0298] In one embodiment, a siRNA or dsRNA includes at least two sequences that are complementary to each other. The dsRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding the target gene, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length. Generally, the dsRNA is 19 to 25, 19 to 24 or 19 to 21 nucleotides in length. In some embodiments, the dsRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the dsRNA is from about

25 to about 30 nucleotides in length. In some embodiments, the dsRNA is about 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides in length,

26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, or 30 nucleotides in length.

[0299] In some embodiments, the siRNA or the ASO is conjugated to the ligand directly. In some embodiments, the siRNA or the ASO is conjugated to the ligand via a linker, e.g., a cross-linker. In some embodiments, the crosslinker comprises succinimidyl-4-(N-maleimidomethyl) and/or a saturated or unsaturated hydrocarbon chain (e.g., cyclohexane-l-carboxylate). In some embodiments, the crosslinker comprises succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate. In some embodiments, the ligand is conjugated to the RNAi agent via a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate. In some embodiments, the ligand is conjugated directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent. In some embodiments, the ligand is conjugated, e.g., directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent. In some embodiments, the ligand is conjugated, e.g., directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent. In some embodiments, the ligand is conjugated to the sense strand. In other embodiments, the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the siRNA agent, e.g., as described in WO2021207189; W02004065601; US8034376; WO2019217459; Brown et al. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nature Biotechnology. 2022; Eyford et al. A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood Brain Barrier to Attenuate Ischemic Stroke. Front Mol Biosci 2021 8:611367; which are hereby incorporated by reference in their entirety.

[0300] In some embodiments, the RNAi agent, e.g., the siRNA or ASO, further comprises a lipophilic moiety. In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or poly alicyclic compound. In some embodiments, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain, e.g., a saturated or unsaturated C16 hydrocarbon chain. In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) of the double stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone. In some embodiments, the lipophilic moiety is conjugated to the RNAi agent, e.g., the siRNA or ASO, via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate. In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage. In some embodiments, the lipophilic moiety is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof. In some embodiments, the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent. In some embodiments, the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent. In some embodiments, the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent. In some embodiments, the lipophilic moiety is conjugated, e.g., directly or indirectly via a linker to the sense strand. In some embodiments, lipophilic moiety is conjugated, e.g., directly or indirectly via a linker, to the antisense strand. In some embodiments, the lipophilic moiety and the ligand are present on the same strand, e.g., the sense strand. In some embodiments, lipophilic moiety and the ligand are present on different strands. In some embodiments, the lipophilic moiety is as described in WO2021207189; W02004065601; US8034376; WO2019217459; Brown et al. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nature Biotechnology. 2022 (the contents of which are hereby incorporated in their entirety).

[0301] In some embodiments, the RNAi agent, e.g., the siRNA or the ASO, further comprises an N-acetylgalactosamine (GalNAc) conjugate. In some embodiments, the GalNAc conjugate is attached through a monovalent linker; or a bivalent, trivalent, or tetravalent branched linker. In some embodiments, the GalNAc conjugate is as described in WO2013155204, which is hereby incorporated by reference in its entirety.

[0302] In some embodiments, the RNAi agent, e.g., an RNAi agent described herein inhibits the expression of the gene, mRNA, and/or protein by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method known in the art. In some embodiments, the RNAi agent inhibits expression of a gene, mRNA, and protein by 50-100%, e.g., by 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0303] In some embodiments, the AAV particle described herein, comprising a viral genome encoding an RNAi agent targeting a gene of interest is administered to a subject in need for treating and/or ameliorating a disease, e.g., a neurological disorder of any disease associated with the central or peripheral nervous systems.

Design of siRNA

[0304] An AAV particle described herein (e.g., an AAV particle comprising an AAV capsid variant described herein) may comprise a viral genome encoding a siRNA molecule (e.g., siRNA duplex or encoded dsRNA) that target a gene of interest and suppress target gene expression, mRNA expression, and protein production. In some aspects, the siRNA molecules are designed and used to knock out target gene variants in cells, e.g., transcripts that are identified in neurological disease. In some aspects, the siRNA molecules are designed and used to knock down target gene variants in cells. [0305] Some guidelines for designing siRNAs (for insertion into a viral genome of the AAV particles described herein) have been proposed in the art. These guidelines generally recommend generating a 19-nucleotide duplexed region, symmetric 2-3 nucleotide 3’overhangs, 5-phosphate and 3-hydroxyl groups targeting a region in the gene to be silenced. Other rules that may govern siRNA sequence preference include, but are not limited to, (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nucleotides in length. In accordance with such considerations, together with the specific sequence of a target gene, highly effective siRNA molecules essential for suppressing mammalian target gene expression may be readily designed. In some embodiments, an RNAi agent described herein, e.g., a siRNA or an ASO, is chemically modified to enhance one or more properties of the RNAi agent, e.g. stability.

[0306] In one embodiment, the sense and/or antisense strand is designed based on the method and rules outlined in European Patent Publication No. EP1752536, the contents of which are herein incorporated by reference in their entirety. As a non-limiting example, the 3 ’-terminal base of the sequence is adenine, thymine or uracil. As a non-limiting example, the 5 ’-terminal base of the sequence is guanine or cytosine. As a non-limiting example, the 3’ -terminal sequence comprises seven bases rich in one or more bases of adenine, thymine and uracil.

[0307] In one embodiment, a siRNA molecule comprises a sense strand and a complementary antisense strand in which both strands are hybridized together to form a duplex structure. The antisense strand has sufficient complementarity to the target mRNA sequence to direct target-specific RNAi, e.g., the siRNA molecule has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

[0308] In some embodiments, the antisense strand and target mRNA sequences have 100% complementarity. The antisense strand may be complementary to any part of the target mRNA sequence. Neither the identity of the sense sequence nor the homology of the antisense sequence need be 100% complementary to the target.

[0309] In other embodiments, the antisense strand and target mRNA sequences comprise at least one mismatch. As a non-limiting example, the antisense strand and the target mRNA sequence have at least 50-90%, 50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70-80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% complementary.

[0310] The siRNA molecule may have a length from about 10-50 or more nucleotides, e.g., each strand comprising 10-50 nucleotides (or nucleotide analogs). Preferably, the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. In one embodiment, the siRNA molecule has a length from about 19 to 25, 19 to 24 or 19 to 21 nucleotides.

[0311] In some embodiments, the siRNA molecule can be a synthetic RNA duplex comprising about 19 nucleotides to about 25 nucleotides, and two overhanging nucleotides at the 3'-end.

[0312] The siRNA molecule may comprise an antisense sequence and a sense sequence, or a fragment or variant thereof. As a non-limiting example, the antisense sequence and the sense sequence have at least 50-90%, 50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70- 80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99% or 95-99% complementary.

[0313] The sense and antisense sequences may be completely complementary across a substantial portion of their length. In other embodiments, the sense sequence and antisense sequence may be at least 70, 80, 90, 95 or 99% complementary across independently at least 50, 60, 70, 80, 85, 90, 95, or 99% of the length of the strands.

[0314] In some embodiments, the sense and antisense strands of a siRNA duplex are linked by a short spacer sequence leading to the expression of a stem-loop structure termed short hairpin RNA (shRNA). The hairpin is recognized and cleaved by Dicer, thus generating mature siRNA molecules. [0315] In some embodiments, the siRNA molecules, as well as associated spacer and/or flanking regions once designed, can be encoded by the viral genome of the AAV particles described herein, for delivery to a cell. siRNA modification

[0316] In some embodiments, the RNAi agents, e.g., siRNA molecules or ASOs may be chemically modified to modulate some features of RNA molecules, such as, but not limited to, increasing the stability of siRNAs in vivo. The chemically modified siRNA molecules can be used in human therapeutic applications, and are improved without compromising the RNAi activity of the siRNA molecules. As a non-limiting example, the siRNA molecules modified at both the 3' and the 5' end of both the sense strand and the antisense strand.

[0317] In some aspects, the RNAi agent, e.g., siRNA or ASO, may contain one or more modified nucleotides such as, but not limited to, sugar modified nucleotides, nucleobase modifications and/or backbone modifications. In some aspects, the siRNA molecule may contain combined modifications, for example, combined nucleobase and backbone modifications. In some embodiments, the RNAi agent, e.g., siRNA or ASO comprises at least one modified nucleotide. In some embodiments, not more than five of the sense strand nucleotides of the siRNA and not more than five of the nucleotides of the antisense strand of the siRNA are unmodified nucleotides. In some embodiments, all of the nucleotides of the sense strand of the siRNA and all of the nucleotides of the antisense strand of the siRNA are modified. In some embodiments, not more than five of the nucleotides of ASO are unmodified nucleotides. In some embodiments, all of the nucleotides of the ASO are modified.

[0318] In one embodiment, the modified nucleotide may be a sugar-modified nucleotide. Sugar modified nucleotides include, but are not limited to 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotides, e.g. 2'-fluoro modified ribonucleotides. Modified nucleotides may be modified on the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles. In one embodiment, the modified nucleotide may be a nucleobase-modified nucleotide. [0319] In one embodiment, the modified nucleotide may be a backbone-modified nucleotide. In some embodiments, the RNAi agent may further comprise other modifications on the backbone. In some embodiments, the phosphodiester bonds/linker (PO linkage) may be modified as “phosphorothioate backbone (PS linkage). In some cases, the natural phosphodiester bonds may be replaced by amide bonds but the four atoms between two sugar units are kept. Such amide modifications can facilitate the solid phase synthesis of oligonucleotides and increase the thermodynamic stability of a duplex formed with siRNA complement. See e.g. Mesmaeker et al., Pure & Appl. Chem., 1997, 3, 437-440; the content of which is incorporated herein by reference in its entirety.

[0320] Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of modifications on the nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5- propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N, -dimethyladenine, 2- propyladenine, 2-propylguanine, 2-aminoadenine, 1 -methylinosine, 3-methyluridine, 5- methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5-(2- amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1 -methyladenosine, 2- methyladenosine, 3 -methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2- dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7- deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2 -thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides.

[0321] In some embodiments, the 3’ end of the sense strand of the RNAi agent, e.g., the siRNA agent, is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0322] In some embodiments, the siRNA or ASO comprises a modification, e.g., as described in Table 1 of WO2021207189, the contents of which are hereby incorporated by reference in its entirety. In some embodiments, the siRNA or ASO comprises a modification, e.g., as described in WO2012/037254, US9587240, US7786290, or W02009086558, which are incorporated by reference herein. In some embodiments, the siRNA or ASO comprises a modification to increase stability, e.g., a 2’-O-methoxyethyl sugar modification.

Molecular Scaffold

[0323] In some embodiments, the siRNA molecules may be encoded in a modulatory polynucleotide which also comprises a molecular scaffold.

[0324] In some embodiments, the modulatory polynucleotide which comprises the payload (e.g., siRNA, miRNA or other RNAi agent described herein) includes a molecular scaffold which comprises a 5’ flanking sequence, a loop region, and/or a 3’ flanking region. In some embodiments a 5’ or 3’ flanking region may be of any length and may a wild type microRNA sequence or a portion thereof, or may be completely artificial. A 3’ flanking sequence may mirror the 5’ flanking sequence in size and origin. Either flanking sequence may be absent. In one embodiment, both the 5’ and 3’ flanking sequences are absent. The 3’ flanking sequence may optionally contain one or more CNNC motifs, where “N” represents any nucleotide. In some embodiments, the loop comprises at least one UGUG motif. In some embodiments, the UGUG motif is located at the 5’ terminus of the loop. In some embodiments the 5’ and 3’ flanking sequences are the same sequence. In some embodiments they differ by 2%, 3%, 4%, 5%, 10%, 20% or more than 30% when aligned to each other.

[0325] In some embodiments, modulatory polynucleotide comprises a stem loop structure. In some embodiments, the modulatory polynucleotide comprises in 5’ to 3’ order: a 5’ flanking sequence, a guide strand sequence, a loop region, a passenger strand sequence, and a 3’ flanking sequence. In some embodiments, the modulatory polynucleotide comprises in 5’ to 3’ order: a 5’ flanking sequence, a passenger strand sequence, a loop region, a guide strand sequence, and a 3’ flanking sequence.

[0326] In one embodiment, the molecular scaffold comprises a dual-function targeting modulatory polynucleotide.

[0327] In one embodiment, the molecular scaffold may comprise one or more linkers known in the art. The linkers may separate regions or one molecular scaffold from another. As a non-limiting example, the molecular scaffold may be polycistronic.

[0328] In one embodiment, the modulatory polynucleotide is designed using at least one of the following properties: loop variant, seed mismatch/bulge/wobble variant, stem mismatch, loop variant and basal stem mismatch variant, seed mismatch and basal stem mismatch variant, stem mismatch and basal stem mismatch variant, seed wobble and basal stem wobble variant, or a stem sequence variant.

Other Active Agents

[0329] In some embodiments the active agent comprises a diagnostic agent. In some embodiments, the diagnostic agent is or comprises an imaging agent (e.g., a protein or small molecule compound coupled to a detectable moiety). In some embodiments, the imaging agent comprises a PET or MRI ligand, or an antibody molecule coupled to a detectable moiety. In some embodiments, the detectable moiety is or comprises a radiolabel, a fluorophore, a chromophore, or an affinity tag. In some embodiments, the radiolabel is or comprises tc99m, iodine-123, a spin label, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen- 15, oxygen-17, gadolinium, manganese, or iron.

[0330] In some embodiments, the active agent is a small molecule. In some embodiments, the active agent is a ribonucleic acid complex (e.g., a Cas9/gRNA complex), a plasmid, a closed-end DNA, a circ-RNA, or an mRNA.

Therapeutic Applications

[0331] The present disclosure provides a method for treating a disease, disorder and/or condition in a subject, including a human subject, comprising administering to the subject a composition described herein, e.g., a composition comprising a ligand that binds to a GPI anchor protein fused or coupled (e.g., covalently or non-covalently) to an active agent (e.g., a therapeutic agent or a diagnostic agent).

[0332] In some embodiments, a composition described herein is administered to a subject prophylactically, to prevent on-set of disease. In another embodiment, the composition is administered to treat (e.g., lessen the effects of) a disease or symptoms thereof. In yet another embodiment, the composition is administered to cure (eliminate) a disease. In another embodiment, the composition is administered to prevent or slow progression of disease. In yet another embodiment, the composition is used to reverse the deleterious effects of a disease. Disease status and/or progression may be determined or monitored by standard methods known in the art.

[0333] In some embodiments, a composition described herein is useful for treatment, prophylaxis, palliation or amelioration of a genetic disorder, e.g., an autosomal dominant genetic disorder, an autosomal recessive disorder, X-linked dominant genetic disorder, an X-linked recessive genetic disorder, or a Y-linked genetic disorder. In some embodiments, the genetic disorder is a monogenetic disorder or a polygenic disorder. In some embodiments, treatment of a genetic disorder, e.g., a monogenic disorder, comprises the use of a composition described herein for a gene replacement therapy.

[0334] In some embodiments, provided herein is method for treating a neurological disorder and/or neurodegenerative disorder in a subject, comprising administering to the subject an effective amount of a composition described herein. In some embodiments, treatment of a neurological disorder and/or neurodegenerative disorder comprises prevention of said neurological disorder and/or neurological disorder.

[0335] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of neurological diseases and/or disorders. In some embodiments, the composition is useful for the treatment, prophylaxis, palliation or amelioration of tauopathy.

[0336] In some embodiments, a composition described herein is for the treatment, prophylaxis, palliation or amelioration of Alzheimer’s Disease. In some embodiments, treatment of Alzheimer’s Disease comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ApoE2 protein, ApoE4 protein, an ApoE3 protein, BDNF protein, CYP46A1 protein, Klotho protein, fractalkine (FKN) protein, neprilysin protein (NEP), CD74 protein, caveolin-1, or a combination or variant thereof. In some embodiments, treatment of Alzheimer’s Disease comprises the use of the composition for a reduction in the expression of a tau gene and/or protein, a synuclein gene and/or protein, or a combination or variant thereof. In some embodiments, the encoded payload or active agent comprises an antibody molecule that binds to tau or synuclein, an RNAi agent for inhibiting tau or synuclein, a gene editing system (e.g., a CRISPR-Cas system) for altering tau or synuclein expression, or a combination thereof.

[0337] In some embodiments, a composition described herein is useful the treatment, prophylaxis, palliation or amelioration of Friedreich’s ataxia, or any disease stemming from a loss or partial loss of frataxin protein.

[0338] In some embodiments, a composition described herein is for the treatment, prophylaxis, palliation or amelioration of frontal temporal dementia. In some embodiments, treatment of frontal temporal dementia comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a progranulin protein or variant thereof. [0339] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of Parkinson’s Disease. In some embodiments, treatment of Parkinson’ s disease comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an AADC protein, GAD protein, GDNF protein, TH-GCH1 protein, GBA protein, AIMP2-DX2 protein, or a combination or variant thereof. In some embodiments, treatment of Parkinson’s disease comprises the composition for a gene knockdown therapy or a gene editing therapy (e.g., knock-out, repression, or correction). In some embodiments, the encoded payload or active agent comprises a modulator, e.g., an RNAi agent or a CRISPR-Cas system, for altering expression of an alpha-synuclein gene, mRNA, and/or protein, or variant thereof. In some embodiments, the composition is useful for the treatment, prophylaxis, palliation or amelioration of an AADC deficiency. In some embodiments, treatment of AADC deficiency comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an AADC protein or variant thereof.

