METHODS AND COMPOSITIONS FOR DELIVERY OF AGENTS ACROSS THE BLOOD-BRAIN BARRIER

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Serial No. 62/696,422, filed on July 11, 2018. The entire contents of the foregoing are incorporated herein by reference. SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 11, 2019, is named 296l8-0200WOl_SL.txt and is 58,834 bytes in size. TECHNICAL FIELD

Described herein are sequences that enhance permeation of agents across the blood brain barrier, compositions comprising the sequences, and methods of use thereof.

BACKGROUND

Delivery of therapeutic agents, including gene therapy reagents, is an impediment to development of treatments for a number of conditions. The blood-brain barrier (BBB) is a key obstacle for drug delivery to the mammalian central nervous system (CNS), particularly for delivery to the human brain, to treat conditions including neurodegenerative diseases such as Parkinson’s disease; Alzheimer’s disease; Huntington's disease; Amyotrophic lateral sclerosis; and Multiple sclerosis.

SUMMARY

The present invention is based on the development of artificial targeting sequences that enhance permeation of agents into cells and across the blood brain barrier.

Thus provided herein is an AAV capsid protein, e.g., an engineered AAV capsid protein, comprising a targeting sequence that comprises at least four contiguous amino acids from the sequence TVSALFK (SEQ ID NO:8); TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84). In some embodiments, the AAV capsid protein comprises a targeting sequence that comprises at least five contiguous amino acids from the sequence TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84). In some embodiments, the AAV capsid protein comprises a targeting sequence that comprises at least six contiguous amino acids from the sequence TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

In some embodiments, the AAV is AAV9; other AAV as known in the art (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof and others as known in the art or described herein) can also be used.

In some embodiments, the AAV capsid protein comprises AAV9 VP1 (e.g., SEQ ID NO:85).

In some embodiments, the targeting sequence is inserted in the capsid protein at a position corresponding to between amino acids 588 and 589 of SEQ ID NO:85.

Also provided herein are nucleic acids encoding the AAV capsid proteins comprising a targeting sequence as described herein.

In addition, provided herein is an AAV comprising a capsid protein comprising a targeting sequence as described herein. In some embodiments, AAV further comprises a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes can include, e.g., cDNAs that restore protein function, guide RNAfor gene editing, RNA, or miRNA.

Also provided herein are targeting sequences comprising V[S/p][A/m/t/]L (SEQ ID NO:79), TV[S/p][A/m/t/]L (SEQ ID NO:80), TV[S/p][A/m/t/]LK (SEQ ID NO:8l), or TV[S/p][A/m/t/]LFK. (SEQ ID NO:82). In some embodiments, the targeting sequence comprises VPALR (SEQ ID NO: l); VSALK (SEQ ID NO:2); TVPALR (SEQ ID NO:3); TVSALK (SEQ ID NO:4); TVPMLK (SEQ ID NO: 12); TVPTLK (SEQ ID NO: 13); FT VSALK (SEQ ID NO:5); LTVSALK (SEQ ID NO:6); TVSALFK (SEQ ID NO: 8); TVPALFR (SEQ ID NO: 9); TVPMLFK (SEQ ID NO: lO) or TVPTLFK (SEQ ID NO: ll). Also provided are fusion proteins comprising the targeting sequences linked to a heterologous (e.g., non-AAV VP1) sequence, and AAV capsid proteins (e.g., AAV9 VP1) comprising the targeting sequence. In some embodiments, the targeting sequence is inserted in a position corresponding to amino acids 588 and 589 of SEQ ID NO:85.

Additionally provided herein are nucleic acids encoding the targeting sequences, fusion proteins or AAV capsid proteins described herein, as well as AAV comprising the capsid proteins comprising a targeting sequence. In some

embodiments, the AAV further comprises a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes can include, e.g., cDNAs that restore protein function, guide RNAfor gene editing, RNA, or miRNA.

Further, provided herein are methods of delivering a transgene to a cell, the method comprising contacting the cell with an AAV or fusion protein described herein. In some embodiments, the cell is in a living subject, e.g., a mammalian subject. In some embodiments, the cell is in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, or muscle, and a combination thereof. In some embodiments, the cell is a neuron (optionally a dorsal root ganglion neuron), astrocyte, cardiomyocyte, or myocyte.

In some embodiments, the subject has a neurodegenerative disease, epilepsy; stroke; spinocerebellar ataxia; Canavan's disease; Metachromatic leukodystrophy; Spinal muscular atrophy; Friedreich's ataxia; X-linked centronuclear myopathy; Lysosomal storage disease; Barth syndrome; Duchenne muscular dystrophy; Wilson's disease; or Crigler-Najjar syndrome type 1. In some embodiments, the

neurodegenerative disease is Parkinson’s disease; Alzheimer’s disease; Huntington's disease; Amyotrophic lateral sclerosis; and Multiple sclerosis.

In some embodiments, the subject has a brain cancer, and the method includes administering an AAV encoding an anti-cancer agent. In some embodiments, the anti- cancer agent is HSV.TK1, and the method further comprises administering ganciclovir.

In some embodiments, the cell is in the brain of the subject, and the AAV is administered by parenteral delivery (e.g., via intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery); intracerebral; or intrathecal delivery (e.g., via lumbar injection, cisternal magna injection, or intraparenchymal injection).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Figs. 1 A-1B depict an exemplary strategy of engineering AAV9 by inserting cell-penetrating peptides (CPPs) into its capsid. Fig. 1 A is a 3D model of an AAV9 virus. Individual CPP inserted into the capsid between amino acids 588 and 589 (VP1 numbering) will be displayed at the 3-fold axis where receptor binding presumably occurs. Fig. 1B illustrate the method of individual AAV production. Three plasmids including pRC (engineered or not), pHelper and p AAV are co-transfected into HEK 293T cells, with AAVs harvested and purified using iodixanol gradient.

Figs. 2A-2B depict representative images of mouse brain sections and their quantitative analysis after intravenous administration of low-dose candidate AAVs. Mice with mixed genetic background are used. Candidate AAVs differs in their inserted CPPs (see Table 3), but all express nuclear red fluorescent protein (RFP) as reporter. Candidate AAVs with low production yields are excluded for further screening. The dose of AAV is 1 x 10 ¹⁰ vg (viral genome) per animal. Each white dot in Fig. 2A represents a RFP -labeled cell. In Fig. 2B, * P< 0.05, vs. AAV9, ANOVA.

Figs. 2C-2D depict representative images of mouse brain sections and their quantitative analysis after intravenous administration of AAV. CPP.11 and AAV. CPP.12 in a repeat experiment. AAV. CPP.11 and AAV. CPP.12 contain CPPs BIP1 and BIP2 respectively (see Table 3). The doses of the AAVs are increased to 1 ^c 10 ¹¹ vg per animal. Candidate AAVs express nuclear red fluorescent protein (RFP) as reporter. Each white dot in Fig. 2C represents a RFP-labeled cell. In Fig. 2D, * P< 0.05, ** P< 0.01, vs. AAV9, ANOVA.

