Regulatory Polynucleotides

Abstract

To provide modulatory polynucleotides encoding at least one siRNA molecule for treatment or prevention of disease, AAV particles including the modulatory polynucleotides, and compositions including the AAV particles.SOLUTION: An adeno-associated viral (AAV) viral genome comprises a nucleic acid sequence positioned between two inverted terminal repeats (ITRs), wherein the nucleic acid sequence when expressed inhibits or suppresses the expression of a target gene in a cell. The nucleic acid sequence comprises, in a 5' to 3' order: a region encoding a first sense strand sequence, a region encoding a first antisense strand sequence, a region encoding a second sense strand sequence, and a region encoding a second antisense strand sequence. The first and second antisense strand sequences are complementary to an mRNA produced by the target gene. The first sense strand sequence and the first antisense strand sequence, and the second sense strand sequence and the second antisense strand sequence have complementary regions, respectively.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions, methods and processes for designing, preparing, manufacturing, using and / or formulating regulatory polynucleotides, such as polynucleotides encoding at least one small interfering RNA (siRNA) molecule that targets at least one gene of interest.Targeting a gene of interest can interfere with gene expression and resulting protein production.AAV particles containing regulatory polynucleotides encoding at least one siRNA molecule can be inserted into a recombinant adeno-associated virus (AAV) vector.A method for using AAV particles to inhibit the expression of a gene of interest in a subject is also disclosed. [Background technology]

[0002] MicroRNAs (or miRNAs or miRs) are small, non-coding, single-stranded ribonucleic acid molecules (RNAs), typically 19–25 nucleotides in length. More than 1,000 microRNAs have been identified in mammalian genomes. Mature microRNAs primarily bind to the 3'-untranslated region (3'-UTR) of target messenger RNAs (mRNAs) by partially or fully pairing with complementary sequences in the target mRNA, promoting the degradation of the target mRNA at the post-transcriptional level and, in some cases, inhibiting translation initiation. MicroRNAs play crucial roles in numerous key biological processes, including cell cycle and growth control, apoptosis, cell proliferation, and tissue development.

[0003] miRNA genes are generally transcribed as long primary miRNA transcripts (i.e., pri-miRNAs), which are cleaved into precursors of the miRNAs (i.e., pre-miRNAs) and further processed to generate mature and functional miRNAs.

[0004] Although many targeted expression strategies use nucleic acid-based modalities, there remains a need for improved nucleic acid modalities with higher specificity and fewer off-target effects. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides such improved modalities in the form of artificial pri-, pre-, and mature microRNA constructs, as well as methods for their design. These novel constructs can be synthetic standalone molecules or encoded in plasmids or expression vectors for delivery to cells. Such vectors include, but are not limited to, adeno-associated virus vectors, such as the vector genome of any AAV serotype, or other viral delivery vehicles, such as lentivirus. [Means for solving the problem]

[0006] Described herein are methods, processes, compositions, kits, and devices for administering AAV particles comprising a regulatory polynucleotide encoding at least one siRNA molecule to treat, prevent, alleviate, and / or ameliorate diseases and / or disorders.

[0007] Details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. Representative, non-limiting embodiments illustrating the subject matter of this specification are set forth below.

[0008] 1. An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence located between two inverted terminal repeats (ITRs), wherein, upon expression, the nucleic acid inhibits or suppresses expression of a target gene in a cell, the nucleic acid sequence comprising, in 5' to 3' order, a first region encoding a first sense strand sequence, a second region encoding a first antisense strand sequence, a third region encoding a second sense strand, and a fourth region encoding a second antisense strand sequence, wherein the first and second sense strand sequences comprise at least 15 contiguous nucleotides, and the first and second antisense strand sequences are complementary to mRNA produced by the target gene and comprise at least 15 contiguous nucleotides, and the first sense strand sequence and the first antisense strand sequence share a region of complementarity at least 4 nucleotides in length, and the second sense strand sequence and the second antisense strand sequence share a region of complementarity at least 4 nucleotides in length.

[0009] 2. An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence located between two inverted terminal repeats (ITRs), the nucleic acid inhibiting or suppressing expression of a first target gene and a second target gene in a cell when expressed, the nucleic acid sequence comprising, in 5' to 3' order, a first region encoding a first sense strand sequence, a second region encoding a first antisense strand sequence, a third region encoding a second sense strand, and a fourth region encoding a second antisense strand sequence, the first and second sense strand sequences comprising at least 15 contiguous nucleotides; and an adeno-associated virus (AAV) viral genome, wherein the first antisense strand sequence is complementary to an mRNA produced by the first target gene, and the second antisense strand sequence is complementary to an mRNA produced by the second target gene and comprises at least 15 contiguous nucleotides, and the first sense strand sequence and the first antisense strand sequence share a region of complementarity that is at least 4 nucleotides in length, and the second sense strand sequence and the second antisense strand sequence share a region of complementarity that is at least 4 nucleotides in length.

[0010] 3. The AAV viral genome of embodiment 2, further comprising, in 5' to 3' order, a fifth region encoding a third sense strand sequence and a sixth region encoding a third antisense strand sequence, wherein the third sense strand sequence comprises at least 15 contiguous nucleotides, and the third antisense strand sequence is complementary to an mRNA produced by a third target gene and comprises at least 15 contiguous nucleotides, and the third sense strand sequence and the third antisense strand sequence share a region of complementarity of at least 4 nucleotides.

[0011] 4. The AAV viral genome of embodiment 3, further comprising, in 5' to 3' order, a seventh region encoding a fourth sense strand sequence and an eighth region encoding a fourth antisense strand sequence, wherein the fourth sense strand sequence comprises at least 15 contiguous nucleotides, and the fourth antisense strand sequence is complementary to an mRNA produced by a fourth target gene and comprises at least 15 contiguous nucleotides, and the fourth sense strand sequence and the fourth antisense strand sequence share a region of complementarity of at least 4 nucleotides.

[0012] 5. The AAV viral genome of embodiment 2, wherein the first target gene is the same as the second target gene. 6. The AAV viral genome of embodiment 3, wherein the third target gene is the same as the first target gene.

[0013] 7. The AAV viral genome of embodiment 3, wherein the third target gene is the same as the second target gene. 8. The AAV viral genome of embodiment 3, wherein the first target gene, the second target gene, and the third target gene are the same.

[0014] 9. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the first target gene. 10. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the second target gene.

[0015] 11. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the third target gene. 12. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the first target gene and the second target gene.

[0016] 13. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the second target gene and the third target gene. 14. The AAV viral genome of embodiment 4, wherein the fourth target gene is the same as the first target gene, the second target gene, and the third target gene.

[0017] 15. The AAV viral genome of any one of embodiments 1 to 14, wherein the first target gene, the second target gene, the third target gene and / or the fourth target gene is huntingtin.

[0018] 16. The AAV viral genome of any one of embodiments 1 to 14, wherein the first target gene, the second target gene, the third target gene and / or the fourth target gene is SOD1.

[0019] 17. The AAV viral genome of any one of embodiments 1 to 14, wherein the first target gene, the second target gene, the third target gene and / or the fourth target gene is huntingtin or SOD1.