[0340] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of Amyotrophic lateral sclerosis. In some embodiments, treatment of ALS comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or the active agent comprises a TDP-43 protein, UPF1 protein, C9orf72 protein, CCNF protein, HSF1 protein, Factor H protein, NGF protein, ADAR2 protein, GDNF protein, VEGF protein, HGF protein, NRTN protein, AIMP2-DX2 protein, or a combination or variant thereof. In some embodiments, treatment of ALS comprises the use of the composition for a gene knock-down therapy or a gene editing therapy (e.g., knock-out, repression, or correction). In some embodiments, the encoded payload or the active agent comprises a modulator, e.g., an RNAi agent or a CRISPR-Cas system, for altering expression of a SOD1 or C9ORF72 gene, mRNA, and/or protein, or a combination or variant thereof.

[0341] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of Huntington’s Disease. In some embodiments, treatment of ALS comprises the use of the composition for a gene knock-down (e.g., knock-out) therapy or a gene editing therapy (e.g., knock-out, repression, or correction). In some embodiments, the encoded payload or active agent comprises a modulator, e.g., an RNAi agent or a CRISPR-Cas system, for altering expression of an HTT gene, mRNA, and/or protein, or a variant thereof.

[0342] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of spinal muscular atrophy. In some embodiments, treatment of spinal muscular atrophy comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an SMN1 protein, an SMN2 protein, or a combination or variant thereof.

[0343] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of multiple system atrophy. In some embodiments, treatment of multiple system atrophy comprises the use of the composition for a gene replacement therapy. [0344] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of Gaucher disease (GD) (e.g., Type 1 GD, Type 2 GD, or Type 3 GD). In some embodiments, the composition is useful for the treatment, prophylaxis, palliation or amelioration of Parkinson’s disease associated with a GBA mutation. In some embodiments, the composition is useful for the treatment, prophylaxis, palliation or amelioration of dementia with Lewy Bodies (DLB).

[0345] In some embodiments, the a composition described herein is useful for treatment, prophylaxis, palliation or amelioration of a leukodystrophy, e.g., Alexander disease, autosomal dominant leukodystrophy with autonomic diseases (ADLD), Canavan disease, cerebrotendinous xanthomatosis (CTX), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease, or Refsum disease. In some embodiments, treatment of MLD comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ARSA protein or variant thereof. In some embodiments, treatment of ALD comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ABCD-1 protein or variant thereof.

[0346] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of megalencephalic leukoencephalopathy (MLC). In some embodiments, treatment of MLC comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload encoded or active agent comprises an MLC1 protein or variant thereof.

[0347] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Krabbe disease. In some embodiments, treatment of Krabbe disease comprises the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a GALC protein or variant thereof.

[0348] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Mucopolysaccharidosis, e.g., a Type I (MPS I), Type II (MPS II), Type IIIA (MPS IIIA), Type IIIB (MPS IIIB), or Type IIIC (MPS IIIC). In some embodiments, treatment of Mucopolysaccharidosis comprises the use of the composition for a gene replacement therapy or a gene editing therapy (e.g., enhancement or correction). In some embodiments, the encoded payload or active agent comprises an IDUA protein, IDS protein, SGSH protein, NAGLU protein, HGSNAT protein, or a combination or variant thereof.

[0349] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Batten/NCL. In some embodiments, treatment of Batten/NCL comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a CLN1 protein, CLN2 protein, CLN3 protein, CLN5 protein, CLN6 protein, CLN7 protein, CLN8 protein, or a combination or variant thereof.

[0350] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of Rett Syndrome. In some embodiments, treatment of Rett Syndrome comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload comprises a capsid variant described herein comprises an MeCP2 protein or variant thereof.

[0351] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Angelman Syndrome. In some embodiments, treatment of Angelman Syndrome comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a UBE3A protein or variant thereof.

[0352] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Fragile X Syndrome. In some embodiments, treatment of Fragile X Syndrome comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a Reelin protein, a DgkK protein, a FMRI protein, or a combination or variant thereof.

[0353] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Canavan Disease. In some embodiments, treatment of Canavan Disease comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ASPA protein or variant thereof. [0354] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Gangliosidosis, e.g., a GM1 Gangliosidosis or a GM2 Gangliosidosis (e.g., Tay Sachs Sandhoff). In some embodiments, treatment of a Gangliosidosis, e.g., a GM1 Gangliosidosis or a GM2 Gangliosidosis (e.g., Tay Sachs Sandhoff), comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprising a capsid variant described herein comprises a GLB 1 protein, a HEXA protein, a HEXB protein, a GM2A protein, or a combination or variant thereof.

[0355] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of GM3 Synthase Deficiency. In some embodiments, treatment of GM3 Synthase Deficiency comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ST3GAL5 protein or variant thereof.

[0356] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Niemann-Pick disorder, e.g., a Niemann-Pick A or a Niemann-Pick Cl (NPC-1). In some embodiments, treatment of a Niemann-Pick disorder, e.g., a Niemann-Pick A or a Niemann-Pick Cl (NPC-1) comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an ASM protein, an NPC1 protein, or variant thereof.

[0357] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Schwannoma (e.g., Neuroma). In some embodiments, treatment of Schwannoma (e.g., Neuroma) comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a Caspase- 1 protein or variant thereof.

[0358] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Tuberous Sclerosis, e.g., Tuberous Sclerosis Type 1 or Tuberous Sclerosis Type 2. In some embodiments, treatment of Tuberous Sclerosis, e.g., Tuberous Sclerosis Type 1 or Tuberous Sclerosis Type 2 comprises the use the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a TSC1 protein, a TSC2 protein, or variant thereof. [0359] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a CDKL5 Deficiency. In some embodiments, treatment of a CDKL5 Deficiency comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a CDKL5 protein or variant thereof.

[0360] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Charcot-Marie -Tooth disorder, e.g., a Charcot-Marie- Tooth Type IX (CMT1X) disorder, a Charcot-Marie-Tooth Type 2A (CMT2A) disorder, or a Charcot-Marie-Tooth Type 4J (CMT4J) disorder. In some embodiments, treatment of a Charcot- Marie -Tooth disorder, e.g., a Charcot-Marie -Tooth Type IX (CMT1X) disorder, a Charcot-Marie- Tooth Type 2A (CMT2A) disorder, or a Charcot-Marie-Tooth Type 4J (CMT4J) disorder, comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a GJB 1 protein, a MFN2 protein, a FIG4 protein, or variant thereof.

[0361] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of an Aspartylglucosaminuria (AGU). In some embodiments, treatment of an AGU comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an AGA protein or variant thereof.

[0362] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Leigh Syndrome. In some embodiments, treatment of a Leigh Syndrome comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a SURF1 protein or variant thereof. [0363] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of epilepsy. In some embodiments, treatment of epilepsy comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an NPY/Y2 protein, a Galanin protein, a Dynorphin protein, an AIMP2-DX2 protein, an SLC6A1 protein, an SLC13A5 protein, a KCNQ2 protein, or variant thereof.

[0364] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Dravet Syndrome. In some embodiments, treatment of Dravet Syndrome comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises an SCNla protein, or variant thereof. [0365] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of a Duchenne muscular dystrophy (DMD). In some embodiments, treatment of DMD comprises the use of the composition for a gene replacement therapy or enhancement (e.g., correction of exon-skipping), or a gene editing therapy (e.g., enhancement or correction). In some embodiments, the encoded payload or active agent comprises a Dystrophin gene and/or protein, an Utrophin gene and/or protein, or a GALGT2 gene and/or protein, or a Follistatin gene and/or protein, or a combination or variant thereof.

[0366] In some embodiments, a compositions described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Pompe Disease. In some embodiments, treatment of Pompe Disease comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a GAA protein, or variant thereof. [0367] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation, or amelioration of Limb-Girdle Muscular Dystrophy (LGMD2A). In some embodiments, treatment of LGMD2A comprises the use of the composition for a gene replacement therapy. In some embodiments, the encoded payload or active agent comprises a CAPN-3 protein, DYSF protein, a SGCG protein, a SGCA protein, a SGCB protein, a FKRP protein, a ANO5 protein, or a combination or variant thereof.

[0368] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of chronic or neuropathic pain.

[0369] In some embodiments, a composition described herein is useful for treatment, prophylaxis, palliation or amelioration of a disease associated with the central nervous system.

[0370] In some embodiments, a composition described herein is useful for treatment, prophylaxis, palliation or amelioration of a disease associated with the peripheral nervous system.

[0371] In some embodiments, provided herein is a method for treating a neuro-oncological disorder in a subject, comprising administering to the subject an effective amount of a composition described herein. In some embodiments, treatment of a neuro-oncological disorder comprises prevention of said neuro-oncological disorder. In some embodiments, a neuro-oncological disorder comprises a cancer of a primary CNS origin (e.g., a CNS cell, a tissue, or a region), or a metastatic cancer in a CNS cell, tissue, or region. Examples of primary CNS cancers could be gliomas (which may include glioblastoma (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, and ependymomas, and mixed gliomas), meningiomas, medulloblastomas, neuromas, and primary CNS lymphoma (in the brain, spinal cord, or meninges), among others. Examples of metastatic cancers include those originating in another tissue or organ, e.g., breast, lung, lymphoma, leukemia, melanoma (skin cancer), colon, kidney, prostate, or other types that metastasize to brain.

[0372] In some embodiments, a composition described herein is useful for the treatment, prophylaxis, palliation or amelioration of a disease associated with expression of HER2, e.g., a disease associated with overexpression of HER2. In some embodiments, the composition is useful for the treatment, prophylaxis, palliation or amelioration of a HER2 -positive cancer. In some embodiments, the HER2 -positive cancer is a HER2 -positive solid tumor. Additionally, or alternatively, the HER2 -positive cancer may be a locally advanced or metastatic HER2 -positive cancer. In some instances, the HER2 -positive cancer is a HER2 -positive breast cancer or a HER2- positive gastric cancer. In some embodiments, the HER2 -positive cancer is selected from the group consisting of a HER2- positive gastroesophageal junction cancer, a HER2 -positive colorectal cancer, a HER2 -positive lung cancer (e.g., a HER2 -positive non-small cell lung carcinoma), a HER2 -positive pancreatic cancer, a HER2 -positive colorectal cancer, a HER2 -positive bladder cancer, a HER2- positive salivary duct cancer, a HER2 -positive ovarian cancer (e.g., a HER2 -positive epithelial ovarian cancer), or a HER2-positive endometrial cancer. In some instances, the HER2 -positive cancer is prostate cancer. In some embodiments, the HER2 -positive cancer has metastasized to the central nervous system (CNS). In some instances, the metastasized HER2-cancer has formed CNS neoplasms.

[0373] In some embodiments, a composition described herein is administered to a subject having at least one of the diseases or symptoms described herein. In some embodiments, the composition is administered to a subject having or diagnosed with having a disease or disorder described herein.

[0374] In some embodiments, provided herein is a method for treating a muscular disorder and/or neuromuscular disorder in a subject, comprising administering to the subject an effective amount of a composition described herein. In some embodiments, treatment of a muscular disorder and/or neuromuscular disorder comprises prevention of said muscular disorder and/or neuromuscular disorder.

[0375] In some embodiments, a composition described herein is administered to a subject having at least one of the diseases or symptoms described herein. In some embodiments, the composition is administered to a subject having or diagnosed with having a disease or disorder described herein. [0376] Any neurological disease or disorder, neurodegenerative disorder, muscular disorder, neuromuscular disorder, and/or neuro-oncological disorder may be treated with compositions described herein, or pharmaceutical compositions thereof.

Pharmaceutical Composition and Formulations

[0377] According to the present disclosure, an AAV particle comprising an AAV capsid variant described herein may be prepared as a pharmaceutical composition. In some embodiments a composition described herein, e.g., a composition comprising a ligand that binds to a GPI anchor protein fused or coupled (e.g., covalently or non-covalently) to an active agent (e.g., a therapeutic agent or a diagnostic agent) can be prepared as pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises at least one active ingredients. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0378] In some embodiments, an AAV particle or composition described herein can be formulated using an excipient to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed expression of the payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein; and/or (7) allow for regulatable expression of the payload. Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.

[0379] In some embodiments, the relative amount of the active ingredient (e.g. an AAV particle or a composition described herein), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[0380] The present disclosure also provides in some embodiments, a pharmaceutical composition suitable for administration to a subject, e.g., a human. In some embodiments, the pharmaceutical composition is administered to a subject, e.g., a human.

Administration

[0381] In some embodiments, a composition described herein may be administered to a subject by a delivery route, e.g., a localized delivery route or a systemic delivery route.

[0382] In some embodiments, a composition described herein may be administered via such a route that it is able to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. In some embodiments, a composition described herein may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution. In some embodiments, a composition described herein may be formulated with any appropriate and pharmaceutically acceptable excipient.

[0383] In some embodiments, a composition described herein is administered intramuscularly, intravenously, intracerebrally, intrathecally, intratumorally, intracerebroventricularly, via intraparenchymal administration, or via intra-cisterna magna injection (ICM). In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered via intra-cisterna magna injection (ICM). In some embodiments, the composition is administered intratumorally. In some embodiments, the composition is administered intraarterially. [0384] In some embodiments, a composition described herein may be delivered to a subject via a single route administration. In some embodiments, the composition may be delivered to a subject via a multi-site route of administration. In some embodiments, a subject may be administered at 2, 3, 4, 5, or more than 5 sites.

[0385] In some embodiments, a composition described herein is administered via a bolus infusion. In some embodiments, the composition is administered via sustained delivery over a period of minutes, hours, or days. In some embodiments, the infusion rate may be changed depending on the subject, distribution, formulation, and/or another delivery parameter. In some embodiments, the composition is administered using a controlled release. In some embodiments, the composition is administered using a sustained release, e.g., a release profile that conforms to a release rate over a specific period of time.

[0386] In some embodiments, a composition described herein may be delivered by more than one route of administration. As non-limiting examples of combination administrations, the composition may be delivered by intrathecal and intracerebroventricular, or by intravenous and intraparenchymal administration.

Intravenous administration

[0387] In some embodiments, a composition described herein may be administered to a subject by systemic administration. In some embodiments, the systemic administration is intravenous administration. In another embodiment, the systemic administration is intraarterial administration. In some embodiments, the composition is administered to a subject by intravenous administration. In some embodiments, the intravenous administration may be achieved by subcutaneous delivery. In some embodiments, the composition is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS -MB) or MRI-guided FUS coupled with intravenous administration, e.g., as described in Terstappen et al. (Nat Rev Drug Discovery, doi.org/10.1038/s41573-021-00139-y (2021)), the contents of which are incorporated herein by reference in its entirety. In some embodiments, the composition is administered to the subject intravenously. In some embodiments, the subject is a human.

Administration to the CNS

[0388] In some embodiments, a composition described herein may be delivered by direct injection into the brain. As a non-limiting example, the brain delivery may be by intrahippocampal administration. In some embodiments, the composition is administered to a subject by intraparenchymal administration. In some embodiments, the intraparenchymal administration is to tissue of the central nervous system. In some embodiments, the composition is administered to a subject by intracranial delivery (See, e.g., US Pat. No. 8119611; the content of which is incorporated herein by reference in its entirety). In some embodiments, the composition is delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration. In some embodiments, the composition is administered via intracisternal magna (ICM) injection.

[0389] In some embodiments, a composition described herein is delivered to the brain by systemic delivery. As a non-limiting example, the systemic delivery may be by intravascular administration. As a non-limiting example, the systemic or intravascular administration may be intravenous.

[0390] In some embodiments, a composition described herein is delivered by an intraocular delivery route. A non-limiting example of an intraocular administration includes an intravitreal injection.

Intramuscular administration

[0391] In some embodiments, a composition described herein is delivered by intramuscular administration. Without wishing to be bound by theory, it is believed in some embodiments, that the multi-nucleated nature of muscle cells provides an advantage to gene transduction subsequent to delivery. In some embodiments, cells of the muscle are capable of expressing recombinant proteins with the appropriate post-translational modifications. Without wishing to be bound by theory, it is believed in some embodiments, the enrichment of muscle tissue with vascular structures allows for transfer to the blood stream and whole-body delivery. Examples of intramuscular administration include systemic (e.g., intravenous), subcutaneous or directly into the muscle. In some embodiments, more than one injection is administered. In some embodiments, an AAV particle of the present disclosure may be delivered by an intramuscular delivery route. (See, e.g., U. S. Pat. No. 6506379; the content of which is incorporated herein by reference in its entirety). Non-limiting examples of intramuscular administration include an intravenous injection or a subcutaneous injection.

[0392] In some embodiments, a composition described herein is administered to a subject and transduces the muscle of a subject. As a non-limiting example, the composition is administered by intramuscular administration. In some embodiments, the composition is administered to a subject by subcutaneous administration. In some embodiments, the intramuscular administration is via systemic delivery. In some embodiments, the intramuscular administration is via intravenous delivery. In some embodiments, the intramuscular administration is via direct injection to the muscle.

[0393] In some embodiments, the muscle is transduced by administration, e.g., intramuscular administration. In some embodiments, an intramuscular delivery comprises administration at one site. In some embodiments, an intramuscular delivery comprises administration at more than one site. In some embodiments, an intramuscular delivery comprises administration at two, three, four, or more sites. In some embodiments, intramuscular delivery is combined with at least one other method of administration.

[0394] In some embodiments, a composition described herein is administered to a subject by peripheral injections. Non-limiting examples of peripheral injections include intraperitoneal, intramuscular, intravenous, conjunctival, or joint injection. It was disclosed in the art that the peripheral administration of AAV vectors can be transported to the central nervous system, for example, to the motor neurons (e.g., U. S. Patent Publication Nos. US20100240739 and US20100130594; the content of each of which is incorporated herein by reference in their entirety). [0395] In some embodiments, a composition described herein is administered to a subject by intraparenchymal administration. In some embodiments, the intraparenchymal administration is to muscle tissue. In some embodiments, an AAV particle or composition described herein is delivered as described in Bright et al 2015 (Neurobiol Aging. 36(2):693-709), the contents of which are herein incorporated by reference in their entirety. In some embodiments, the composition is administered to the gastrocnemius muscle of a subject. In some embodiments, the composition is administered to the bicep femorii of the subject. In some embodiments, the composition is administered to the tibialis anterior muscles. In some embodiments, the composition is administered to the soleus muscle. Depot administration

[0396] In some embodiments, a composition as described herein is formulated in depots for extended release. Generally, specific organs or tissues are targeted for administration.