Fig. 3 A depicts the optimization of the BIP targeting sequence in order to further engineer AAV9 towards better brain transduction. BIP1 (VPALR, SEQ ID

NO: 1), which enables AAV9 to transduce brain more efficiently (as in AAV. CPP.11), is derived from the protein Ku70 in rats. Human, mouse and rat Ku70 proteins differ in their exact amino acid sequences. BIP2 (VSALK, SEQ ID NO:2) as in AAV.CPP.12 is a“synthetic” peptide related to BIP1. Further engineering focuses on the VSALK sequence in the hope of minimizing species specificity of final engineered AAV. To generate new targeting sequence, amino acids of interest are added to the VSALK sequence, and in other cases positions of individual amino acids are switched. All new BIP2-derived sequences are again inserted into the AAV9 capsid to generate new candidate AAVs for screening. Sequences appearing in order are SEQ ID NOs: 69,

70, 71, 1-6, 72, 7, and 8.

Figs. 3B-3C depict representative images of mouse brain sections and their quantitative analysis after intravenous administration of more candidate AAVs. All candidate AAVs express nuclear red fluorescent protein (RFP) as reporter. The dose of AAV is 1 x 10 ¹¹ vg per animal. Each white dot in Fig. 3B represents a RFP-labeled cell. AAV.CPP.16 and AAV.CPP.21 were identified as top hits with their robust and widespread brain transduction. In Fig. 3C, * P< 0.05, ** P< 0.01, *** P< 0.001, vs. AAV9, ANOVA.

Fig. 3D depicts quantitative analysis of transduction efficiency in the liver after intravenous administration of candidate AAVs. Percentage of transduced liver cells is presented. The dose of AAV is l x lO ¹¹ vg per animal. *** P< 0.001, vs.

AAV9, ANOVA.

Figs. 4A-4E depict screening of selected candidate AAVs in an in vitro spheroid model of human blood-brain barrier. Fig. 4A illustrates the spheroid comprising human microvascular endothelial cells, which forms a barrier at the surface, and human pericyte and astrocytes inside the spheriod. Candidate AAVs were assessed for their ability to penetrate from the surrounding medium into the inside of the spheroid and to transduce the cells inside. Fig. 4B-4D shows images of AAV9, AAV.CPP.16 and AAV.CPP.21 treated spheroids. Fig. 4E shows relative RFP intensity of different AAV treated spheroids. *** P< 0.001, vs. AAV9, ANOVA.

Figs. 5A-5B depict representative images of brain sections and their quantitative analysis after intravenous administration of AAV9, AAV.CPP.16 and AAV.CPP.21 in C57BL/6J inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as reporter. The dose of AAV is 1 ^c 10 ¹² vg per animal. Each white dot in Fig. 5A represents a RFP -labeled cell. In Fig. 5B, * P< 0.05, *** P< 0.001, ANOVA.

Figs. 6A-6B depict representative images of brain sections and their quantitative analysis after intravenous administration of AAV9, AAV.CPP.16 and AAV.CPP.21 in BALB/cJ inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as reporter. The dose of AAV is 1 ^c 10 ¹² vg per animal. Each white dot in Fig. 6A represents a RFP -labeled cell. In Fig. 6B, *** P< 0.001, ANOVA.

Figs. 7A-7B depict representative images of brain sections and their quantitative analysis after intravenous administration of high-dose AAV.CPP.16 and AAV.CPP.21 in C57BL/6J inbred mice. Both candidate AAVs express nuclear red fluorescent protein (RFP) as reporter. The dose of AAV is 4x 10 ¹² vg per animal. Each white dot in Fig. 7A represents a RFP -labeled cell. In Fig. 7B, * P< 0.05, Student test.

Fig. 8 A shows AAV.CPP.16 and AAV.CPP.21 transduce adult neurons (labeled by a NeuN antibody) across multiple brain regions in mice including the cortex, midbrain and hippocampus. Transduced neurons are co-labeled by NeuN antibody and RFP. AAVs of 4x 10 ¹² vg were administered intravenously in adult C57BL/6J mice (6 weeks old).

Fig. 8B depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs. AAV9 in targeting the spinal cord and motor neurons in mice. AAVs of 4x 10 ¹⁰ vg were administered intravenously into neonate mice (1 day after birth). Motor neurons in the ventral horn of the spinal cord were visualized using CHAT antibody staining. Co-localization of RFP and CHAT signals suggests specific transduction of the motor neurons.

Fig. 9A depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 in targeting the heart in adult mice. AAVs of 1 x 10 ¹¹ vg were administered intravenously in adult C57BL/6J mice (6 weeks old). Percentage of RFP-labeled cells relative to all DAPI-stained cells is presented. * P< 0.05, Student test.

Fig. 9B depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 in targeting the skeletal muscle in adult mice. AAVs of 1 ^c 10 ¹¹ vg were administered intravenously in adult C57BL/6J mice (6 weeks old). Percentage of RFP-labeled cells relative to all DAPI-stained cells is presented. * P< 0.05, Student test.

Fig. 9C depicts that AAV.CPP.16 shows enhanced ability vs. AAV9 in targeting the dorsal root ganglion (DRG) in adult mice. AAVs of 1 x 10 ¹¹ vg were administered intravenously in adult C57BL/6J mice (6 weeks old). Percentage of RFP-labeled cells relative to all DAPI-stained cells is presented. * P< 0.05, Student test.

Fig. 10A depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs. AAV9 to transduce brain cells in primary visual cortex after intravenous

administration in non-human primates. 2 x 10 ¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injected intravenously into 3 months old cynomolgus monkeys with low pre-existing neutralizing antibody. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. Squared areas in the left panels are enlarged as in the right panels. AAV.CPP.16 transduced significantly more cells vs. AAV9. AAV.CPP.21 also transduced more cell vs. AAV9 although its effect was less evident in comparison with AAV.CPP.16.

Fig. 10B depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs. AAV9 to transduce brain cells in parietal cortex after intravenous administration in non-human primates. 2 x 10 ¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injected intravenously into 3 months old cynomolgus monkeys with low pre-existing neutralizing antibody. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. Squared areas in the left panels are enlarged as in the right panels. AAV.CPP.16 transduced significantly more cells vs. AAV9.

AAV.CPP.21 also transduced more cell vs. AAV9 although its effect was less evident in comparison with AAV.CPP.16.

Fig. 10C depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs. AAV9 to transduce brain cells in thalamus after intravenous administration in non human primates. 2 ^c 10 ¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injected intravenously into 3 months old cynomolgus monkeys with low pre-existing neutralizing antibody. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. Squared areas in the left panels are enlarged as in the right panels. AAV.CPP.16 transduced significantly more cells vs. AAV9.

AAV.CPP.21 also transduced more cell vs. AAV9 although its effect was less evident in comparison with AAV.CPP.16.

