REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing submitted March 13, 2023 as an XML file named “38013_0024PlSequenceListing,” created on March 13, 2023, and having a size of 117,630 bytes is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Muscle-directed gene transfer has proven advantageous in the treatment of muscle disorders, such as dystrophinopathies. The use of regulatory elements to drive gene expression within specific tissues is advantageous, yet highly complex. The Spc5-12 promoter has been shown to be effective in promoting muscle-specific expression from adeno-associated viral (AAV) vectors. However, the need for higher yield of full genome AAV vectors exists as many AAV vectors using a Spc5-12 promoter for transgene expression can result in a high yield of AAV vectors with partial genomes.

[0003] Thus, there remains a need for tissue-targeted gene expression elements for use in vectors that are highly productive and manufacturable, especially muscle-specific elements.

BRIEF SUMMARY

[0004] The inventors have unexpectedly discovered that modifying certain musclespecific promoter sequences can improve packaging of AAV vectors having these muscle specific promoters within AAV virions, and, thus, result in enhanced production and recovery of fully packaged viral (capsids) particles, as well as provide AAV vectors exhibiting efficient expression of genes encoding therapeutic proteins from the AAV genomes in muscle tissue in the treatment of muscle disorders. Although not intending to be bound by any particular theory, promoter sequences forming hairpin loop secondary structure may inhibit genome packaging into AAV capsids. Thus, provided are modified muscle-specific promoter sequences that have reduced hairpin loop secondary structure, compared to parental promoter sequences. AAV genomes incorporating these modified promoter sequences into an expression cassette exhibit improved packaging efficiency as compared to AAV genomes with the parental promoter sequence in comparable expression cassettes, while still exhibiting muscle-specific expression of the therapeutic protein encoded by the expression cassettes. In embodiments, provided are modified Spc-5-12 promoter sequences, for example, promoter sequences having a nucleotide sequence of SEQ ID NO: 1 (vl) or SEQ ID NO: 2 (v2). Also provided are expression cassettes, recombinant AAV genomes, and recombinant AAV virus particles in which a nucleotide sequence encoding a gene of interest for expression in muscle tissue is operably linked to a modified Spc5-12 promoter, including a promoter having a nucleotide sequence of SEQ ID NO: 1 (vl) or SEQ ID NO: 2 (v2), and methods of manufacturing or using the same.

[0005] Embodiment 1. A nucleic acid comprising a nucleotide sequence of SEQ ID

NO: I.

[0006] Embodiment 2. A nucleic acid comprising a nucleotide sequence having muscle specific promoter activity, at least 80% sequence identity to SEQ ID NO: 1, and 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 1, which promotes expression of an operably linked nucleotide sequence in muscle cells.

[0007] Embodiment 3. A nucleic acid comprising the nucleotide sequence of SEQ ID NO:2.

[0008] Embodiment 4. A nucleic acid comprising a nucleotide sequence having musclespecific promoter activity, at least 80% sequence identity to SEQ ID NO:2, and 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO:2 which promotes expression of an operably linked nucleotide sequence in muscle cells.

[0009] Embodiment 5. A nucleic acid comprising a nucleotide sequence of SEQ ID NO:38.

[0010] Embodiment 6. A nucleic acid comprising a nucleotide sequence of SEQ ID NO:39.

[0011] Embodiment 7. A nucleic acid comprising a nucleotide sequence of SEQ ID NO:40.

[0012] Embodiment 8. A nucleic acid comprising a nucleotide sequence of SEQ ID NO:41.

[0013] Embodiment 9. A recombinant expression cassette comprising the nucleic acid of any one of embodiments 1-8.

[0014] Embodiment 10. A recombinant expression cassette comprising a transgene operably linked to a promoter comprising the nucleic acid sequence of SEQ ID NO: I or SEQ ID NO:2

[0015] Embodiment 11. The recombinant expression cassette of embodiment 10, wherein the transgene comprises a nucleotide sequence encoding one of the therapeutics listed in Tables 7A-7B.

[0016] Embodiment 12. The recombinant expression cassette of any one of embodiments 9-11, wherein the transgene encodes a therapeutic antibody or antigen binding fragment thereof. [0017] Embodiment 13. The recombinant expression cassette of any one of embodiments 9-12, wherein the transgene is a muscle-specific disease therapeutic.

[0018] Embodiment 14. The recombinant expression cassette of embodiment 13, wherein the muscle-specific disease therapeutic is microdystrophin.

[0019] Embodiment 15. The recombinant expression cassette of embodiment 14, wherein the microdystrophin is Dyslhaving an amino acid sequence of SEQ ID NO:35, Dys3 having an amino acid sequence of SEQ ID NO: 36, Dys5 having an amino acid sequence of SEQ ID NO: 37, MD1, having an amino acid sequence of SEQ ID NO: 56, human microdystrophin, having an amino acid sequence of SEQ ID NO: 57, Dys3978, having an amino acid sequence of SEQ ID NO: 58, MD3 having an amino acid sequence of SEQ ID NO: 59, MD4 having an amino acid sequence of SEQ ID NO: 60, or MD5, having an amino acid of SEQ ID NO: 72.

[0020] Embodiment 16. The recombinant expression cassette of embodiment 15, wherein the microdystrophin is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

[0021] Embodiment 17. The recombinant expression cassette of embodiment 16, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

[0022] Embodiment 18. A vector comprising the recombinant expression cassette of any one of embodiments 9-17.

[0023] Embodiment 19. The vector of embodiment 18, wherein the vector is a viral vector.

[0024] Embodiment 20. The vector of embodiment 19, further comprising AAV ITRs flanking the expression cassette.

[0025] Embodiment 21 The vector of any one of embodiments 17 to 20, wherein the cassette is suitable for packaging in an AAV capsid.

[0026] Embodiment 22. The vector of any one of embodiments 17-21, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 39 or SEQ ID NO: 41. [0027] Embodiment 23. A vector comprising an artificial genome comprising (1) AAV inverted terminal repeats (ITRs) flanking an expression cassette; (2) the expression cassette comprising (a) a nucleic acid regulatory element comprising a nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO:1, and 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO:2, and 100% sequence identity' over nucleotides 113-131 and 191-212 of SEQ ID NO:2, b) a poly A signal, and c) a transgene coding for one or more RNA or protein products to which the nucleic acid regulatory element is operably linked.

[0028] Embodiment 24. The vector of embodiment 23, wherein the nucleic acid regulatory element comprises a nucleotide sequence of SEQ ID NO: 1.

[0029] Embodiment 25. The vector of embodiment 23, wherein the nucleic acid regulatory element comprises a nucleotide sequence of SEQ ID NO: 2.

[0030] Embodiment 26. The vector of embodiment 23, further comprising an intron operably linked to the nucleotide sequence encoding the transgene.

[0031] Embodiment 27. The vector of embodiment 26, wherein the transgene is a musclespecific disease therapeutic.

[0032] Embodiment 28. The vector of embodiment 27, wherein the muscle-specific disease therapeutic is microdystrophin.

[0033] Embodiment 29. The vector of embodiment 28, wherein the micrody strophin is Dy si having an amino acid sequence of SEQ ID NO: 35, Dys3 having an amino acid sequence of SEQ ID NO: 36, Dys5 having an amino acid sequence of SEQ ID NO: 37, MD1, having an amino acid sequence of SEQ ID NO: 56, human microdystrophin, having an amino acid sequence of SEQ ID NO: 57), Dys3978, having an amino acid sequence of SEQ ID NO: 58, MD3 having an amino acid sequence of SEQ ID NO: 59, MD4 having an amino acid sequence of SEQ ID NO: 60, or MD5, having an ammo acid sequence of SEQ ID NO: 72. [0034] Embodiment 30. The vector of claim 29, wherein the microdystrophin is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

[0035] Embodiment 31. The vector of embodiment 30, wherein the micrody strophin is Dysl having an amino acid sequence of SEQ ID NO:35.

[0036] Embodiment 32. A rAAV particle comprising the vector of any one of embodiments 17-29 and a capsid protein from an AAV capsid serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7,

AAV.HSC8, AAV.HSC9, AAV.HSCIO, AAV.HSC11, AAV.HSC12, AAV.HSC13,

AAV HSC14, AAV HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.

[0037] Embodiment 33. The rAAV particle of embodiment 32 wherein the AAV capsid serotype is AAV8 or AAV9.

[0038] Embodiment 34. The rAAV particle of embodiment 33, wherein the AAV capsid serotype is AAV8.

[0039] Embodiment 35. A rAAV particle comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 38.

[0040] Embodiment 36. A rAAV particle comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 39.

[0041] Embodiment 37. A rAAV particle comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 40.

[0042] Embodiment 38. A rAAV particle comprising a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 41.

[0043] Embodiment 39. The rAAV particle of any one of embodiments 35-38 further comprising an AAV 8 or AAV9 capsid protein.

[0044] Embodiment 40. A cell comprising the nucleic acid of any one of embodiments 1- 8, the expression cassette of any one of embodiments 9-17, or the vector of any one of embodiments 17-31.

[0045] Embodiment 41. A method for enhancing expression of a transgene in muscle, comprising delivery of viral vectors comprising a recombinant expression cassette comprising a promoter comprising the nucleotide sequence of SEQ ID NO: I or SEQ ID NO:2 operably linked to the transgene.

[0046] Embodiment 42. The method of embodiment 41, wherein the transgene comprises a gene or nucleic acid encoding any of the therapeutics listed in Tables 7A-7D.

[0047] Embodiment 43. The method of embodiment 41, wherein the transgene is a muscle-specific disease therapeutic.

[0048] Embodiment 44. The method of embodiment 43, wherein the muscle-specific disease therapeutic is a microdystrophin. [0049] Embodiment 45. The method of embodiment 44, wherein the microdystrophin is Dysl (SEQ ID NO:35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MD1 (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59), MD4 (SEQ ID NO: 60), or MD5 (SEQ ID NO: 72).

[0050] Embodiment 46. The method of embodiment 45, wherein the microdystrophin is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

[0051] Embodiment 47. The method of any one of embodiments 41-46, wherein the viral vector is administered intravenously or intramuscularly.

[0052] Embodiment 48. The method of any one of embodiments 41-47, wherein transgene expression is enhanced in the circulation or systemically.

[0053] Embodiment 49. The method of any one of embodiments 41-48, wherein the transgene expression is enhanced in the skeletal muscle or cardiac muscle.

[0054] Embodiment 50. A method for treating a disease or disorder listed in Tables 7A- 7D or a pharmaceutical composition for use in treating a disease or disorder listed in Tables 7A-7D in a subject in need thereof comprising delivering rAAVs comprising the recombinant expression cassettes of any one of embodiments 9-17, any one of the vectors of embodiments 18-31 or any one of the rAAVs of embodiments 32-39.

[0055] Embodiment 51. A method for treating a disease or disorder listed in Tables 7A- 7D or a pharmaceutical composition for use in treating a disease or disorder listed in Tables 7A-7D in a subject in need thereof comprising the administration of recombinant AAV particles comprising an expression cassette comprising a promoter comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 operably linked to a transgene encoding a therapeutic for treatment of said disease or disorder.

[0056] Embodiment 52 The method or pharmaceutical composition of any one of embodiments 50-51, wherein the transgene is selected from Tables 7A-7D.

[0057] Embodiment 53. The method or pharmaceutical composition of any one of embodiments 50-52, wherein the transgene encodes a therapeutic antibody, or antigen binding fragment thereof.

[0058] Embodiment 54. The method or pharmaceutical composition of any one of embodiments 50-53, wherein the transgene encodes a muscle-specific disease therapeutic. [0059] Embodiment 55. The method or pharmaceutical composition of embodiment 54, wherein the muscle-specific disease therapeutic is microdystrophin.

[0060] Embodiment 56. The method or pharmaceutical composition of embodiment 55, wherein the microdystrophin is Dysl (SEQ ID NO:35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MDI (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59), MD4 (SEQ ID NO: 60), or MD5 (SEQ ID NO: 72).

[0061] Embodiment 57. The method or pharmaceutical composition of claim 56, wherein the microdystrophin is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

[0062] Embodiment 58. A method for treating a disease or disorder listed in Tables 7A- 7D or a pharmaceutical composition for use in treating a disease or disorder listed in Tables 7A-7D in a subject in need thereof comprising administering rAAV particles comprising an expression cassette operably linked to a transgene, wherein the expression cassette comprises SEQ ID NO: 38.

[0063] Embodiment 59. A method for treating a disease or disorder listed in Tables 7A- 7D or a pharmaceutical composition for use in treating a disease or disorder listed in Tables 7A-7D in a subject in need thereof comprising administering rAAV particles comprising an expression cassette operably linked to a transgene, wherein the expression cassette comprises SEQ ID NO: 40.