[0397] In some embodiments, a composition described herein is spatially retained within or proximal to target tissues. Provided are methods of providing a composition described herein to target tissues of mammalian subjects by contacting target tissues (which comprise one or more target cells) with the composition, under conditions such that they are substantially retained in target tissues, e.g., such that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissues. In some embodiments, retention is determined by measuring the amount of the composition that enter a target cell or a plurality of target cells. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or greater than 99.99% of a pharmaceutical composition and/or an AAV particle, administered to a subject are present intracellularly at a period of time following administration. For example, intramuscular injection to a subject may be performed using aqueous compositions comprising a composition described herein and a transfection reagent, and retention is determined by measuring the amount of the composition, present in the muscle cell or plurality of muscle cells.

[0398] In some embodiments, disclosed herein are methods of providing a composition described herein to a tissue of a subject, by contacting the tissue (comprising a cell, e.g., a plurality of cells) with the composition under conditions such that they are substantially retained in the tissue. In some embodiments, a composition described herein comprises a sufficient amount of an active ingredient such that the effect of interest is produced in at least one cell. In some embodiments, a composition described herein generally comprises one or more cell penetration agents. In some embodiments, the disclosure provides a naked formulation (such as without cell penetration agents or other agents), with or without pharmaceutically acceptable carriers.

Methods of Treatment

[0399] Provided in the present disclosure are methods for introducing (e.g., delivering) a composition described herein into cells. In some embodiments, the method comprises introducing into said cells an AAV particle or vector described herein in an amount sufficient to modulate, e.g., increase, the production of a target gene, mRNA, and/or protein. In some embodiments, the method comprises introducing into said cells a composition or vector described herein in an amount sufficient to modulate, e.g., decrease, expression of a target gene, mRNA, and/or protein. In some aspects, the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes .

[0400] Disclosed in the present disclosure are methods for treating a neurological disease/disorder or a neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder associated with aberrant, e.g., insufficient or increased, function/presence of a protein, e.g., a target protein in a subject in need of treatment.

[0401] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition described herein.

[0402] In some embodiments, the composition comprising the AAV particles of the present disclosure (e.g., an AAV particle comprising an AAV capsid variant described herein) is administered to the central nervous system of the subject via systemic administration. In some embodiments, the systemic administration is intravenous (IV) injection. In some embodiments, the AAV particle described herein or a pharmaceutical composition comprising an AAV particle described herein is administered by focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB) or MRI-guided FUS coupled with intravenous administration.

[0403] In some embodiments, a composition described herein is administered to the central nervous system of the subject via intraventricular administration. In some embodiments, the composition comprising the AAV particle of the present disclosure (e.g., an AAV particle comprising an AAV capsid variant) is administered via intra-cisterna magna injection (ICM).

[0404] In some embodiments, a composition described herein is administered to the central nervous system of the subject via intraventricular injection and intravenous injection.

[0405] In some embodiments, a composition described herein is administered to the central nervous system of the subject via ICM injection and intravenous injection at a specific dose per subject. As a non-limiting example, the AAV particles are administered via ICM injection at a dose of IxlO ⁴ VG per subject. As a non-limiting example, the AAV particles are administered via IV injection at a dose of 2xl0 ¹³ VG per subject.

[0406] In some embodiments, a composition described herein is administered to the central nervous system of the subject. In other embodiments, the composition comprising the AAV particles of the present disclosure is administered to a CNS tissue of a subject (e.g., putamen, hippocampus, thalamus, or cortex of the subject). [0407] In some embodiments, a composition described herein is administered to the central nervous system of the subject via intraparenchymal injection. Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.

[0408] In some embodiments, a composition described herein is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.

[0409] In some embodiments, a composition described herein is administered to the central nervous system of the subject via intraventricular injection, intraparenchymal injection and intravenous injection.

[0410] In some embodiments, a composition described herein is administered to a muscle of the subject via intravenous injection.

[0411] In some embodiments, a composition described herein is delivered into specific types of cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells. In some embodiments, a composition described herein is delivered to a cell or region of the midbrain. In some embodiments, a composition described herein is delivered to a cell or region of the brains stem. In some embodiments, a composition described herein is delivered to neurons in the putamen, hippocampus, thalamus and/or cortex.

[0412] In some embodiments, administration of a composition described herein to a subject may increase target gene, mRNA, and/or protein levels in a subject, relative to a control, e.g., the gene, mRNA, and/or mRNA levels in the subject prior to the composition. The target gene, mRNA, and/or protein levels may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS, or a muscle, a region of a muscle, or a cell of a muscle, of a subject. In some embodiments, cell of the CNS comprises an astrocyte, microglia, cortical neuron, hippocampal neuron, DRG and/or sympathetic neuron, sensory neuron, oligodendrocyte, motor neuron, or combination thereof. As a non-limiting example, the composition may increase the gene, mRNA, and/or protein levels of a target protein by fold increases over baseline. In some embodiments, the composition leads to 5-6 times higher levels of a target gene, mRNA, or protein.

[0413] In some embodiments, administration of a composition described herein e.g., a comprising a siRNA molecule, to a subject may decrease target gene, mRNA, and/or protein levels in a subject, relative to a control, e.g., the gene, mRNA, and/or mRNA levels in the subject prior to receiving the composition. The target gene, mRNA, and/or protein levels may be decreased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20- 70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30- 95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70- 90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS, or a muscle, a region of a muscle, or a cell of a muscle, of a subject. In some embodiments, cell of the CNS comprises an astrocyte, microglia, cortical neuron, hippocampal neuron, DRG and/or sympathetic neuron, sensory neuron, oligodendrocyte, motor neuron, or combination thereof.

[0414] In some embodiments, a composition described herein may be used to increase target protein and reduce symptoms of neurological disease in a subject. In some embodiments, the composition may be used to decrease target protein and reduce symptoms of neurological disease in a subject.

[0415] In some embodiments, a composition described herein may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.

[0416] In some embodiments, a composition described herein may be used to improve performance on any assessment used to measure symptoms of neurological disease. Such assessments include, but are not limited to ADAS-cog (Alzheimer Disease Assessment Scale - cognitive), MMSE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activities Questionnaire), ADL (Activities of Daily Living), GPCOG (General Practitioner Assessment of Cognition), Mini-Cog, AMTS (Abbreviated Mental Test Score), Clockdrawing test, 6-CIT (6-item Cognitive Impairment Test), TYM (Test Your Memory), MoCa (Montreal Cognitive Assessment), ACE-R (Addenbrookes Cognitive Assessment), MIS (Memory Impairment Screen), BADLS (Bristol Activities of Daily Living Scale), Barthel Index, Functional Independence Measure, Instrumental Activities of Daily Living, IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly), Neuropsychiatric Inventory, The Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and/or MBR Caregiver Strain Instrument, or any of the other tests as described in Sheehan B (Ther Adv Neurol Disord. 5(6):349-358 (2012)), the contents of which are herein incorporated by reference in their entirety.

[0417] In some embodiments, the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of a neurological disease/disorder or a neurodegenerative disorder, a muscular disorder or neuromuscular disorder, and/or a neuro-oncological disorder. [0418] A composition described herein may be used in combination with one or more other therapeutic agents. In some embodiments, compositions can be administered concurrently with, prior to, or subsequent to, additional therapeutic or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

[0419] Therapeutic agents that may be used in combination with the composition can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, anti-glutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation. As a non-limiting example, the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.

[0420] Compounds tested for treating neurological disease which may be used in combination with the AAV particles described herein include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMD A receptor antagonists such as memantine, antipsychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n- phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3P (lithium) or PP2A, immunization with Ap peptides or tau phospho-epitopes, anti-tau or anti-amyloid antibodies, dopamine -depleting agents (e.g., tetrabenazine for chorea), benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity, and/or spasticity), , amino acid precursors of dopamine (e.g., levodopa for rigidity), skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and/or spasticity), inhibitors for acetylcholine release at the neuromuscular junction to cause muscle paralysis (e.g., botulinum toxin for bruxism and/or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and/or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and/or irritability, clozapine for treatment-resistant psychosis, aripiprazole for psychosis with prominent negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtazapine, venlafaxine for depression, anxiety, obsessive compulsive behavior and/or irritability), hypnotics (e.g., xopiclone and/or zolpidem for altered sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania) and mood stabilizers (e.g., lithium for mania or hypomania).

[0421] Neurotrophic factors may be used in combination therapy with a composition described herein for treating neurological disease. Generally, a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron. In some embodiments, the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment. Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophinreleasing hormone and ADNF, and variants thereof.

[0422] In one aspect, a composition described herein may be co-administered with AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the contents of which are incorporated herein by reference in their entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).

[0423] In some embodiments, administration of a composition described herein to a subject will modulate, e.g., increase or decrease, the expression of a target protein in a subject and the modulation, e.g., increase or decrease of the presence, level, activity, and/or expression of the target protein will reduce the effects and/or symptoms of a neurological disease/disorder or a neurodegenerative disorder, a muscular disorder or neuromuscular disorder, and/or a neuro-oncological disorder in a subject.

DEFINITIONS

[0424] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. [0425] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0426] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ and “consisting essentially thereof’ is thus also encompassed and disclosed.

[0427] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0428] Adeno-associated virus: As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependovirus genus or a variant, e.g., a functional variant, thereof. In some embodiments, the AAV is wildtype, or naturally occurring. In some embodiments, the AAV is recombinant.

[0429] AAV Particle'. As used herein, an “AAV particle” refers to a particle or a virion comprising an AAV capsid, e.g., an AAV capsid variant, and a polynucleotide, e.g., a viral genome or a vector genome. In some embodiments, the viral genome of the AAV particle comprises at least one payload region and at least one ITR. In some embodiments, an AAV particle of the disclosure is an AAV particle comprising an AAV variant. In some embodiments, the AAV particle is capable of delivering a nucleic acid, e.g., a payload region, encoding a payload to cells, typically, mammalian, e.g., human, cells. In some embodiments, an AAV particle of the present disclosure may be produced recombinantly. In some embodiments, an AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In some embodiments, the AAV particle may be replication defective and/or targeted. It is to be understood that reference to the AAV particle of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.

[0430] Administering: As used herein, the term "administering" refers to providing a pharmaceutical agent or composition to a subject.

[0431] Amelioration-. As used herein, the term "amelioration" or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of neurodegeneration disorder, amelioration includes the reduction of neuron loss.

[0432] Amplicon: As used herein, “amplicon” may refer to any piece of RNA or DNA formed as the product of amplification events, e.g. PCR. In some embodiments, full-length capsid amplicons may be used as templates for next generation sequencing (NGS) library generation. Full-length capsid amplicons may be used for cloning into a DNA library for any number of additional rounds of AAV selection as described herein.

[0433] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically engineered animal, or a clone.

[0434] Antisense strand: As used herein, the term “the antisense strand” or “the first strand” or “the guide strand” of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of a gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.

[0435] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0436] Biopanning: As used herein, the term “biopanning” refers to an AAV capsid library selection process comprising administration of an AAV particle with enhanced tissue- and/or cell type-specific transduction to a cell and/or subject; extraction of nucleotides encoded by said AAV particle from said transduced tissue- and/or cell type-specific; and, use of the extracted nucleotides for cloning into a nucleotide library for the generation of AAV particles for subsequent rounds of the same.

[0437] Capsid'. As used herein, the term “capsid” refers to the exterior, e.g., a protein shell, of a virus particle, e.g., an AAV particle, that is substantially (e.g., >50%, >60%, >70%, >80%, >90%, >95%, >99%, or 100%) protein. In some embodiments, the capsid is an AAV capsid comprising an AAV capsid protein described herein, e.g., a VP1, VP2, and/or VP3 polypeptide. The AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and/or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.” In some embodiments, the AAV capsid variant described herein has the ability to enclose, e.g., encapsulate, a viral genome and/or is capable of entry into a cell, e.g., a mammalian cell. In some embodiments, the AAV capsid variant described herein may have modified tropism compared to that of a wild-type AAV capsid, e.g., the corresponding wild-type capsid.

[0438] Complementary and substantially complementary: As used herein, the term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenine. However, when a U is denoted in the context of the present disclosure, the ability to substitute a T is implied, unless otherwise stated. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form a hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity. The term “complementary” as used herein can encompass fully complementary, partially complementary, or substantially complementary. As used herein, the term “substantially complementary” means that the siRNA has a sequence (e.g., in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA. “Fully complementary”, “perfect complementarity”, or “100% complementarity” refers to the situation in which each nucleotide unit of one polynucleotide or oligonucleotide strand can base-pair with a nucleotide unit of a second polynucleotide or oligonucleotide strand.

[0439] Control Elements: As used herein, “control elements”, “regulatory control elements” or “regulatory sequences” refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and/or translated in an appropriate host cell.

[0440] Delivery: As used herein, “delivery” refers to the act or manner of delivering an AAV particle, a compound, substance, entity, moiety, cargo or payload.

[0441] Element: As used herein, the term “element” refers to a distinct portion of an entity. In some embodiments, an element may be a polynucleotide sequence with a specific purpose, incorporated into a longer polynucleotide sequence.

[0442] Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein, e.g., an AAV capsid variant, often encapsulates a viral genome. In some embodiments, encapsulate within a capsid, e.g., an AAV capsid variant, encompasses 100% coverage by a capsid, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60% or less. For example, gaps or discontinuities may be present in the capsid so long as the viral genome is retained in the capsid, e.g., prior to entry into a cell.

[0443] Effective Amount: As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent. [0444] Expression-. As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0445] Formulation-. As used herein, a “formulation” includes at least one AAV particle (active ingredient) and an excipient, and/or an inactive ingredient.

[0446] Fragment: A “fragment,” as used herein, refers to a portion. For example, an antibody fragment may comprise a CDR, or a heavy chain variable region, or a scFv, etc.

[0447] Homology. As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

[0448] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; the contents of each of which are incorporated herein by reference in their entirety. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0449] Inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.

[0450] Inverted terminal repeat: As used herein, the term “inverted terminal repeat” or “ITR” refers to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids. [0451] Isolated'. As used herein, the term “isolated” refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of component with which it is associated in the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant, e.g., incorporated into a vector.

[0452] Library: As used herein, the term “library” refers to a diverse collection of linear polypeptides, polynucleotides, viral particles, or viral vectors. As examples, a library may be a DNA library or an AAV capsid library.

[0453] Ligand'. As used herein, the term “ligand” refers to molecule that binds to a target, e.g., a receptor. In some embodiments, the receptor is a GPI-anchored protein, e.g., as described herein. In some embodiments, the receptor is alkaline phosphatase (ALPL), e.g., human ALPL, NHP ALPL, or murine ALPL. In some embodiments, the ligand is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule, optionally in isolation or as part of a fusion or a conjugate, e.g., with an active agent.

[0454] Molecular scaffold: As used herein a “molecular scaffold” is a framework or starting molecule that forms the sequence or structural basis against which to design or make a subsequent molecule.

[0455] Neurological disease: As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).

[0456] Orthogonal evolution: As used herein, the term “orthogonal evolution” refers to a method wherein AAV particles are administered for a first round of AAV selection as described herein across a set of any number of cell- and/or subject-types that may be from different species and/or strains, and wherein any number of additional, i.e., subsequent, AAV selection rounds are performed either across a set of any number of cell- and/or subject-types that may be from different species and/or strains, or across a set of any number of cell- and/or subject-types that may be from the same species and/or strain.

[0457] Open reading frame: As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.

[0458] Particle'. As used herein, a “particle” is a virus comprised of at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.

[0459] Payload region: As used herein, a “payload region” is any nucleic acid sequence (e.g., within the viral genome) which encodes one or more “payloads” of the disclosure. As non-limiting examples, a pay load region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is an RNAi agent or a polypeptide. Payloads of the present disclosure may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc. [0460] Polypeptide: As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0461] Polypeptide variant: The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. In some embodiments, a variant comprises a sequence having at least about 50%, at least about 80%, or at least about 90%, identical (homologous) to a native or a reference sequence.

[0462] Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0463] Pharmaceutically acceptable'. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0464] Preventing'. As used herein, the term “preventing” or “prevention” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

[0465] Prophylactic. As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease. [0466] Prophylaxis: As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.

[0467] Region: As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and/or a cluster of epitopes. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini.

[0468] In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5’ and/or 3’ termini.

[0469] RNA orRNA molecule-. As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). The term “mRNA” or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.

[0470] RNA interfering orRNAi: As used herein, the term “RNA interfering” or “RNAi” refers to a sequence specific regulatory mechanism mediated by RNA molecules which results in the inhibition or interfering or “silencing” of the expression of a corresponding protein-coding gene. RNAi has been observed in many types of organisms, including plants, animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. RNAi is controlled by the RNA-induced silencing complex (RISC) and is initiated by short/small dsRNA molecules in cell cytoplasm, where they interact with the catalytic RISC component argonaute. The dsRNA molecules can be introduced into cells exogenously. Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves dsRNAs to produce doublestranded fragments of 21-25 base pairs with a few unpaired overhang bases on each end. These short double stranded fragments are called small interfering RNAs (siRNAs).

[0471] RNAi agent: As used herein, the term “RNAi agent” refers to an RNA molecule, or its derivative, that can induce inhibition, interfering, or “silencing” of the expression of a target gene and/or its protein product. An RNAi agent may knock-out (virtually eliminate or eliminate) expression, or knock-down (lessen or decrease) expression. The RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, IncRNA, piRNA, or snoRNA.