Fig. 10D depicts that AAV.CPP.16 and AAV.CPP.21 show enhanced ability vs. AAV9 to transduce brain cells in cerebellum after intravenous administration in non human primates. 2 ^c 10 ¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injected intravenously into 3 months old cynomolgus monkeys with low pre-existing neutralizing antibody. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. Squared areas in the left panels are enlarged as in the right panels. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly more cells vs. AAV9.

Figs. 11 A-l 1B depict that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A. LY6A serves as a receptor for AAYPHP.B and its variants including AAV.PHP.eB (as in US9102949, US20170166926) and mediates AAYPHP.eB’s robust effect in crossing the BBB in certain mouse strains (Hordeaux et al. Mol Ther 2019 27(5):9l2- 921; Huang et al. 2019, dx.doi.org/l0.H0l/53842l). Over-expressing mouse LY6A in cultured 293 cells significantly increases binding of AAV.PHP.eB to the cell surface (Fig. 11 A). On the contrary, over-expressing LY6A does not increase viral binding for AAV9, AAV.CPP 16 or AAV.CPP.21 (Fig. 11B). This suggests AAV.CPP.16 or

AAV.CPP.21 does not share LY6A with AAV.PHP.eB as a receptor.

Figs. 12A-12C depict that AAV.CPP.21 can be used to systemically deliver a therapeutic gene into brain tumor in a mouse mode of glioblastoma (GBM). As in Fig. 11 A, intravenously administered AAV. CPP.2l-H2BmCherry was shown to target tumor mass, especially the tumor expanding frontier. In Fig. 11B-11C, using

AAV.CPP.21 to systemically deliver the“suicide gene” HSV.TK1 results in shrinkage of brain tumor mass, when combined with the pro-drug ganciclovir. HSV.TK1 turns the otherwise“dormant” ganciclovir into a tumor-killing drug. * P< 0.05, Student test.

Fig. 13 depicts that when injected locally into adult mouse brain, AAV.CPP.21 resulted in more widespread and robust transduction of brain tissue in comparison with AAV9. Intracerebral injection of AAVs (1 x 10 ¹¹ vg) was performed in adult mice (>6 weeks old) and brain tissues were harvested and examined 3 weeks after AAV injection. ** P< 0.01, Student test.

DETAILED DESCRIPTION

Difficulties associated with delivery across the BBB have hindered

development of therapeutic agents to treat brain disorders including cancer and neurodegenerative disorders. Adeno-associated virus (AAV) has emerged as an important research and clinical tool for delivering therapeutic genes to the brain, spinal cord and the eye; see, e.g., US9102949; US 9585971; and US20170166926. However, existing AAVs including AAV9 have either limited efficiency in crossing the BBB, or only work in some non-primate species.

Through rational design and targeted screening on the basis of known cell- penetrating peptides (CPPs) (see, e.g., Gomez et ah, Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem. Soc. Trans. 2007;35(Pt

4):797-80l), targeting sequences have been discovered that, when engineered into the capsid of an AAV, improved the efficiency of gene delivery to the brain by up to three orders of magnitude. These methods were used to engineer one such AAV vector that dramatically reduced tumor size in an animal model of glioblastoma.

Targeting Sequences

The present methods identified a number of potential targeting peptides that enhance permeation through the BBB, e.g., when inserted into the capsid of an AAV, e.g., AAV1, AAV2, AAV8, or AAV9, or when conjugated to a biological agent, e.g., an antibody or other large biomolecule, either chemically or via expression as a fusion protein.

In some embodiments, the targeting peptides comprise sequences of at least 5 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, contiguous amino acids of the sequences VPALR (SEQ ID NO: l) and VSALK (SEQ ID NO:2).

In some embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3

X ₄ X5, wherein:

(i) Xi, X ₂ , X ₃ , X ₄ are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and

(ii) X ₅ is K, R, H, D, or E (SEQ ID NO:73).

In some embodiments, the targeting peptides comprise sequences of at least 6 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5 or 6 contiguous amino acids of the sequences TVPALR (SEQ ID NO:3),

T VSALK (SEQ ID NO:4), TVPMLK (SEQ ID NO: 12) and TVPTLK (SEQ ID NO: l3).

In some embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3

X4 X5 ¾, wherein:

(i) Xi is T; (ii) X ₂ , X ₃ , X ₄ , X ₅ are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and

(iii) X ₆ is K, R, H, D, or E (SEQ ID NO: 74).

In some embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3 X ₄ X5 Xe, wherein:

(i) Xi, X ₂ , X ₃ , X ₄ are any four non-identical amino acids from V, A, L, I, G, P, S, T, or M;

(ii) X5 is K, R, H, D, or E; and

(iii) X ₆ is E or D (SEQ ID NO:75).

In some embodiments, the targeting peptides comprise sequences of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of the sequences FTVSALK (SEQ ID NO:5), LTVSALK (SEQ ID NO: 6), TVSALFK (SEQ ID NO: 8), TVPALFR (SEQ ID NO: 9), TVPMLFK (SEQ ID NO: 10) and TVPTLFK (SEQ ID NO: 11). In some other embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3 X4 X5 Xe X ₇ , wherein:

(i) Xi is F, L, W, or Y;

(ii) X ₂ is T;

(iii) X3, X4, X5, Xe are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and

(iv) Xv is K, R, H, D, or E (SEQ ID NO:76).

In some embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3 X ₄ X5 Xe X7, wherein:

(i) Xi is T;

(ii) X ₂ , X3, X4, X5 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M;

(iii) X ₆ is K, R, H, D, or E; and

(iv) X ₇ is E or D (SEQ ID NO:77).

In some embodiments, the targeting peptides comprise a sequence of Xi X ₂ X3 X ₄ X5 Xe X7, wherein:

(i) Xi, X ₂ , X3, X ₄ are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M;

(ii) X ₅ is K, R, H, D, or E; (iii) X ₆ is E or D; and

(iv) Xv is A or I (SEQ ID NO:78).

In some embodiments, the targeting peptides comprise a sequence of

V[S/p][A/m/t/]L (SEQ ID NO:79), wherein the upper case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]L (SEQ ID NO:80). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LK (SEQ ID NO:8l). In some embodiments, the targeting peptides comprise a sequence of TV[S/p][A/m/t/]LFK. (SEQ ID NO:82).

In some embodiments, the targeting peptide does not consist of VPALR (SEQ ID NO: l) or VSALK (SEQ ID NO:2).

Specific exemplary amino acid sequences that include the above mentioned 5, 6, or 7-amino acid sequences are listed in Table 1.

TABLE 1 - Targeting Sequences

Targeting peptides including reversed sequences can also be used, e.g., KLASVT (SEQ ID NO: 83) and KFLASVT (SEQ ID NO: 84). Targeting peptides disclosed herein can be modified according to the methods known in the art for producing peptidomimetics. See, e.g., Qvit et al., Drug Discov Today. 2017 Feb; 22(2): 454-462; Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12: 1239-1254; Avan et al., Chem. Soc. Rev., 2014,43, 3575-3594; Pathak, et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmierski, W.M., ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., eds., Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 (2003). In some cases, these modified peptidomimetic versions of the peptides and fragments disclosed herein exhibit enhanced stability in vivo , relative to the non-peptidomimetic peptides.