[0020] 18. The AAV viral genome of embodiment 1 or 2, wherein the region of complementarity between the first sense strand and the first antisense strand is at least 12 nucleotides in length. 19. The AAV viral genome of embodiment 18, wherein the region of complementarity between the first sense strand and the first antisense strand is 14 to 21 nucleotides in length.

[0021] 20. The AAV viral genome of embodiment 19, wherein the region of complementarity between the first sense strand and the first antisense strand is 19 nucleotides in length. 21. The AAV viral genome of embodiment 1 or 2, wherein the region of complementarity between the second sense strand and the second antisense strand is at least 12 nucleotides in length.

[0022] 22. The AAV viral genome of embodiment 21, wherein the region of complementarity between the second sense strand and the second antisense strand is 14 to 21 nucleotides in length. 23. The AAV viral genome of embodiment 22, wherein the region of complementarity between the second sense strand and the second antisense strand is 19 nucleotides in length.

[0023] 24. The AAV viral genome of embodiment 3, wherein the region of complementarity between the third sense strand and the third antisense strand is at least 12 nucleotides in length. 25. The AAV viral genome of embodiment 24, wherein the region of complementarity between the third sense strand and the third antisense strand is 14 to 21 nucleotides in length.

[0024] 26. The AAV viral genome of embodiment 25, wherein the region of complementarity between the third sense strand and the third antisense strand is 19 nucleotides in length. 27. The AAV viral genome of embodiment 4, wherein the region of complementarity between the fourth sense strand and the fourth antisense strand is at least 12 nucleotides in length.

[0025] 28. The AAV viral genome of embodiment 27, wherein the region of complementarity between the fourth sense strand and the fourth antisense strand is 14 to 21 nucleotides in length. 29. The AAV viral genome of embodiment 25, wherein the region of complementarity between the fourth sense strand and the fourth antisense strand is 19 nucleotides in length.

[0026] 30. The AAV viral genome of embodiment 1 or 2, wherein the first sense strand sequence, the second sense strand sequence, the first antisense strand sequence, and the second antisense strand sequence are independently 30 nucleotides or less.

[0027] 31. The AAV viral genome of embodiment 3, wherein the first sense strand sequence, the second sense strand sequence, the third sense strand sequence, the first antisense strand sequence, the second antisense strand sequence, and the third antisense strand sequence are independently 30 nucleotides or less.

[0028] 32. The AAV viral genome of embodiment 4, wherein the first sense strand sequence, the second sense strand sequence, the third sense strand sequence, the fourth sense strand sequence, the first antisense strand sequence, the second antisense strand sequence, the third antisense strand sequence, and the fourth antisense strand sequence are independently 30 nucleotides or less.

[0029] 33. The AAV viral genome of embodiment 1 or 2, wherein at least one of the first sense strand sequence and the first antisense strand sequence or the second sense strand sequence and the second antisense strand sequence comprises a 3' overhang of at least one nucleotide.

[0030] 34. The AAV viral genome of embodiment 1 or 2, wherein at least one of the first sense strand sequence and the first antisense strand sequence or the second sense strand sequence and the second antisense strand sequence comprises a 3' overhang of at least two nucleotides.

[0031] 35. The AAV viral genome of embodiment 3, wherein the third sense strand sequence and the third antisense strand sequence comprise a 3' overhang of at least one nucleotide. 36. The AAV viral genome of embodiment 3, wherein the third sense strand sequence and the third antisense strand sequence comprise a 3' overhang of at least 2 nucleotides.

[0032] 37. The AAV viral genome of embodiment 4, wherein the fourth sense strand sequence and the fourth antisense strand sequence comprise a 3' overhang of at least one nucleotide. 38. The AAV viral genome of embodiment 4, wherein the fourth sense strand sequence and the fourth antisense strand sequence comprise a 3' overhang of at least 2 nucleotides.

[0033] 39. The AAV viral genome of any one of embodiments 1-38, wherein the first region comprises a promoter 5' of the first sense strand sequence followed by the first sense strand sequence, and the second region comprises the first antisense strand sequence followed by a promoter terminator 3' of the first antisense strand sequence, or the third region comprises a promoter 5' of the second sense strand sequence followed by the second sense strand sequence, and the fourth region comprises the second antisense strand sequence followed by a promoter terminator 3' of the second antisense strand sequence.

[0034] 40. The AAV viral genome of any one of embodiments 1-38, wherein the first region comprises a promoter 5' of the first sense strand sequence followed by the first sense strand sequence, and the second region comprises the first antisense strand sequence followed by a promoter terminator 3' of the first antisense strand sequence, and the third region comprises a promoter 5' of the second sense strand sequence followed by the second sense strand sequence, and the fourth region comprises the second antisense strand sequence followed by a promoter terminator 3' of the second antisense strand sequence.

[0035] 41. The AAV viral genome of any one of embodiments 3-40, wherein the fifth region comprises a promoter 5' of the third sense strand sequence followed by the third sense strand sequence, and the sixth region comprises the third antisense strand sequence followed by a promoter terminator 3' of the third antisense strand sequence.

[0036] 42. The AAV viral genome of any one of embodiments 4-41, wherein the seventh region comprises a promoter 5' of the fourth sense strand sequence followed by the fourth sense strand sequence, and the eighth region comprises the fourth antisense strand sequence followed by a promoter terminator 3' of the fourth antisense strand sequence.

[0037] 43. The AAV viral genome of embodiment 3, wherein the fifth region is 3' to the fourth region. 44. The AAV viral genome of embodiment 4, wherein the seventh region is 3' to the sixth region.

[0038] 45. The AAV viral genome of any one of embodiments 39 to 44, wherein the promoter is a Pol III promoter and the promoter terminator is a Pol III promoter terminator.

[0039] 46. ​​The AAV viral genome of embodiment 45, wherein the Pol III promoter is U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter, and the Pol III promoter terminator is U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter terminator, respectively.

[0040] 47. The AAV viral genome of embodiment 46, wherein the Pol III promoter is an H1 promoter and the Pol III promoter terminator is an H1 promoter terminator.

[0041] 48. The AAV viral genome of any one of embodiments 1 to 47, which is a monospecific polycistronic AAV viral genome. 49. The AAV viral genome of any one of embodiments 1 to 47, which is a bispecific polycistronic AAV viral genome.

[0042] 50. The AAV viral genome of embodiment 1 or 2, wherein the first region and the second region encode a first siRNA molecule, and the third region and the fourth region encode a second siRNA molecule, and the first and second siRNA molecules target different target genes.

[0043] 51. The AAV viral genome of embodiment 3, wherein the fifth region and the sixth region encode a third siRNA molecule, and the first siRNA molecule, the second siRNA molecule, and the third siRNA molecule each target a different target gene.

[0044] 52. The AAV viral genome of embodiment 4, wherein the seventh region and the eighth region encode a fourth siRNA molecule, and the first siRNA molecule, the second siRNA molecule, the third siRNA molecule, and the fourth siRNA molecule each target a different target gene.