[0064] Embodiment 60. The method or pharmaceutical composition of any one of embodiments 50-59, wherein the rAAV is administered intravenously or intramuscularly. [0065] Embodiment 61. The method or pharmaceutical composition of any one of embodiments 50-60, wherein the disease or disorder is a muscle specific disorder.

[0066] Embodiment 62. The method or pharmaceutical composition of embodiment 61, wherein the muscle-specific disease or disorder is a muscular dystrophy.

[0067] Embodiment 63. The method or pharmaceutical composition of embodiment 62, wherein the muscular dystrophy is DMD.

[0068] Embodiment 64. A method for treating DMD or a pharmaceutical composition for use in treating DMD in a subject in need thereof comprising administering rAAV particles comprising an expression cassette, wherein the expression cassette comprises a promoter comprising a nucleotide sequence of SEQ ID NO: 1 operably linked to a transgene encoding Dysl having an amino acid sequence of SEQ ID NO:35, and wherein the rAAV particles are AAV8.

[0069] Embodiment 65. A method for treating DMD or a pharmaceutical composition for use in treating DMD in a subject in need thereof comprising administering rAAV particles comprising an expression cassette, wherein the expression cassette comprises a promoter comprising a nucleotide sequence of SEQ ID NO: 2 operably linked to a transgene encoding Dysl having an amino acid sequence of SEQ ID NO:35, and wherein the rAAV particles are AAV8.

[0070] Embodiment 66. The method of any one of embodiments 50-65, wherein the transgene is expressed in skeletal muscle or cardiac muscle.

[0071] Embodiment 67. A host cell comprising a plasmid comprising a cis expression cassette, wherein the cis expression cassette comprises a promoter operably linked to a transgene, wherein the promoter comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0072] Embodiment 68. A host cell comprising a plasmid comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 38.

[0073] Embodiment 69. A host cell comprising a plasmid, wherein the plasmid comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 39.

[0074] Embodiment 70. A host cell comprising a plasmid comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 40.

[0075] Embodiment 71. A host cell comprising a plasmid, wherein the plasmid comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 41.

[0076] Embodiment 72. A method of producing recombinant AAVs comprising: (a) culturing a host cell containing: (i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a promoter operably linked to a transgene coding for one or more RNA or protein products, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2; (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans; (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture.

[0077] Embodiment 73. The method of embodiment 72, wherein at least 40% of the rAAVs produced encapsidate a full genome.

[0078] Embodiment 74. The method of embodiment 72, wherein the production results in recombinant AAVs having at least two-fold more full capsids than rAAVs comprising a recombinant expression cassette comprising a Spc5-12 promoter produced using the same method.

[0079] Embodiment 75. The method of embodiment 72, wherein the production results in rAAVs having at least two-fold more full capsids than partial capsids.

[0080] Embodiment 76. A method of increasing packaging efficiency of intact AAV genomes in recombinant AAVs comprising (a) cultunng a host cell containing: (i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a promoter operably linked to a transgene coding for one or more RNA or protein products, wherein the promoter comprises the nucleotide sequence of SEQ ID NOT or SEQ ID NO:2; (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans; (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture; wherein the presence of the promoter comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 allows for increased packaging efficiency of intact AAV genomes.

[0081] Embodiment 77. The method of embodiment 76, wherein the increased packaging efficiency of intact AAV genomes is compared to production of recombinant AAVs comprising a wild-type Spc5-12 promoter.

[0082] Embodiment 78. The method of embodiment 76, wherein an increased packaging efficiency is the production of at least 30%, at least 50%, at least 70% or at least 90% of the recombinant AAVs having the full AAV genome.

[0083] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0085] FIG. 1 shows predicted secondary structure of the represented (boxed) nucleotide sequences of Spc5-12 Mutantl (SEQ ID NO: 1) in which the positions of the MEF-2 and SRE elements are modified (labelled as STEM1-STEM1’ (SEQ ID NO:61) and STEM2-LOOP2- STEM2’ (SEQ ID NO:62)) as compared to the Spc5-12 promoter (SEQ ID NO: 3).

[0086] FIG. 2 shows predicted secondary structure of the represented (boxed) nucleotide sequences of Spc5-12 Mutant2 (SEQ ID NO:2) which modifies the orientation of the MEF-2 and SRE elements (labelled as STEMl ’-STEME (SEQ ID NO:63) and STEM2-LOOP2- STEM2, SEQ ID NO: 64)) compared to the Spc5-12 promoter.

[0087] FIG. 3 shows the predicted first and second hairpin structures for the Spc5-12 promoter (SEQ ID NOs: 65 and 66, respectively) and the predicted change in secondary' structure for the first and second hairpins of the Spc5-12vl promoter (“mutant 1 ”) (SEQ ID NOs: 61 and 62, respectively) and for the first and second hairpins of the Spc5-12v2 promoter (“mutant 2”) (SEQ ID NOs: 63 and 64, respectively).

[0088] FIGs. 4A and 4B show the impact of the different promoters (Spc5-12vl, Spc5- 12v2, synlOO, and CK7) on the production process titers and quality, including viable cell density (FIG. 4A) and cell viability (FIG. 4B) for production of AAV virions having a microdystrophin transgene as compared to AAV virions having an Spc5-12 promoter operably linked to the microdystrophin transgene

[0089] FIG. 5 shows the total capsids produced from constructs having the different promoters operably linked to the microdystrophin encoding sequence after an affinity column purification step.

[0090] FIGs. 6A-6C show the results of a DNA TapeStation analysis (Agilent Technologies, Santa Clara, CA) for analyzing annealed double strand DNA. For example, a representative graph (FIG. 6A) provides a plot of sequence reads from analysis of vector particles packaging a genome containing Spc5-12 promoter, and a representative graph (FIG. 6B) provides a plot of sequence reads from analysis of vectors packaging a genome containing Spc5-12v2 promoter. The relative amounts of full genome vectors and partial genome vectors are represented by TapeStation analysis (FIG. 6C) for constructs having Spc5-12 promoter compared to the modified Spc5-12vl and Spc5-12v2 were used to generate AAV particles.

[0091] FIGs. 7A and 7B show an example of the sedimentation profile of ultracentrifugation separation of capsids generated from cis plasmids having the microdystrophin coding sequence operably linked to the control (Spc5-12, A) and the Spc5- 12v2 promoter (“mutant 2”, B), labeling peaks representing empty, partial and full capsids. The mutant promoter Spc5-12v2 resulted in a higher percentage of full genome vector compared to the control.

[0092] FIGs. 8A-8C show long read next generation sequencing (NGS) (by Oxford Nanopore method) of AAV virions generated from constructs in which the transgene is operably linked to the Spc5-12 promoter (FIG. 8A), Mutant 1 (Spc5-12vl) (FIG. 8B) and Mutant 2 (Spc5-12v2) (FIG. 8C) promoters.

[0093] FIG. 9 shows microdystrophin RNA copies in different tissues (heart, gastrocnemius, tibialis anterior, diaphragm and liver) of mice administered recombinant AAV virions wherein the microdystrophin coding sequence is operably linked to control (Spc5-12), Spc5-12vl or Spc5-12v2. Gas = gastrocnemius, TA = tibialis anterior.

[0094] FIG. 10 shows an alternative representation of the normalized data (in copies/pg RNA) in heart, gastrocnemius, tibialis anterior, diaphragm and liver tissues from Fig. 9. [0095] FIG. 11 shows AAV DNA biodistribution in tissues (gastrocnemius, tibialis anterior, diaphragm and liver) from mice administered recombinant AAV virions generated from constructs having a micrody strophin encoding transgene operably linked to indicated promoter sequences. Values normalized to diploid genomes. Gas = gastrocnemius, TA = tibialis anterior, DIA = diaphragm.

[0096] FIG. 12 shows AAV DNA biodistribution in mouse tissues (gastrocnemius, tibialis anterior, diaphragm and liver). Values normalized to micrograms DNA. Gas = gastrocnemius, TA = tibialis anterior, DIA = diaphragm.

[0097] FIG. 13 shows the ratio of microdystrophin RNA to AAV DNA copies in a diploid cell in tissues (gastrocnemius, tibialis anterior, diaphragm and liver)of mice administered AAV virions generated from constructs having microdystrophin encoding transgene operably linked to the indicated promoter sequences. Gas = gastrocnemius, TA = tibialis anterior, DIA = diaphragm.

DETAILED DESCRIPTION

[0098] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0099] The inventors have unexpectedly discovered that modifying regulatory elements to reduce the incidence of hairpin formation within muscle-specific promoter sequences can improve packaging of AAV vectors with full length AAV genomes and thus result in enhanced production and recovery of fully packaged viral (capsids) particles, which provides higher titers of the final product thus reducing overall cost of goods for the production of gene therapies for the treatment of muscle disorders.

[00100] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary . It is also to be understood that the terminology' used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

[00101] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary'. It is also to be understood that the terminology' used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[00102] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.

Thus, for example, reference to "a sequence" includes a plurality of such sequences, reference to "the promoter" is a reference to one or more promoters and equivalents thereof known to those skilled in the art, and so forth.

[00103] The term “regulatory element” or “nucleic acid regulatory element” are noncoding nucleic acid sequences that control the transcription of neighboring genes. Cis regulatory elements typically regulate gene transcription by binding to transcription factors. This includes “composite nucleic acid regulatory elements” comprising more than one enhancer or promoter elements as described herein. Also included are introns, polyadenylation sequences and other transcription enhancers.

[00104] The term “expression cassette” or "nucleic acid expression cassette" refers to nucleic acid molecules that include one or more transcriptional control elements including, but not limited to promoters, enhancers and/or regulatory elements, introns and poly adenylation sequences. The enhancers and promoters typically function to direct (trans)gene expression in one or more desired cell types, tissues or organs.

[00105] The term “operably linked” and “operably linked to” refers to nucleic acid sequences being linked and typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked and still be functional while not directly contiguous with a downstream promoter and transgene.

[00106] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene. An example of the latter includes a rAAV having a capsid protein comprising a peptide insertion into or modification of the amino acid sequence of the naturally-occurring capsid.

[00107] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.

[00108] The term “rep-cap helper plasmid” refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.

[00109] The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus. For AAV, the capsid protein may be VP1, VP2, or VP3.

[00110] The term “rep gene” refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.

[00111] The terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.

[00112] The terms “subject”, “host”, and “patient” are used interchangeably. As used herein, a subject is preferably a mammal such as anon-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.

[00113] The terms “therapeutic agent,” “biotherapeutic agent,” or “therapeutic,” refer to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. As used herein, a “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom. Further, a therapeutically effective amount with respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[00114] The phrase “muscle-specific” or “muscle-directed” refers to nucleic acid elements that have adapted their activity in muscle cells or tissue due to the interaction of such elements with the intracellular environment of the muscle cells. Muscle cells include skeletal muscle as well as cardiac muscle. Secretion of transgene product into the muscle, and/or bloodstream may also be enhanced following various routes of administration, such as intravenous or intramuscular administration, due to intramuscular expression where musclespecific promoters are present. Various therapeutics benefit from muscle-specific expression of the transgene, or from both muscle-specific and liver-specific expression of the transgene (such as in combining the new synthetic muscle-specific promoter with a liver-specific promoter in a tandem promoter). Muscle production of a biotherapeutic agent (such as produced by the delivered transgene) may provide also provide the host with increased immunotolerance to the agent, as compared to direct inj ection of an equivalent protein agent to the host. Muscle specific or muscle directed activity may be assessed, for example, by administering to an animal model, such as a mouse or NHP, a recombinant AAV in which the muscle specific promoter is operably linked to a transgene, after a period of time sufficient for the transgene in the recombinant AAV to be expressed, harvesting muscle tissue and assaying for levels of transgene mRNA and/or protein product, including relative to the number of vector genomes in the muscle tissue.

[00115] A “variant” or “variant thereof’ can mean a difference in some way from the reference sequence. For example, a variant can be different from the reference sequence due to a substitution, deletion or modification to a nucleic acid or amino acid. Where the variant includes a substitution of an amino acid residue or nucleic acid base, the substitution can be considered conservative or non-conservative. Conservative substitutions are those within the following groups: Ser, Thr, and Cys; Leu, He, and Vai: Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gin, Asn, Glu, Asp, and His. Variants can include at least one substitution and/or at least one addition, there may also be at least one deletion. Variants can also include one or more non-naturally occurring residues. For example, they may include sei enocysteine (e.g., seleno-L- cysteine) at any position, including in the place of cysteine. Many other “unnatural” amino acid substitutes are known in the art and are available from commercial sources. Examples of non-naturally occurring amino acids include D-amino acids, amino acid residues having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, and omega amino acids of the formula NH2(CH2)nCOOH wherein n is 2-6 neutral, nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N- methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties of proline.