Methods for creating a peptidomimetic include substituting one or more, e.g., all, of the amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as“retro” sequences. In another method, the N- terminal to C-terminal order of the amino acid residues is reversed, such that the order of amino acid residues from the N-terminus to the C-terminus of the original peptide becomes the order of amino acid residues from the C-terminus to the N-terminus in the modified peptidomimetic. Such sequences can be referred to as“inverso” sequences.

Peptidomimetics can be both the retro and inverso versions, i.e., the“retro- inverso” version of a peptide disclosed herein. The new peptidomimetics can be composed of D-amino acids arranged so that the order of amino acid residues from the N-terminus to the C-terminus in the peptidomimetic corresponds to the order of amino acid residues from the C-terminus to the N-terminus in the original peptide.

Other methods for making a peptidomimetic include replacing one or more amino acid residues in a peptide with a chemically distinct but recognized functional analog of the amino acid, i.e., an artificial amino acid analog. Artificial amino acid analogs include b-amino acids, b-substituted b-amino acids (‘^ ³ -amino acids”), phosphorous analogs of amino acids, such as V-amino phosphonic acids and V-amino phosphinic acids, and amino acids having non-peptide linkages. Artificial amino acids can be used to create peptidomimetics, such as peptoid oligomers (e.g., peptoid amide or ester analogues), b-peptides, cyclic peptides, oligourea or oligocarbamate peptides; or heterocyclic ring molecules. Exemplary retro-inverso targeting peptidomimetics include KLASVT and KFLASVT, wherein the sequences include all D-amino acids. These sequences can be modified, e.g., by biotinylation of the amino terminus and amidation of the carboxy terminus. A A Vs

Viral vectors for use in the present methods and compositions include recombinant retroviruses, adenovirus, adeno-associated virus, alphavirus, and lentivirus, comprising the targeting peptides described herein and optionally a transgene for expression in a target tissue.

A preferred viral vector system useful for delivery of nucleic acids in the present methods is the adeno-associated virus (AAV). AAV is a tiny non-enveloped virus having a 25 nm capsid. No disease is known or has been shown to be associated with the wild type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.7 kb. An AAV vector such as that described in Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et ah, Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et ah, Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et ah, J. Virol. 51 :611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). There are numerous alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desirable characteristics. In some embodiments, the

AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.lO, rh.39, rh.43 or CSp3; for CNS use, in some embodiments the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As one example, AAV9 has been shown to somewhat efficiently cross the blood-brain barrier. Using the present methods, the AAV capsid can be genetically engineered to increase permeation across the BBB, or into a specific tissue, by insertion of a targeting sequence as described herein into the capsid protein, e.g., into the AAV9 capsid protein VP1 between amino acids 588 and 589. An exemplary wild type AAV9 capsid protein VP1 (Q6JC40-1) sequence is as follows:

10 20 30 40 50

MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

60 70 80 90 100

KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

110 120 130 140 150

QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

160 170 180 190 200

QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

210 220 230 240 250

LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

260 270 280 290 300

TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

310 320 330 340 350

LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

360 370 380 390 400

QLPYVLGSAH EGCLPPFPAD VEMIPQYGYL TLNDGSQAVG RSSFYCLEYF

410 420 430 440 450

PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT

460 470 480 490 500

INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

510 520 530 540 550

FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

560 570 580 590 600

DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG

610 620 630 640 650

ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK

660 670 680 690 700

NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ

710 720 730

YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL (SEQ ID

NO: 85)

Thus provided herein are AAV that include one or more of the targeting peptide sequences described herein, e.g., an AAV comprising a capsid protein comprising a targeting sequence described herein, e.g., a capsid protein comprising SEQ ID NO: 1 wherein a targeting peptide sequence has been inserted into the sequence, e.g., between amino acids 588 and 589.

In some embodiments, the AAV also includes a transgene sequence (i.e., a heterologous sequence), e.g., a transgene encoding a therapeutic agent, e.g., as described herein or as known in the art, or a reporter protein, e.g., a fluorescent protein, an enzyme that catalyzes a reaction yielding a detectable product, or a cell surface antigen. The transgene is preferably linked to sequences that promote/drive expression of the transgene in the target tissue.

Exemplary transgenes for use as therapeutics include neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino acid decorboxylase (AADC), aspartoacylase (ASP A), blood factors, such as b-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), and the like; soluble receptors, such as soluble TNF-a receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-l receptors and soluble type II IL-l receptors), soluble gamma/delta T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a-glucosidase, imiglucarase, b- glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP- 10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIR-Ia, MPMb, MCP-l, PF-4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121,

VEGF165, VEGF-C, VEGF-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti-angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha-l antitryp sin; leukemia inhibitory factor (LIF); transforming growth factors (TGFs); tissue factors, luteinizing hormone; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitors of

metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-l receptor antagonists; and the like. Some other examples of protein of interest include ciliary neurotrophic factor (CNTF); neurotrophins 3 and 4/5 (NT-3 and 4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia related clotting proteins, such as Factor VIII, Factor IX, Factor X; dystrophin or nini-dystrophin; lysosomal acid lipase;

phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, b-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N- acetylhexosaminidase A); and any variants thereof.

The transgene can also encode an antibody, e.g., an immune checkpoint inhibitory antibody, e.g., to PD-L1, PD-l, CTLA-4 (Cytotoxic T-Lymphocyte- Associated Protein-4; CD 152); LAG-3 (Lymphocyte Activation Gene 3; CD223);

TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3; HAVCR2); TIGIT (T- cell Immunoreceptor with Ig and GPM domains); B7-H3 (CD276); VSIR (V-set immunoregulatory receptor, aka VISTA, B7H5, Cl0orf54); BTLA 30 (B- and T- Lymphocyte Attenuator, CD272); GARP (Glycoprotein A Repetitions; Predominant; PVRIG (PVR related immunoglobulin domain containing); or VTCN1 (Vset domain containing T cell activation inhibitor 1, aka B7-H4).

Other transgenes can include small or inhibitory nucleic acids that alter/reduce expression of a target gene, e.g., siRNA, shRNA, miRNA, antisense oligos, or long non-coding RNAs that alter gene expression (see, e.g., WO2012087983 and

US20140142160), or CRISPR Cas9/casl2a and guide RNAs .