[0045] 53. An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence located between two inverted terminal repeats (ITRs), wherein the nucleic acid sequence comprises a first molecular scaffold region and a second molecular scaffold region, and the first molecular scaffold region comprises: (a) a first stem and loop forming a first stem-loop structure, the sequence of the first stem-loop structure being, from 5' to 3', i. a first UG motif at or near the base of the first 5' stem of said first stem-loop structure; ii. a first 5' stem arm comprising a first sense strand and optionally a first 5' spacer region, wherein, if present, said first 5' spacer region is located between said first UG motif and said first sense strand; iii. a first loop region comprising a first UGUG motif at its 5' end; iv. a first 3' stem arm comprising a first antisense strand and optionally a first 3' spacer region, wherein a uridine is present at the 5' end of the first antisense strand, and, if present, the first 3' spacer region has a length sufficient to form one helical turn. a first stem and loop comprising: (b) a first 5' flanking region located 5' of the first stem-loop structure; and (c) a first 3' flanking region located 3' of the first stem-loop structure, the first 3' flanking region comprising a CNNC motif; a first molecular scaffold nucleic acid sequence encoding the second molecular scaffold region comprises: (d) a second stem and loop forming a second stem-loop structure, the sequence of the second stem-loop structure being, from 5' to 3', v. a second UG motif at or near the base of the second 5' stem of said second stem-loop structure; vi. a second 5' stem arm comprising a second sense strand and optionally a second 5' spacer region, wherein, if present, the second 5' spacer region is located between the second UG motif and the second sense strand; vii. a second loop region containing a second UGUG motif at its 5' end; viii. a second 3' stem arm comprising a second antisense strand and optionally a second 3' spacer region, wherein a uridine is present at the 5' end of the second antisense strand, and, if present, the second 3' spacer region has a length sufficient to form one helical turn; ix. a second stem and loop comprising a second 5' flanking region located 5' of the second stem-loop structure; and (e) a second 3' flanking region located 3' of the second stem-loop structure, the second 3' flanking region comprising a CNNC motif; a second molecular scaffold nucleic acid sequence encoding an adeno-associated virus (AAV) viral genome, wherein the first antisense strand and the first sense strand form a first siRNA duplex, and the second antisense strand and the second sense strand form a second siRNA duplex, the first siRNA duplex inhibits or suppresses expression of a first target gene in a cell when expressed, and the second siRNA duplex inhibits or suppresses expression of a second target gene in a cell when expressed, the first and second sense strand sequences comprise at least 15 nucleotides, the first antisense strand sequence is complementary to an mRNA produced by the first target gene, and the second antisense strand sequence is complementary to an mRNA produced by the second target gene, and the first sense strand sequence and the first antisense strand sequence share a region of complementarity at least 4 nucleotides in length, and the second sense strand sequence and the second antisense strand sequence share a region of complementarity at least 4 nucleotides in length.

[0046] 54. An adeno-associated virus (AAV) viral genome comprising a nucleic acid sequence located between two inverted terminal repeats (ITRs), wherein the nucleic acid sequence comprises a first molecular scaffold region and a second molecular scaffold region, and the first molecular scaffold region comprises: (a) a first stem and loop forming a first stem-loop structure, the sequence of the first stem-loop structure being, from 5' to 3', i. a first UG motif at or near the base of the first 5' stem of said first stem-loop structure; ii. a first 5' stem arm comprising a first antisense strand and optionally a first 5' spacer region, wherein, if present, said first 5' spacer region is located between said first UG motif and said first antisense strand; iii. a first loop region comprising a first UGUG motif at its 5' end; iv. a first 3' stem arm comprising a first sense strand and optionally a first 3' spacer region, wherein a uridine is present at the 5' end of the first sense strand, and when present, the first 3' spacer region has a length sufficient to form one helical turn; a first stem and loop comprising: (b) a first 5' flanking region located 5' of the first stem-loop structure; and (c) a first 3' flanking region located 3' of the first stem-loop structure, the first 3' flanking region including a CNNC motif; a first molecular scaffold nucleic acid sequence encoding the second molecular scaffold region comprises: (d) a second stem and loop forming a second stem-loop structure, the sequence of the second stem-loop structure being, from 5' to 3', v. a UG motif at or near the base of the second 5' stem of said second stem-loop structure; vi. a second 5' stem arm comprising a second antisense strand and optionally a second 5' spacer region, wherein, if present, the 5' spacer region is located between the second UG motif and the second antisense strand; vii. a second loop region containing a second UGUG motif at its 5' end; viii. A second 3' stem arm comprising a second sense strand and optionally a second 3' spacer region, wherein a uridine is present at the 5' end of the second sense strand, and, if present, the second 3' spacer region has a length sufficient to form one helical turn. a second stem and loop comprising: (e) a second 5' flanking region located 5' of the second stem-loop structure; and (f) a second 3' flanking region located 3' of the second stem-loop structure, the second 3' flanking region comprising a CNNC motif; a second molecular scaffold nucleic acid sequence encoding an adeno-associated virus (AAV) viral genome, wherein the first antisense strand and the first sense strand form a first siRNA duplex, and the second antisense strand and the second sense strand form a second siRNA duplex, the first siRNA duplex inhibits or suppresses expression of a first target gene in a cell when expressed, and the second siRNA duplex inhibits or suppresses expression of a second target gene in a cell when expressed, the first and second sense strand sequences comprise at least 15 nucleotides, the first antisense strand sequence is complementary to an mRNA produced by the first target gene, and the second antisense strand sequence is complementary to an mRNA produced by the second target gene, and the first sense strand sequence and the first antisense strand sequence share a region of complementarity at least 4 nucleotides in length, and the second sense strand sequence and the second antisense strand sequence share a region of complementarity at least 4 nucleotides in length.

[0047] 55. The AAV viral genome of embodiment 53 or 54, wherein the first antisense strand sequence or the second antisense strand sequence inhibits or suppresses the expression of huntingtin.

[0048] 56. The AAV viral genome of embodiment 53 or 54, wherein the first antisense strand sequence and the second antisense strand sequence inhibit or suppress the expression of huntingtin.

[0049] 57. The AAV viral genome of embodiment 53 or 54, wherein the first antisense strand sequence or the second antisense strand sequence inhibits or suppresses the expression of SOD1.

[0050] 58. The AAV viral genome of embodiment 53 or 54, wherein the first antisense strand sequence and the second antisense strand sequence inhibit or suppress the expression of SOD1.

[0051] 59. The AAV viral genome of embodiment 53 or 54, wherein the first 5' flanking region is selected from the sequences listed in Table 10. 60. The AAV viral genome of embodiment 53 or 54, wherein the second 5' flanking region is selected from the sequences listed in Table 10.

[0052] 61. The AAV viral genome of embodiment 59, wherein the second 5' flanking region is selected from the sequences listed in Table 10. 62. The AAV viral genome of embodiment 53 or 54, wherein the first loop region is selected from the sequences listed in Table 11.

[0053] 63. The AAV viral genome of embodiment 53 or 54, wherein the second loop region is selected from the sequences listed in Table 11. 64. The AAV viral genome of embodiment 62, wherein the second loop region is selected from the sequences listed in Table 11.