B. Modified Muscle-Specific Promoters and other Regulatory Elements

[00116] As detailed in the Examples, single stranded versions of the Spc5-12 promoter may form hairpin structures which, although not intended to be bound by any theory, may inhibit the production of full length AAV genomes in the production and packaging of recombinant AAV virions from cis plasmids encoding a transgene operably linked to the Spc5-12 promoter, resulting in an increase in the production of AAV vectors with fragmented genomes and non-transgene-related DNA contaminants, referred to as “partially-filled capsids” to be removed by purification processes. MEF-2, TEF and SRE elements are shown in FIGs. 1 and 2. Spc5-12 (having a nucleotide sequence of SEQ ID NO: 3) comprises a specific order to the MEF-2, TEF, and SRE elements. In some aspects, the promoter of SEQ ID NO: 1 has the MEF-2 in steml and the SRE of stem2 switched in position (see Figure 1). In some aspects, the promoter of SEQ ID NO:2 has loopl (MEF1) and steml (MEF2) missing (see Figure 2). Modification of the promoter sequence, including to change the positions of the MEF-2 and SRE elements (Mutant 1) or to reverse the orientations of the MEF-2 and SRE elements (Mutant 2), results in a new active promoter which reduces the formation of hairpin structures while maintaining the elements of the Spc5-12 promoter, and increases the production of full capsids containing the complete genome encoded by the cis plasmid while maintaining the muscle-specific promoter function. It was unexpected that such a modification to the promoter would result in an improved product by reducing the burden and cost of purification methods during the rAAV production process.

[00117] Accordingly, provided are regulatory elements comprising a modified Spc5-12 promoter having a nucleotide sequence of SEQ ID NO: 1 (Spc5-12vl) or SEQ ID NO:2 (Spc5-12v2) or a composite nucleic acid sequence comprising a modified Spc5-12 promoter of SEQ ID NO: 1 (Spc5-12vl) or SEQ ID NO:2 (Spc5-12v2), in combination with one or more other regulatory elements (e.g. enhancers). These modified promoters promote gene expression in muscle. The provided regulatory elements are included in cis plasmids in which the regulatory element is operably linked to the transgene for production of recombinant AAV virions.

[00118] Provided are nucleic acid regulatory elements for enhancing gene expression in the muscle comprising nucleic acid sequences SEQ ID NO: 1 or SEQ ID NO:2. Also included are regulatory elements that enhance gene expression in the muscle which have 99%, 95%, 90%, 85% or 80% sequence identity with one of nucleic acid sequences SEQ ID NO: 1 or SEQ ID NO:2, including wherein there is 100% identity at nucleotides 121-129 and 197-209 to those positions of SEQ ID NOT or wherein there is 100% identity at nucleotides 113-131 and 191-212 to those positions of SEQ ID NO:2.

[00119] In some aspects, variants of SEQ ID NO: 1 are provided. In some aspects, the disclosed nucleic acids can comprise a nucleotide sequence having muscle specific promoter activity, at least 80% sequence identity to SEQ ID NOT, including with 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NOT, which generate a greater percentage of full capsids or yield of full capsids compared to a construct that is identical except for having the Spc5-12 promoter (SEQ ID NOT). In some aspects, the disclosed nucleic acids can comprise a nucleotide sequence having muscle specific promoter activity, at least 85, 90, 95, 99 or 100% sequence identity to SEQ ID NOT, including in some aspects with 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NOT, which generate a greater percentage of full capsids or yield of full capsids compared to a comparable construct having the Spc5-12 promoter (SEQ ID NO:3) (i.e., a construct identical except for the form of the Spc5-12 promoter). A variant of SEQ ID NO: 1 can be the sequence of SEQ ID NO:1 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleic acid substitutions. In some aspects, the variants have 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 1, have muscle specific promoter activity and generate a greater percentage of full capsids or yield of full capsids compared to a comparable construct identical except for having the Spc5-12 promoter (SEQ ID NO:3) (i.e., a construct identical except for the form of the Spc5-12 promoter). [00120] In some aspects, variants of SEQ ID NO:2 are provided. In some aspects, the disclosed nucleic acid can comprise a nucleotide sequence having muscle-specific promoter activity, at least 80% sequence identity to SEQ ID NO:2, including, in some aspects, 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO:2, and which generate a greater percentage of full capsids or yield of full capsids compared to a construct identical except for having the Spc5-12 promoter (SEQ ID NO: 3). In some aspects, the disclosed nucleic acid can comprise a nucleotide sequence having muscle-specific promoter activity, at least 85, 90, 95, 99 or 100% sequence identity to SEQ ID NO:2, including, in some aspects, 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO:2, and which generate a greater percentage of full capsids or yield of full capsids compared to a construct that is identical except for having the Spc5-12 promoter (SEQ ID NO:3) (i.e., a construct identical except for the form of the Spc5-12 promoter). A variant of SEQ ID NO:2 can be the sequence of SEQ ID NO:2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleic acid substitutions. In some aspects, the variants have 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO:2 and retain muscle specific promoter activity and generate a greater percentage of full capsids or yield of full capsids compared to a construct having the Spc5-12 promoter (SEQ ID NO: 3) (i.e., a construct identical except for the form of the Spc5-12 promoter).

[00121] Also provided are nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette where at least one of the elements is a modified Spc5-12 promoter, including either Spc5-12vl (SEQ ID NO: 1) or Spc5-12v2 (SEQ ID NO: 2). Regulatory elements, in general, have multiple functions such as acting as recognition sites for transcription initiation or regulation, coordinating with cellspecific machinery to drive expression upon signaling, and enhancing expression of downstream gene(s). [00122] In some aspects, disclosed are expression cassetes comprising regulatory elements designed to confer or enhance muscle-specific expression (including skeletal or cardiac muscle specific expression) comprising solely a promoter or the combination of a promoter (e.g. SEQ ID NO:1 or SEQ ID NO:2) and one or more other regulatory elements, such as enhancers.

[00123] In some aspects, the promoters of SEQ ID NO: 1 or SEQ ID NO:2 can be combined with, or operably linked to, one or more of the regulatory elements provided in PCT Application Publication No. WO 2021/021661, hereby incorporated by reference in its entirety herein. The combination of the promoters of SEQ ID NO: 1 or SEQ ID NO:2 with one or more of the disclosed regulatory elements can result in a muscle-liver specific composite regulatory element.

[00124] Provided are arrangements of combinations of nucleic acid regulatory elements that promote transgene expression in muscle (skeletal and/or cardiac) tissue that include the modified Spc5-12 promoters, Spc5-12vl (mutant 1) and Spc5-12v2 (mutant 2). Exemplary nucleotide sequences of the individual promoter and enhancer elements are provided in Table 1. In certain embodiments, the disclosed promoters have fewer repeat sequences prone to hairpin loop formations compared to the unmodified version of the same promoter. Muscle specific enhancers include the Mck Enhancer (MckE) and MhcE enhancers and tandem arrangements thereof arranged with a modified Spc5-12 promoter are described herein. Accordingly, provided are nucleic acid regulatory elements that comprise or consist of promoters and/or other nucleic acid elements, such as enhancers, that promote muscle expression such as one or more copies, for example two copies, of the MckE element, which may be arranged as two or more copies in tandem or an MckE and MhcE elements arranged in tandem arranged in combination with a modified Spc5-12 promoter (e.g., SEQ ID NO:1 or 2). These may be present as single copies or with two or more copies in tandem.

[00125] In other embodiments, provided are arrangements of combinations of nucleic acid regulatory elements that promote transgene expression in muscle (skeletal and/or cardiac) tissue. In particular, certain elements are arranged with two or more copies of the individual enhancer and promoter elements arranged in tandem and operably linked to a transgene to promote expression, particularly muscle specific expression. Exemplary nucleotide sequences of the individual promoter and enhancer elements are provided in Table 1. In certain embodiments the downstream promoter is a modified Spc5-12 (version 1 or version 2) promoter and the other promoter is a hAAT promoter or is a TBG promoter or vice versa. [00126] Accordingly, with respect to expression in both the muscle and liver expression, provided are nucleic acid regulatory elements that comprise or consist of promoters and/or other nucleic acid elements, such as one or more elements that promote muscle specific expression (including skeletal and/or cardiac muscle), including a modified Spc5-12 promoter (SEQ ID NO:1 or SEQ ID NO:2), and may, for example, also include one or more copies, for example two copies, of the MckE element, which may be arranged as two or more copies in tandem or an MckE and MhcE elements arranged in tandemand liver specific regulatory elements such as ApoE enhancers, Mic/BiKE elements or hAAT promoters . These may be present as single copies or with two or more copies in tandem. In certain embodiments where there are 2 promoter elements (one of which is a modified Spc5-12 promoter), one promoter element is deleted for the initiation codon to prevent translation initiation at that site, and preferably, the promoter with the modified start codon is at the 3’ end or the downstream end of the nucleic acid regulatory element. In certain embodiments, the composite nucleic acid regulatory element comprises a hAAT promoter, in embodiments a hAAT promoter which is start-codon modified (AATG) as the downstream promoter, and a second promoter in tandem with the hAAT promoter, which a modified Spc5-12 promoter (version 1 or version 2). In certain embodiments, the composite nucleic acid regulatory element comprises a modified Spc5-12 promoter (version 1 or version 2) which is start-codon modified (AATG) as the downstream promoter, and a second promoter in tandem with the modified Spc5-12 promoter, which a hAAT promoter. The nucleic acid regulatory element may also comprise, in addition to the modified Spc5-12 promoter sequences, one or more elements that promote liver specific expression, for example, such as ApoE enhancers, or Mic/BiKE elements. The recombinant expression cassettes provided herein comprise i) a nucleic acid regulatory element comprising a) one copy of ApoE, two or three copies of MckE arranged in tandem, one copy of each MckE, MhcE, and ApoE arrange in tandem, or two or three copies of MckE arranged in tandem with one copy of ApoE, b) a promoter comprising at least one copy of a modified Spc5-12 promoter, and ii) a transgene. In certain embodiments, the promoter is a tandem promoter with at least 2 promoter elements, one of which is a modified Spc5-12 promoter, which may be start-codon modified (AATG) if it is the dow nstream promoter and a second and upstream promoter is a CK8 promoter, a hAAT promoter or a TBG promoter.

[00127] The unique promoter sequences provided herein maintain tissue specificity and increase full capsid yield. The novel regulatory element nucleic acids were generated by modifying the tandem repeats and/or the likelihood of stem-loop-stem formation. This approach was employed to ameliorate potential genome instability that may interfere with rAAV vector replication. Ultimately, these designs improved the packaging efficiency to optimize the yield of full capsids containing an rAAV genome incorporating the regulatory elements operably linked to a nucleotide sequence encoding a therapeutic protein of interest. [00128] In some aspects, rAAV vectors containing these mutant promoters have yield and/or efficiency of packaging full capsids with complete genomes, such as about 2-fold to 5- fold higher than comparable constructs (having the same transgene and other regulatory elements as well as ITR sequences) comprising the control Spc5-12 promoter. In some aspects, AAV constructs comprising the disclosed mutant promoters have an increased packaging efficiency as seen by a decrease in partial capsids relative to the number of full capsids as compared to AAV constructs comprising the Spc5-12 promoter.

Table 1: Examples of regulatory elements.

[00129] In some embodiments, the disclosed regulatory elements can be a part of the recombinant expression cassettes provided herein, examples of which are provided in Table 6. [00130] The recombinant expression cassettes provided herein comprise a nucleic acid regulatory element operably linked to a transgene, wherein the regulatory element comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or variants thereof. The transgene may be any one of the genes or nucleic acids encoding the therapeutic proteins listed in, but not limited to, Tables 7A-7B, including microdystrophin which is made up of domains of dystrophin that are listed in Table 4. Several examples of microdystrophin proteins that may be incorporated (encoded for) as transgenes are listed in Table 5. In certain embodiments, the transgene is a therapeutic antibody, including a full length antibody or antigen binding fragment thereof, such as, a Fab fragment. In embodiments, the transgenes encode therapeutic proteins for treatment, amelioration or prevention of a muscle disease. In embodiments, the transgene encodes a microdystrophin. In embodiments, the transgene encodes Dysl (having an amino acid sequence of SEQ ID NO:35), Dys3 (having an amino acid sequence of SEQ ID NO: 36), Dys5 (having an amino acid sequence of SEQ ID NO: 37), MD1 (having an amino acid sequence of SEQ ID NO: 56), human microdystrophin (having an amino acid sequence of SEQ ID NO: 57), Dys3978 (having an amino acid sequence of SEQ ID NO: 58), MD3 (having an ammo acid sequence of SEQ ID NO: 59), MD4 (having an amino acid sequence of SEQ ID NO: 60), or MD5 (having an amino acid sequence of SEQ ID NO: 72). In embodiments, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. Accordingly, with respect to muscle specific expression, provided are nucleic acid regulatory elements that comprise or consist of promoters and/or other nucleic acid elements, such as enhancers that promote muscle expression, such as MckE enhancers. These may be present as single copies or with two or more copies in tandem.