The virus can also include one or more sequences that promote expression of a transgene, e.g., one or more promoter sequences; enhancer sequences, e.g., 5’ untranslated region (UTR) or a 3’ UTR; a polyadenylation site; and/or insulator sequences. In some embodiments, the promoter is a brain tissue specific promoter, e.g.,a neuron-specific or glia-specific promoter. In certain embodiments, the promoter is a promoter of a gene selected to from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis coli (APC), ionized calcium- binding adapter molecule 1 (Iba-l), synapsin I (SYN), calcium/calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain. In some embodiments, the promoter is a pan-cell type promoter, e.g., cytomegalovirus (CMV), beta glucuronidase, (GUSB), ubiquitin C (UBC), or rous sarcoma virus (RSV) promoter. The woodchuck hepatitis virus

posttranscriptional response element (WPRE) can also be used.

In some embodiments, the AAV also has one or more additional mutations that increase delivery to the target tissue, e.g., the CNS, or that reduce off-tissue targeting, e.g., mutations that decrease liver delivery when CNS, heart, or muscle delivery is intended (e.g., as described in Pulicherla et al. (2011) Mol Ther 19: 1070- 1078); or the addition of other targeting peptides, e.g., as described in Chen et al.

(2008) Nat Med 15: 1215-1218 or Xu et ak, (2005) Virology 341 :203-214 or

US9102949; US 9585971; and US20170166926. See also Gray and Samulski (2011) “Vector design and considerations for CNS applications,” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso T, editor. (Washington, DC: Society for Neuroscience; ) 1-9, available at

sfn.org/~/media/SfN/Documents/Short%20Courses/20l l%20Short%20Course%20I/2 01 l_SCl_Gray.ashx.

Targeting Peptides as Tags/Fusions

The targeting peptides described herein can also be used to increase permeation of other (heterologous) molecules across the BBB, e.g., by conjugation to the molecule, or by expression as part of a fusion protein, e.g., with an antibody or other large biomolecule. These can include genome editing proteins or complexes (e.g., TALEs, ZFNs, Base editors, and CRISPR RNPs comprising a gene editing protein such as Cas9 or Casl2a, fused to a peptide described herein (e.g., at the N terminus, C terminus, or internally) and a guide RNA), in addition to therapeutic agents or reporters as described herein as well as those listed in Table 2. The fusions/complexes do not comprise any other sequences from Ku70, e.g., comprise heterologous non-Ku70 sequences, and are not present in nature. In some embodiments, targeting sequences used as part of a non-AAV fusion protein do not comprise or consist of VPALR (SEQ ID NO:l) or VSALK (SEQ ID NO:2).

Methods of Use

The methods and compositions described herein can be used to deliver any composition, e.g., a sequence of interest to a tissue, e.g., to the central nervous system (brain), heart, muscle, or dorsal root ganglion or spinal cord (peripheral nervous system). In some embodiments, the methods include delivery to specific brain regions, e.g., cortex, cerebellum, hippocampus, substantia nigra, amygdala. In some embodiments, the methods include delivery to neurons, astrocytes, glial cells, or cardiomyocytes.

In some embodiments, the methods and compositions, e.g., AAVs, are used to deliver a nucleic acid sequence to a subject who has a disease, e.g., a disease of the CNS; see, e.g., US9102949; US 9585971; and US20170166926. In some

embodiments, the subject has a condition listed in Table 2; in some embodiments, the vectors are used to deliver a therapeutic agent listed in Table 2 for treating the corresponding disease listed in Table 2. The therapeutic agent can be delivered as a nucleic acid, e.g. via a viral vector, wherein the nucleic acid encodes a therapeutic protein or other nucleic acid such as an antisense oligo, siRNA, shRNA, and so on; or as a fusion protein/complex with a targeting peptide as described herein.

TABLE 2 - Diseases

Examples of diseases _ Tissue Therapeutic agent

Parkinson’s disease CNS GDNF, A A DC

Tau antibody, APP

Alzheimer’s disease CNS

antibody

Huntington's disease CNS miRNA targeting HTT Amyotrophic lateral sclerosis CNS shRNA targeting SOD Multiple sclerosis CNS IFN-beta

Epilepsy CNS Neuropeptide Y

Stroke CNS IGF-l, osteopontin

HSV.TK1, PD-l/PD-

Brain cancer CNS

Ll antibody

Spinocerebellar ataxia CNS RNAi targeting ataxin Canavan disease CNS ASPA

Metachromatic leukodystrophy Nervous systems ARSA, PSAP

Spinal muscular atrophy Neuromuscular system SMN1

Friedreich's ataxia Nervous systems, heart Frataxin

X-linked myotubular MTM1

Neuromuscular system

myopathy

Lysosome (global GAA

Pompe disease

including CNS)

Barth syndrome Heart, muscle TAZ

Duchenne muscular dystrophy Muscle dystrophin

Wilson’s disease Brain, liver ATP7B

Crigler-Najjar syndrome type 1 Liver UGT1A1

In some embodiments, the compositions and methods are used to treat brain cancer. Brain cancers include gliomas (e.g., glioblastoma multiforme (GBM)), metastases (e.g., from lung, breast, melanoma, or colon cancer), meningiomas, pituitary adenomas, and acoustic neuromas. The compositions include a targeting peptide linked to an anticancer agent, e.g., a“suicide gene” that induces apoptosis in a target cell (e.g., HSV.TK1, Cytosine Deaminase (CD) from Herpes simplex virus or Escherichia coli , or Escherichia coli purine nucleoside phosphorylase

(PNPyfludarabine; see Krohne et ak, Hepatology. 2001 Sep;34(3):5l 1-8; Dey and

Evans,“Suicide Gene Therapy by Herpes Simplex Virus-l Thymidine Kinase (HSV- TK)” (2011) DOI: 10.5772/18544) an immune checkpoint inhibitory antibody as known in the art or described herein. For example, an AAV vector comprising a targeting peptide as described herein can be used to deliver the“suicide gene” HSV.TK1 to a brain tumor. HSV.TK1 turns the otherwise“dormant” ganciclovir into a tumor-killing drug. Thus the methods can include systemically, e.g., intravenously, administering an AAV (e.g., AAV9) comprising a targeting peptide as described herein and encoding HSV.TK1, and the pro-drug ganciclovir, to a subject who has been diagnosed with brain cancer.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the use of pharmaceutical compositions comprising the targeting peptides as an active ingredient.

Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion administration. Delivery can thus be systemic or localized.

Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21 st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline,

bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. For example, the

compositions comprising an AAV comprising a targeting peptide as described herein and a nucleic acid encoding HSV.TK1 can be provided in a kit with ganciclovir.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples below.