[0054] 65. The AAV viral genome of embodiment 53 or 54, wherein the first 3' flanking region is selected from the sequences listed in Table 12. 66. The AAV viral genome of embodiment 53 or 54, wherein the second 3' flanking region is selected from the sequences listed in Table 12.

[0055] 67. The AAV viral genome of embodiment 65, wherein the second 3' flanking region is selected from the sequences listed in Table 12. 68. The AAV viral genome of embodiment 53 or 54, wherein the nucleic acid sequence comprises a promoter sequence between the first molecular scaffold nucleic acid sequence and the second molecular scaffold nucleic acid sequence.

[0056] 69. The AAV viral genome of embodiment 53 or 54, further comprising: (b) a promoter 5' of the first 5' flanking region followed by the first 5' flanking region; (c) a first 3' flanking region followed by a promoter terminator 3' of the first 3' flanking region; (d) a promoter 5' of the second 5' flanking region followed by the second 5' flanking region; and (e) a second 3' flanking region followed by a promoter terminator 3' of the second 3' flanking region.

[0057] 70. The AAV viral genome of embodiment 69, wherein the promoter is a Pol III promoter. 71. The AAV viral genome of embodiment 70, wherein the Pol III promoter sequence is U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter.

[0058] 72. The AAV viral genome of embodiment 71, wherein the Pol III promoter is an H1 promoter. 73. The nucleic acid sequence: (g) a third stem and loop forming a third stem-loop structure, the sequence of the third stem-loop structure being, from 5' to 3', ix. a UG motif at or near the base of the third 5' stem of said third stem-loop structure; x. a third 5' stem arm comprising a third sense strand and optionally a third 5' spacer region, wherein, if present, the third 5' spacer region is located between the third UG motif and the third sense strand; xi. a third loop region containing a third UGUG motif at its 5' end; xii. A third 3' stem arm comprising a third antisense strand and optionally a third 3' spacer region, wherein a uridine is present at the 5' end of the third antisense strand, and, if present, the third 3' spacer region has a length sufficient to form one helical turn. a third stem and loop, including: (h) a third 5' flanking region located 5' of the third stem-loop structure; and (i) a third 3' flanking region located 3' of the third stem-loop structure, the third 3' flanking region including a CNNC motif; and a third molecular scaffold region comprising a third molecular scaffold nucleic acid sequence encoding 54. The AAV viral genome of embodiment 53, wherein the third antisense strand and the third sense strand form a third siRNA duplex, which, when expressed, inhibits or suppresses expression of a third target gene in a cell, the third sense strand sequence comprises at least 15 nucleotides, the third antisense strand sequence is complementary to mRNA produced by the third target gene, and the third sense strand sequence and the third antisense strand sequence share a region of complementarity at least 4 nucleotides in length.

[0059] 74. The AAV viral genome of embodiment 73, further comprising a promoter 5' of the third 5' flanking region followed by the third 5' flanking region in (h), and a promoter terminator 3' of the third flanking region followed by the third flanking region in (i).

[0060] 75. The AAV viral genome of embodiment 74, wherein the promoter is a Pol III promoter. 76. The AAV viral genome of embodiment 75, wherein the Pol III promoter sequence is U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter.

[0061] 77. The AAV viral genome of embodiment 76, wherein the Pol III promoter is an H1 promoter. 78. The nucleic acid sequence: (j) a fourth stem and loop forming a fourth stem-loop structure, the sequence of the fourth stem-loop structure being, from 5' to 3', xiii. a fourth UG motif at or near the base of the fourth 5' stem of the fourth stem-loop structure; xiv. a fourth 5' stem arm comprising a fourth sense strand and optionally a fourth 5' spacer region, wherein, if present, the fourth 5' spacer region is located between the fourth UG motif and the fourth sense strand; xv. a fourth loop region containing a fourth UGUG motif at its 5' end; xvi. A fourth 3' stem arm comprising a fourth antisense strand and optionally a fourth 3' spacer region, wherein a uridine is present at the 5' end of the fourth antisense strand, and, if present, the fourth 3' spacer region has a length sufficient to form one helical turn. a fourth stem and loop, including; (k) a fourth 5' flanking region located 5' of the fourth stem-loop structure; and (l) a fourth 3' flanking region located 3' of the fourth stem-loop structure, the fourth 3' flanking region including a CNNC motif; and a fourth molecular scaffold region comprising a fourth molecular scaffold nucleic acid sequence encoding 74. The AAV viral genome of embodiment 73, wherein the fourth antisense strand and the fourth sense strand form a fourth siRNA duplex, which, when expressed, inhibits or suppresses expression of a fourth target gene in a cell, the fourth sense strand sequence comprises at least 15 nucleotides, the fourth antisense strand sequence is complementary to mRNA produced by the fourth target gene, and the fourth sense strand sequence and the fourth antisense strand sequence share a region of complementarity at least 4 nucleotides in length.

[0062] 79. The AAV viral genome of embodiment 78, further comprising in (k) a promoter 5' of the fourth 5' flanking region followed by the fourth 5' flanking region, and in (l) a fourth 3' flanking region followed by a promoter terminator 3' of the fourth 3' flanking region.

[0063] 80. The AAV viral genome of embodiment 79, wherein the promoter is a Pol III promoter. 81. The AAV viral genome of embodiment 80, wherein the Pol III promoter sequence is U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter.

[0064] 82. The AAV viral genome of embodiment 81, wherein the Pol III promoter is an H1 promoter. 83. The AAV viral genome of any one of embodiments 53 to 82, wherein the first target gene is the same as the second target gene.

[0065] 84. The AAV viral genome of any one of embodiments 53 to 82, wherein the third target gene is the same as the first target gene. 85. The AAV viral genome of any one of embodiments 53 to 82, wherein the third target gene is the same as the second target gene.

[0066] 86. An AAV viral genome described in any one of embodiments 53 to 82, wherein the first target gene, the second target gene, and the third target gene are the same. 87. The AAV viral genome of any one of embodiments 53 to 82, wherein the fourth target gene is the same as the first target gene.

[0067] 88. The AAV viral genome of any one of embodiments 53 to 82, wherein the fourth target gene is the same as the second target gene. 89. The AAV viral genome of any one of embodiments 53 to 82, wherein the fourth target gene is the same as the third target gene.

[0068] 90. An AAV viral genome described in any one of embodiments 53 to 82, wherein the fourth target gene is the same as the first target gene and the second target gene. 91. An AAV viral genome described in any one of embodiments 53 to 82, wherein the fourth target gene is the same as the second target gene and the third target gene.

[0069] 92. The AAV viral genome of any one of embodiments 53 to 82, wherein the fourth target gene is the same as the first target gene and the third target gene. 93. The AAV viral genome of any one of embodiments 53 to 82, wherein the fourth target gene is the same as the first target gene, the second target gene, and the third target gene.

[0070] 94. The AAV viral genome of any one of embodiments 53 to 93, wherein the first target gene, the second target gene, the third target gene, and / or the fourth target gene is huntingtin.