1. Enhancers

[00132] In some aspects, the disclosed regulatory elements can comprise, in addition to the modified, muscle specific promoter, one or more enhancers. Specifically, muscle-specific enhancers can be used in combination with one or more of the muscle-specific promoters disclosed herein.

[00133] Accordingly, provided are muscle-specific enhancer (MckE) nucleic acid elements as a single copy, or two or three copies arranged in tandem (SEQ ID NOs: 6, 7 and 8, respectively, in Table 1) from the mouse muscle creatine kinase gene (Jaynes, J.B., Johnson, J.E., Buskin, J.N., Gartside, C.L., and Hauschka, S.D that can be arranged in tandem with the modified Spc5-12 promoter. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer. Mol. Cell. Biol., 8: 62-70, 1988; and GenBank Accn. No. AF188002.1). The 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment of this sequence confers high-level expression in skeletal myocytes but is inactive in myocardiocytes (Amacher, et al. 1993 Molecular and Cellular Biology 13(5):2753-64).

[00134] Also provided are Myosin heavy chain enhancer (MhcE) nucleic acids (SEQ ID NO: 9, in Table I) placed in tandem with additional regulatory elements, including a modified Spc5-12 promoter element. Myosin is the most abundant protein in muscle, which is the most abundant tissue in the body. Enhancement of muscle production of transgene, including skeletal and cardiac muscle expression, would greatly benefit the biotherapeutic effect of many transgenes.

[00135] Other enhancers are well known to the skilled person in the art.

2. Introns

[00136] Another aspect of the present invention relates to nucleic acid expression cassettes comprising an intron within the regulatory cassette. In some embodiments, the intron nucleic acid is a chimenc mtron denved from human P-globm and Ig heavy chain (also known as [3- globin splice donor/immunoglobulin heavy chain splice acceptor intron, or 0-globin/IgG chimeric intron, Reed, R., et al. Genes and Development, 1989). Use of an intron may further induce efficient splicing in eukaryotic cells. Although use of an intron may not indicate increases in expression to an already strong promoter, the presence of an intron may increase the expression level of transgene and can also increase the duration of expression in vivo.

[00137] In some embodiments, the intron is a VH4 intron. The VH4 intron nucleic acid can comprise SEQ ID NO: 11 as shown in Table 2 below. The VH4 intron 5’ of the coding sequence may enhance proper splicing and, thus, transgene expression. Accordingly, in some embodiments, an intron is coupled to the 5’ end of a transgene sequence, including positioned between the coding sequence and the promoter sequence. In other embodiments, the intron is less than 100 nucleotides in length.

Table 2: Nucleotide sequences for different introns [00138] In other embodiments, the intron is a chimeric intron derived from human 0- globin and Ig heavy chain (also know n as 0-globin splice donor/immunoglobulin heavy chain splice acceptor intron, or 0-globin/IgG chimeric intron) (Table 2, SEQ ID NO: 10). Other introns well known to the skilled person may be employed, such as the chicken 0-actin intron, minute virus of mice (MVM) intron, human factor IX mtron (e.g., FIX truncated intron 1), 0- globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, and/or SV40 late splice donor /splice acceptor (19S/16S) intron (Table 2, SEQ ID NO: 12).

[00139] Other introns well known to the skilled person may be employed.

3. Other regulatory elements i. polyA

[00140] Another aspect of the present disclosure relates to expression cassettes comprising a polyadenylation (polyA) site downstream of the coding region of the transgene. Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure. Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit 0-globin gene (SEQ ID NO: 14), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57. In one embodiment, the polyA signal comprises SEQ ID NO: 14 as shown in Table 3.

[00141] Table 3: Nucleotide Sequence of the PolyA Signal

C. Recombinant Expression Cassettes

[00142] Disclosed are recombinant expression cassettes comprising a regulatory element comprising a modified Spc5-12 promoter disclosed herein operably linked to a transgene. In some aspects, the regulatory element can be any one or more of the regulatory elements disclosed herein. For example, the nucleic acid regulatory element comprises one of the promoters of Table 1, for example a promoter having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. Thus, in some aspects, disclosed are recombinant expression cassettes comprising a transgene operably linked to a promoter comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[00143] In some embodiments, the expression cassette can comprise one or more of the disclosed nucleic acid regulatory elements operably linked to one or more of the disclosed transgenes. For example, the transgene can be any one of the genes or nucleic acids encoding the therapeutic proteins listed in, but not limited to, Tables 7A-7B. Thus, in some aspects, the transgene can encode a therapeutic antibody, including a full length antibody or an antigen binding fragment, such as a Fab fragment.

[00144] In some aspects, the disclosed recombinant expression cassettes can comprise one or more of the disclosed nucleic acid regulatory elements operably linked to a transgene, wherein the transgene is a muscle-specific disease therapeutic. For example, a musclespecific disease therapeutic can be, but is not limited to, a microdystrophin (see also Table 5), growth and development factor 8 (GDF-8) inhibitor, SMN, huFollistatin344, hSGCA, human-alpha-sarcoglycan, GALGT2, hSGCB, or ravulizumab.

[00145] Encoded by the trans genes provided herein for the methods of the invention are microdystrophins that consist of dystrophin domains arranged amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is a hinge 4 region of dystrophin, CR is a cysteine-rich region of dystrophin and CT is the C terminal domain (and comprises at least the portion of the CT domain containing the al-syntrophin binding site, including SEQ ID NO:33). Table 4 below has the amino acid sequences for these components, in particular from the full length human DMD protein (UniProtDB- 11532, which is incorporated by reference herein).

[00146] To overcome the packaging limitation that is typical of AAV vectors, many of the microdystrophin genes developed for clinical use are lacking the CT domain. Several researchers have indicated that the DAPC does not even require the C-terminal domain in order to assemble or that the C-terminus is non-essential [Crawford, et al., J Cell Biol, 2000, 150(6): 1399-1409; and Ramos, J.N, et al. Molecular Therapy 2019 , 27(3): 1-13], However, overexpression of a microdystrophin gene containing helix 1 of the coiled-coil motif of the CT domain in skeletal muscle of mdx mice increased the recruitment al-syntrophin and a- dystrobrevin, which are members of DAP complex, serving as modular adaptors for signaling proteins recruited to the sarcolemma membrane [Koo, T., et al., Delivery of AAV2/9- microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of al-syntrophin and a- dystrobrevin in skeletal muscles of mdx mice. Hum Gene Ther, 2011. 22(11): p. 1379-88], Overexpression of the longer version of microdystrophin also improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice as compared with the shorter version [Koo, T , et al. 2011, supra}. The CT domain assists in the formation of the Dystrophin Associated Protein Complex (DAPC).

[00147] The CT domain of dystrophin contains two polypeptide stretches that are predicted to form a-helical coiled coils similar to those in the rod domain (see Hl indicated by single underlining and H2 indicated by double underlining in SEQ ID NO:29 in Table 4 below). Each coiled coil has a conserved repeating heptad (a,b,c,d,e,f,g) _n similar to those found in leucine zippers where leucine predominates at the “d” position. This domain has been named the CC (coiled coil) domain. The CC region of dystrophin forms the binding site for dystrobrevin and may modulate the interaction between al-syntrophin and other dystrophin-associated proteins.

[00148] Both syntrophin isoforms, al-syntrophin and i -syntrophin are thought to interact directly with dystrophin through more than one binding site in dystrophin exons 73 and 74 (Yang et al, JBC 270(10):4975-8 (1995)). al- and pi-syntrophin bind separately to the dystrophin C-terminal domain, and the binding site for al-syntrophin reportedly resides at least within the ammo acid residues 3447 to 3481, while that for |31 -syntrophin has been reported to reside within the amino acid residues 3495 to 3535 (as numbered in the DMD protein of UniProtDB-11532 (SEQ ID NO:34), see also Table 4, SEQ ID NO:29, italic). Alphal- (al-) syntrophin and alpha-syntrophin are used interchangeably throughout.

[00149] Microdystrophins disclosed herein were found to bind to and recruit nNOS, as well as alpha-syntrophin, alpha-dystrobrevin and beta-dystroglycan. Binding to nNOS, in the context of a microdystrophin including a C-terminal domain of dystrophin binding to nNOS, means that the microdystrophin expressed in muscle tissue was determined by immunostaining with appropriate antibodies to identify each of alpha-syntrophin, alpha- dystrobrevin, and nNOS in or near the sarcolemma in a section of the transduced muscle tissue. In certain embodiments, the microdystrophin protein has a C-terminal domain that "increases binding” to al-syntrophin, P-syntrophin and/or dystrobrevin compared to a comparable microdystrophin that does not contain the C-terminal domain (but has the same amino acid sequence otherwise, that is a “reference microdystrophin protein”). Increased binding of microdystrophin to al-syntrophin, P-syntrophin and/or dystrobrevin means that the DAPC is stabilized or anchored to the sarcolemma, to a greater extent than a reference microdystrophin protein , as determined by greater levels of one or more DAPC components (including al-syntrophin, P-syntrophin, a-dystrobrevin, P-dystroglycan or nNOS) in the muscle membrane as measured by immunostaining of muscle sections or in muscle tissue lysates or muscle membrane preparations as measured by western blot analysis.

[00150] In some embodiments, the microdystrophin including a C-terminal domain of dystrophin comprises an al-syntrophin binding site and/or a dystrobrevin binding site in the C-terminal domain. In some embodiments, the C-terminal domain comprising an al-syntrophin binding site is a truncated C-terminal domain. The al-syntrophin binding site functions in part to recruit and anchor nNOS to the sarcolemma through al -syntrophin. [00151] The microdystrophins described herein can comprise all or a portion of the CT domain comprising the Helix 1 of the coiled-coil motif. The C Terminal sequence may be defined by the coding sequence of the exons of the / /J gene, in particular exons 70 to 74, and a portion of exon 75 (in particular, the nucleotide sequence encoding the first 36 amino acids of the amino acid sequence encoded by exon 75, or by the sequence of the human DMD protein, for example, the sequence of UniProtKB-P11532 (SEQ ID NO:34) (the CT is amino acids 3361 to 3554 of the UniProtKB-Pl 1532 sequence), or comprising or consisting of binding sites for dystrobrevin and/or al-syntrophin (indicated in Table 4, SEQ ID NO:29). In certain embodiments, the CT domain consists or comprises the 194 C-terminal amino acids of the DMD protein, for example, residues 3361 to 3554 of the amino acid sequence of UniProtKB-Pl 1532 (SEQ ID NO:34), the amino acids encoded by exons 70 to 74, and the nucleotide sequence encoding the first 36 nucleotides of the nucleotide sequence of exon 75 of the DMD gene, or the amino acid sequence of SEQ ID NO:29 (see Table 4). For example, DYS1 (SEQ ID NO: 35) has the 194 amino acid CT sequence of SEQ ID NO:29. In other embodiments, the amino acid sequence of the C-terminal domain is truncated and comprises at least the binding sites for dystrobrevin and/or al-syntrophin. In certain embodiments, the truncated C-terminal domain comprises the amino acid sequence

MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (al-syntrophin binding site) (SEQ ID NO:33). In certain embodiments, the truncated C-terminal domain comprises an al-syntrophin binding site, wherein the binding site has amino acid sequence MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (SEQ ID NO:33). In particular embodiments, the CT domain sequence has the amino acid sequence of SEQ ID NO:30 or amino acids 3361 to 3500 of the UniProtKB-Pl 1532 human DMD sequence. For example, RGX-DYS5 has a CT domain having the amino acid sequence of SEQ ID NO:30. In alternative embodiments, the microdystrophin lacks a CT domain, and may have the domains arranged as follows: ABD1-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR, for example RGX- DYS3 (SEQ ID NO:36).

[00152] The NH ₂ terminus and a region in the rod domain of dystrophin bind directly to but do not cross-link cytoskeletal actin. The rod domain of wild type dystrophin is composed of 24 repeating units that are similar to the triple helical repeats of spectrin. This repeating unit accounts for the majority of the dystrophin protein and is thought to give the molecule a flexible rod-like structure similar to P-spectrin. These a-helical coiled-coil repeats are interrupted by four proline-rich hinge regions. At the end of the 24th repeat is the fourth hinge region that is immediately followed by the WW domain [Blake, D. et al, Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle. Physiol. Rev. 82: 291- 329, 2002], Microdystrophins disclosed herein do not include R4 to R23, and only include 3 of the 4 hinge regions or portions thereof. In some embodiments, no new amino acid residues or linkers are introduced into the microdystrophin.