1. Generation of capsid variants

To generate the capsid variant plasmids, DNA fragments that encode the cell- penetrating peptides (Table 3) were synthesized (GenScript), and inserted into the backbone of the AAV9 Rep-cap plasmid (pRC9) between amino acid position 588 and 589 (VP1 amino acid numbering), using CloneEZ seamless cloning technology (GenScript). CPPs BIPl(VPALR, SEQ ID NO: l) and BIP2 (VSALK, SEQ ID NO:2), as well as their derivatives such as TVSALK (SEQ ID NO:4) in AAV.CPP.16 and TVSALFK (SEQ ID NO:8) in AAV.CPP.21, are derived from the Ku70 proteins, of which the sequences are provided as below:

Human Ku70 MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLI FLVDASKAMFESQSEDELTPF 60 Mouse Ku70 MSEWESYYKTEGEEEEEE—EES PDTGGEYKYSGRDSLI FLVDASRAMFESQGEDELTPF 58 Rat Ku70 MSEWESYYKTEGEEEEEE—EQSPDTNGEYKYSGRDSLI FLVDASRAMFESQGEDELTPF 58

Human Ku70 DMSIQCIQSVYISKIISSDRDLLAWFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELD 120 Mouse Ku70 DMSIQCIQSVYTSKI ISSDRDLLAWFYGTEKDKNSVNFKNIYVLQDLDNPGAKRVLELD 118 Rat Ku70 DMSIQCIQSVYTSKI I SSDRDLLAWFYGTEKDKNSVNFKSIYVLQDLDNPGAKRVLELD 118

Human Ku70 QFKGQQGQKRFQDMMGHGSDYSLSEVLWVCANLFSDVQFKMSHKRIMLFTNEDNPHGNDS 180 Mouse Ku70 QFKGQQGKKHFRDTVGHGSDYSLSEVLWVCANLFSDVQLKMSHKRIMLFTNEDDPHGRDS 178 Rat Ku70 RFKGQQGKKHFRDTIGHGSDYSLSEVLWVCANLFSDVQFKMSHKRIMLFTNEDDPHGNDS 178

Human Ku70 AKASRARTKAGDLRDTGIFLDLMHLKKPGGFDI SLFYRDII SIAEDEDLRVHFEESSKLE 240 Mouse Ku70 AKASRARTKASDLRDTGIFLDLMHLKKPGGFDVSVFYRDIITTAEDEDLGVHFEESSKLE 238 Rat Ku70 AKASRARTKASDLRDTGI FLDLMHLKKRGGFDVSLFYRDI I S IAEDEDLGVHFEES SKLE 238 Human Ku70 DLLRKVRAKETRKRALSRLKLKLNKDIVI SVGIYNLVQKALKPPPIKLYRETNEPVKTKT 300 Mouse Ku70 DLLRKVRAKETKKRVLSRLKFKLGEDWLMVGIYNLVQKANKPFPVRLYRETNEPVKTKT 298 Rat Ku70 DLLRKVRAKETKKRVLSRLKFKLGKDVALMVGVYNLVQKANKPFPVRLYRETNEPVKTKT 298

Human Ku70 RTFNTSTGGLLLPSDTKRSQIYGSRQI ILEKEETEELKRFDDPGLMLMGFKPLVLLKKHH 360 Mouse Ku 70 RTFNVNTGSLLLPSDTKRSLTYGTRQIVLEKEETEELKRFDEPGLILMGFKPTVMLKKQH 358 Rat Ku 70 RTFNVNTGSLLLPSDTKRSLTFGTRQIVLEKEETEELKRFDEPGLILMGFKPMVMLKNHH 358

Human Ku70 YLRPSLFVYPEESLVIGSSTLFSALLIKCLEKEVAALCRYTPRRNIPPYFVALVPQEEEL 420 Mouse Ku70 YLRPSLFVYPEESLVSGSSTLFSALLTKCVEKEVIAVCRYTPRKNVSPYFVALVPQEEEL 418 Rat Ku70 YLRPSLFLYPEESLVNGSSTLFSALLTKCVEKEVIAVCRYTARKNVSPYFVALVPQEEEL 418

Human Ku70 DDQKIQVTPPGFQLVFLPFADDKRKMPFTEKIMATPEQVGKMKAIVEKLRFTYRSDSFEN 480 Mouse Ku70 DDQNIQVTPGGFQLVFLPYADDKRKVPFTEKVTANQEQIDKMKAIVQKLRFTYRSDSFEN 478 Rat Ku70 DDQNIQVTPAGFQLVFLPYADDKRKVPFTEKVMANPEQIDKMKAIVQKLRFTYRSDSFEN 478

Human Ku70 PVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRLGSLVDEFKELVYPPDYNPEGKV 540 Mouse Ku70 PVLQQHFRNLEALALDMMESEQWDLTLPKVEAIKKRLGSLADEFKELVYPPGYNPEGKV 538 Rat Ku70 PVLQQHFRNLEALALDMMESEQWDLTLPKVEAIKKRLGSLADEFKELVYPPGYNPEGKI 538

Human Ku70 TKRKHDNEGSGSKRPKVEYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKSGLKKQELL 600 Mouse Ku70 AKRKQDDEGSTSKKPKVELSEEELKAHFRKGTLGKLTVPTLKDICKAHGLKSGPKKQELL 598 Rat Ku70 AKRKADNEGSASKKPKVELSEEELKDLFAKGTLGKLTVPALRDICKAYGLKSGPKKQELL 598

Human Ku70 EALTKHFQD- 609 (SEQ ID NO: 86)

Mouse Ku70 DALIRHLEKN 608 (SEQ ID NO: 87)

Rat Ku70 EALSRHLEKN 608 (SEQ ID NO: 88)

In addition, VP1 protein sequences for AAV9, AAV.CPP.16 and

AAV.CPP.21 are provided as below:

AAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60

AAV. CPP16 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60

AAV. CPP21 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLD 60

AAV9 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AAV. CPP16 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AAV. CPP21 KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ

AAV9 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180

AAV. CPP16 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180

AAV. CPP21 AKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTE 180 AAV9 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240

AAV. CPP16 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240 AAV. CPP21 SVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVI 240

AAV9 TTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300

AAV. CPP16 TTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300 AAV. CPP21 TTSTRTWALPTYNNHLYKQI SNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR 300

AAV9 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360

AAV. CPP16 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360 AAV. CPP21 LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAH 360

AAV9 EGCLPPFPADVFMI PQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420

AAV. CPP16 EGCLPPFPADVFMI PQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420 AAV. CPP21 EGCLPPFPADVFMI PQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV 420

AAV9 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480

AAV. CPP16 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480 AAV. CPP21 PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIP 480

AAV9 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540

AAV. CPP16 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540 AAV. CPP21 GPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGS 540

AAV9 LI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQ- AQAQT 593

AAV. CPP16 LIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTVSAL-KAQAQT 599 AAV. CPP21 LI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTVSALFKAQAQT 600

AAV9 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP 653

AAV. CPP16 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP 659 AAV. CPP21 GWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTP 660

AAV9 VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEF 713

AAV. CPP16 VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEF 719 AAV. CPP21 VPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEF 720

AAV9 AVNTEGVYSEPRPIGTRYLTRNL 736 (SEQ ID NO: 85)

AAV. CPP16 AVNTEGVYSEPRPIGTRYLTRNL 742 (SEQ ID NO: 89)

AAV. CPP21 AVNTEGVYSEPRPIGTRYLTRNL 743 (SEQ ID NO: 90) 2. Recombinant AAV production