[0071] 95. The AAV viral genome of any one of embodiments 53 to 93, wherein the first target gene, the second target gene, the third target gene and / or the fourth target gene is SOD1.

[0072] 96. The AAV viral genome of any one of embodiments 53 to 93, wherein the first target gene, the second target gene, the third target gene and / or the fourth target gene is huntingtin or SOD1.

[0073] 97. A method for inhibiting expression of a target gene in a cell, the method comprising administering to the cell a composition comprising an AAV viral genome described in any one of embodiments 1 to 96.

[0074] 98. The method of embodiment 97, wherein the cell is a mammalian cell. 99. The method of embodiment 98, wherein the mammalian cell is a medium spiny neuron. 100. The method of embodiment 98, wherein the mammalian cell is a cortical neuron.

[0075] 101. The method of embodiment 98, wherein the mammalian cell is a motor neuron. 102. The method of embodiment 98, wherein the mammalian cells are astrocytes. 103. A method for treating a disease and / or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising an AAV viral genome described in any one of embodiments 1 to 96.

[0076] 104. The method of embodiment 103, wherein expression of the target gene is inhibited or suppressed. 105. The method of embodiment 104, wherein expression of the target gene of interest is inhibited or suppressed by about 30% to about 70%.

[0077] 106. The method of embodiment 104, wherein expression of the target gene is inhibited or suppressed by about 50% to about 90%. 107. A method for inhibiting expression of a target gene in a cell, wherein the target gene causes a gain-of-function effect in the cell, the method comprising administering to the cell a composition comprising an AAV viral genome described in any one of embodiments 1 to 96.

[0078] 108. The method of embodiment 107, wherein the cell is a mammalian cell. 109. The method of embodiment 108, wherein the mammalian cell is a medium spiny neuron. 110. The method of embodiment 108, wherein the mammalian cell is a cortical neuron.

[0079] 111. The method of embodiment 108, wherein the mammalian cell is a motor neuron. 112. The method of embodiment 108, wherein the mammalian cells are astrocytes. The foregoing and other objects, features, and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, which are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. [Brief explanation of the drawings]

[0080] [Figure 1] Schematic diagram of the viral genome of the present invention. [Figure 2] Schematic diagram of the viral genome of the present invention. [Figure 3] Schematic diagram of the viral genome of the present invention. [Figure 4] Schematic diagram of the viral genome of the present invention. [Figure 5] Schematic diagram of the viral genome of the present invention. [Figure 6] Schematic diagram of the viral genome of the present invention. [Figure 7] Schematic diagram of the viral genome of the present invention. [Figure 8] Schematic diagram of the viral genome of the present invention. [Figure 9] Schematic diagram of the viral genome of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0081] The details of one or more embodiments of the present invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects, and advantages of the present invention will become apparent from this description. In the description, the singular includes the plural unless the context clearly dictates otherwise. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of a conflict, the present description shall control.

[0082] I. Compositions of the Invention In accordance with the present invention, compositions are provided for delivery of regulatory polynucleotides and / or regulatory polynucleotide-based compositions by adeno-associated virus (AAV). The AAV particles of the present invention can be provided to cells, tissues, organs, or organisms in vivo, ex vivo, or in vitro via any of several routes of administration.

[0083] As used herein, an "AAV particle" is a virus that comprises a viral genome having at least one payload region and at least one inverted terminal repeat (ITR) region.

[0084] As used herein, "viral genome" or "vector genome" or "viral vector" refers to a nucleic acid sequence encapsulated in an AAV particle. The viral genome includes at least one payload region encoding a polypeptide or fragment thereof.

[0085] As used herein, a "payload" or "payload region" refers to any nucleic acid molecule encoding one or more polypeptides of the present invention. The payload region includes, at a minimum, nucleic acid sequences encoding sense and antisense sequences, siRNA-based compositions, or fragments thereof, but may also optionally include one or more functional or regulatory elements that facilitate transcriptional expression and / or polypeptide translation.

[0086] The nucleic acid sequences and polypeptides disclosed herein may be engineered to contain modular elements and / or sequence motifs assembled to enable expression of the regulatory polynucleotides and / or compositions based on the regulatory polynucleotides of the present invention. In some embodiments, the nucleic acid sequence comprising the payload region may include one or more of a promoter region, an intron, a Kozak sequence, an enhancer, or a polyadenylation sequence. The payload region of the present invention typically encodes at least one sense and antisense sequence, an siRNA-based composition, or fragments of the foregoing, in combination with each other or with other polypeptide moieties.

[0087] The payload region of the present invention within the viral genome of an AAV particle can be delivered to one or more target cells, tissues, organs or organisms. Adeno-associated virus (AAV) and AAV particles Parvoviridae viruses are small, non-enveloped, icosahedral-capsid viruses characterized by a single-stranded DNA genome. Parvoviridae viruses are composed of two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect invertebrates. This virus family is useful as a biological tool because of its relatively simple structure and ease of manipulation using standard molecular biology techniques. The viral genome can be modified to contain the minimum components for assembly of functional recombinant viruses or viral particles carrying or engineered to express or deliver a desired payload, which can then be delivered to target cells, tissues, organs, or organisms.

[0088] Parvoviruses and other members of the Parvoviridae family are reviewed in Chapter 69 of Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," FIELDS VIROLOGY (3rd ed., 1996), the contents of which are incorporated by reference in their entirety.

[0089] The Parvoviridae family includes the genus Dependovirus, which comprises the adeno-associated viruses (AAV) that are capable of replication in vertebrate hosts, including but not limited to, humans, primates, bovine, canine, equine, and ovine species.

[0090] The AAV viral genome is a linear single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) long. The AAV viral genome may contain a payload region and at least one inverted terminal repeat (ITR) or ITR region. Traditionally, ITRs flank the coding nucleotide sequences for nonstructural proteins (encoded by the Rep gene) and structural proteins (encoded by the capsid or Cap gene). Without wishing to be bound by theory, the AAV viral genome typically contains two ITR sequences. The AAV viral genome contains a characteristic T-shaped hairpin structure defined by self-complementary terminal 145 nt at the 5' and 3' ends of ssDNA, which form an energetically stable double-stranded region. The double-stranded hairpin structure has multiple functions, including, but not limited to, acting as a primer for the endogenous DNA polymerase complex of the host viral replicating cell, thereby serving as an origin of DNA replication.

[0091] In addition to encoding a heterologous payload, AAV vectors can contain, in whole or in part, the viral genome of any naturally occurring and / or recombinant AAV serotype nucleotide sequence or variant. AAV variants can have sequences that are significantly homologous at the nucleic acid level (genome or capsid) and amino acid level (capsid) so as to produce constructs that are generally physically and functionally equivalent, replicate by similar mechanisms, and assemble by similar mechanisms. Chiorini et al., J. Vir., Vol. 71, pp. 6823-6833 (1997); Srivastava et al., J. Vir., Vol. 45, pp. 555-5564 (1983); Chiorini et al., J. Vir., Vol. 73, pp. 1309-1319 (1999); Rutledge et al., J. Vir., Vol. 72, pp. 309-319 (1998); and Wu et al., J. Vir., Vol. 74, pp. 8635-8647 (2000). the contents of each of which are incorporated herein by reference in their entirety.