[00153] In some embodiments, microdystrophin comprises H3 (e.g, SEQ ID NOS: 35, 36, or 37). In embodiments, H3 can be a full endogenous H3 domain from N-terminal to C- terminal, e.g., SEQ ID NO:23. Stated another way, some microdystrophin embodiments do not contain a fragment of the H3 domain but contain the entire H3 domain. In some embodiments, the C-terminal amino acid of the R3 domain is coupled directly (or covalently bonded to) the N-terminal amino acid of the H3 domain. In some embodiments, the C- terminal amino acid of the R3 domain coupled to the N-terminal amino acid of the H3 domain is Q. In some embodiments, the 5' amino acid of the H3 domain coupled to the R3 domain is Q.

[00154] Without being bound by any one theory, a full hinge domain may be appropriate in any microdystrophin in order to convey full activity upon the derived microdystrophin protein. Hinge segments of dystrophin have been recognized as being proline-rich in nature and may therefore confer flexibility to the protein product (Koenig and Kunkel, 265(6):4560- 4566, 1990). Any deletion of a portion of the hinge, especially removal of one or more proline residues, may reduce its flexibility and therefore reduce its efficacy by hindering its interaction with other proteins in the DAP complex.

[00155] Microdystrophins disclosed herein comprise the wild-type dystrophin H4 sequence (which contains the WW domain) to and including the CR domain (which contains the ZZ domain, represented by a single underline (UniProtKB-Pl 1532 aa 3307-3354) in SEQ ID NO:27). The WW domain is a protein-binding module found in several signaling and regulatory molecules. The WW domain binds to proline-rich substrates in an analogous manner to the src homology-3 (SH3) domain. This region mediates the interaction between [3- dystroglycan and dystrophin, since the cytoplasmic domain of -dystroglycan is proline rich. The WW domain is in the Hinge 4 (H4 region). The CR domain contains two EF-hand motifs that are similar to those in a-actinin and that could bind intracellular Ca ²⁺ . The ZZ domain contains a number of conserved cysteine residues that are predicted to form the coordination sites for divalent metal cations such as Zn ²⁺ . The ZZ domain is similar to many types of zinc finger and is found both in nuclear and cytoplasmic proteins. The ZZ domain of dystrophin binds to calmodulin in a Ca ²⁺ -dependent manner. Thus, the ZZ domain may represent a functional calmodulin-binding site and may have implications for calmodulin binding to other dystrophin-related proteins.

[00156] Microdystrophin embodiments can further comprise linkers (LI, L2, L3, L4, L4.1 and/or L4.2) or portions thereof connected the domains as shown as follows: ABD1-L1-H1- L2-R1-R2-L3-R3-H3-L4-R24-H4-CR-CT (e.g., SEQ ID NOs: 35, 37, or 91) or ABD1-L1- H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR (e.g., SEQ ID NO: 36) LI can be an endogenous linker LI (e.g., SEQ ID NO: 16) that can couple ABDI to HL L2 can be an endogenous linker L2 (e.g., SEQ ID NO: 18) that can couple Hl to Rl. L3 can be an endogenous linker L3 (e.g., SEQ ID NO:21) that can couple R2 to R3.

[00157] L4 can also be an endogenous linker that can couple H3 and R24. In some embodiments, L4 is 3 amino acids, e.g. TLE (SEQ ID NO:24) that precede R24 in the native dystrophin sequence. In other embodiments, L4 can be the 4 amino acids that precede R24 in the native dystrophin sequence (SEQ ID NO:31) or the 2 amino acids that precede R24 (SEQ ID NO:32). In other embodiments, there is no linker, L4 or otherwise, in between H3 and R24. On the 5’ end of H3, as mentioned above, no linker is present, but rather R3 is directly coupled to H3, or alternatively H2.

[00158] The above described components of microdystrophin other domains not specifically described can have the amino acid sequences as provided in Table 4 below. The amino acid sequences for the domains provided herein correspond to the dystrophin isoform of UniProtKB-Pl 1532 (DMD_HUMAN) (SEQ ID NO:34), which is herein incorporated by reference. Other embodiments can comprise the domains from naturally-occurring functional dystrophin isoforms known in the art, such as UniProtKB-A0A075B6G3

(A0A075B6G3 HUMAN), (incorporated by reference herein) wherein, for example, R24 has an R substituted for the Q at amino acid 3 of SEQ ID NO:25.

[00159] Additional embodiments are disclosed in International Application PCT/US2020/062484, filed November 27, 2020, which is hereby incorporated by reference in its entirety.

Table 4: Microdystrophin segment amino acid sequences

[00160] The present disclosure also contemplates variants of these sequences so long as the function of each domain and linker is substantially maintained and/or the therapeutic efficacy of microdystrophin comprising such variants is substantially maintained. Functional activity includes (1) binding to one of, a combination of, or all of actin, P-dystroglycan, al- syntrophin, a-dystrobrevin, and nNOS; (2) improved muscle function in an animal model (for example, in the mdx mouse model described herein) or in human subjects; and/or (3) cardioprotective or improvement in cardiac muscle function in animal models or human patients. In particular, microdystrophin can comprise ABD consisting of SEQ ID NO: 15 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15; Hl consisting of SEQ ID NO: 17 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:17; R1 consisting of SEQ ID NO: 19 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:19; R2 consisting of SEQ ID NO:8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20; H3 consisting of SEQ ID NO: 11 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23; R24 consisting of SEQ ID NO:25 or an ammo acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:25; H4 consisting of SEQ ID NO:26 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26; CR consisting of SEQ ID NOs:27 or 28 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:27 or 28; CT consisting of SEQ ID NOs:29 or 30 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:29 or 30, or CT comprising SEQ ID NO:33. In addition to the foregoing, microdystrophin can comprise linkers in the locations described above that comprise or consist of sequences as follows: LI consisting of SEQ ID NO: 16 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; L2 consisting of SEQ ID NO: 18 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18; L3 consisting of SEQ ID NO:21 or an amino acid sequence with at least 50% identity to SEQ ID NO:21 or a variant with conservative substitutions for both L3 residues; and L4 consisting of SEQ ID NOs:24, 31, or 32 or an amino acid sequence with at least 50%, at least 75% sequence identity to SEQ ID NOs:24, 31, or 32.

[00161] Table 5 includes a representative list of microdystrophin proteins (by their amino acid sequences). It is also contemplated that other embodiments are provided, for example substituted variants of microdystrophin, such as defined by SEQ ID NOs:35 (RGX-DYS1), 36 (RGX-DYS3), or 37 (RGX-DYS5). For example, conservative substitutions can be made to SEQ ID NOs:35, 36, or 37 and substantially maintain its functional activity. In embodiments, microdystrophin may have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:35, 36, or 37 and maintain functional microdystrophin activity.

Table 5: Amino acid sequences of microdystrophin proteins

[00162] Examples of a recombinant expression cassetes and portions of a recombinant expression cassete having a promoter operably linked to a transgene are shown in Table 6, including the genome sequences with 5’ and 3’ ITR sequences. Also provided are recombinant expression cassetes comprising a nucleotide sequence coding for any of the microdystrophins provided in Table 5 operably linked to either Spc5-12 VI or Spc5-12 V2 and any other regulatory elements, such as polyA signal sequences.

Table 6

D. Vectors for Gene Delivery

[00163] Another aspect of the present invention relates to the genetic engineering of nucleic acid regulatory elements and incorporating these nucleic acid sequences in a vector expression system. In one embodiment, the vector is a viral vector, including but not limited to recombinant adeno-associated viral (rAAV) vectors (e.g. Gao G., et al 2003 Proc. Natl. Acad. Sci. U.S.A. 100(10): 6081 -6086), lentiviral vectors (e.g. Matrai, J, et al. 2011, Hepatology 53, 1696-707), retroviral vectors (e.g. Axelrod, JH, et al. 1990. Proc Natl Acad Sci USA; 87, 5173-7), adenoviral vectors (e.g. Brown et al., 2004 Blood 103, 804-10), herpes-simplex viral vectors (Marconi, P. et al. Proc Natl Acad Sci U S A. 1996 93(21): 11319-11320; Baez, MV, et al. Chapter 19 - Using Herpes Simplex Virus Type 1-Based Amplicon Vectors for Neuroscience Research and Gene Therapy of Neurologic Diseases, Ed.: Robert T. Gerlai, Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research, Academic Press, 2018:Pages 445-477), and retrotransposon-based vector systems (e.g. Soifer, 2004, Current Gene Therapy 4(4):373-384). In another embodiment, the vector is a non-viral vector. rAAV vectors have limited packaging capacity of the vector particles (i.e. approximately 4.7 kb), constraining the size of the transgene expression cassette to obtain functional vectors (Jiang et al., 2006 Blood. 108:107-15). The length of the transgene and the length of the regulatory nucleic acid sequences comprising tandem enhancer(s) and promoter(s) are taken into consideration when selecting a regulatory region suitable for a particular transgene and target tissue.

[00164] Another aspect of the present invention relates to a viral vector comprising an expression cassette comprising a nucleic acid regulatory element of SEQ ID NO:1 or SEQ ID NO:2 operably linked to a transgene. In some embodiments, the expression cassette enhances expression of the transgene in muscle.

[00165] In another aspect, the expression cassettes are suitable for packaging in an AAV capsid, as such the cassette comprises (1) AAV inverted terminal repeats (ITRs) flank the expression cassette; (2) regulatory control elements, a) modified Spc5-12 promoter having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 alone or in combination with an enhancer (such as those listed in Table 1), b) a poly A signal, and c) optionally an intron; and (3) a transgene providing (e.g., coding for) one or more RNA or protein products of interest, including a microdystrophin protein. In certain embodiments, the expression cassette comprises a nucleotide sequence comprising SEQ ID NO: 38. In certain embodiments, the expression cassette comprises a nucleotide sequence comprising SEQ ID NO: 40.

[00166] In certain embodiments, the transgene is from Tables 7A-7B. In embodiments for expressing an intact or substantially intact mAb, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) regulatory control elements comprising a) a promoter having a nucleotide sequence of SEQ ID NO or SEQ ID NO: 2 alone or in combination with an enhancer (such as those listed in Table 1), b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for any therapeutic protein used to treat muscle specific diseases such as muscular dystrophies. In certain embodiments, the therapeutic protein is Dysl, having an amino acid sequence of SEQ ID NO: 35.

[00167] In certain embodiments, the rAAV comprise an artificial genome comprising a nucleic acid sequence comprising SEQ ID NO: 39. In certain embodiments, the rAAV comprise an artificial genome comprising a nucleic acid sequence comprising SEQ ID NO: 41.

[00168] In the various embodiments, the target tissue may be muscle or heart. The transgenes expressed in muscle are considered systemic expression, since enhanced delivery of muscle-expressed protein may be sufficient to cross into other tissues including crossing the blood brain barrier to the CNS and delivering therapeutics for treating neurological disorders or neurological symptoms of a systemic disorder.

1. AAV

[00169] Another aspect of the present invention relates to expression cassettes suitable for packaging in an AAV capsid, as such the expression cassette comprises (1) AAV inverted terminal repeats (ITRs) flank the expression cassette; (2) regulatory control elements, comprising a) a promoter having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 provided herein, b) a poly A signal, and c) optionally, an intron; and (3) a transgene providing (e.g., coding for) one or more RNA or protein products of interest, particularly a therapeutic for muscle-specific diseases or disorders, including a microdystrophin.

[00170] The provided nucleic acids and methods are suitable for use in the production of any isolated recombinant AAV particles, in the production of a composition comprising any isolated recombinant AAV particles, or in the method for treating a disease or disorder in a subject in need thereof comprising the administration of any isolated recombinant AAV particles. As such, the rAAV may be of any serotype, modification, or derivative, known in the art, or any combination thereof (e.g., a population of rAAV particles that comprises two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles) known in the art. In some embodiments, the rAAV particles are AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV- 14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV PHP B, AAV2.5, AAV21YF. AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof.

[00171] In some embodiments, rAAV particles have a capsid protein from an AAV serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2FYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[00172] In some embodiments, rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[00173] In some embodiments, rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety . In certain embodiments, the rAAV particles comprise the capsid with one of the following amino acid insertions: LGETTRP (SEQ ID NO:42) or LALGETTRP (SEQ ID NO:43), as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAV-PHP.B. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.R1174 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10 , HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[00174] In some embodiments, rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States PatentNos. 7,282,199; 7,906,111; 8,524,446;

8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803;

2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335.