Recombinant AAVs were packaged using standard three-plasmid co- transfection protocol (pRC plasmid, pHelper plasmid and pAAV plasmid). pRC9 (or its variant), pHelper and pAAV carrying a transgene (e.g. nucleus-directed RFP H2B- mCherry driven by an ubiquitous EFla promoter) were co-transfected into HEK 293 T cells using polyethylenimine (PEI, Polysciences). rAAVs vectors were collected from serum-free medium 72h and l20h post transfection and from cell at l20h post transfection. AAV particles in the medium were concentrated using a PEG- precipitation method with 8% PEG-8000 (wt/vol). Cell pellets containing viral particles were resuspended and lysed through sonication. Combined viral vectors from PEG-precipitation and cell lysates were treated with DNase and RNase at 37 °C for 30mins and then purified by iodixanol gradient (15%, 25%, 40% and 60%) with ultracentrifugation(VTi 50 rotor, 40,000 r.p.m, l8°C, lh). rAAVs were then concentrated using Millipore Amicon filter unit (UFC910008, 100K MWCO) and formulated in Dulbecco's phosphate buffered saline (PBS) containing 0.001%

Pluronic F68 (Gibco).

3. AAV titering

Virus titer was determined by measuring DNase-resistant genome copies using quantitative PCR. pAAV-CAG-GFP was digested with PVETII(NEB) to generate free ends for the plasmid ITRs, and was used for generating a standard curve. Virus samples were incubated with DNase I to eliminate contaminating DNA, followed by sodium hydroxide treatment to dissolve the viral capsid and to release the viral genome. Quantitative PCR was performed using an ITR Forward primer 5'- GGAACCCCTAGTGATGGAGTT (SEQ ID NO:9l) and an ITR Reverse primer 5'- CGGCCTCAGTGAGCGA (SEQ ID NO:92). Vector titers were normalized to the rAAV-2 reference standard materials (RSMs, ATCC, cat No:VR-l6l6, Manassas, VA). 4. Administration of AAV in mice

For intravenous administration, AAV diluted in sterile saline (0.2 ml) was administered through tail vein injection in adult mice (over 6 weeks of age). Animals then survived for three weeks before being euthanized for tissue harvesting. For intracerebral injection, AAV diluted in PBS (10 ul) was injected using a Hamilton syringe with coordinates from bregma: 1.0 mm right, 0.3 backward, 2.6 mm deep. All animal studies were performed in an A AAL AC -accredited facility with IACUC approval.

5. Mouse tissue processing

Anesthetized animals were transcardially perfused with cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Tissues were post- fixed in 4% PFA overnight, and then immersed in 30% sucrose solutions for two days prior to embedding and snap-freezing in OCT. Typically, 80 um thick brain sections were cut for imaging of native fluorescence, 40um thick brain sections for IHC.

6. In vitro human BBB spheroid model

Hot 1% agarose (w/v, 50 ul) was added in a 96-well plate to cool/solidify. Primary human astrocytes (Lonza Bioscience), human brain microvascular pericytes (HBVP, ScienCell Research Laboratories) and human cerebral microvascular endothelial cells (hCMEC/D3; Cedarlane) were then seeded onto the agarose gel in a 1 : 1 : 1 ratio (1500 cells of each type). Cells were cultured at 37 °C in a 5% C02 incubator for 48-72 hours to allow for spontaneous assembly of multicellular BBB spheroids. A multicellular barrier was reported to form at the periphery of the spheroid, mimicking the BBB. AAVs-H2B-mCherry were added to the culture medium, and 4 days later all spheroids were fixed using 4% PFA, transferred into a Nunc Lab-Tek II thin-glass 8-well chambered coverglass (Thermo Scientific), and imaged using a Zeiss LSM710 confocal microscope. The intensity of RFP signal inside the spheroids was examined and used as a“read-out”.

7. AAV administration in non-human primate (NHP)

All NHP studies were performed by a CRO in an AAALAC-accredited facility with IACUC approval. Cynomolgus monkeys were pre-screened for little or no pre- existing neutralizing antibody against AAV9 (titer of <1 :5). AAV diluted in

PBS/0.00l%F68 was injected intravenously (via cephalic vein or femoral vein) using a peristaltic pump. 3 weeks later, animals were subject to transcardial perfusion with PBS, followed by 4% PFA. Tissues were then collected and processed for paraffin embedding and sectioning.

8. Immunohistochemistry

Floating staining was performed for mouse tissue sections with primary antibodies diluted in PBS containing 10% donkey serum and 2% Triton X-100.

Primary antibodies used include: chicken anti-GFP (1 : 1000); rabbit anti-RFP

(1 : 1000); mouse anti-NeuN (1 :500); rat anti-GFAP(l :500); Goat anti-GFAP(l :500); mouse anti-CD31(1 :500). Secondary antibodies conjugated to fluorophores of Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 647 were applied against the primary antibody’s host species at a dilution of 1 :200.

For paraffin sections of NHP tissue, DAB staining was performed to visualize cells transduced by AAV-AADC. Rabbit anti-AADC antibody (1 :500, Millipore) was used as primary antibody.

9. AAV binding assay

HEK293T cells were cultured at 37 °C in a 5% C02 incubator. One day after seeding of HEK293T cells in a 24-well plate at a density of 250,000 cells per well, a cDNA plasmid of LY6A was transiently transfected into the cells using a transfection mixture of 200ul DMEM (31053028; Gibco), 1 ug DNA plasmid and 3ug of PEI. 48 hours post transfection, cells were placed on ice to chill down for 10 mins. The medium was then changed with 500ul ice-cold serum-free DMEM medium containing rAAVs-mCherry at MOI of 10000. After incubating on ice for one hour, cells with presumably AAVs bound to their surface were washed with cold PBS for three times and were then subject to genomic DNA isolation. Cell-binding viral particles were quantified by using qPCR with primers specific to mCherry and normalized to HEK293T genomes using human GCG as reference. 10. Mouse model of glioblastoma

All experiments were performed in compliance with protocols approved by the Animal Care and ETse Committees (IACUC) at the Brigham and Women’s Hospital and Harvard Medical School. Syngeneic immuno-competent C57BL/6 female mice weighing 20 +/- 1 g (Envigo) were used. GL26l-Luc (100,000 mouse glioblastoma cells) resuspended in 2pL phosphate buffered saline (PBS) was injected intracranially using 10m1 syringe with a 26-gauge needle (80075; Hamilton). A stereotactic frame was used to locate the implantation site (coordinates from bregma in mm: 2 right, 0.5 forward, at a depth of 3.5 into cortex). 7 days later, 200 ul AAV-HSV-TK1 (1E+12 viral genomes, IV) was administered once and ganciclovir (50 mg/kg) was administered daily for 10 days.