[0472] miR binding site: As used herein, a “miR binding site” comprises a nucleic acid sequence (whether RNA or DNA, e.g., differ by “U” of RNA or “T” in DNA) that is capable of binding, or binds, in whole or in part to a microRNA (miR) through complete or partial hybridization . Typically, such binding occurs between the miR and the miR binding site in the reverse complement orientation. In some embodiments, the miR binding site is transcribed from the AAV vector genome encoding the miR binding site.

[0473] In some embodiments, a miR binding site may be encoded or transcribed in series. Such a “miR binding site series” or “miR BSs” may include two or more miR binding sites having the same or different nucleic acid sequence.

[0474] Spacer: As used here, a “spacer” is generally any selected nucleic acid sequence of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive miR binding site sequences. Spacers may also be more than 10 nucleotides in length, e.g., 20, 30, 40, or 50 or more than 50 nucleotides.

[0475] Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells, nucleic acids, or component parts (e.g. body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).

[0476] Self-complementary viral particle: As used herein, a “self-complementary viral particle” is a particle comprised of at least two components, a protein capsid and a self-complementary viral genome enclosed within the capsid.

[0477] Sense Strand: As used herein, the term “the sense strand” or “the second strand” or “the passenger strand” of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand. The antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure. As used herein, a “siRNA duplex” includes a siRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a siRNA strand having sufficient complementarity to form a duplex with the other siRNA strand.

[0478] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. [0479] Short interfering RNA or siRNA: As used herein, the terms “short interfering RNA,” “small interfering RNA” or “siRNA” refer to an RNA molecule (or RNA analog) comprising between about 5-60 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi. Preferably, a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogs, such as between about 16-25 nucleotides (or nucleotide analogs), between about 18-23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nucleotides (or nucleotide analogs), and between about 19-24 nucleotides (or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising 5-23 nucleotides, preferably 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi or translational repression absent further processing, e.g., enzymatic processing, to a short siRNA. siRNAs can be single stranded RNA molecules (ss-siRNAs) or double stranded RNA molecules (ds- siRNAs) comprising a sense strand and an antisense strand which hybridized to form a duplex structure called an siRNA duplex.

[0480] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

[0481] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0482] Target Cells: As used herein, “target cells” or “target tissue” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

[0483] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. [0484] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose.

[0485] Therapeutically effective outcome'. As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[0486] Treating'. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

[0487] Conservative amino acid substitution: As used herein, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0488] Variant: As used herein, the term “variant” refers to a polypeptide or polynucleotide that has an amino acid or a nucleotide sequence that is substantially identical, e.g., having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a reference sequence. In some embodiments, the variant is a functional variant.

[0489] Functional Variant'. As used herein, the term “functional variant” refers to a polypeptide variant or a polynucleotide variant that has at least one activity of the reference sequence.

[0490] Insertional Variant'. "Insertional variants" when referring to polypeptides are those with one or more amino acids inserted, e.g., immediately adjacent or subsequent, to a position in an amino acid sequence. "Immediately adjacent" or “immediately subsequent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

[0491] Deletional Variant'. "Deletional variants" when referring to polypeptides, are those with one or more amino acids in deleted from a reference protein.

[0492] Vector: As used herein, the term “vector” refers to any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. In some embodiments, vectors may be plasmids. In some embodiments, vectors may be viruses. An AAV particle is an example of a vector. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequences. The heterologous molecule may be a polynucleotide and/or a polypeptide.

[0493] Viral Genome: As used herein, the terms “viral genome” or “vector genome” refer to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

Equivalents and Scope

[0494] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to certain embodiments, it is apparent that further embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0495] The present disclosure is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1. High-throughput screen of TRACER AAV library in NHP and Mice

[0496] A TRACER based method as described in W02020072683, WO 2021/202651, and WO2021230987, the contents of which are herein incorporated by reference in their entirety, was used to generate the AAV capsid variants described herein. An orthogonal evolution approach was combined with a high throughput screening by NGS. Briefly, the library of AAV capsid variants was generated using a sliding window approach, where 6 amino acid sequences were inserted into 8 different positions across loop IV of AAV9, including immediately subsequent to positions 453, 454, 455, 456, 457, 458, 459, and 460, relative to a reference sequence numbered according to SEQ ID NO: 138. The initial library was passed twice through non-human primates (NHP, 2-4 years of age). After the second passage (e.g., 28 days post injection into two NHPs), RNA was extracted from six brain regions. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate fold enrichment relative to an AAV9 wild-type control.

Following these two passages, approximately 21195 variants were identified with an average fold change greater than wild-type. Of the 21195 variants, 1558 demonstrated a fold-change of greater than 6 compared to wild-type and were detected across all brain regions investigated. Of these 1558, approximately 1470 variants were selected for constructing a synthetic library and a third passage through two NHPs. Within the 1470 variants selected for further characterization and investigation, there was a relatively even distribution for each insertion position of the sliding window used to generate the initial library.

[0497] After creation of the synthetic library with the sub-selected variants, the synthetic library was screened (passage 3) in two NHPs (2-4 years of age) and two strains of mice, BALB/c (n=3, 6-8 weeks of age) and C57B1/6 mice (n=3, 6-8 weeks of age), in a first cross-species evolution screen. The animals were injected intravenously with the synthetic library. After a period in vivo, (e.g., 28- days) RNA was extracted from nervous tissue, e.g., brain, spinal cord, and DRG of the NHPs and the brains of mice. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed, and the peptides comprised within the variants were identified and the capsid enrichment ratio for each variant compared to the wild- type AAV9 control was calculated (fold enrichment relative to wild-type AAV9) (Table 9). Values above 1 indicate an increase in expression relative to AAV9. All animals were dosed intravenously at 2-3 VG/kg across the screen.

[0498] As shown in Table 9, approximately 700 variants demonstrated an increase in expression relative to AAV9, and several variants demonstrated a greater than 10-fold enrichment relative to AAV9 in the brain of NHPs. Further, the variants demonstrating the greatest fold enrichment in the brain also demonstrated the greatest fold enrichment in the spinal cord relative to AAV9 in NHPs. These variants also demonstrated de -targeting in the DRG (data not shown). For instance, the variant comprising GSGSPHSKAQNQQT (SEQ ID NO: 200) demonstrated a 76.6 fold enrichment in the brain, a 29.4 fold enrichment in the spinal cord, and 0.4 fold enrichment in the DRG of NHPs relative to AAV9; and GHDSPHKSGQNQQT (SEQ ID NO: 201) demonstrated a 62.6 fold enrichment in the brain, a 15.6 fold enrichment in the spinal cord, and 0.0 fold enrichment in the DRG of NHPs relative to AAV9. Also, across the peptides comprised within the AAV capsid variants with the greatest foldenrichment in the NHP brain relative wild-type AAV9, it was observed that each of these peptides comprised an SPH motif in the same position (e.g., immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138), regardless of the insertion position within the variant capsid, as well as a positive amino acid (e.g., K or R) in one of the next three residues subsequent to the SPH motif.

[0499] Those variants with the greatest fold enrichment in the brains of NHPs also had the greatest fold enrichment in the brains of both mouse species. Also, when comparing the fold enrichment relative to wild-type for each variant between the two species of mice investigated (C57B1/6 and BALB/c mice), they were highly correlated (R ² = 0.8591). Table 9. NGS fold-enrichment of AAV capsid variants in NHPs and mice

[0500] A second cross-species evolution screen was performed using an AAV capsid variant library with a modification in loop IV introduced as described above and passaging it once through NHPs (passage 1) and then subsequently injected it into two different strains of mice (passage 2), C57B1/6 and BALB/c. The fold-enrichment for each variant in the brain of each mouse species was calculated by systematic NGS enrichment analysis following RNA recovery and RT-PCR amplification. The fold enrichment values in the second passage in mice were compared to those fold enrichment values from the second pass that was performed in NHPs as described above. As shown in Table 10, when comparing the second pass fold enrichment values in the mice versus NHPs, 12 variants were identified that had a fold-enrichment value greater than 10 in all three animal groups. Further, 10 of these 12 variants comprised the SPH motif and a positive residue in one of the next three subsequent residues (Table 10).

Table 10. NGS fold-enrichment of AAV capsid variants from a second passage (P2) in NHPs or mice (C57B1/6 or BALB/c) following a first passage in NHPs

[0501] Following the second passage in mice, a synthetic library was generated using those variants that demonstrated a fold-change in enrichment relative to wild-type AAV9 that was above 10 in the brain of either strain of mice, as measured by systematic NGS enrichment analysis following RNA recovery and RT-PCR amplification. There were approximately 500 variants in this synthetic library. This synthetic library was then injected back into both strains of mice (C57B1/6 and BALB/c; passage 3). RNA was recovered from the mouse brains, RT-PCR amplification was performed, and foldenrichment relative to wild-type AAV9 was calculated by NGS analysis, which is provided in Table 11. As shown in Table 11, the variants with the greatest fold-enrichment in the brain in each strain, were highly correlated across strains (R ² =0.8458).

Table 11. NGS fold-enrichment of AAV capsid variants in the brain from a third passage (P3) in mice (C57B1/6 or BALB/c) following a first and second passage in mice

[0502] Taken together, these results demonstrate that after 3 rounds of screening of this AAV9 variant library with loop IV modifications in NHP and mice, many AAV capsid variants outperformed the wild-type AAV9, for example, in penetration of the blood brain barrier (BBB) and spinal cord expression. These capsid variants were able to cross-species, evidenced by expression and tropism in the NHP brain/spinal cord as well as in the brain of two different mouse species.

Example 2. Individual Capsid Characterization in Mice

[0503] The goal of these experiments was to determine the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of 2 capsid variants selected from the study described in Example 1 relative to AAV9 following intravenous injection in mice. The 2 capsid variants were TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2), as outlined in Table 3 above. The amino acid and DNA sequences of TTM-001 and TTM-002 are provided, e.g., in Tables 4 and 5, respectively.

[0504] AAV particles were generated with each of these capsid variants encapsulating a luciferase-EGFP transgene driven by a CMV/chicken beta actin promoter in a single stranded viral genome. Each capsid variant and AAV9 control were tested by intravenously administering by tail vein injection, the AAV particle formulation at 5el l VG/dose (2.5E13 vg/kg) to three female BALB/c mice. The in-life period was 28 days and then various CNS and peripheral tissues were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution).

[0505] At 28 days post-injection of the AAV particles encapsulated in the TTM-001 capsid variant (AAV_TTM-001), mice were injected with luciferin and their brains were harvested for IVIS imaging. Robust luciferase signal was observed in mice injected with AAV particles encapsulated in the TTM-001 capsid variant, and this was greatly increased relative to AAV particles encapsulated in the wild- type AAV9 control capsid.

[0506] The brains isolated from mice injected with the AAV particles encapsulated in the TTM- 001 capsid variant (AAV_TTM-001) or the TTM-002 capsid variant (AAV_TTM-002) were assayed by qPCR for the presence of transgene RNA as a measure of transgene expression, and the presence of viral DNA as a measure of viral genome levels. Data were provided as fold over AAV9 (Table 12). As shown in Table 12, when compared to the wild-type AAV9 capsid control, TTM-001 and TTM- 002 demonstrated a 30-fold and 66-fold increase, respectively, in transgene mRNA levels and expression in the brain, indicative of enhanced payload delivery. This correlated with a 32-fold (TTM-001) and 47-fold (TTM-002) increase, respectively, in viral genome (DNA) concentrations in the brain relative to the AAV9 capsid control, which is indicative of enhanced CNS tropism and transduction (Table 12).

Table 12. Transgene mRNA and viral genome levels (DNA) in mice relative to the AAV9 control

[0507] The brain tissues and spinal cords of the mice were also subjected to anti-GFP immunohistochemistry staining to evaluate overall CNS tropism and biodistribution.

Immunohistochemical staining correlated with the qPCR analysis, as TTM-001 and TTM-002 showed significantly stronger staining and payload expression in the brain and spinal cord, as compared to the AAV9 control. More specifically, TTM-001 and TTM-02 demonstrated localization and strong payload expression and transduction in the mid-brain region, with increased staining observed in the hippocampus and thalamus, as well as in the brain stem, compared to AAV9. Less staining was observed in the cortical regions of the brain compared to the midbrain. However, staining in these cortical regions was stronger for TTM-001 and TTM-002 compared to the AAV9 control. It also appeared that the TTM-001 and TTM-002 capsid variants were able to transduce non-neuronal cells, including glial cells and oligodendrocytes. With respect to the spinal cord, staining and payload expression for TTM-01 and TTM-002 were localized to the ventral horns of the grey matter.

[0508] Peripheral tissues were also isolated from the mice intravenously injected with the AAV particles encapsulated in the TTM-001 capsid variant or the TTM-002 capsid variant for analysis by qPCR and/or GFP immunohistochemical staining. Transgene mRNA levels and viral genome DNA levels were quantified in the liver by qPCR and the fold over AAV9 was calculated for each capsid variant (Table 12). TTM-001 resulted in similar levels of payload expression (mRNA levels) as compared to wild-type AAV9, but only half as much viral genome DNA was quantified in the liver compared to AAV9. TTM-002 demonstrated greatly reduced mRNA and viral genome DNA levels in the liver compared to AAV9. GFP immunohistochemical staining of the spleen, heart, skeletal muscle, kidneys, and lungs of mice injected with AAV particles encapsulated in the TTM-001 capsid variant or the TTM-002 capsid variant showed similar levels of payload expression as compared to those mice injected with AAV particles encapsulated in the wild-type AAV9 control capsid.

[0509] Taken together, these data demonstrate that TTM-001 and TTM-002 are enhanced CNS tropic capsids in mice that can infect non-neuronal cells. Additionally, these capsid variants were able to successfully penetrate the blood brain barrier following intravenous injection.

Example 3. Maturation of TTM-001 and TTM-002 Capsid in Mice

[0510] This Example describes maturation of the TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2) capsid variants to further enhance their transduction and biodistribution in the central nervous system and evolve the AAV capsid variants to provide further cross-species compatibility. Two approaches were used to mature the TTM-001 and TTM-002 capsid sequences in order to randomize and mutate within and around the peptide insert comprised within loop IV of the capsid variant. As many of the AAV capsid variants that demonstrated the greatest fold-enrichment in the NHP brain relative wild-type AAV9 comprised an SPH motif in the same position (e.g., immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138) (see Example 1), the SPH motif was not mutated in either approach to mature the TTM-001 and TTM-002 capsid variants. In the first maturation approach, sets of three contiguous amino acids were randomized across the mutagenesis region in the TTM-001 and TTM-002 sequences, which spanned from position 450 to position 466, numbered according to SEQ ID NO: 981 and 982. In the second maturation approach, mutagenic primers were used to introduce point mutations at a low frequency, scattered across the mutagenesis region in the TTM-001 and TTM-002 sequences ranging from position 449 to position 466, numbered according to SEQ ID NO: 981 and 982. AAV capsid variants arising from each maturation approach for TTM-001 were pooled together and AAV capsid variants arising from each maturation approach for TTM-002 were also pooled together, for subsequent testing and characterization in mice.

[0511] The library of pooled matured AAV capsid variants generated from TTM-001 or library of pooled matured AAV capsid variants generated from the TTM-002 matured AAV capsid variant each were intravenously injected into the tail vein of three female CD-I Outbred mice (Charles River) at a dose of 1.0 x 10 ¹² VG/dose. After 14-days in life, the brains of the mice were isolated and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the corresponding TTM-001 or TTM-002 control, and the peptides comprised within the variants were identified. The data for the TTM-001 matured capsid variants is provided in Table 13 and the data for the TTM-002 matured capsid variants is provided in Table 14.

[0512] As shown in Table 13, approximately 714 TTM-001 matured capsid variants demonstrated at least a 2-fold increase in expression relative to the non-matured TTM-001 control, and several variants demonstrated greater than a four-fold enrichment relative to the non-matured TTM-001 control. Also, across the peptides comprised within the TTM-001 matured capsid variants with the greatest fold-enrichment relative to the non-matured TTM-001 capsid in the brain, it was observed that the modifications in the variant sequences appeared in the region C-terminal to the SPH motif present within the capsid variant. This indicates that modifications that appeared to improve TTM-001 capsid tropism in the CNS of mice were skewed to the C-terminal portion of the peptide insertion in loop IV of the sequence. Additionally, a number of these C-terminal modifications were the incorporation of an arginine (R) or leucine (L) residue.

Table 13. NGS fold-enrichment of TTM-001 matured AAV capsid variants in the brain of CD-I Outbred mice

[0513] As shown in Table 14, approximately 72 TTM-002 matured capsid variants demonstrated at least a 2-fold increase in expression relative to the non-matured TTM-002 control, with a few variants demonstrating greater than a three- to five-fold enrichment relative to the non-matured TTM- 002 control. Also, across the peptides comprised within the TTM-002 matured capsid variants with the greatest fold-enrichment relative to the non-matured TTM-002 capsid in the brain, it was observed that the modifications in the variant sequences appeared in the region N-terminal to the SPH motif present within the capsid variant. This indicates that modifications that appeared to improve TTM-002 capsid tropism in the CNS of mice were skewed to the N-terminal portion of the peptide insertion in loop IV of the sequence. Additionally, a number of these N-terminal modifications that were incorporated into the matured TTM-002 capsid variants were negatively charged amino acids (particularly glutamic acid (E)).