[00175] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 in ’051 publication), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 in ’321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 in ’397 publication), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 in ’888 publication), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38 in ’689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 in ’964 publication), W02010/127097 (see, e.g., SEQ ID NOs: 5-38 in ’097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 in ’508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 in ’924 publication), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 in ’051 publication), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 in ’321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 in ’397 publication), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 in ’888 publication), WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38 in ’689 publication) W02009/104964 (see, e g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 in ’964 publication), W0 2010/127097 (see, e.g., SEQ ID NOs: 5-38 in ’097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of in ’508 publication), and U.S. Appl. Publ. No.

20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 in ’924 publication).

[00176] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024;

2017/0051257; International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335; WO 2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689, W02009/104964, W0 2010/127097, and WO 2015/191508, and U.S. Appl. Publ. No. 20150023924.

[00177] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 or rAAV2/hu.32 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin et al., Methods 28: 158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[00178] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV-11, AAV-12, AAV-2/13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[00179] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[00180] In some embodiments, rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV-8 or AAV-9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-1 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-4 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-5 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-8 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV-9 or a derivative, modification, or pseudotype thereof. [00181] In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV-8 or AAV-9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV-8 capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV-8 capsid protein.

[00182] In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV-9 capsid protein. In some embodiments, rAAV particles in the clarified feed comprise a capsid protein that has an AAV-8 capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV-9 capsid protein.

[00183] In additional embodiments, rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[00184] In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV-1, AAV -2, AAV-5, AAV-6, AAV-7, AAV- 8, AAV-9, AAV-10, AAVrh.8, and AAVrh.10.In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16). In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV- 14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.R1174, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC1O , AAV.HSCll, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle comprised of AAV-8 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle comprised of AAV-9 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAVhu.32 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 or hu.32 particles are rAAV2/8 or rAAV2/9 or rAAV2/hu.32 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[00185] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu.32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSCll, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAVrh.8, and AAVrh.10.

[00186] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV-8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV-8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[00187] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV-9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAVhu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[00188] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV-9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[00189]

2. Methods of Making rAAV Vectors

[00190] Another aspect of the present invention involves making molecules disclosed herein. In some embodiments, a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding an AAV capsid protein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid proteins described herein, and retains (or substantially retains) biological function of the capsid protein and the inserted peptide from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to a particular sequence of the AAV capsid protein, while retaining (or substantially retaining) biological function of the AAV capsid protein.

[00191] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector, for example, those disclosed herein in Table 6. In some embodiments, the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene. In certain embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome.

[00192] In some embodiments, the nucleic acid encoding the capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package an rAAV genome into the capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Nonlimiting examples include HEK293 cells or derivatives thereof, HELA cells, or insect cells.

[00193] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e.g., in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.

[00194] In preferred embodiments, the rAAVs provide trans gene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below. The rAAV vector also includes the regulatory control elements discussed supra, including the modified Spc5-12 promoters of SEQ ID NOs: 1 and 2, to influence and promote the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells, including muscle cells, of the subject. [00195] Provided in particular embodiments are AAV vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV capsid protein.

[00196] The recombinant adenovirus can be a first generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene generally is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.

[00197] The rAAV vector for delivering the transgene to target tissues, cells, or organs, may also have a tropism for that particular target tissue, cell, or organ, e.g. muscle, in conjunction with the use of tissue-specific promoters as described herein. The construct can further include additional expression control elements such as introns that enhance expression of the transgene (e.g., introns such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), -globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron, and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals such as the rabbit P-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(I02):49-57.

[00198] In certain embodiments, nucleic acids sequences disclosed herein may be codon- optimized, for example, via any codon-optimization technique know n to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).

[00199] In a certain embodiment, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a) one or more regulatory elements at least including one or more of the promoters of SEQ ID NO: 1 or SEQ ID NO:2, alone or in combination with one or more enhancers, b) a poly A signal, and c) optionally an intron; (3) transgene providing (e.g., coding for) one or more RNA or protein products of interest. In another embodiment, the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) one or more regulatory elements at least including one or more of the promoters of SEQ ID NO: I or SEQ ID NO:2, alone or in combination with one or more enhancers, b) a rabbit |3-globin poly A signal and e) optionally a chimeric intron derived from human [3-globin and Ig heavy chain, or other intron; and (3) transgene providing (e.g., coding for) one or more RNA or protein products of interest, such as those in Tables 7A-7B or a microdystrophin protein. In certain embodiments, the transgene is a muscle-specific therapeutic. In certain embodiments, the muscle-specific therapeutic is a microdystrophin. In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MD1 (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59), MD4 (SEQ ID NO: 60) or MD5 (SEQ ID NO: 71). In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35). In embodiments, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 38. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 40. In certain embodiments, the constructs described herein comprise SEQ ID NO: 39. In certain embodiments, the constructs described herein comprise SEQ ID NO: 40. The expression cassettes may be within a plasmid that is appropriate for propagation in the target host cells and used for manufacturing of the recombinant AAV particles described herein.

[00200] The viral vectors provided herein may be manufactured using host cells, e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters. Nonlimiting examples include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells. Typically, the host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and genetic components for producing viruses in the host cells, such as the replication and capsid genes (e.g., the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsC12 sedimentation. Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102: 1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.

[00201] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. Cell lines derived from muscle or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma FIT-1080, HKB-11, C2C12 myoblasts, and CAP cells. Once expressed, characteristics of the expressed product (transgene product) can also be determined, including serum half-life, functional activity of the protein (e.g. enzymatic activity or binding to a target), determination of the glycosylation and tyrosine sulfation patterns, and other assays known in the art for determining protein characteristics. Provided are methods of manufacturing a recombinant AAV comprising culturing a host cell capable of producing a recombinant AAV described herein under conditions appropriate for production of the recombinant AAV comprising an artificial genome with an expression cassette comprising a synthetic promoter operably linked to a transgene. In particular, the method provides (1) culturing a host cell containing (i) an artificial genome comprising AAV ITRs flanking a recombinant cis expression cassette which comprises a nucleic acid regulatory element comprising a promoter having a nucleotide sequence of SEQ ID NO: 1 or 2, as disclosed herein operably linked to a transgene; (ii) a trans expression cassette lacking AAV ITRs which encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans; and (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and (2) recovering recombinant AAV encapsidating the artificial genome from the cell culture.

[00202] Also provided are host cells containing (i) an artificial genome comprising AAV ITRs flanking a recombinant cis expression cassette which comprises one or more regulatory elements comprising a promoter having a nucleotide sequence of SEQ ID NO: 1 or 2, as disclosed herein operably linked to a transgene; (ii) a trans expression cassette lacking AAV ITRs which encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans; and, optionally, (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein. In particular embodiments, the composite nucleic acid regulatory element consists of SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the artificial genome comprises a transgene encoding one of the therapeutics listed in Tables 7A- 7B or a micrody strophin.

[00203] Also provided are host cells comprising a plasmid comprising a cis expression cassette, wherein the cis expression cassette comprises a promoter operably linked to a transgene, wherein the promoter comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, the artificial genome comprises a transgene encoding one of the therapeutics listed in Tables 7A-7B or a microdystrophin. In certain embodiments, the transgene is a muscle-specific therapeutic. In certain embodiments, the muscle-specific therapeutic is a microdystrophin. In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MD1 (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59), MD4 (SEQ ID NO: 60) or MD5 (SEQ ID NO: 71). In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35). In embodiments, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 38. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 40. In certain embodiments, the plasmid comprises SEQ ID NO: 39. In certain embodiments, the plasmid comprises SEQ ID NO: 40.

[00204] In some aspects, the disclosed methods of manufacturing or producing recombinant AAV vectors having an AAV genome with a transgene operably linked to a promoter having a nucleotide sequence of SEQ ID NO: 1 or 2 results in a higher packaging efficiency, including resulting in a higher percentage of capsids with intact or complete genomes than a recombinant AAV vector with a comparable AAV genome comprising an unmodified Spc5-12 promoter. In other words, the recombinant AAVs disclosed herein comprise a promoter having the sequence of SEQ ID NO: 1 or SEQ ID NO:2, or a variant thereof, and the manufacturing process results in 40, 45, 50, 55, 60, 65, 70, or 75% full capsids of the capsid population recovered. Thus, in some aspects, at least 30%, 40%, 50%, 60%, 70% 80%, 90% or 95% of the rAAVs produced encapsidate a full AAV genome. In some aspects, the production results in recombinant AAVs having at least 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, or more full capsids than recombinant AAVs comprising a comparable recombinant expression cassette (i.e., having all of the same elements except the promoter) comprising an unmodified Spc5-12 promoter produced using the same method. In some aspects, the production results in rAAVs having at least 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold or more full capsids than partial capsids. In some aspects, the percent of full capsids produced can be 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80- 100%, 90-100%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%, 10-50%, 20-50%, 30-50%, or 40-50%.

E. Therapeutic and Prophylactic Uses

[00205] Another aspect relates to therapies which involve administering a transgene via a rAAV vector according to the invention to a subject in need thereof, for delaying, preventing, treating, and/or managing a disease or disorder, and/or ameliorating one or more symptoms associated therewith, wherein the transgene is operably linked to one or more of the promoters described herein, such as the promoter having a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2. A subject in need thereof includes a subject suffering from the disease or disorder, or a subject pre-disposed thereto, e.g., a subject at risk of developing or having a recurrence of the disease or disorder. Generally, a rAAV carrying a particular transgene will find use with respect to a given disease or disorder in a subject where the subject’s native gene, corresponding to the transgene, is defective in providing the correct gene product, or correct amounts of the gene product. The transgene then can provide a copy of a gene that is defective in the subject.

[00206] Generally, the transgene comprises cDNA that restores protein function to a subject having a genetic mutation(s) in the corresponding native gene. In embodiments, the transgene encodes the heavy and light chains of a therapeutic antibody, or an antigen binding fragment thereof. In some embodiments, the cDNA comprises associated RNA for performing genomic engineering, such as genome editing via homologous recombination. In some embodiments, the transgene encodes a therapeutic RNA, such as a shRNA, artificial miRNA, or element that influences splicing.

[00207] In some aspects, the therapeutic encoded by one or more of the disclosed transgenes can by any of the microdystrophins disclosed herein. Microdystrophins include those having the amino acid sequence of SEQ ID NOs: 35-37, 57-60 (Table 5) and/or encoded by the nucleotide sequence of SEQ ID NOs: 38-41 (Table 6, which lists the nucleotide sequences of expression cassettes encoding a microdystrophin operably linked to regulatory elements), such as the microdystrophin of Dysl (SEQ ID NO: 35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MD1 (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59), MD4 (SEQ ID NO: 60) or MD5 (SEQ ID NO: 71). In embodiments, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68 SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. Provided are methods of treating human subjects for any muscular dystrophy disease that can be treated by providing a functional dystrophin. In some aspects, the functional dystrophin is one or more of the microdystrophins disclosed herein. DMD is the most common muscular dystrophy disease, but other diseases can be treated such as, but not limited to, Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert’s disease), Facioscapulohumeral disease (FSHD), limb-girdle muscular dystrophy, X-linked dilated cardiomyopathy, or oculopharyngeal muscular dystrophy.

[00208] Tables 7A-7B below provides a list of transgenes that may be used in any of the rAAV vectors described herein in which the transgene sequence is operably linked to the modified Spc5-12 promoter (Spc5-12Vl or Spc5-12V2), preferably to treat or prevent the disease with which it is associated, also listed in Tables 7A-7B. The appropriate AAV serotype may be chosen to engineer to optimize the tissue tropism and transduction of the vector.

Table 7A

Table 7B

[00209] Generally, the rAAV vector is administered systemically, and following transduction, the vector’s production of the protein product is enhanced by an expression cassette employing a modified Spc5-12 promoter, for example, the promoter of SEQ ID NO:1 or SEQ ID NO:2 can be operably linked to the transgene. In some aspects, the rAAV vector may be provided by intravenous, intramuscular, and/or intra-peritoneal administration. [00210] With respect to the therapeutic antibodies in Table 7B expression in both muscle and liver may be desirable to deliver the expressed therapeutic antibody systemically. Accordingly, provided are expression cassettes in which the regulatory sequences operably linked to the transgene encoding the therapeutic antibody are composite or tandem regulatory sequences which, in addition to one of the Spc5-12 promoters (Spc5-12 VI or Spc5-12 V2) comprise an additional liver specific promoter in tandem (e g., hAAT promoter or TBG promoter) and/or liver specific enhancer elements, such as, ApoE enhancers, or Mic/BiKE elements, the sequences of which are provided in Table 1. In addition, the expression cassettes comprising the regulatory sequences operably linked to the transgene encoding the therapeutic antibody may be packaged in an rAAV for delivery that preferably has an AAV8 capsid, an AAV9 capsid or an AAVrhlO capsid for targeting or expression in liver and/or muscle cells.