[0092] In one embodiment, the AAV particles of the present invention are replication-deficient recombinant AAV vectors that lack sequences encoding functional Rep and Cap proteins within their viral genome. These defective AAV vectors may lack most or all of the parent coding sequences and may essentially contain only one or two AAV ITR sequences and a nucleic acid of interest for delivery to a cell, tissue, organ, or organism.

[0093] In one embodiment, the viral genome of the AAV particle of the present invention comprises at least one control element that provides replication, transcription, and translation of the coding sequence encoded therein. Not all control elements need to be present, as long as the coding sequence can be replicated, transcribed, and / or translated in a suitable host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion.

[0094] According to the present invention, AAV particles used in therapy and / or diagnosis comprise a virus that has been purified or reduced to the minimum components necessary for transduction of a desired nucleic acid payload or cargo. In this manner, AAV particles are engineered as specific delivery vehicles while lacking the deleterious replication and / or integration characteristics seen in wild-type virus.

[0095] The AAV vectors of the present invention may be recombinantly produced or may be based on an adeno-associated virus (AAV) parent or reference sequence. As used herein, "vector" refers to any molecule or moiety that transports, transduces, or otherwise acts as a carrier of a heterologous molecule, such as a nucleic acid described herein.

[0096] In addition to single-stranded AAV viral genomes (e.g., ssAAV), the present invention also provides self-complementary AAV (scAAV) viral genomes. scAAV viral genomes contain DNA strands that anneal together to form double-stranded DNA. By skipping second-strand synthesis, scAAV can be rapidly expressed in cells.

[0097] In one embodiment, the AAV particles of the invention are scAAV. In one embodiment, the AAV particles of the invention are ssAAV. Methods for producing and / or modifying AAV particles, such as pseudotyped AAV vectors, have been disclosed in the art (WO 200028004, WO 200123001, WO 2004112727, WO 2005005610, and WO 2005072364, the contents of each of which are incorporated herein by reference in their entirety).

[0098] AAV particles can be modified to enhance delivery efficiency.Such modified AAV particles can be efficiently packaged and can be used to successfully infect target cells at high frequency and with minimal toxicity.In some embodiments, the capsid of AAV particles is engineered according to the method described in US Patent Application Publication No. 20130195801.The contents of this application are incorporated herein by reference in their entirety.

[0099] In one embodiment, AAV particles comprising a payload region encoding a polypeptide of the invention can be introduced into mammalian cells. AAV serotype The AAV particles of the present invention may comprise or be derived from any natural or recombinant AAV serotype. According to the present invention, the AAV particles may be any of the following: AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV17.4, AAV18.4, AAV19.4, AAV20.4, AAV21.4, AAV22.4, AAV23.4, AAV24.4, AAV25.4, AAV26.4, AAV27.4, AAV28.4, AAV29.4, AAV29.5, AAV29.6, AAV29.7, AAV29.8, AAV29.9, AAV30.4, AAV31.4, AAV32.4, AAV33.4, AAV34.4, AAV35.4, AAV36.4, AAV37.4, AAV38.4, AAV39.4, AAV39.5, AAV39.6, AAV39.7, AAV39.8, AAV39.9, AAV40.4, AAV41.4, AAV42.4, AAV43.4, AAV44.4, AAV45.4, AAV46.4, AAV47.4, A AV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-1 1, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV 44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV33.8 / hu.16、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV33.8 / hu.16 AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh6. 0、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.4 0、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3 、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu. 16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29、AAVhu.31、AAVhu. Vhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu. u.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R、AAVrh8R R533A、AAAV、BAAV、ヤギAAV、ウシAAV、ヒツジAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35 、AAVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、A AV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-L K16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-P AEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV BC 10-2、AAVシャッフル100-1、AAVシャッフル100-3、AAVシャッフル100-7、AAVシ10-2 AAV 10-6 AAV 10-8 AAV 100-2 AAV SM 10-1、AAV SM 10-8、AAV SM 100-3、AAV SM 100-10、BNP61 AAV、BNP62 AAV、BNP63. AAV、AAVrh.50、AAVrh.43、AAVrh.62、AAVrh.48、AAVhu.19、AAVhu.11、AAVhu.53、AAV4-8 / rh.64、AAVLG-9 / hu.39、AAV54.5 / hu.23、AAV54.2 / hu.22、AAV54.7 / hu.24、AAV54.1 / hu.21、AAV54.4R / hu.27、AAV46.2 / hu.28、AAV46.6 / hu.29、AAV128.1 / hu.43、トゥルータップAAV(ttAAV)、UPENN AAV10、ジャパニーズAAV10 serotype、AAV CBr-7.1、AAV CBr-7.10、AAV CBr-7.2、AAV CBr-7.3、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV.hu.48R3、AAV.VR-355、AAV3B、AAV4、AAV5、AAVF1 / HSC1、AAVF11 / HSC11、AAVF12 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AA Serotypes selected from VF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5 and variants thereof may be utilized or may be based thereon.