Table 14. NGS fold-enrichment of TTM-002 matured AAV capsid variants in the brain of CD-I Outbred mice

[0514] These data demonstrate that following two maturation approaches, matured TTM-001 and TTM-002 capsid variants with loop IV modifications were generated with significantly enhanced CNS tropism in mice compared to the corresponding non-matured TTM-001 and TTM-002 capsid variants, which already exhibited a significant fold enrichment over AAV9 in the mouse brain.

Example 4. Maturation of TTM-001 and TTM-002 Capsid in NHPs

[0515] This Example describes maturation of the AAV9 capsid variants, TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 (encoded by SEQ ID NO: 942)) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2 (encoded by SEQ ID NO: 944)) in NHPs to further enhance their transduction and biodistribution in the central nervous system as well as other tissues, and evolve the AAV capsid variants to provide further crossspecies compatibility. Two approaches were used to mature the TTM-001 and TTM-002 capsid sequences in order to randomize and mutate within and around the peptide insert comprised within loop IV of the capsid variant. As many of the AAV capsid variants that demonstrated the greatest fold-enrichment in the NHP brain relative wild-type AAV9 comprised an SPH motif in the same position (e.g., immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138) (see Example 1), the SPH motif was not mutated in either approach to mature the TTM-001 and TTM-002 capsid variants. In the first maturation approach, sets of three contiguous amino acids were randomized across the mutagenesis region in the TTM-001 and TTM-002 sequences, which spanned from position 450 to position 466, numbered according to SEQ ID NO: 981 and 982. In the second maturation approach, mutagenic primers were used to introduce point mutations at a low frequency, scattered across the mutagenesis region in the TTM-001 and TTM-002 sequences ranging from position 449 to position 466, numbered according to SEQ ID NO: 981 and 982. AAV capsid variants arising from each maturation approach for TTM-001 and TTM-002 were pooled together, for subsequent testing and characterization in NHPs.

[0516] The library of pooled matured AAV capsid variants generated using the first maturation approach and the second maturation approach for the TTM-001 and TTM-002 AAV capsid variants were injected into two NHPs. After a period in life, the brains, heart, liver, muscle, and DRG of the NHPs were isolated and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to an AAV9 control, and the peptides comprised within the variants were identified.

[0517] Following the RNA recovery and NGS analysis from the second maturation approach, approximately 680,000 capsid variants were identified. The 680,000 matured capsid variants were then filtered based on samples with a raw virus count greater than 10 and a coefficient of variance (CV) of less than 1 , which was calculated for each peptide across the brain samples taken from the two NHPs. Those that had a CV value <1 were identified, as these were the peptides that were reliably detected in the majority of samples isolated from the brains of the two NHPs. Using this filtering criteria, this led to approximately 64,000 matured capsid variants.

[0518] Table 15 provides the peptide sequences of the matured capsid variants having a raw virus count greater than 10, a CV of less than 1 for the brain samples isolated, and that also demonstrated a 50-fold or greater fold-increase in expression in the brain relative to the AAV9 control in both mice and NHPs. The matured variants in Table 15, were also those variants that had a fold-change in expression that was less than 2 relative to the AAV9 control in the liver and the DRG. Applying these criteria, approximately 350 matured capsid variants were identified that demonstrated high transduction in the brain in NHPs and mice, cross-species compatibility in mice and NHPs, and were de -targeted in the liver and DRG, relative to the AAV9 control. Several variants as shown in Table 15, led to greater than 100-fold increase in expression relative to AAV9 in the NHP and/or mouse brain, with one variant resulting in a greater than 200-fold increase in expression relative to AAV9 in both species.

[0519] Fold-change in expression for the TTM-001 and TTM-002 matured variants in Table 15 that showed increased expression in the brain of the NHPs and mice, were also calculated for the DRG, muscle, liver (RNA and DNA), and heart of the NHPs following each maturation approach. As shown in Table 15, many variants were de -targeted in the peripheral tissues with a lower fold-change in expression relative to the AAV9 control, demonstrating CNS-specific tropism and a preferential transduction of the brain and CNS. Some variants demonstrated increased expression to AAV9 in multiple tissues, including the brain and peripheral tissues, demonstrating pan-tropism.

Table 15. NGS fold-enrichment of TTM-001 and TTM-002 matured AAV capsid variants in the brain of NHPs and mice

[0520] Table 16 provides the peptide sequence of 341 matured capsid variants, and the fold enrichment of these matured capsid variants relative to the AAV9 control that demonstrated a 75-fold or greater increase in expression in the brain of NHPs relative to the AAV9 control and had a foldchange in expression that was less than 2 relative to the AAV9 control in the liver and the DRG.

Table 16. NGS fold-enrichment of TTM-001 and TTM-002 matured AAV capsid variants in the brain of NHPs

[0521] Table 17 provides the sequences of 216 matured capsid variants having a CV of less than

1 for the liver RNA samples isolated and a 10-fold or greater increase in expression relative to AAV9 in the liver of NHPs. These matured variants showed preferential transduction of the liver over other tissues as shown by a low value for fold-enrichment relative to AAV9 in the other tissues investigated including the brain, DRG, heart and muscle. As such, Table 17 provides TTM-001 and TTM-002 matured AAV capsid variants with liver-specific tropism. Across the peptides within the matured capsid variants in Table 17, approximately 175 of them comprised the sequence GSGSPH (SEQ ID NO: 4695) and further comprised additional modifications in the C-terminal region of the sequence.

Table 17. NGS fold-enrichment of TTM-001 and TTM-002 matured AAV capsid variants in the liver of NHPs

[0522] Table 18 provides the peptide sequences of 43 matured capsid variants having a raw virus count greater than 10, a CV of less than 1 for the heart samples isolated, and that also demonstrated a 4-fold or greater fold-increase in expression in the heart relative to the AAV9 control. A number of the matured variants shown in Table 18 also demonstrated increased expression in other tissues isolated from the NHPs, including the brain, muscle, and/or liver, and are therefore pan-tropic.

Table 18. NGS fold-enrichment of TTM-001 and TTM-002 matured AAV capsid variants in the heart of NHPs

[0523] Table 19 provides the peptide sequences of 14 matured capsid variants having a raw virus count greater than 10, a CV of less than 1 for the muscle samples isolated (e.g., quadriceps), and that also demonstrated a 4-fold or greater fold-increase in expression in the muscle relative to the AAV9 control. A number of the matured variants shown in Table 19 also demonstrated increased expression in other tissues isolated from the NHPs, including the brain, heart, and/or liver, and are therefore pantropic.

Table 19. NGS fold-enrichment of TTM-001 and TTM-002 matured AAV capsid variants in the muscle (e.g., quadriceps) of NHPs

[0524] These data demonstrate that following two maturation approaches, matured TTM-001 and TTM-002 capsid variants (AAV9 capsid variants) with loop IV modifications were generated with significantly enhanced CNS tropism over wild-type AAV9 controls in both NHPs and mice, while also exhibiting de-targeting in peripheral tissues (e.g., the liver and DRG). These resulting matured variants therefore demonstrated cross-species CNS tropism in both NHPs and mice. Matured TTM- 001 and TTM-002 capsid variants with liver-specific tropism were also generated with at least 10 times the expression compared to wild-type AAV9 in the liver of NHPs. Several matured variants were also generated with increased expression in the heart and skeletal muscle (e.g., quadriceps) relative to wild-type AAV9 in NHPs.

Example 5. Evaluation of TTM-001 and TTM-002 AAV capsid variants in Diverse Primate Species

[0525] This Example evaluates the tropism and cross-species compatibility of the TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2) capsid variants in two diverse primate species, marmosets (Callithrixjacchus') and African green monkeys (Chlorocebits sabaeus), as compared to their tropism in cynomolgus macaques (Macaca fascicularis) provided in Example 1. The cross-species compatibility and tropism of an AAV9 capsid variant comprising the amino acid sequence of SPHKYG (SEQ ID NO: 966) was also investigated in this example. The amino acid and DNA sequences of TTM-001 and TTM-002 are provided, e.g., in Tables 4 and 5, respectively.

[0526] To investigate tropism in African green monkeys, AAV particles comprising the TTM-001 capsid variant, the TTM-002 capsid variant, an AAV9 capsid variant comprising SEQ ID NO: 966, or an AAV9 control under the control of a synapsin promoter, were intravenously injected into NHPs (n=2, 3-12 years of age) at a dose of 2E13 vg/kg. After 14-days in life, the brains and tissues (liver, DRG, quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV9 wild-type control.

[0527] To investigate tropism in marmoset monkeys, AAV particles comprising the TTM-001 capsid variant, the TTM-002 capsid variant, an AAV9 capsid variant comprising SEQ ID NO: 966, or an AAV9 control, were intravenously injected into NHPs (n=2, >10 months of age) at a dose of 2E13 vg/kg (8.75E12 vg/mL). After 28-days in life, the brains and tissues (liver quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV9 wild-type control. [0528] As provided in Table 20 (African green monkeys) and Table 21 (marmosets), both the TTM-001 and TTM-002 capsid variants demonstrated increased CNS tropism in diverse primate species. The TTM-001 capsid variant demonstrated a 73.6-fold increase in expression relative to AAV9 in the brain of cynomolgus macaques (Table 9, Example 1), a 43.5-fold increase in expression relative to AAV9 in the brain of African green monkeys, and a 703.3-fold increase in expression relative to AAV9 in the brain of marmosets. The TTM-002 capsid variant demonstrated a 62.6-fold increase in expression relative to AAV9 in the brain of cynomolgus macaques (Table 9), a 13.8-fold increase in expression relative to AAV9 in the brain of African green monkeys, and a 366.6-fold increase in expression relative to AAV9 in the brain of marmosets. Both TTM-001 and TTM-002 led to a significant increase in expression relative to AAV9 in the heart of both African green monkeys and marmosets (Table 20 and Table 21). The AAV9 capsid variant comprising SEQ ID NO: 966 also demonstrated in increase in expression relative to AAV9 in the brain and heart of both African green monkeys and marmosets. Furthermore, TTM-001, TTM-002, and the AAV9 capsid variant comprising SEQ ID NO: 966, also all led to increased expression in the brain of both B ALB/c and C57B1/6 mice (Table 11, Example 1), demonstrating an average fold change in expression relative to AAV9 across both species of mice of 63.1, 66.8, and 126.97, respectively.

Table 20. NGS-fold enrichment of TTM-001 (comprises SEQ ID NO: 941), TTM-002 (comprises SEQ ID NO: 2), and an AAV9 capsid variant comprising SEQ ID NO: 966 in African green monkeys

Table 21. NGS-fold enrichment of TTM-001 (comprises SEQ ID NO: 941), TTM-002 (comprises SEQ ID NO: 943) ), and an AAV9 capsid variant comprising SEQ ID NO: 966 in marmosets

[0529] Taken together, these data demonstrate that the AAV9 capsid variants of TTM-001 and TTM-002 demonstrated increased CNS tropism relative to the AAV9 control in the CNS across three diverse primate species and two species of mice, providing evidence of strong cross-species capacity. The AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 966 also demonstrated strong CNS expression relative to the AAV9 control in two species of NHPs and two species of mice, also showing strong cross-species capacity. Example 6. Advanced maturation of TTM-002 capsid variant in mice

[0530] This Example describes additional maturation of the TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2) capsid variant in mice. In order to mature the TTM-002 capsid variant, sets of three contiguous amino acids were randomized across the mutagenesis region in TTM-002 sequence, which spanned from position 450 to position 466, numbered according to SEQ ID NO: 982. Unlike the maturation performed in in Example 3, where the SPH motif that was observed in the AAV capsid variants that demonstrated the greatest foldenrichment in the NHP brain relative wild- type AAV9 was not disrupted, in the maturation approach used in this Example, the SPH motif was not held constant to further explore the role of this motif in the capsid variant. The matured TTM-002 capsid variants that resulted from the maturation approach were pooled together for subsequent testing and characterization in mice.

[0531] The library of matured AAV capsid variants generated from the TTM-002 matured AAV capsid variant were intravenously injected into the tail vein of three CD-I Outbred mice (Charles River; 6-8 weeks of age) at a dose of 1.0 x 10 ¹² VG/dose. After about 28 days in life, the brains of the mice were isolated, and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the corresponding TTM-002 non-matured control, and the peptides comprised within the variants were identified. Variants were filtered by those with a raw virus count in the sample above 10 and a coefficient of variance (CV) that was greater than 1 (identifies the peptides/variants reliably detected in the majority of the samples isolated from the three mice).

[0532] Following the advanced maturation screen and filtering of the variants, 1302 variants demonstrated an increase in expression relative to the non-matured TTM-002 capsid variant in the brain of the outbred mice. Of the 1302 variants with improved tropism relative to the non-matured TTM-002, 1283 comprised the SPH motif in the same position as the non-matured TTM-002 capsid variant (e.g., immediately subsequent to position 455, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982). Mutations in the region of the SPH motif present in the non-matured TTM-002 capsid variant only consistently appear in those variants with a fold change of 0.2 or 0.1 or lower relative to the non-matured TTM-002 control in the brain of the mice. This indicates that the SPH motif may be important to the increased brain tropism that observed for the TTM-002 capsid variant. In instances when the SPH motif was disrupted, the fold change of the matured variants of TTM-002 decreased considerably in relation to the nonmatured TTM-002 variant which comprised the SPH motif.

Example 7. Tropism of TTM-002 AAV capsid variant

[0533] This Example further investigates the tropism and CNS cells transduced by the TTM-002 capsid variant (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2), as outlined in Table 3 above. The amino acid and DNA sequences of TTM-002 are provided, e.g., in Tables 4 and 5, respectively.

[0534] AAV particles were generated with the TTM-002 capsid variant encapsulating a GFP transgene (AAV_TTM-002.GFP) or a payload driven by a heterologous CBA constitutive promoter (AAV_TTM-002.Payload).

[0535] Two tandem single cell RNA sequencing runs (scRNA-Seq) of mouse cells derived from the midbrain area were performed. In the first run, cells were pooled from two mice at day 28 post treatment with AAV_TTM-002.Payload particles. In the second run we treated with AAV_TTM- 002.GFP particles, in the same manner but without xenografts. Orthotopic xenografts of MDA-MB- 361-Luc#l high passage cells grown as tumorspheres (in tumorsphere media; Sigma # C-28070) were injected (250,000 cells/2pL/mouse) intracranially into 2-month old female SCID CB17 (Mutation: Icr-Prkdcscid/IcrlcoCrl) congenic immunodeficient mice (Charles River Laboratories). The injections were 2.5mm (lateral), -1mm (posterior) with respect to bregma, lowered -3mm ventral and raised +.5 mm dorsal to a final -2.5mm ventral position. Two days later, dilutions of the AAV_TTM- 002.Payload particles (run 1), or in the case without xenografts, dilutions of AAV_TTM-002.GFP particles (run 2) were prepared. IV injections of lOOpL (2.5el l VG/animal) of the AAV_TTM- 002.payload particles or AAV_TTM-002.GFP particles were administered through the tail veins of mice (n=5 mice per groups). At 7 days post-injection, mice from run 1 were imaged in an AmiHTX (Spectral Imager) for bioluminescence of the human tumor cells due to expression of luciferase in response to intraperitoneal luciferin injections.

[0536] At 28 days post-injection with the AAV_TTM-002.payload particles or AAV_TTM- 002.GFP particles, two mice from each run were necropsied, brain samples were isolated, and the midbrain was dissected and isolated. The midbrain samples were then exposed to a cold protease inhibitor (Creative Biomart #NATE-0633) and were dissociated at 6 degrees centigrade. For the samples collected from the mice of run 1 ( A AV_TTM-002. Payload particles), myelin depletion was performed (Miltenyi, #130-096-731), cells were filtered through a 40pM mesh to filter out neurons) and loaded on a 10X chromium G chip. scRNA-Seq was performed (10X Genomics) and samples were sequenced on a NextGen500 Sequencing machine (Illumina). For the samples collected from run 2 (AAV_TTM-002.GFP particles and no xenografts), the cells were not myelin depleted or filtered through 40pM mesh to include neurons. The cells isolated after run 2 were FACS sorted for GFP+/7AAD- (live GFP+ cells). The resultant cells were loaded on a 10X chromium G chip and the scRNA-Seq was run and processed (10X Genomics).

[0537] For run 1, the scRNA-Seq data was filtered to include cells with only greater than 1000 genes per cell and less than 5000, and less than 20 percent mitochondrial gene expression. For run 2, the scRNA-Seq data was filtered to include cells with only greater than 200 genes per cell and less than 5000, and less than 20 percent mitochondrial gene expression. The data were normalized, scaled, and integrated into one combined dataset. Clusters were generated with a resolution of 0.3 and each cluster identity was determined using a panel of cell type specific genes (e.g., as described in Brown et al., 2021. “Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing”. Front. Immunol. 12:730825; the contents of which are hereby incorporated by reference in its entirety). The percentage of GFP sorted cells per cluster was calculated as was the percentage of payload expressing genes per cluster as parallel measures of TTM-002 transduction.

[0538] For payload expressing cells, endothelial cells had the highest proportion of payload positive cells, followed by astrocytes (Table 22). For GFP+ sorted cells, endothelial cells had the highest proportion of GFP positive cells, and astrocytes were the third highest cell type when sorting by proportion of cells expressing GFP (Table 22). These data indicate TTM-002 transduction exhibits an endothelial and astrocytic tropism. Furthermore, the astrocytic cluster had the second highest level of expression of Olig2 (oligodendrocytes demonstrated the greatest Olig2 expression). IHC staining was performed on brain samples isolated from AAV_TTM-002.GFP infected mice and demonstrated that GFP co-localized with some but not all Olig2+ cells. No co-staining was observed with mylein basic protein (MBP), a marker of oligodendrocytes. Co-staining with GFP was also not observed in NeuN positive cells (neurons), GFAP positive cells (astrocytes), and Ibal positive cells (microglia). GFP staining was observed throughout the sagittal section of the mouse brain, which was demonstrative of increased staining in the midbrain. The GFP expressing cells observed did not have a bipolar morphology like oligodendrocyte progenitor (OPC) cells and therefore, together with the scRNA-Seq data, these results indicated that at day 28 post AAV treatment, Olig2+ astrocytes in the midbrain are being transduced by AAV particles comprising a TTM-002 capsid, in a cell type specific tropism.