[00211] In some aspects, the rAAVs of the present invention find use in delivery to target tissues associated with the disorder or disease to be treated/prevented. A disease or disorder associated with a particular tissue or cell type is one that largely affects the particular tissue or cell type, in comparison to other tissue of cell types of the body, or one where the effects or symptoms of the disorder appear in the particular tissue or cell type. Methods of delivering a transgene to a target tissue of a subject in need thereof involve administering to the subject the an rAAV where the expression cassette comprises a nucleic acid regulatory element comprising SEQ ID NO: 1 or SEQ ID NO:2 operably linked to a transgene.

[00212] In some aspects, the transgene is a muscle-specific disease therapeutic. One or more of the transgene listed in Tables 7A-7B are muscle-specific disease therapeutics. A muscle-specific disease therapeutic can be a therapeutic that treats a muscle-specific disease. A muscle-specific disease can be diseases such as myopathies. In some aspects, a myopathy includes muscular dystrophies (e.g. Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), human limb girdle muscular dystrophy (LGMD)), X-linked dilated cardiomyopathy, Myasthenia Gravis, Rhabdomyolysis, Amyotrophic Lateral Sclerosis (ALS), and Sarcopenia.

[00213] Disclosed are methods for enhancing expression of a transgene, comprising delivery of viral vectors comprising one or more of the disclosed recombinant expression cassettes. Thus, in some aspects, disclosed are methods for enhancing expression of a transgene, comprising delivery of viral vectors comprising a promoter comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 operably linked to the transgene. In some aspects, the transgene can be any one or more of the transgenes disclosed herein, for example, a gene or nucleic acid encoding any of the therapeutics listed in Tables 7A-7B or a microdystrophin (for example, having an amino acid sequence of Table 5).

[00214] In some aspects, the delivery of viral vectors is to a cell. In some aspects, the cell can be in a subject and therefore the delivery can be administering to a subject. In some aspects, the viral vector is administered intravenously or intramuscularly.

[00215] In some aspects of the disclosed methods for enhancing expression of a transgene, the transgene expression is enhanced in the circulation or systemically. In some aspects of the disclosed methods for enhancing expression of a transgene, the transgene expression is enhanced in the skeletal muscle or cardiac muscle.

[00216] In some aspects, disclosed are methods of treatment by delivery of rAAVs comprising the one or more of the recombinant expression cassettes disclosed herein, one or more vectors disclosed herein or one or more of the disclosed rAAV. Also provided are methods for treating a disease or disorder in a subject in need thereof comprising the administration of recombinant AAV particles comprising an expression cassette comprising a promoter comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 operably- linked to a transgene encoding a therapeutic for treatment of said disease or disorder. In some aspects, the transgene can be any one or more of the transgenes disclosed herein, for example, a gene or nucleic acid encoding any of the therapeutics listed in Tables 7A-7B. In some aspects, the transgene encodes a muscle-specific disease therapeutic, such as a Microdystrophin, for example the microdystrophin Dysl, having an amino acid sequence of SEQ ID NO: 35, Dys3 (having an amino acid sequence of SEQ ID NO: 36), Dys5 (having an amino acid sequence of SEQ ID NO: 37), MD1 (having an amino acid sequence of SEQ ID NO: 56), human microdystrophin (having an amino acid sequence of SEQ ID NO: 57), Dys3978 (having an amino acid sequence of SEQ ID NO: 58), MD3 (having an amino acid sequence of SEQ ID NO: 59),MD4 (having an amino acid sequence of SEQ ID NO: 60) or MD5 (SEQ ID NO: 71). In some aspects, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In some aspects, the recombinant AAV particles comprise an expression cassette of SEQ ID NO:38 or SEQ ID NO: 40. In some aspects, the recombinant AAV particles comprise SEQ ID NO:39 or SEQ ID NO: 41. In some aspects of the treatment methods, the disease or disorder is any of the diseases or disorders listed in Tables 7A-7B. In some aspects, the disease or disorder is a muscle-specific disease or disorder, such as, but not limited to, a muscular dystrophy.

[00217] Following transduction of target cells, the expression of the protein product is enhanced by employing such muscle-specific expression cassettes. Such enhancement may be measured by the following non-limiting list of determinations such as 1) protein titer by assays known to the skilled person, not limited to sandwich ELISA, Western Blot, histological staining, and liquid chromatography tandem mass spectrometry (LC-MS/MS); 2) protein activity, by assays such as binding assays, functional assays, enzymatic assays and/or substrate detection assays; and/or 3) serum half-life or long-term expression; and/or (4) detection of mRNA encoding the therapeutic protein of the transgene. Enhancement of transgene expression may be determined as efficacious and suitable for human treatment (Hintze, J.P. et al, Biomarker Insights 2011:6 69-78). Assessment of the quantitative and functional properties of a transgene using such in vitro and in vivo cellular, blood and tissue studies have been shown to correlate to the efficacy of certain therapies (Hintze, J.P. et al, 2011, supra), and are utilized to evaluate response to gene therapy treatment of the transgene with the vectors described herein.

[00218] rAAV vectors of the invention also can facilitate deliver, in particular, targeted delivery, of transgenes operably linked to the regulatory sequences described herein, including but not limited to oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, antibodies to target cells or tissues. The rAAV vectors also can facilitate delivery, in particular, targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.

[00219] The agents may be provided as pharmaceutically acceptable compositions as known in the art and/or as described herein. In some embodiments, the rAAV molecule may be administered alone or in combination with other prophylactic and/or therapeutic agents. [00220] The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease, the route of administration, as well as age, body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56th ed., 2002). Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic agents, and whether such agents are administered separately or as an admixture. [00221] The amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00222] Prophylactic and/or therapeutic agents, as well as combinations thereof, can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. Such model systems are widely used and well known to the skilled artisan. In some preferred embodiments, animal model systems for a muscle condition are used that are based on rats, mice, or other small mammal other than a primate, such as, but not limited to, mdx mouse models.

[00223] Once the prophylactic and/or therapeutic agents of the invention have been tested in an animal model, they can be tested in clinical trials to establish their efficacy. Establishing clinical trials will be done in accordance with common methodologies known to one skilled in the art, and the optimal dosages and routes of administration as well as toxicity profiles of agents of the invention can be established. For example, a clinical trial can be designed to test a rAAV molecule of the invention for efficacy and toxicity in human patients.

[00224] Toxicity and efficacy of the prophylactic and/or therapeutic agents of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00225] A rAAV molecule of the invention generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit. The data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[00226] A therapeutically effective dosage of an rAAV vector for patients is generally from about 0.1 ml to about 100 ml of solution containing concentrations of from about IxlO ⁹ to about IxlO ¹⁶ genomes rAAV vector, or about IxlO ¹⁰ to about IxlO ¹⁵ , about IxlO ¹² to about IxlO ¹⁶ , or about IxlO ¹⁴ to about IxlO ¹⁶ AAV genomes. Levels of expression of the transgene can be monitored to determine/adjust dosage amounts, frequency, scheduling, and the like.

[00227] Treatment of a subject with a therapeutically or prophy tactically effective amount of the agents of the invention can include a single treatment or, in certain embodiments, can include one or more administrations.

[00228] Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.).

[00229] In certain embodiments, the agents of the invention are administered intravenously or intramuscularly and may be administered together with other biologically active agents.

[00230] In another specific embodiment, agents of the invention may be delivered in a sustained release formulation, e.g., where the formulations provide extended release and thus extended half-life of the administered agent. Controlled release systems suitable for use include, without limitation, diffusion-controlled, solvent-controlled, and chemically- controlled systems. Diffusion controlled systems include, for example reservoir devices, in which the molecules of the invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers.

Monolithic (matrix) device are a second type of diffusion controlled system, wherein the dual antigen-binding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix). Agents of the invention can be homogeneously dispersed throughout a ratecontrolling matrix and the rate of release is controlled by diffusion through the matrix. Polymers suitable for use in the monolithic matrix device include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers, as well as polymer derivatives.

[00231] Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents described herein. See, e.g. U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al., “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology, 39: 179 189, 1996; Song et al., “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology, 50:372 397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Inti. Symp. Control. Rel. Bioact. Mater., 24:853 854, 1997; and Lam et al., “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Infl. Symp. Control Rel. Bioact. Mater., 24:759 760, 1997, each of which is incorporated herein by reference in its entirety . In one embodiment, a pump may be used in a controlled release system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321:574, 1989). In another embodiment, polymeric materials can be used to achieve controlled release of agents comprising dual antigen-binding molecule, or antigen-binding fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds ), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science, 228: 190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7 1: 105, 1989); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)). Other controlled release systems are discussed in the review by Langer, Science, 249: 1527 1533, 1990.

[00232] In addition, the rAAVs can be used for in vivo delivery of transgenes for scientific studies such as gene knock-down with miRNAs, recombinase delivery' for conditional gene deletion, gene editing with CRISPRs, and the like.

F. Methods of Increasing Packaging Efficiency

[00233] Disclosed are methods of increasing packaging efficiency of recombinant AAVs by using one or more of the modified Spc5-12 promoters described herein, Spc5-12v l (SEQ ID NO: 1) or Spc5-12v2 (SEQ ID NO: 2) in place of an unmodified Spc5-12 promoter. Packaging efficiency includes increasing the proportion or percentage of produced capsids that encapsidate a full rAAV genome, with reduced proportion of produced capsids being empty or containing genome fragments or contaminating non-genome sequences.

[00234] For example, disclosed are methods of increasing packaging efficiency of recombinant AAVs by making the recombinant AAVs using one or more of the methods described herein. For example, disclosed are methods of increasing packaging efficiency of recombinant AAVs comprising (a) culturing a host cell containing: (i) an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a promoter operably linked to a transgene coding for one or more RNA or protein products, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2; (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans,- (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein; and (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture; wherein the presence of the promoter comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2 allows for increased packaging efficiency. In certain embodiments, the transgene is a muscle-specific therapeutic. In certain embodiments, the muscle-specific therapeutic is a microdystrophin. In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35), Dys3 (SEQ ID NO: 36), Dys5 (SEQ ID NO: 37), MD1 (SEQ ID NO: 56), human microdystrophin (SEQ ID NO: 57), Dys3978 (SEQ ID NO: 58), MD3 (SEQ ID NO: 59),MD4 (SEQ ID NO: 60) or MD5 (SEQ ID NO: 72). In certain embodiments, the microdystrophin is Dysl (SEQ ID NO: 35). In embodiments, the microdystrophin transgene is encoded by a nucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or a nucleotide sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 38. In certain embodiments, the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 40. In certain embodiments, the artificial genome comprises SEQ ID NO: 39. In certain embodiments, the artificial genome comprises SEQ ID NO: 40.

[00235] In some aspects, the increased packaging efficiency is compared to production of recombinant AAVs comprising an unmodified Spc5-12 promoter (e.g., the only difference in the cis plasmid construct being the promoter).

[00236] In some aspects, increased packaging efficiency is production of a population of rAAVs in which at least 30% of the recombinant AAVs have the full AAV genome. In some aspects, increased packaging efficiency is production of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the recombinant AAVs having the full (e.g. intact) AAV genome rather than an empty capsid or containing a fragment of the AAV genome or contaminating nucleic acid. In some aspects, increased packaging efficiency means there are at least one-fold, two-fold, three-fold, 4-fold or 5 -fold more full capsids produced than rAAVs comprising a comparable genome having a recombinant expression cassette comprising an unmodified Spc5-12 promoter produced using the same method.

[00237] In some aspects, increased packaging efficiency means the recombinant AAVs have at least two-fold more full capsids than partial capsids.

G. Pharmaceutical Compositions and Kits

[00238] The invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent of the invention, said agent comprising a rAAV molecule of the invention comprising a transgene cassette wherein the transgene expression is driven by the chimeric regulatory elements described herein. In preferred embodiments, the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously or intramuscularly. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ as known in the art. The pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[00239] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising a therapeutically and/or prophy lactically effective amount of an agent of the invention along with a pharmaceutically acceptable carrier.

[00240] In preferred embodiments, the agent of the invention is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). In a specific embodiment, the host or subject is an animal, preferably a mammal such as nonprimate (e g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e g., monkey such as, a cynomolgus monkey and a human). In a preferred embodiment, the host is a human.

[00241] The invention provides further kits that can be used in the above methods. In one embodiment, a kit comprises one or more agents of the invention, e.g., in one or more containers. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a condition, in one or more containers.

[00242] The invention also provides agents of the invention packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent or active agent. In one embodiment, the agent is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject. Typically, the agent is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent should be stored at between 2 and 8°C in its original container and the agent should be administered within 12 hours, usually within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, an agent of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of agent or active agent. Typically, the liquid form of the agent is supplied in a hermetically sealed container at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml.