[0100] In some embodiments, the AAV serotypes include, but are not limited to, AAV1 (SEQ ID NOs: 6 and 64 in US Patent Application Publication No. 20030138772), AAV2 (SEQ ID NOs: 7 and 70 in US Patent Application Publication No. 20030138772), AAV3 (SEQ ID NOs: 8 and 71 in US Patent Application Publication No. 20030138772), AAV4 (SEQ ID NO: 63 in US Patent Application Publication No. 20030138772), AAV5 (SEQ ID NO: 114 in US Patent Application Publication No. 20030138772), AAV6 (SEQ ID NO: 115 in US Patent Application Publication No. 20030138772), AAV7 (SEQ ID NO: 116 in US Patent Application Publication No. 20030138772), AAV8 (SEQ ID NO: 117 in US Patent Application Publication No. 20030138772), AAV9 (SEQ ID NO: 119 in US Patent Application Publication No. 20030138772), AAV10 (SEQ ID NO: 119 in US Patent Application Publication No. 20030138772), AAV11 (SEQ ID NO: 119 in US Patent Application Publication No. 20030138772), AAV12 (SEQ ID NO: 120 in US Patent Application Publication No. 20030138772), AAV13 (SEQ ID NO: 121 in US Patent Application Publication No. 20030138772), AAV14 (SEQ ID NO: 122 in US Patent Application Publication No. 20030138772), AAV15 (SEQ ID NO: 123 in US AAV7 (SEQ ID NO: 65 in U.S. Patent Application Publication No. 20030138772), AAV8 (SEQ ID NOs: 4 and 95 in U.S. Patent Application Publication No. 20030138772), AAV9 (SEQ ID NOs: 5 and 100 in U.S. Patent Application Publication No. 20030138772), AAV10 (SEQ ID NO: 117 in U.S. Patent Application Publication No. 20030138772), AAV11 (SEQ ID NO: 118 in U.S. Patent Application Publication No. 20030138772), AAV12 (SEQ ID NO: 120 in U.S. Patent Application Publication No. 20030138772), AAV13 (SEQ ID NO: 121 in U.S. Patent Application Publication No. 20030138772), AAV14 (SEQ ID NO: 122 in U.S. Patent Application Publication No. 20030138772), AAV15 (SEQ ID NO: 123 in U.S. Patent Application Publication No. 20030138772), AAV16 (SEQ ID NO: 124 in U.S. Patent Application Publication No. 20030138772), AAV17 (SEQ ID NO: 125 in U.S. Patent Application Publication No. 20030138772), AAV18 (SEQ ID NO: 126 in U.S. Patent Application Publication No. 20030138772), AAV19 (SEQ ID NO: 127 in U.S. Patent Application Publication No. 20030138772), AAV20 (SEQ ID NO: 128 in U.S. Patent Application Publication No. 20 U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 119), AAVrh10 (amino acids 1 to 738 of SEQ ID NO: 81 of U.S. Patent Application Publication No. 20030138772), AAV16.3 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 10), AAV29.3 / bb.1 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 11), AAV29.4 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 12), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 13), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 14), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 15), AAV29.5 / bb.1 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 16), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 17), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 18), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 19), AAV29.5 / bb.1 (U.S. Patent Application Publication No. Sequence No. 13), AAV1.3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 14), AAV13.3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 15), AAV24.1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 16), AAV27.3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 17), AAV7.2 (US Patent Application Publication No. 20030138772, SEQ ID NO: 18), AAVC1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 19),AAVC3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 20), AAVC5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 21), AAVF1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 22), AAVF3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 23), AAVF5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 24), AAVH6 (US Patent Application Publication No. 20030138772, SEQ ID NO: 25), AAVH2 (US Patent Application Publication No. 20030138772, SEQ ID NO: 26), 8772, SEQ ID NO: 26), AAV42-8 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 27), AAV42-15 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 28), AAV42-5b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 29), AAV42-1b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 30), AAV42-13 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 31), AAV42-3a (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 32), AAV42-5b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 33), AAV42-1b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 34), AAV42-13 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 35), AAV42-3a (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 36), AAV42-5b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 37), AAV42-1b (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 38), AAV42-13b (U.S. Patent Application Publication No. 2003013877 SEQ ID NO: 32), AAV42-4 (US Patent Application Publication No. 20030138772, SEQ ID NO: 33), AAV42-5a (US Patent Application Publication No. 20030138772, SEQ ID NO: 34), AAV42-10 (US Patent Application Publication No. 20030138772, SEQ ID NO: 35), AAV42-3b (US Patent Application Publication No. 20030138772, SEQ ID NO: 36), AAV42-11 (US Patent Application Publication No. 20030138772, SEQ ID NO: 37), AAV42-6b (US Patent Application Publication No. 20030138772, SEQ ID NO: 38), AAV42-5b (US Patent Application Publication No. 20030138772, SEQ ID NO: 39), AAV42-6c (US Patent Application Publication No. 20030138772, SEQ ID NO: 40), AAV42-7d (US Patent Application Publication No. 20030138772, SEQ ID NO: 41), AAV42-8e (US Patent Application Publication No. 20030138772, SEQ ID NO: 42), AAV42-9e (US Patent Application Publication No. 20030138772, SEQ ID NO: 43), AAV42-10f (US Patent Application Publication No. 20030138772, SEQ ID NO: 44), AAV42-11f (US Patent Application Publication No. 20030138772, SEQ ID NO: 45), AAV42-12f (US Patent Application Publication No. 20030138772, SEQ ID NO: No. 38), AAV43-1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 39), AAV43-5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 40), AAV43-12 (US Patent Application Publication No. 20030138772, SEQ ID NO: 41), AAV43-20 (US Patent Application Publication No. 20030138772, SEQ ID NO: 42), AAV43-21 (US Patent Application Publication No. 20030138772, SEQ ID NO: 43), AAV43-23 (US Patent Application Publication No. 20030138772, SEQ ID NO: 44),AAV43-25 (US Patent Application Publication No. 20030138772, SEQ ID NO: 45), AAV44.1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 46), AAV44.5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 47), AAV223.1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 48), AAV223.2 (US Patent Application Publication No. 20030138772, SEQ ID NO: 49), AAV223.3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 50), AAV223.4 (US Patent Application Publication No. 20030138772, SEQ ID NO: 51), AAV223.5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 52), AAV223.6 (US Patent Application Publication No. 20030138772, SEQ ID NO: 53), AAV223.7 (US Patent Application Publication No. 20030138772, SEQ ID NO: 54), AAV223.8 (US Patent Application Publication No. 20030138772, SEQ ID NO: 55), AAV223.9 (US Patent Application Publication No. 20030138772, SEQ ID NO: 56), AAV223.10 (US Patent Application Publication No. 20030138772, SEQ ID NO: 57), AAV223.21 (US Patent Application Publication No. 20030138772, SEQ ID NO: 58), AAV223.3 8772, SEQ ID NO: 49), AAV223.4 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 50), AAV223.5 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 51), AAV223.6 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 52), AAV223.7 (U.S. Patent Application Publication No. 20030138772, SEQ ID NO: 53), AAVA3.4 (US Patent Application Publication No. 20030138772, SEQ ID NO: 54), AAVA3.5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 55), AAVA3.7 (US Patent Application Publication No. 20030138772, SEQ ID NO: 56), AAVA3.3 (US Patent Application Publication No. 20030138772, SEQ ID NO: 57), AAV42.12 (US Patent Application Publication No. 20030138772, SEQ ID NO: 58), The AAV42-2 vector may be or have a sequence as described in U.S. Patent Application Publication No. 20030138772 (the contents of which are incorporated by reference in their entirety), such as AAV44.2 (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 58), AAV44.2 (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 59), AAV42-2 (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 9), or a variant thereof.