Table 22. Quantification of payload positive cells and GFP positive cells Example 8. Identification of a receptor for TTM-001 and TTM-002 capsid variants

[0539] This example investigates the tropism and receptor of the TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2) capsid variants for crossing the blood brain barrier. Without wishing to be bound by theory, it is believed that identification of a receptor of these AAV capsid variants provides a better understand of translatability of these variants to different species, as well as the mechanism used for crossing the blood brain barrier that results in an increase in CNS transduction relative to AAV9.

A. Binding of TTM-001 and TTM-002 capsid variants to N-linked galactose

[0540] Primary glycan receptors have been identified for various AAV serotypes, including AAV9 which binds N-linked galactose. In order to investigate the ability of TTM-001 and TTM-002 AAV9 variants to retain this natural glycan binding, HeLa cells were treated with increasing concentrations of Neuraminidase (0, 5, 50, 500, and 100 mU/mL), which cleaves N-sialic acid and exposes N-galactose. The treated cells were then transduced with AAV particles comprising the TTM-001 capsid variant (AAV_TTM-001), the TTM-002 capsid variant (AAV_TTM-002), or an AAV9 control (AAV_AAV9cntl), transduction was measured by quantification of Luc2 activity (RLU), and data was normalized relative to the no neuraminidase control. As shown in Table 25, enzymatic removal of N-sialic acid and exposure of N-galactose on HeLa cells, resulted in a dose dependent increase, more specifically a 9- to 14-fold increase, in transduction by AAV particles comprising the TTM-001 capsid variant and AAV particles comprising the TTM-002 capsid variant. This was analogous to what was observed with the AAV9 control (Table 25). These data demonstrate that the TTM-001 and TTM-002 AAV9 capsid variants retained the natural binding affinity to terminal N-linked galactose observed with AAV9 wild-type.

Table 25. Quantification of HeLa cell transduction post-neuraminidase treatment and transduction with AAV_TTM-001 particles, AAV_TTM-002 particles, or AAV_AAV9cntrl particles. Data measured as fold change in Luc2 activity (RLU) relative to the no neuraminidase control

B. Receptor Identification

[0541] A cell binding array assay was then used to identify a receptor for the TTM-001 and TTM- 002 capsid variants. Briefly, a library of over 5,500 cDNAs was overexpressed in human cells. Cells were contacted with a test ligand, e.g., AAV viral particles comprising a TTM-001 capsid variant or an AAV9 control capsid, which was applied to the array. Binding of the TTM-001 capsid variant or the AAV9 control capsid to the cells was detected using an anti- A A V9 antibody followed by a labeled anti-IgG detection antibody. A comparison of the proteins contacted using AAV particles comprising a wild type AAV9 control capsid and AAV particles comprising a TTM-001 capsid variant revealed a unique interaction with the TTM-001 capsid variant but not the AAV9 wild-type control capsid. This interacting protein was identified as the GPI-anchored protein, alkaline phosphatase issue-nonspecific isozyme (NM_000478.4, which is incorporated by reference herein) (ALPL). ALPL is part of a family of membrane-bound glycoproteins that hydrolyze monophosphate esters at a high pH (see, e.g., Weiss et al., Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc. Natl. Acad. Sci., 83: 7182-7186 (1986), the contents of which are hereby incorporated by reference in their entirety).

[0542] ALPL is highly conserved across humans, mice, and cynomolgus macaques (Macaca fascicidaris) when compared by sequence alignment (Table 26). Additionally, in humans ALPL is expressed on endothelial cells and neurons, and at a low level on astrocytes. The highest level of ALPL expression in human is on endothelial cells. In mice, ALPL is more highly expressed on astrocytes, oligodendrocyte progenitor cells (OPCs), and to a lesser extent on endothelial cells.

Table 26. Identity and similarity of ALPL receptor between different, species

[0543] Furthermore, as shown in Example 7 and Table 22, when mice were treated intravenously with AAV particles comprising the TTM-002 capsid variant expressing a payload, payload expression as measured by RNA-seq was the highest in a subset of endothelial cells (FIG. 1A). This same subset of endothelial cells also showed high expression of ALPL by RNA-seq (FIG. IB). These data indicated a correlation between expression of ALPL and the TTM-002 tropism in mice.

[0544] Taken together, these data indicate that the TTM-001 and TTM-002 capsid variants are capable of binding ALPL, which could serve as a receptor for crossing the blood brain barrier and CNS transduction.

C. Characterization of interaction of the TTM-001 and/or TTM-002 with ALPL

[0545] In order to further characterize the interaction between the TTM-001 and TTM-002 capsid variants and the ALPL protein, it was investigated whether increased expression of the ALPL protein resulted in increased transduction of AAV particles comprising the TTM-001 or TTM-002 capsid variant. Briefly, a transduction assay was performed in that HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing ALPL, an AAVR positive control, or a pCMV6 negative control (250 ng or 500 ng of plasmid). AAVR is a universal AAV entry factor involved in AAV transduction. At 24 hours post-transfection, the HEK 293T cells expressing the ALPL protein or other controls were transduced with an AAV particle comprising the TTM-001 capsid variant, the TTM-002 capsid variant, another AAV capsid variant (TTD-001), or an AAV9 control capsid protein, expressing a GFP payload. At 24-hours post-transduction, GFP expression and luciferase activity were measured to quantify and observe AAV cellular transduction. By immunofluorescence microscopy, expression of the ALPL protein resulted in a significant increase in the transduction of AAV particles comprising the TTM-002 capsid variant compared to particles comprising the AAV9 wild-type control capsid. Additionally, the increase in transduction of the AAV particles comprising the TTM-002 capsid variant was specific to ALPL expression, as expression of the AAVR control did not result in the same increase in transduction of AAV particles comprising the TTM-002 capsid variant. As summarized in Table 27, expression of ALPL led to a 35 and 45-fold increase in transduction the TTM-001 and TTM-002 AAV9 capsid variants, respectively, when measured by a luciferase assay. Transduction of AAV9 wild-type control as well as the AAV9 capsid variant TTD-001 was not affected by expression of ALPL, indicating the specific role of ALPL in transduction of TTM-001 and TTM-002. TTD-001 is an AAV9 capsid variant comprising a loop VIII modification, and the sequence and capsid characterized can be found in WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety). Table 28 provides the results of a second experiment performed as described above, where HEK 293T cells expressing the ALPL protein or the other controls were transduced with AAV particles comprising the TTM-002 capsid variant or one of three AAV9 capsid variants also comprising a modification in loop IV: TTM- 006 (SEQ ID NO: 39), TTM-018 (SEQ ID NO: 51), and TTM-019 (SEQ ID NO: 52). The TTM-002, TTM-006, TTM-018, and TTM-019 capsid variants all comprised the SPH motif immediately subsequent to position 455, numbered relative to SEQ ID NO: 138 and a positive residue in one of the next three residues subsequent to the SPH motif. The TTM-002, TTM-006, TTM-018, and TTM-019 capsid variants all led to an increase in transduction in cells expressing ALPL, which was not observed with the AAV9 control (Table 28).

Table 27. Transduction of TTM-001 and TTM-002 capsid variants as measured by luciferase assay relative to the AAV9 control and AAV variant TTD-001 with loop VIII modification (data shown as fold change relative to the pCMV6 transfected negative control cells)

Table 28. Transduction of TTM-002, TTM-006, TTM-018, and TTM-019 capsid variants as measured by luciferase assay relative to the AAV9 control (data shown as fold change relative to the pCMV6 transfected negative control cells)

[0546] Binding and internalization of AAV capsid variants comprising the TTM-001 capsid variant, the TTM-002 capsid variant, or the AAV9 control capsid was also investigated in cells engineered to express ALPL. HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing ALPL, an AAVR positive control, or a pCMV6 negative control. At 24 hours post-transfection, the HEK 293T cells expressing the ALPL receptor were incubated with an AAV particle comprising the TTM-001 capsid variant, the TTM-002 capsid variant, or an AAV9 control capsid protein, expressing a GFP payload. At 2 or 3-hours post-incubation, cells were washed to remove unbound AAV particles and DNA was extracted to quantify viral genomes. As shown in Table 29, expression of ALPL led to a 3-fold and 6-fold increase in binding/internalization by TTM- 001 and TTM-002, respectively. This effect was specific to TTM-001 and TTM-002, as the binding/internalization of the wild-type AAV9 control was unaffected by ALPL expression.

Table 29. Relative viral gene expression (2 ^AACT ) of cells transfected with a plasmid expressing pCMV6 control, an AAVR control, or ALPL and subsequently transduced with AAV particles comprising the TTM-001 capsid variant, TTM-002 capsid variant, or AAV9 control

[0547] Three isoforms of ALPL exist, isoform 1 (Alkaline phosphatase, placental-like 2 (ALPPL2), NM_031313, which is incorporated by reference herein), isoform 2 (alkaline phosphatase, placental (ALPP), NM_001632, which is incorporated by reference herein), and isoform 3 (alkaline phosphatase, intestinal (ALPLI), NM_001631, which is incorporated by reference herein), that can also be expressed on cell surfaces via a GPI-anchor. Isoform 1 is 56.25% identical and 72.54% similar (gaps: 4.17%) to ALPL, isoform 2 is 54.96% identical and 71.37% similar (gaps: 2.29%) to ALPL, and isoform 3 is 55.98% identical and 72.11% similar (gaps: 3.04%) to ALPL. The transduction assay described above was repeated with the three isoforms. HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing ALPL, isoform 1 of ALPL, isoform 2 of ALPL, isoform 3 of ALPL, an AAVR positive control, or a pCMV6 negative control. At 24 hours posttransfection, the HEK 293T cells expressing the ALPL receptor were transduced with an AAV particle comprising the TTM-001 capsid variant or the TTM-002 capsid variant, expressing a Luc2- GFP payload. At 24-hours post-transduction, luciferase activity (RLU) was measured to quantify AAV cellular transduction. As shown in Table 30, the increase in transduction observed for TTM-001 and TTM-002 when cells express ALPL did not occur in cells expressing isoform 1, 2, or 3. This demonstrates that the significant increase in TTM-001 and TTM-002 transduction is a specific function of ALPL.

Table 30. Transduction of TTM-001 and TTM-002 capsid variants as measured by luciferase assay (RLU) in cells expressing ALPL or isoforms thereof

[0548] Endogenous ALPL was also removed from the surface of HeLa cells by treatment with increasing concentrations phosphatidylinositol-specific phospholipase C (PI/PLC), which cleaves GPI anchored proteins (0, 1, 3, 6, or 10 U/mL), for 1.5 hours at 37°C. Following PI/PLC treatment, cells were incubated with 1E4 VG/cell for three hours of AAV particles comprising the TTM-002 capsid variant or AAV particles comprising an AAV9 control capsid, cells were then washed to remove free virus, and luciferase activity was measured 24 hours post-transduction (RLU). As shown in Table 31, treatment with PI/PLC and removal of the GPI-anchored proteins, significantly decreased transduction by the TTM-002 capsid variant, indicating that increased transduction by TTM-002 in HeLa cells is dependent on a GPI-anchored protein.

Table 31. Transduction of TTM-002 capsid variant or AAV9 control in HeLa as measured by luciferase assay (RLU) following treatment with PI/PLC

[0549] To determine if deletion of the endoplasmic reticulum (ER) localization signal of ALPL affected transduction of the TTM-001 and TTM-002 capsid variants, HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing ALPL, ALP with a deletion of the ER localization signal (ALPL transcript variant 2 that lacks the ER signal (NM_001127501, which is incorporated by reference herein)), or a pCMV6 negative control. At 24 hours posttransfection, the HEK 293T cells expressing the ALPL receptor were transduced with an AAV particle comprising the TTM-001 capsid variant, the TTM-002 capsid variant, or an AAV9 capsid control, expressing a GFP payload. At 24-hours post-transduction, luciferase activity (RLU) was measured to quantify AAV cellular transduction. Data was normalized to fold change in luciferase activity (RLU) compared to the pCMV6 control. As shown in Table 33, the increase in transduction observed for TTM-001 and TTM-002 when cells express ALPL did not occur in cells expressing ALPL comprising a deleted ER localization signal, and therefore did not express ALPL on the surface of the cells. Similar results were observed by immunofluorescence microscopy staining for GFP expression, as no GFP staining was observed in cells transfected with the ALPL mutant comprising a deletion of the ER localization signal that were transduced with AAV particles comprising the TTM- 001 and TTM-002 capsid variants. These data demonstrate that the ER localization signal may play an important role in effect ALPL has on the transduction of the TTM-001 and TTM-002 capsid variants.

Table 33. Transduction of TTM-001 and TTM-002 capsid variants as measured by luciferase assay (RLU) in cells expressing ALPL

[0550] To determine if the TTM-001 and TTM-002 capsid variants could bind to both the human ALPL protein (NM_000478.6, which is incorporated by reference herein) and the mouse ALPL ortholog (NM_001287172.1, which is incorporated by reference herein), HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing human ALPL, the murine ortholog of ALPL, or a pCMV6 negative control. At 24 hours post-transfection, the HEK 293T cells expressing the ALPL receptor were transduced with an AAV particle comprising the TTM-001 capsid variant, the TTM-002 capsid variant, or an AAV9 control capsid protein, expressing a Luc2-GFP payload. At 24-hours post-transduction, luciferase activity (RLU) was measured to quantify AAV cellular transduction. As shown in Table 34, the increase in transduction observed for TTM-001 and TTM-002 when cells express human ALPL was also observed in cells expressing the murine ALPL ortholog. These luciferase results were also confirmed by immunofluorescence microscopy staining for GFP. These data indicate that the murine ALPL protein is also a receptor for the TTM-001 and TTM-002 capsid variants.

Table 34. Transduction of TTM-001 and TTM-002 capsid variants as measured by luciferase assay (RLU) in cells expressing ALPL

[0551] To determine if the TTM-002 capsid variants could bind also bind to the cynomolgus ALPL protein (XM_005544525, which is incorporated by reference herein), HEK 293T cells were transfected via calcium phosphate transfection with a plasmid expressing human ALPL, the ortholog of ALPL in cynomolgus macaques (Macacafascicularis~), the murine ortholog of ALPL (NM_001287172.1, which is incorporated by reference herein), an AAVR positive control (universal AAV entry factor involved in AAV transduction), or a pCMV6 negative control. At 24 hours posttransfection, the HEK 293T cells expressing the ALPL receptor were transduced with an AAV particle comprising the TTM-002 capsid variant or an AAV9 control capsid protein, expressing a Luc2-GFP payload. At 24-hours post-transduction, luciferase activity (RLU) was measured to quantify AAV cellular transduction. As shown in Table 35, the increase in transduction observed for TTM-002 when cells express human ALPL and murine ortholog was also observed in cells expressing the ALPL ortholog in cynomolgus macaques. These luciferase results were also confirmed by immunofluorescence microscopy staining for GFP. These data indicate that the ALPL protein in cynomolgus macaques is also a receptor for the TTM-002 capsid variant.

Table 35. Transduction of the TTM-002 capsid variant as measured by luciferase assay (RLU) in cells expressing human ALPL, murine ALP, and cynomolgus ALPL

[0552] Direct binding and specific interaction of the TTM-002 capsid variant and the AAV9 capsid control to ALPL was measured by Surface Plasmon Resonance (SPR) on a Biacore 8K instrument. His-tagged ALPL was first captured on a CM5 sensor chip pre-immobilized with anti-His antibody by passing 5 pg/ml of ALPL for 240 seconds (FIGs. 2A-2B). AAV9 or TTM-002 and buffer were then passed over ALPL to monitor rates of association and dissociation, respectively. The concentration of AAV used was from 0.0625 to 1 nM (e.g., 0.0625 nM, 0.125 nM, 0.25 nM, 0.5 nM, and 1 nM; FIGs. 2A-2B) and association/dissociation rates were monitored for 120 seconds. The surface was regenerated using two pulses of 10 mM glycine pH 1.7 for 30 seconds. A flow rate of 30 pl/min was used for all steps and the running buffer used was PBS-P+. In a second experiment, the AAV9 control or TTM-002 capsid variant were immobilized on the CM5 sensor chip (FIGs. 2C-2D). His-tagged ALPL and buffer were then passed over the AAV9 control or TTM-002 capsid variant to monitor rates of association and dissociation. The concentration of ALPL used was from 15.625 to 250 nM (e.g., 15.625 nM, 32.25 nM, 62.5 nM, 125 nM, and 250 nM; FIGs. 2C-2D).

[0553] As shown in FIG 2A and FIG. 2C, TTM-002 was capable of directly and specifically binding to ALPL in a dose dependent manner, where as AAV9 showed no binding (FIG. 2B and FIG. 2D). The dissociation constant (KD) was quantified for the TTM-002 capsid variant binding to ALPL, and was determined to be approximately 32 nM (k _on 3.2c4 1/Ms; k _O ff. 1.27e-3 1/s) (Table 41). For this experiment, the density of TTM-002 on the chip was approximately 6300RU.

[0554] Additional experiments were performed varying the density of the TTM-002 capsid variant on the chip. His-tagged ALPL and buffer were then passed over the AAV9 control or TTM- 002 capsid variant to monitor rates of association and dissociation. The concentration of ALPL used was from 15.625 to 250 nM (e.g., 15.625 nM, 32.25 nM, 62.5 nM, 125 nM, and 250 nM, As shown in Table 41, despite varying the density of the TTM-002 capsid variant, the affinity values of TTM-002 to ALPL were similar.