Example 1. Modification of AAV9 capsid

To identify peptide sequences that would enhance permeation of a

biomolecule or virus across the blood brain barrier an AAV peptide display technique was used. Individual cell-penetrating peptides, as listed in Table 3, were inserted into the AAV9 capsid between amino acids 588 and 589 (VP1 numbering) as illustrated in FIG. 1 A. The insertion was carried out by modifying the RC plasmid, one of the three plasmids co-transfected for AAV packaging; FIG. 1B shows an exemplary schematic of the experiments. Individual AAV variants were produced and screened separately. See Materials and Methods #1-3 for more details.

TABLE 3

#, SEQ ID NO:

Syn, synthetic

Example 2. First Round of in vivo screening

AAVs expressing nuclear RFP (H2B-RFP) were injected intravenously in adult mice with mixed C57BL/6 and BALB/c genetic background. 3 weeks later, brain tissues were harvested and sectioned to reveal RFP-labelled cells (white dots in FIGs. 2A and 2C, quantified in FIGs. 2B and 2D, respectively). CPPs BIP1 and BIP2 were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12, respectively. See Materials and Methods #4-5 for more details. Example 3. Optimization of modified AAV9 capsids

AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIP targeting sequences. BIP inserts were derived from the protein Ku70 (See FIG.

3A and Material/Methods #1 for full sequence). The BIP sequence VSALK, which is of“synthetic” origin, was chosen as a study focus to minimize potential species specificity of engineered AAV vectors. AAVs were produced and tested separately for brain transduction efficiency as compared with AAV9 (see FIGs. 3B-C).

Percentages of cell transduction in the mouse liver 3 weeks after IV injection of some AAV variants delivering the reporter gene RFP are shown in FIG. 3D. See Materials and Methods #1-5 for more details.

Example 4. In vitro model - BBB permeation screening

Some of the AAV variants were screened for the ability to cross the human BBB using an in vitro spheroid BBB model. The spheroid contains human

microvascular endothelial cells, which form a barrier at the surface, and human pericytes and astrocytes. AAVs carrying nuclear RFP as reporter were assessed for their ability to penetrate from the surrounding medium into the inside of the spheroid and to transduce the cells inside. FIG. 4A shows an experimental schematic. FIGs. 4B-D show results for wt AAV9, AAV.CPP.16, and AAV.CPP.21, respectively, those and other peptides are quantified in FIG. 4E. In this model, peptides 11, 15, 16, and 21 produced the greatest permeation into the spheroids. See Materials and Methods #6 for more details.

Example 5. In vivo BBB permeation screening

AAV.CPP.16 and AAV.CPP.21 were selected for further evaluation in an in vivo model, in experiments performed as described above for Example 2. All AAVs carried nuclear RFP as reporter. Both showed enhanced ability vs. AAV9 to transduce brain cells after intravenous administration in C57BL/6J adult mice (white dots in brain sections in FIG. 5A, quantified in FIG. 5B) and in BALB/c adult mice (white dots in brain sections in FIG. 6A, quantified in FIG. 6B).

High doses of AAV.CPP.16 and AAV.CPP.21 (4 x 10 ¹² vg per mouse, administered IV) resulted in widespread brain transduction in mice. Both AAVs carried nuclear RFP as reporter (white dots in brain sections in FIG. 7A, quantified in FIG. 7B). Example 6. In vivo distribution of modified AAVs

As shown in FIG. 8A, AAV.CPP.16 and AAV.CPP.21 preferentially targeted neurons (labeled by a NeuN antibody) across multiple brain regions in mice including the cortex, midbrain and hippocampus. Both AAVs carried nuclear RFP as a reporter.

AAV.CPP.16 and AAV.CPP.21 also showed enhanced ability vs. AAV9 in targeting the spinal cord and motor neurons in mice. All AAVs carry nuclear RFP as reporter and were administered intravenously into neonate mice (4 x 10 ¹⁰ vg). Motor neurons were visualized using CHAT antibody staining. Co-localization of RFP and CHAT signals in FIG. 8B suggested specific transduction of the motor neurons.

The relative abilities of AAV-CAG-H2B-RFP and AAV.CPP.16-CAG-H2B-

RFP to transduce various tissues in mice was also evaluated. 1 ^c 10 ¹¹ vg was injected intravenously. The number of cells transduced was normalized to the number of total cells labeled by DAPI nuclear staining. The results showed that AAV.CPP.16 was more efficient than AAV9 in targeting heart (FIG. 9A); skeletal muscle (FIG. 9B), and dorsal root ganglion (FIG. 9C) tissue in mice.

Example 7. BBB permeation in a non-human primate model

2 x 10 ¹³ vg/kg AAVs-CAG-AADC (as reporter gene) were injected intravenously into 3 -month-old cynomolgus monkeys. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. As shown in FIGs. 10A-D, AAV.CPP.16 and AAV.CPP.21 showed enhanced ability vs. AAV9 to transduce brain cells after intravenous administration in non-human primates.

AAV.CPP.16 transduced significantly more cells then wt AAV9 in the primary visual cortex (FIG. 10 A), parietal cortex (FIG. 10B), thalamus (FIG. 10C), and cerebellum (FIG. 10D). See Materials and Methods #7-8 for more details.

Example 8. AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A

LY6A serves as a receptor for AAV.PHP.eB and mediates AAV.PHP.eB’s robust effect in crossing the BBB in certain mouse strains. Over-expressing mouse LY6A in cultured 293 cells significantly increased binding of AAV.PHP.eB to the cell surface (see FIG. 11 A). On the contrary, over-expressing LY6A does not increase viral binding for AAV9, AAV.CPP.16 or AAV.CPP.21 (see FIG. 11B). This suggests AAV.CPP.16 or AAV.CPP.21 does not share LY6A with AAV.PHP.eB as a receptor. See Materials and Methods #9 for more details.

Example 9. Delivering therapeutic proteins to the brain using

AAV.CPP.21

AAV.CPP.21 was used to systemically deliver the“suicide gene” HSV.TK1 in a mouse model of brain tumor. HSV.TK1 turns the otherwise“dormant” ganciclovir into a tumor-killing drug. Intravenously administered AAV.CPP.2l-H2BmCherry (FIG. 12 A, bottom left and middle right panel) was shown to target tumor mass, especially the tumor expanding frontier. As shown in FIGs. 12B-C, using

AAV.CPP.21 to systemically deliver the“suicide gene” HSV.TK1 resulted in shrinkage of brain tumor mass, when combined with the pro-drug ganciclovir. These results show that AAV.CPP.21 can be used to systemically deliver a therapeutic gene into brain tumor. See Materials and Methods #10 for more details.

Example 10. Intracerebral administration of AAV.CPP.21

In addition to systemic administration (such as in Example 2), an AAV as described herein was administered locally into the mouse brain. Intracerebral injection of AAV9-H2B-RFP and AAV.CPP.21-H2B-RFP (FIG. 13) resulted in more widespread and higher-intensity RFP signal in AAV.CPP.21 -treated brain sections vs. AAV9-treated ones. See Materials and Methods #4 for more details.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.