[00243] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) as well as pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient). Bulk drug compositions can be used in the preparation of unit dosage forms, e.g., comprising a prophy tactically or therapeutically effective amount of an agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier. [00244] The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of the target disease or disorder can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[00245] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of agent or active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Examples

A. Example 1: Analysis of promoter chimeric repeat sequences and loop formation [00246] Synthetic Promoter c5-12 (Li, X. et al. Nature Biotechnology' Vol. 17, pp. 241- 245, MARCH 1999), known as the SPc5-12 promoter, has been shown to have cell type restricted expression, specifically muscle-cell specific expression. At less than 350 bp in length, the SPc5-12 promoter is smaller in length than most endogenous promoters, which can be advantageous when the length of the nucleic acid encoding the therapeutic protein is relatively long. The nucleotide sequence of one strand of the Spc5-12 promoter is disclosed herein as SEQ ID NO: 3 (see Table 1, supra).

[00247] Xie et al (2017, Molecular Therapy, 25, 1363-1375) hypothesized that hairpin loops, such as shRNA transgenes, within or proximal to inverted terminal repeats (ITRs) of an AAV genome packaging cassette may produce a population of truncated or defective genomes. The SPc5-12 promoter sequence was analysed to identify chimeric repeat sequences that may be prone to hairpin loop formation. MEF-2 tandem repeats in opposite onentation (5 -CGCTCTAAAAAATAACTCCCGGGAGTTATTTTTAGAGCG-3’; SEQ ID NO: 4) within the Spc5-12 synthetic promoter were, for example, analysed for predictive thermodynamic ensemble (hairpin assembly). The free energy of the thermodynamic ensemble is -22.84 kcal/mol, the frequency of the minimum free energy (MFE) structure in the ensemble is 79.08%, and the ensemble diversity is 0.36 for this particular tandem TFRE orientation of MEF-2 repeats. In some aspects, RNAfold can be used to calculate the free energy. The RNAfold program was used to calculate secondary structures of RNAs. The free energy of the thermodynamic ensemble (each potential structure), as performed by the RNAfold server, http://ma.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, is calculated to determine the lowest energy structure, i.e. minimum free energy (MFE), that is the most stable thermodynamically (Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH (2004)) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101(19):7287-92.; Gruber AR, Lorenz R, Bemhart SH, Neubock R, Hofacker IL. The Vienna RNA Websuite. Nucleic Acids Res. 2008). The frequency of the MFE structure is the probability of a single structure in the Boltzmann weighted ensemble of all structures. The ensemble diversity reveals the average base-pair distance between all structures in the thermodynamic ensemble. Low frequency of the MFE structure and low diversity indicate the favorability of a thermodynamically stable structure.

[00248] Analysis of SRE-TEF-l-SRE tandem sequences (5’- GGACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATATTTGGGTGTCC-3 ’ ; SEQ ID NO: 14) within the Spc5-12 synthetic promoter revealed that the free energy of the thermodynamic ensemble is -43.25 kcal/mol, the frequency of the MFE structure in the ensemble is 92.80%, and the ensemble diversity is 0.29 for this particular tandem arrangement of transcription factor regulatory elements (TFREs) containing SRE repeats in opposite orientation.

[00249] Based on the calculated energies, stem-stem formation of MEF-2 tandem repeats and stem-loop-stem formation of SRE-TEF-l-SRE elements were predicted, and modifications were sought to ameliorate potential genome instability that may interfere with rAAV vector replication.

B. Example 2: Characterization of partially-filled capsids

[00250] Recombinant AAV vectors were produced by triple transfection of three plasmids 1) a plasmid containing a polynucleotide encoding a functional rep gene and a polynucleotide encoding a capsid gene, 2) a plasmid containing a genome to be packaged into a capsid (comprises at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a therapeutic gene product operably linked to sequences which direct expression of the gene product in a target cell, such as the Spc5-12 promoter (SEQ ID NO:3), and 3) a polynucleotide comprising sufficient helper functions to permit packaging of the genome into the AAV capsid protein under conditions which permit packaging of the genome into the AAV capsid. Following transfection in HEK293 cells, the cells were maintained in cell culture under conditions that allow production of the rAAV particles.

1. Analytical Ultracentrifugation (AUC) Characterization

[00251] The presence of vectors with fragmented genomes and non-transgene-related DNA contaminants, often referred to as partially -filled capsids, can be resolved by Analytical Ultracentrifugation (AUC). Separate purification fractions were tested: ultracentrifugation (UC) Pool 1, UC Pool 3 and UC Pool 4. UC Pool 1 (contained 98% full particles, whereas, UC Pool 3 contained the highest number of partially -filled capsids at 51%, and UC Pool 3 contained 87% empty capsids.

2. Next-Generation Sequencing (NGS)

[00252] Next generation sequencing of the genomes within the produced AAV capsids can be used to assess the completeness of the packaged genomes. Long read information for AAV capsids containing genomes with the Spc-5-12 promoter was generated using an Oxford Nanopore technique.

[00253] The partially-filled and empty capsids do not support transgene expression and NGS data suggests that the DNA fragments in these partially-filled capsids primarily consist of chimeric DNA with repeated sequences of various sizes from the SPc5-12 promoter region (not the therapeutic gene). However, overall low ratio of full particles compromises total yield of viral particles in the production process.

C. Example 3: Improved design for Spc5-12 promoter to minimize secondary structure and reduce partial AAV genome

[00254] To minimize the formation of hairpin loops in the Spc5-12 promoter, the position of MEF-2 and SRE regulatory sequences were modified and resulted in two variant promoters, Spc5-12vl (SEQ ID NO: 1) and Spc5-12v2 (SEQ ID NO: 2).

[00255] FIG. 1 shows Spc5-12 Mutantl (SEQ ID NO: 1) in which the MEF-2 and SRE elements are exchanged relative to the Spc5-12 promoter sequence. Results for thermodynamic ensemble prediction of the structure on the left shows the free energy of the thermodynamic ensemble is -8.40 kcal/mol. The frequency of the MFE structure in the ensemble is 72.26%. The ensemble diversity is 0.84. Results for thermodynamic ensemble prediction of the structure on the right shows the free energy of the thermodynamic ensemble is -21.42 kcal/mol. The frequency of the MFE structure in the ensemble is 30.84%. The ensemble diversity is 2.55.

[00256] FIG. 2 shows Spc5-12 Mutant2 (SEQ ID NO:2) with MEF-2 and SRE in reverse orientation relative to Spc5-12. Results for thermodynamic ensemble prediction of the structure on the left shows the free energy of the thermodynamic ensemble is -3. 11 kcal/mol. The frequency of the MFE structure in the ensemble is 26.99%. The ensemble diversity is 9.65. Results for thermodynamic ensemble prediction of the structure on the right shows the free energy of the thermodynamic ensemble is -7.79 kcal/mol. The frequency of the MFE structure in the ensemble is 6.49%. The ensemble diversity is 13.48.

[00257] FIG. 3 summarizes the assessment of the secondary' structures of Spc5-12 promoter and variants 1 and 2, from FIGs. 1 and 2. Switching position or reversing orientation of the SRE and MEF2 elements as shown is predicted to reduce hairpin formation while not impacting TFRE binding and, therefore, not alter Spc5-12 promoter strength and specificity. Table 8 summarizes the number of nucleotides forming a stem pair, the free energy, and the probability of stem pair formation of the first and second hairpins of Spc5-12, Spc5-12vl and Spc5-12v2. Table 8

D. Example 4: Assessment of modified Spc5-12 promoters on production process and genome integrity [00258] Impact of the modified promoters compared to unmodified Spc5-12 on production of recombinant AAV virions containing a microdystrophin encoding transgene was assessed. [00259] Small scale rAAV production was carried using cis plasmids having a microdystrophin (Dysl) transgene (encoding the amino acid sequence of SEQ ID NO: 35) and varying only by the promoter (VC160: Spc5-12 mutant 1 promoter; VS161 : Spc5-12 mutant 2 promoter; VC162: SynlOO promoter; VS163: CK7 promoter and Control: Spc5-12 promoter). The production was carried out in 2L WV in 5L Thomson Shake Flasks (no baffles) inoculated at p: 1.3xl0 ⁶ vc/mL; dilution a day before transfection; and transfection at p: 5.0xl0 ⁶ vc/mL.

[00260] FIG. 4 shows the viable cell density and cell viability for the cells having the different promoters in the cis plasmids, showing that the VCD and cell viability are comparable. Pre-lysis and post-lysis titers are provided in Table 9:

Table 9 - Titers [00261] The produced capsid samples were then analyzed for total capsid numbers after affinity purification step and at the end of the batch production process (bulk drug substance (BDS)) (FIG. 5 and Tables 10 and 11). Comparing the constructs with the modified promoters compared to the construct with the Spc5-12 promoter, the Spc5-12 mutant promoters had higher amounts of capsid, particularly for the constructs with the mutant 2 (Spc5-12 v2; SEQ ID NO: 2) promoter.

Table 10 - Capsid Number (Fold)

Table 11 - Percentage of Full Capsid

[00262] Genome integrity was also assessed by viral DNA extraction and tape station analysis. FIG. 6 shows the results of a DNA TapeStation analysis (Agilent Technologies, Santa Clara, CA) for analyzing annealed double strand DNA, which data is also summarized in Table 12 below.

Table 12 - Genomic Integrity [00263] The DNA tape kit analysis permits the assessment of the length of viral DNA to provide an assessment of the proportion of partial (containing genome fragments) versus full (intact genomes). The construct having the mutant 2 promoter (Spc5-12 v2) had the highest percentage of full capsids — 65.3% as compared to 13.9% for the rAAVs from the construct with the unmodified Spc5-12 promoter. The mutant 1 construct resulted in 42.5% full capsids.

[00264] The produced capsids were also analyzed by ultracentrifugation on CsCl gradients for the proportion of empty, partial and full capsids in the preparations generated from constructs having the mutant 2 promoter as compared to the unmodified Spc5-12 promoter. FIGs. 7A and 7B show the sedimentation coefficient profile of the Spc5-12 (control) (A) and Mutant 2 (B) samples. The full to partial capsid ratio increased ~4 fold with the Mutant 2 redesign, and resulted in 42% full capsids as compared to 22% full for the control.

[00265] Next generation sequencing (by Oxford Nanopore) was also used to assess the integrity of the genomes in capsids having the redesigned promoters compared to the unmodified Spc5-12 control. FIG. 8 shows that control Spc5-12 promoter has a much lower percent of full length reads compared to the mutant 1 and mutant 2 promoters.

E. Example 5: Transgene expression and biodistribution of rAAV constructs containing the modified Spc5 promoters

[00266] C57B1/6J mice at 5-6 weeks of age were administered constmcts as indicated in

Table 13 below.

Table 13

[00267] Six weeks after injection, mice were euthanized and tissue samples were taken. rAAV genome DNA and transgene expressed mRNA were analyzed in in left TA (tibialis anterior) muscle, left GAS (gastrocnemius) muscle, diaphragm, liver, and/or heart.

Microdystrophin protein levels were assessed by JESS (ProteinSimple) capillary-based Western immunoassay analysis in right gastrocnemius, diaphragm and heart tissue. Immunohistochemistry was also performed in the right quadricep for microdystrophin and degeneration/regeneration (DAPI/eMyHC). This study also compares pDys DNA/RNA levels in select muscles after systemic administration of LIDVS under control of modified Spc5.12, ensures plasma membrane localization of pDys in skeletal muscle, and compares pDys DNA/RNA in non-muscle tissues.

[00268] The expression (as detected by microdystrophin RNA copies by ddPCR) of the microdystrophin transgene was slightly increased from the constructs with the Spc5. 12 mutants VI and V2 in heart and skeletal muscle as compared to Spc5-12 (FIGs. 9 and 10). Liver expression in mice from all three experimental groups is low and remains unaffected by promoter modification. Figures 9 and 10 differ by the normalization of RNA loading, which shows similar results.

[00269] Distribution of the AAV genomes in the tissues of the administered mice was also assessed by ddPCR and normalized to diploid genomes or micrograms of DNA. FIGs. 11 (normalized to diploid genomes) and 12 (normalized to micrograms of DNA) show that Spc5. 12 mutants VI and V2 do not significantly alter AAV-DNA biodistribution compared to constructs having the unmodified Spc5-12 promoter. A large amount of the AAV DNA is present in the liver. FIG. 13 shows ratio of the transgene RNA to DNA copies of the AAV genome per diploid cell based upon the data in FIGs. 9 and 11. The data demonstrate the mutant promoters Spc5-12vl and Spc5-12 v2 have promoter (transcriptional) activity that is essentially equivalent to Spc5-12.

[00270] pDys expression is heterogeneous and present in roughly half of muscle fibers (quadricep) as determined using immunohistochemistry. Expression of the pDys is localized to sarcolemma. The muscle was healthy is all mice treated with the AAV vectors.

[00271] 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 method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.