Table 41. Binding Affinities of varying densities of TTM-002 to ALPL

[0555] The dissociation of the TTM-002 capsid variant from the ALPL receptor was also investigated at a low pH, as lower affinity has been observed at more acidic pH values for other receptors which can promote efficient transcytosis. pH dependence on interaction of the TTM-002 capsid variant and the AAV9 capsid control to ALPL was measured by Surface Plasmon Resonance (SPR) on Biacore 8K instrument at a pH of 7.4 during association phase and a pH of 7.4 or 5.5 during dissociation phase. The TTM-002 capsid variant was immobilized on the CM5 sensor chip. His- tagged ALPL and buffer was then passed over the AAV9 control or TTM-002 capsid variant to monitor the rate of association at pH 7.4 and dissociation, respectively, at a pH of 7.4 (FIG. 3A) or a pH of 5.5 (FIG. 3B). The concentration of ALPL used was from 0 to 250 nM (e.g., 0, 7.8 nM, 15.6 nM, 32.25 nM, 62.5 nM, 125 nM, and 250 nM; FIGs. 3A-3B). As shown in FIGs. 3A-3B, the rate of dissociation between the TTM-002 capsid variant and ALPL increased when the pH was decreased from 7.4 to 5.5, indicating a pH dependent dissociation between the TTM-002 capsid variant and the ALPL receptor.

[0556] Additionally, siRNAs were used to knockdown endogenous levels of ALPL in HeLa cells. HeLa cells were transfected with one of two siRNAs targeting ALPL, both siRNAs targeting ALPL, or a non- ALPL targeting siRNA control using lipofectamine 2000 (5pmol of the siRNA per well of 96-well plate). At 48-hours post transfection, the cells were transduced with 1E4 VG/cell of AAV particles comprising a TTD-002 capsid variant or an AAV9 control capsid and a viral genome encoding a Luc2-GFP payload. At 24-hours post transduction, luciferase activity (RLU) was measured to quantify AAV cellular transduction (FIG 4). siRNA mediated knockdown of ALPL led to a 60% reduction in TTM-002 transduction, indicating that knockdown of endogenous ALPL expression inhibits TTM-002 transduction.

[0557] To determine if blocking the ALPL receptor via an anti- ALPL antibody would reduce cellular transduction of the TTM-002 capsid variant, HeLa cells were incubated with 0, 3.125, 6.25, or 12.5 pg/mL of an antibody against ALPL or an IgG isotype -control antibody for 1 hour at 4°C. Following incubation, the cells were transduced with 1E4 VG/cell of AAV particles comprising the TTM-002 capsid variant or an AAV9 control expressing a GFP-luciferase payload. After a 4 hour incubation period the AAV and media were removed and replaced with fresh media and luciferase activity was measured (RLU) 24 hours post transduction. As shown in Table 36, increasing concentrations of the anti-ALPL antibody resulted in decreased TTM-002 transduction in a dose dependent manner, as compared to the isotype control. Similar results were seen by immunohistochemistry. The AAV9 control showed similar levels of transduction in the presence of the anti-ALPL antibody or the isotype control. These data indicated that blocking access to surface expressed ALPL with this antibody reduces TTM-002 transduction.

Table 36. Transduction of TTM-002 capsid variant in HeLa cells as measured by luciferase assay (RLU) following treatment with an anti-ALPL antibody or IgG isotype control

[0558] An inhibitor of ALPL was also investigated to determine if blocking the ALPL receptor would reduce cellular transduction of the TTM-002 capsid variant. The small molecule Tissue- Nonspecific Alkaline Phosphatase Inhibitor (TNAPi) (CAS 496014-13-2; 2,5-Dimethoxy-N- (quinolin-3-yl)benzenesulfonamide) was selected for use, as kinetic studies such as Dahl et al. (“Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue- Nonspecific Alkaline Phosphatase (TNAP),” J Med Chem, 2009; 52)21):6919-6925), which is incorporated by reference in its entirety, indicate that this inhibitor demonstrated an allosteric inhibition mechanism, and inhibition is uncompetitive with respect to the phosphate donor substrate and non-competitive with respect to the acceptor substrate. The IC50 was measured to be 190 nM. The inhibitor or a vehicle control (equivalent amount of DMSO) was added to HeLa cells expressing ALPL 1 hour prior to transduction with an AAV viral particle comprising the TTM-002 capsid variant or an AAV9 control capsid and expressing a GFP-luciferase transgene under the control of a CAG promoter. The cells were then transduced with 1E4 VG/cell of an AAV particle comprising the TTM- 002 capsid variant or an AAV9 control capsid. Media and virus were removed 4 hours post transduction and luciferase activity was measured (RLU) 24 hours post transduction. As shown in FIGs. 5A and 5B, increasing concentrations of the TNAPi inhibitor resulted in significantly decreased TTM-002 transduction compared to the no inhibitor control (FIG. 5A) and the vehicle control (FIG. 5B). The IC50 of the TNAPi inhibitor in this assay was calculated to be 0.34 nM (FIG 5C). The AAV9 control showed similar levels of transduction in the presence of this inhibitor as compared to the no inhibitor control (FIG. 5A). These experiments were repeated using a second inhibitor, SBI- 425 (5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide; e.g., as described in Pinkerton et al., “Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor,” Bioorg Med Chem Lett., 2018; 28( 1):31 -34, the contents of which are hereby incorporated by reference in their entirety), which is the drug product of TNAPi. Similar to TNAPi, increasing concentrations of SBI-425 significantly inhibited TTM-002 transduction compared to the vehicle control (FIG. 6A), but had no effect on the transduction of AAV9 at the same concentrations (FIG. 6B). The IC50 of the SBI-425 inhibitor in this assay was also calculated to be 0.34 nM (FIG 6C). Similar results were obtained by immunofluorescence microscopy for both inhibitors tested. These data with both the TNAPi and SBI- 425 inhibitors, indicated that blocking access to surface expressed ALPL with a small molecule inhibitor significantly inhibited TTM-002 transduction.

[0559] The ability of ALPL to transport the TTM-002 capsid variant across the cell membrane (transcytosis) was also investigated using a transcytosis assay and Madin-Darby Canine Kidney (MDCK) cells engineered to overexpress ALPL. MDCK cells were used as they demonstrate clear apico-basolateral polarity and well defined tight junctions. The MDCK cells were plated and resistance was measured. The MDCK control cells that did not express ALPL demonstrated resistance levels above 1000 Ohm and the MDCK cells expressing ALPL demonstrated resistance levels between 5000 to 7000 Ohm. AAV particles with the TTM-002 capsid variant were then added to the top of the cells and the ability of these particles to move from the top to the bottom chamber was measured by qPCR. The percent of the AAV particles comprising the TTM-002 capsid variant detected in the bottom chamber relative to the input of the particles to the top was then calculated. No transcytosis was observed in the MDCK cells that did not express ALPL. However, the MDCK cells overexpressing ALPL demonstrated highly effective transcytosis of TTM-002, such that MDCK ALPL overexpressing cells demonstrated 149-fold greater virus detected in the bottom chamber (percent to the initial virus) as compared to the MDCK cells that did not express ALPL. Additionally, MDCK ALPL overexpressing cells demonstrated 252-fold greater virus detected in the bottom chamber (percent to the initial virus) of the TTM-002 capsid variant as compared to AAV9. A single MDCK cell clone engineered to express ALPL was selected from the pool of MDCK cells that were engineered to overexpress ALPL. The transcytosis assay was done using MDCK cells expressing ALPL from this single clone. MDCK cells expressing ALPL from this single clone demonstrated 7478-fold greater virus detected in the bottom chamber (percent to the initial virus) of the TTM-002 capsid variant as compared to AAV9.

D. Conclusions

[0560] Taken together, these data demonstrate the ALPL is a likely surface receptor for the TTM- 001 and TTM-002 capsid variants, as overexpression led to an increase in TTM-001 and TTM-002 transduction as well as cell binding/internalization, which was specific for ALPL. Enzymatic removal of ALPL from the cell surface, mutating the ER localization signal of ALPL, or knockdown of the ALPL receptor by siRNA, also reduced TTM-002 transduction. Without wishing to be bound by theory, it is believed in some embodiments, that the binding of TTM-001 and TTM-002 to ALPL is part of the mechanism leading to increased crossing of the blood brain barrier relative to the AAV9 control. The highly conserved nature of the ALPL receptor protein across species is predictive of cross-species compatibility of the TTM-001 and TTM-002 capsid variants.

Example 9. Identification of Minimal ligand and alternative ALPL-binding moieties

[0561] Biotinylated peptides corresponding to 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid sequences from loop IV of the TTM-001 and TTM-002 AAV9 capsid variants, including tandem/multimers thereof, as well as from the loop IV domain of the control AAV9 capsid, are generated to investigate the minimal peptide sequence needed for binding ALPL. In some embodiments, loop IV comprises positions 449-475 (e.g., amino acids KTINGSGQNQQTLKFSVAGPSNMAVQG (SEQ ID NO: 6404)), numbered according to SEQ ID NO: 138. In some embodiments loop IV comprises positions 449-460 (e.g., amino acids KTINGSGQNQQT (SEQ ID NO: 6405)), numbered according to SEQ ID NO: 138. The AAV9 capsid variants, TTM-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941) and TTM-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2), are outlined in Table 3 above. The amino acid and DNA sequences of TTM-001 and TTM-002 are provided, e.g., in Tables 4 and 5, respectively.

[0562] These biotinylated peptides are first captured on an SA sensor chip pre-immobilized with streptavidin by passing 5 pg/ml of peptide for 240 seconds. Recombinant ALPL and buffer are then passed over these peptides to monitor rates of association and dissociation, respectively. The concentration of ALPL to be used ranges from 0.0625 to 1 nM and association/dissociation rates are monitored for 120 seconds. The surface is regenerated using two pulses of 10 mM glycine pH 1.7 for 30 seconds. A flow rate of 30 pl/min is used for all steps and the running buffer used was PBS-P+. This will identify minimum sequences from loop IV of the TTM-001 and TTM-002 capsid variants needed for binding the ALPL receptor.

[0563] Alternative constrained conformations of peptides isolated from loop IV of the TTM-001, and TTM-002 capsid variants or the AAV9 control capsid are tested for binding to ALPL. Biotinylated cyclic peptides corresponding to 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid sequences from loop IV of the TTM-001 and TTM-002 capsid variants, as well as from the loop IV domain of AAV9 control, are generated. Biotinylated peptides are first captured on an SA sensor chip preimmobilized with streptavidin by passing 5 pg/ml of peptide for 240 seconds. Recombinant ALPL and buffer are then passed over these peptides to monitor rates of association and dissociation, respectively. The concentration of ALPL used ranges from 0.0625 to 1 nM and association/dissociation rates are monitored for 120 seconds. The surface is regenerated using two pulses of 10 mM glycine pH 1.7 for 30 seconds. A flow rate of 30 pl/min is used for all steps and the running buffer used is PBS-P+. This will further identify the minimum sequences from loop IV of the TTM-001 and TTM-002 capsid variants needed for binding the ALPL receptor. [0564] Direct binding of alternative ALPL binding ligands, including antibody molecules and other protein-based aptamers, to ALPL is measured by Surface Plasmon Resonance (SPR) on Biacore 8K instrument. His-tagged ALPL is first captured on a CM5 sensor chip pre-immobilized with anti- His antibody by passing 5 pg/ml of ALPL for 240 seconds. The AAV9 control or the TTM-001 or TTM-002 capsid variant and buffer are then passed over ALPL to monitor rates of association and dissociation, respectively. The concentration of AAV used ranges from 0.0625 to 1 nM and association/dissociation rates are monitored for 120 seconds. The surface is regenerated using two pulses of 10 mM glycine pH 1.7 for 30 seconds. A flow rate of 30 pl/min is used for all steps and the running buffer used was PBS-P+. This will aid in identification of additional binding moieties capable of binding ALPL and the minimum sequences/components needed for binding.

Example 10. Functionalization of antibody molecules through recombinant approaches at or near C-terminus

[0565] Once the minimum sequences isolated from loop IV of the TTM-001 and TTM-002 capsid variants needed to bind ALPL are identified using the techniques described in Example 1 , DNA sequences encoding fusion antibody constructs encoding a therapeutic antibody molecule fused to an ALPL -binding ligand in the CH3 domain of the Fc region are generated. Specifically, the coding sequence for an antibody molecule to a therapeutic protein, is cloned as a full-length antibody, that will be modified to comprise an antigen binding domain (e.g., VH and VL) specific for a therapeutic protein, and an Fc region, wherein peptides comprising sequences of varying lengths isolated from loop IV of the TTM-001 and TTM-002 capsid variants (e.g., ALPL-binding ligands) are fused at or near the C-terminus of the CH3 domain of the Fc region of the antibody molecule. The recombinant antibody molecules fused to the sequences of varying lengths isolated from loop IV of the TTM-001 and TTM-002 capsid variants (e.g., ALPL-binding ligands) or unmodified antibody molecules against the same therapeutic protein are injected into a relevant mouse model. These results will indicate the ability of a recombinant fusion protein, e.g., an antibody molecule, containing an ALPL-binding ligand comprising a portion of the loop IV sequence of the TTM-001 or TTM-002 capsid variants to show improved crossing of the blood brain barrier. Without wishing to be bound by theory, it is believed in some embodiments, that the recombinant antibody molecules fused to the ALPL-binding ligands may exhibit greater biodistribution in the brain and increased efficacy in the brain relative to a control non-modified antibody molecule alone.

[0566] A multispecific antibody molecule comprising a first binding domain for a therapeutic protein and a second binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) is generated. These multispecific antibody molecules are designed to comprise two Fabs for binding a therapeutic protein and ALPL, two scFvs for binding a therapeutic protein and ALPL, or combinations of an Fab and scFv. The multispecific antibody molecule comprising a first binding domain to a therapeutic protein and an anti-ALPL binding domain, a monospecific antibody molecule that binds to the therapeutic protein, or a multispecific antibody comprising a binding domain to the therapeutic protein and IgG control binding domain is intravenously administered to a relevant mouse model. These results will indicate the ability of a recombinant fusion protein, e.g., a multispecific antibody molecule, containing an ALPL-binding domain or an ALPL-binding ligand, in addition to a therapeutic protein or a second binding domain to a therapeutic protein, to show improved crossing of the blood brain barrier. Without wishing to be bound by theory, it is believed in some embodiments that the recombinant multispecific antibody molecule comprising the anti-ALPL-binding domain and the binding domain to a therapeutic protein will exhibit increased biodistribution and efficacy in the brain relative to a monospecific antibody that binds the therapeutic protein or a multispecific antibody comprising binding domain specific for the therapeutic protein and an IgG control binding domain. [0567] Without wishing to be bound by theory, it is believed in some embodiments that these examples will demonstrate that multiple ALPL-binding ligands, including those comprising portions of the amino acid sequence of loop IV of the TTM-001 and TTM-002 capsid variants, can be genetically encoded as fusion proteins to therapeutic proteins, to convey an improved ability to cross the blood brain barrier. Similar methodologies as described in this Example can be used to functionalize other therapeutic proteins and enzymes of interest.

Example 11. Functionalization of therapeutic proteins, antibodies, or enzymes of interest through post-translational linker strategies of ALPL-binding moieties to the protein, antibody, or enzyme of interest.

[0568] Methods for stochastic and site-specific methods of labeling amino acids on protein sequences are disclosed by Shadish JA and DeForest CA, Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials. Matter 2020 2:50-70 (the contents of which are hereby incorporated by reference in their entirety). Similarly, numerous methods have also been employed specifically for conjugation of various payloads to antibodies, including linkage chemistries used for antibody-drug conjugates (e.g., as described in Fu et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy 2022 7:93; and Drago et al. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol 2021 18:327-344; the contents of which are hereby incorporated by reference in their entirety).

[0569] Numerous methods for chemical- or enzyme-mediated site-specific or site-agnostic introduction of ALPL-binding ligands for functionalization of various active agents including but not limited to therapeutic proteins, antibody molecules that bind therapeutic proteins, and enzymes are tested. Peptides of varying lengths of amino acids derived from the loop IV region of the TTM-001 or TTM-002 capsid variants are chemically modified with NHS -esters and are allowed to react with free amines on the surface of an active agent, such as an antibody molecule that binds a therapeutic protein, a therapeutic protein, or an enzyme. The compositions comprising the modified antibody

26 molecules, therapeutic proteins, or enzymes that are chemically linked to the peptides derived from the TTM-001 or TTM-002 capsid variants are injected into a relevant mouse model and the ability of these modified compositions to cross the blood-brain barrier is evaluated.

[0570] Additionally, peptides of varying lengths of amino acids derived from the loop IV region of the TTM-001 or TTM-002 capsid variants are linked by a cleavable linker, e.g., a pH-sensitive hydrazone linker, to an active agent such as an antibody molecule that binds a therapeutic protein, a therapeutic protein, or enzyme. The compositions comprising the modified antibody molecules, therapeutic proteins, or enzymes that are linked to the peptides derived from the TTM-001 or TTM- 002 capsid variants are injected into a relevant mouse model and the ability of these modified antibodies molecule to cross the blood-brain barrier is evaluated. Without wishing to be bound by theory, it is believed in some embodiments, that the peptide derived from the TTM-001 or TTM-002 capsid variants (the ALPL-binding ligand) will be cleaved from the active agent (e.g., an antibody molecule that binds a therapeutic protein, a therapeutic protein, or enzyme) during transcytosis of the composition across the blood brain barrier, resulting in increased biodistribution of the active agent in the brain upon release to the parenchymal side of the blood brain barrier.

[0571] An alternative post-translational method that can be used to link ALPL-binding ligands to an active agent involves the use of click chemistry. Synthesis of a clickable RGD peptide is obtained by reacting Lys side chain with azido acetic acid on an ALPL-binding ligand, which is then linked to another peptide fragment on an active agent, such as a therapeutic protein, antibody molecule that binds a therapeutic protein, or enzyme. The compositions comprising the modified antibody molecules, therapeutic proteins, or enzymes that are linked to the peptides derived from the TTM-001 or TTM-002 capsid variants are injected into a relevant mouse model and the ability of these modified antibodies molecule to cross the blood-brain barrier is evaluated.

[0572] The ability of active agents such as antibody molecules, therapeutic proteins, and enzymes to cross the blood brain barrier is also assessed through chemically-induced dimerization of synthetic proteins containing ALPL-binding interfaces. The coding sequence of the FRB domain of mTORl (mammalian target of Rapamycin complex 1) is cloned as a C-terminal fusion to the Fc domain of an antibody molecule that binds a therapeutic protein, the C-terminus of a therapeutic protein, or the C- terminus of an enzyme. The coding sequence of FKBP12 is cloned as a C-terminal fusion to an antibody molecule that binds ALPL. The modified proteins encoded by these sequences are produced, and allowed to dimerize in the presence of rapamycin or AP20187. These modified proteins are then administered to a relevant mouse model. The ability of the modified proteins to cross the blood brain barrier and accumulate on the parenchymal side of the blood brain barrier is evaluated. [0573] Collectively, this example provides multiple methods that can be used to chemically link an ALPL-binding ligand to an active agent, such that this conjugated composition can be evaluated for improved ability to cross the blood brain barrier relative to the non-conjugated control. Example 12: Con jugation of siRNA molecules to increase blood brain barrier crossing

[0574] Peptides of varying lengths of amino acids isolated from loop IV of the TTM-001 or TTM-002 capsid variants, including tandem or multimer orientations thereof, are synthesized. Similar to the method described, e.g., by Eyford et al. A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood Brain Barrier to Attenuate Ischemic Stroke. Front Mol Biosci 2021 8:611367 (the contents of which are hereby incorporated by reference in their entirety), siRNA molecules against a therapeutic target and conjugates thereof with peptides derived from loop IV of the TTM-001 or TTM-002 capsid variants or control peptides are synthesized. More specifically, the peptides derived from TTM-001 and TTM-002 and the siRNA molecules are produced independently and then are chemically conjugated using the crosslinker succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1- carboxylate. The siRNA-TTMOOl or TTM-002 peptide conjugates and the siRNA-control peptide conjugates are administered intravenously to a relevant mouse model. At 24 hours post-intravenous administration, mice are sacrificed and total RNA is isolated from the brains of each animal. qPCR analysis is performed to measure expression of the target gene in the mice treated with the siRNA- TTM001 or TTM-002 peptide conjugates relative to the mice treated with the siRNA-control peptide conjugates, as a measure of the ability of theses siRNA conjugates to cross the blood brain barrier. Without wishing to be bound by theory, it is believed in some embodiments, that siRNA molecules conjugated to peptides isolated from loop IV of the TTM-001 and TTM-002 capsid variants may demonstrate an increased ability to cross the blood brain barrier and increased efficacy relative to nonconjugated siRNA molecules.

[0575] Additionally, as described, e.g., in Yang et al. A microfluidic method for synthesis of transferrin-lipid nanoparticle loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia. Nanoscale 2014 6(16):9742-9751 (the contents of which are hereby incorporated by reference in their entirety), cationic lipid nanoparticles are prepared by ethanol injection method and comprise an siRNA molecule that binds to a therapeutic target. Peptides of varying lengths of amino acids derived from loop IV of the TTM-001 or TTM-002 capsid variants, including tandem or multimer orientations thereof, are synthesized and conjugated to the surface of the nanoparticles. Mice are injected intravenously with the LNPs comprising the siRNA molecule coated with the peptides isolated from the TTM-001 and TTM-002 capsid variants, or a control non-coated LNP comprising the siRNA molecule. At 24 hours post-intravenous administration, mice are sacrificed and total RNA is isolated from the brains of each animal. qPCR analysis is performed to measure expression of the target gene in the mice treated with the LNPs comprising the siRNA molecule that are coated with the TTM001 or TTM-002 peptides relative to the mice treated with the control non-coated LNPs comprising the siRNA molecule, as a measure of the ability of theses siRNA conjugates to cross the blood brain barrier. Without wishing to be bound by theory, it is believed in some embodiments, that siRNA molecules comprise within LNPs coated with peptides isolated from loop IV of the TTM-001 and TTM-002 capsid variants may demonstrate an increased ability to cross the blood brain barrier and increased efficacy relative to siRNA molecules comprised with in non-coated LNPs.

[0576] Taken together, this example provides methods that can be used to link an ALPL-binding ligand (e.g., a peptide isolated from loop IV of the TTM-001 and TTM-002 capsid variants) to siRNA agent or LNP comprising a siRNA molecule, such that this composition can be evaluated for improved ability to cross the blood brain barrier relative to non-linked controls.

Example 13: Binding of Peptide Ligands to ALPL

[0577] This example investigates the ability of peptides of varying lengths comprising the insert sequences from loop IV of the TTM-001 and TTM-001 capsid variants, which are SPHSKA (SEQ ID NO: 941) and HDSPHK (SEQ ID NO: 2), respectively, plus or minus amino acid residues present N- terminal and/or C-terminal relative to the insert sequences. The peptide sequences tested are provided in Table 37. Each peptide was modified at the N-terminus by biotin followed by a GSGS linker (SEQ ID NO: 6409) and at the C-terminus by amidation.

Table 37. Peptide Ligands (GSGS (SEQ ID NO: 6409) is the linker; bolded sequence is the peptide insert of TTM-002 (HDSPHK (SEQ ID NO: 2)))

[0578] Binding and interaction between the peptides and the ALPL receptor were measured by Surface Plasmon Resonance (SPR) on a Biacore 8K instrument. The biotinylated peptides in Table 37 were captured on the streptavidin biosensor chip and 1000 nM of ALPL and buffer were passed over the peptides to monitor binding. No binding was detected with these peptides. His-tagged ALPL was then captured on the chip pre-immobilized with anti-His antibody, and 50 pM of the peptides in Table 37 or 250 nM of the AAV9 control capsid, the TTM-001 capsid variant (SEQ ID NO: 981), or the TTM-002 capsid variant (SEQ ID NO: 982) were flowed over the ALPL to check for binding. No detectable binding was observed with the peptides or the AAV9 control. Binding to ALPL was detected for the TTM-001 and TTM-002 capsid variants, confirming what was observed in Example 8 above. The longer peptides derived from TTM-002, GSGSLYYLSKTINGHDSPHKSGQNQQTLKF (SEQ ID NO: 19) and GSGSRLMNPLIDQYLYYLSKTINGHDSPHKSGQNQQTLKFSVAGPSNMAV (SEQ ID NO: 20), were also flowed over the his-tagged ALPL captured on the CM5 sensor chip pre-immobilized with anti-His antibody. As was observed with the peptides in Table 37, no binding to ALPL was observed with the longer peptides. [0579] By LC-MS, the overall serine phosphorylation was calculated to be 60% across the TTM- 002 capsid variant (SEQ ID NO: 982), which was much higher than the overall serine phosphorylation levels measured in other AAV9 capsid variants tested comprising modifications in loop IV or loop VIII. The overall serine phosphorylation was calculated to be 4.7% across the TTM- 019 capsid variant (SEQ ID NO: 52), 1.9% across the TTM-018 capsid variant (SEQ ID NO: 51), and 1.5% across the TTD-001 AAV9 capsid variant comprising a loop VIII modification, the sequence and characterization of which can be found in WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety). Additionally, only TTM-002 demonstrated an 80-90% phosphorylation level on the serine in the SPHK (SEQ ID NO: 6398) motif that is present in loop IV of the TTM-002 capsid variant (SEQ ID NO: 982). This serine is present at position 456 in the TTM- 002 capsid variant, numbered according to SEQ ID NO: 982. This increased phosphorylation was detected in the TTM-002 capsid variant encapsulating two different payloads. Without wishing to be bound by theory, it is believed in some embodiments, that this SPHK (SEQ ID NO: 6398) motif present in the TTM-002 capsid variant is a consensus motif for the CDK5 kinase and peptides comprising this motif and a modification, e.g., a phosphate group, on this serine, may demonstrate binding to ALPL, as compared to their non-phosphorylated counterparts.

[0580] Additional peptides derived from the TTM-002 capsid variant and peptide insert of HDSPHK (SEQ ID NO: 2) that is present in loop IV, were then generated that comprised a phosphorylated serine to test binding with the ALPL receptor. These phosphorylated peptides are provided in Table 38. Each peptide was also modified at the N-terminus by biotin followed by a GSGS linker (SEQ ID NO: 6409) and at the C-terminus by amidation.

Table 38. Phosphorylated Peptide Ligands (GSGS (SEQ ID NO: 6409) is the linker)

[0581] Binding and interaction between the phosho-peptides and the ALPL receptor were also measured by SPR. The biotinylated and phosphorylated peptides in Table 38 were captured on the streptavidin biosensor chip along with their non-phosphorylated counterparts, GSGSNGHDSPHKSG (SEQ ID NO: 4500) and GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503). ALPL and buffer were passed over the peptides to monitor binding. The concentration of ALPL used ranged from 0 to 500 nM (0 nM, 125 nM, 250 nM, and 500 nM). As shown in FIGs. 7A-7B, both phospho-peptides demonstrated dose dependent binding to ALPL, whereas no binding was observed with their non- phosphorylated counterparts (FIGs. 7A-7B).

[0582] In a second experiment, his-tagged ALPL was first captured on a CM5 sensor chip preimmobilized with anti-His antibody. The phosphorylated peptides in Table 38 and their non- phosphorylated counterparts, GSGSNGHDSPHKSG (SEQ ID NO: 4500) and GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) and buffer were flowed over the ALPL protein to monitor binding. The concentration of the peptides ranged from 0 to 50 pM (0 pM, 1.56 pM, 3.125 pM, 6.25 pM, 12.5 pM, 25 pM, or 50 pM). Both phospho-peptides showed low signal with dose dependency, but a higher signal was observed for SEQ ID NO: 4513 relative to SEQ ID NO: 4512 (FIGs 8A-8B).

[0583] Binding of the phospho-peptides of SEQ ID NO: 4512 and SEQ ID NO: 4513 to ALPL was also investigated using Bio Layer Interferometry (BLI)/Octet. Biotinylated peptides were first loaded on streptavidin biosensor tips followed by placing tips in tips in varying concentration of his- tagged ALPL ranging from 1.56 to 100 nM to measure binding kinetics. Similar to what was observed by SPR, both of the phospho-peptides of SEQ ID NO: 4512 and SEQ ID NO: 4513 demonstrated binding to ALPL, but no binding was observed with their non-phosphorylated counterparts, GSGSNGHDSPHKSG (SEQ ID NO: 4500) and GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (FIGs. 9A-9B). The loading level of the phosphorylated peptides and the non-phosphorylated peptides was comparable which confirmed that the lack of binding observed for the non- phosphorylated peptides is not due to a lower loading level. Also, as shown in Table 39, the dissociation constant (KD) was quantified for both of the phospho-peptides of SEQ ID NO: 4512 and SEQ ID NO: 4513. The K _D for SEQ ID NO: 4512 was 112 nM and the K _D for SEQ ID NO: 4513 was of 20.7 nM. These binding affinities of the phospho-peptides derived from the TTM-002 capsid variant to ALPL were similar to the binding affinity of the TTM-002 capsid variant to ALPL as provided in Table 41 of Example 8. Additionally, no binding to ALPL was observed with a phosphorylated tau peptide, indicating that the interaction is specific to ALPL and the phosphopeptides derived from TTM-002. The binding of the phospho-peptide of SEQ ID NO: 4513 to ALPL was also tested at a pH of 5.5, and no binding was observed.

Table 39. Binding affinities for SEQ ID NO: 4512 and SEQ ID NO: 4513 to ALPL

[0584] Binding of the phospho-peptides of SEQ ID NO: 4512 and 4513 along with their non- phosphorylated counterparts, GSGSNGHDSPHKSG (SEQ ID NO: 4500) and GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503), respectively, to ALPL was also confirmed by ELISA with ALPL coated on the wells of microplate. (FIGs. 10A-10B). Both of the phosphopeptides of SEQ ID NO: 4512 and 4513 but not their non-phosphorylated counterparts demonstrated dose-dependent binding to ALPL (FIGs. 10A-10B). The EC50 value calculated for the phosphopeptide of SEQ ID NO: 4512 was 1.243 pg/mL and the EC50 value calculated for the phospho-peptide of SEQ ID NO: 4513 was 10.05 pg/mL. [0585] Taken together, these data indicate that phosphorylated peptides derived from the TTM- 002 capsid variant are capable of binding ALPL, as measured by at least three independent methods (Biacore, Octet, and ELISA); and that this binding appeared to be sequence specific as other phosphorylated control peptides showed no binding.

Example 14: Binding and Transcytosis of Antibodies that bind to ALPL

[0586] This example investigates the ability of exemplary antibodies to bind ALPL and to also be transported across the cell membrane (transcytosis).

[0587] Several anti-ALPL antibodies provided in Table 40 were tested to determine if they were able to bind ALPL by ELISA and Surface Plasmon Resonance (SPR) on a Biacore. For the SPR/Biacore assay, the antibodies were captured on the chip and 1.6 to 1000 nM of ALPL was flowed over the chip. Anti-ALPL antibodies #3, #8, #9, #15, #16, #18, #20, and #22 were capable of binding ALPL as measured by ELISA and the anti-ALPL antibodies #5, #8, #9, #15, #16, #19, #22, and #30 were capable of binding ALPL as measured by SPR. Also, as shown in Table 42, the dissociation constant (KD) for binding to human ALPL/binding affinity was quantified for anti-ALPL antibody #9 (Ab 9) and antibody # 22 (Ab 22).

Table 40. Exemplary Anti-ALPL antibodies

Table 42. Binding affinities for anti-ALPL antibody #9 and #22 to human ALPL

[0588] Several anti-ALPL antibodies provided in Table 40 were also tested for their ability to compete for binding with an AAV particle comprising the TTM-002 capsid variant SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 2). hCMEC/D3 cells overexpressing ALPL were incubated with murine and rabbit monoclonal and rabbit polyclonal anti-ALPL antibodies for 1 hour, and then transduced with AAV particles comprising the TTM-002 capsid variant and comprising a viral genome encoding a GFP-luciferase transgene. Luciferase activity was measured (RLU) to determine if the AAV particle was capable of transducing the cells. No luciferase signal was indicative of an antibody that competes with the TTM-002 capsid, suggestive of an identical binding site or pocket on ALPL. As shown in FIG. 12A, anti-ALPL antibody #9 (Ab 9), #22 (Ab 22), and #29 had little to no detectable luciferase activity, indicating competition with the TTM-002 capsid variant. In fact, pre-incubation with Ab 9 led to no luciferase activity.

[0589] The anti-ALPL antibodies Ab 9 and Ab 22 were then added to wild-type hCMEC/D3 that do not overexpress ALPL and hCMEC/D3 cells that were engineered to express ALPL and allowed to internalize for five hours. The cells were then washed to remove any unbound antibodies, fixed, and incubated overnight with an anti-mouse FITC antibody to measure antibody internalization by fluorescence microscopy. Ab 9 and Ab 22 were internalized only in the hCMEC/D3 cells that were engineered to express ALPL. No internalization was observed in the wild-type hCMEC/D3 that did not overexpress ALPL.

[0590] Anti-ALPL antibodies Ab 9 and Ab 22 were then tested to determine if they were capable of binding ALPL and being subsequently transported across the cell membrane (transcytosis). The MDCK ALPL-expressing cells from the single clone generated in Example 8 were plated at a density of 200,000 cells in 250 pl in complete growth media at the apical side of a Transwell® insert (12-well, 0.4 pm pore size Transwell®-65 mm). The cells were incubated for 2-3 days to allow for polarization and electrical resistance was measured to calculate the integrity of tight junctions. The antibodies were then added to the top of the chamber at a concentration of 12 pg in 250 pl of media. The two anti-ALPL antibodies, Ab 9 and Ab 22, were tested along with a PT3 control antibody that does not bind ALPL and an anti-mouse IgGl isotype control (MOPC). The cells and the antibodies were incubated together overnight. Media was then collected from both chambers, and the concentrations of the antibodies in the top and bottom chambers were measured using a mouse IgG alphaLISA, in order to determine the ability of the antibodies to move from the top to the bottom chamber (transcytosis). FIG. 11A shows that the control antibodies as well as the ALPL binding antibodies were loaded at similar levels to the top of the cells. As shown in FIG. 11B, the MDCK cells overexpressing ALPL demonstrated highly effective transcytosis of Ab 9 that binds ALPL as evidenced by the high levels of the antibody quantified in the bottom chamber. Very little of the PT3 antibody, which does not bind ALPL, Ab 22 which is capable of binding ALPL, or the MOPC isotype control antibody, were detected in the bottom chamber. The percentage of the antibody detected in the bottom chamber to the load was also quantified (FIG. 11C) and the percentage of the Ab 9 that binds to ALPL in the bottom chamber relative to the load was greatly increased relative to the PT3 and MOPC controls and Ab 22 (FIG. 11C). Also as shown in FIG. 11C, MDCK cells that were not engineered to express ALPL, demonstrated little to no transcytosis of Ab 9 that binds ALPL or the PT3 control antibody.

[0591] Taken together, these data demonstrate that certain anti-ALPL antibodies are capable of binding ALPL and being transported across the cell membrane in vitro.