[0101] In some embodiments, the AAV serotype may be, but is not limited to, AAV2 (SEQ ID NOs: 7 and 23 of US Patent Application Publication No. 20150159173), rh20 (SEQ ID NO: 1 of US Patent Application Publication No. 20150159173), rh32 / 33 (SEQ ID NO: 2 of US Patent Application Publication No. 20150159173), rh39 (SEQ ID NOs: 3, 20, and 36 of US Patent Application Publication No. 20150159173), rh46 (SEQ ID NOs: 4 and 22 of US Patent Application Publication No. 20150159173), rh73 (SEQ ID NOs: 5 and 6 of US Patent Application Publication No. 20150159173), or AAV2 (SEQ ID NOs: 6 and 7 of US Patent Application Publication No. 20150159173). US Patent Application Publication No. 20150159173, SEQ ID NO: 5), rh74 (US Patent Application Publication No. 20150159173, SEQ ID NO: 6), AAV6.1 (US Patent Application Publication No. 20150159173, SEQ ID NO: 29), rh.8 (US Patent Application Publication No. 20150159173, SEQ ID NO: 41), rh.48.1 (US Patent Application Publication No. 20150159173, SEQ ID NO: 44), hu.44 (US Patent Application Publication No. 20150159173, SEQ ID NO: 45), hu.29 (US Patent Application Publication No. 20150159173, SEQ ID NO: 6 ...7), hu.49 (US Patent Application Publication No. 20150159173, SEQ ID NO: 68), hu.50 (US Patent Application Publication No. 20150159173, SEQ ID NO: 69), hu.60 (US Patent Application Publication No. 20150159173, SEQ ID NO: 70), hu.51 (US Patent No. 20150159173), hu.48 (SEQ ID NO: 38 in U.S. Patent Application Publication No. 20150159173), rh54 (SEQ ID NO: 49 in U.S. Patent Application Publication No. 20150159173), AAV2 (SEQ ID NO: 7 in U.S. Patent Application Publication No. 20150159173), cy.5 (SEQ ID NOs: 8 and 24 in U.S. Patent Application Publication No. 20150159173), rh.10 (SEQ ID NOs: 9 and 25 in U.S. Patent Application Publication No. 20150159173), rh.13 (SEQ ID NO: 10 in U.S. Patent Application Publication No. 20150159173), rh.14 (SEQ ID NO: 11 in U.S. Patent Application Publication No. 20150159173), rh.15 (SEQ ID NO: 12 in U.S. Patent Application Publication No. 20150159173), rh.16 (SEQ ID NO: 13 in U.S. Patent Application Publication No. 20150159173), rh.17 (SEQ ID NO: 14 in U.S. Patent Application Publication No. 20150159173), rh.18 (SEQ ID NO: 15 in U.S. Patent Application Publication No. 20150159173), rh.19 (SEQ ID NO: 16 in U.S. Patent Application Publication No. 20150159173), rh.20 (SEQ ID NO: 17 in U.S. Patent Application Publication No. 20150159173), rh.21 (SEQ ID NO: No. 159173), AAV1 (SEQ ID NOs: 11 and 27 in U.S. Patent Application Publication No. 20150159173), AAV3 (SEQ ID NOs: 12 and 28 in U.S. Patent Application Publication No. 20150159173), AAV6 (SEQ ID NOs: 13 and 29 in U.S. Patent Application Publication No. 20150159173), AAV7 (SEQ ID NOs: 14 and 30 in U.S. Patent Application Publication No. 20150159173), AAV8 (SEQ ID NOs: 15 and 31 in U.S. Patent Application Publication No. 20150159173), hu.13 (SEQ ID NOS: 16 and 32 in U.S. Patent Application Publication No. 20150159173), hu.26 (SEQ ID NOS: 17 and 33 in U.S. Patent Application Publication No. 20150159173), hu.37 (SEQ ID NOS: 18 and 34 in U.S. Patent Application Publication No. 20150159173), hu.53 (SEQ ID NOS: 19 and 35 in U.S. Patent Application Publication No. 20150159173), rh.43 (SEQ ID NOS: 10 and 11 in U.S. Patent Application Publication No. 20150159173), rh.54 (SEQ ID NOS: 11 and 12 in U.S. Patent Application Publication No. 20150159173), rh.63 (SEQ ID NOS: 12 and 13 in U.S. Patent Application Publication No. 20150159173), rh.73 (SEQ ID NOS: 13 and 14 in U.S. Patent Application Publication No. 20150159173), rh.83 (SEQ ID NOS: 14 and 15 in U.S. Patent Application Publication No. 20150159173), rh.93 (SEQ ID NOS: 15 and 16 in U.S. Patent Application Publication No. 20150159173), rh.10 (SEQ ID NOS: 16 and 17 in U.S. Patent Application Publication No. 20150159173), rh.11 (SEQ ID NOS: 17 and 18 in U.S. Patent Application Publication No. 20150159173), rh.12 (SEQ ID No. 59173), rh.2 (SEQ ID NO: 39 in U.S. Patent Application Publication No. 20150159173), rh.37 (SEQ ID NO: 40 in U.S. Patent Application Publication No. 20150159173), rh.64 (SEQ ID NO: 43 in U.S. Patent Application Publication No. 20150159173), rh.48 (SEQ ID NO: 44 in U.S. Patent Application Publication No. 20150159173), ch.5 (U.S. Patent Application Publication No. 20150159173), and rh.67 (SEQ ID NO: 47 of U.S. Patent Application Publication No. 20150159173), rh.58 (SEQ ID NO: 48 of U.S. Patent Application Publication No. 20150159173), or variants thereof, such as, but not limited to, Cy5R1, Cy5R2, Cy5R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.48.1, rh.48.2, rh.48. The AAV6.12, AAV6.2, AAV6.1, AAV6.12, hu.48R1, hu.48R2, and hu.48R3 may be or have a sequence as described in U.S. Patent Application Publication No. 20150159173, the contents of which are incorporated by reference in their entirety.

[0102] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Pat. No. 7,198,951 (the contents of which are incorporated by reference in their entirety), including, but not limited to, AAV9 (SEQ ID NOs: 1-3 of U.S. Pat. No. 7,198,951), AAV2 (SEQ ID NO: 4 of U.S. Pat. No. 7,198,951), AAV1 (SEQ ID NO: 5 of U.S. Pat. No. 7,198,951), AAV3 (SEQ ID NO: 6 of U.S. Pat. No. 7,198,951), and AAV8 (SEQ ID NO: 7 of U.S. Pat. No. 7,198,951).

[0103] In some embodiments, the AAV serotype may be or have mutations in the AAV9 sequence as described by N. Pulicherla et al. (Molecular Therapy, Vol. 19, No. 6, pp. 1070-1078, 2011, herein incorporated by reference in its entirety), including, but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, etc.

[0104] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Pat. No. 6,156,303 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, AAV3B (SEQ ID NOs: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NOs: 2, 7, and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NOs: 3 and 8 of U.S. Pat. No. 6,156,303), AAV3A (SEQ ID NOs: 4 and 9 of U.S. Pat. No. 6,156,303), or derivatives thereof.

[0105] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. 20140359799 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, AAV8 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20140359799), AAVDJ (SEQ ID NOs: 2 and 3 of U.S. Patent Application Publication No. 20140359799), or variants thereof.

[0106] In some embodiments, the serotype may be AAVDJ (AAV-DJ) or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology, Vol. 82, No. 12, pp. 5887-5911, 2008, incorporated herein by reference in its entirety). The amino acid sequence of AAVDJ8 may include two or more mutations to remove the heparin-binding domain (HBD). As a non-limiting example, the AAV-DJ sequence set forth as SEQ ID NO:1 in U.S. Pat. No. 7,588,772 (the contents of which are incorporated by reference herein in their entirety) can contain two mutations: (1) R587Q (arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln)) and (2) R590T (arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr)). Another non-limiting example may include three mutations: (1) K406R (wherein lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg)), (2) R587Q (wherein arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln)), and (3) R590T (wherein arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr)).

[0107] In some embodiments, the AAV serotype may be or have the sequence of AAV4 as described in WO1998011244 (the contents of which are incorporated by reference in their entirety), including, but not limited to, AAV4 (SEQ ID NOs: 1-20 of WO1998011244).

[0108] In some embodiments, the AAV serotype may be or may have a mutation in the AAV2 sequence that gives rise to AAV2G9 as described in WO2014144229 and incorporated by reference herein in its entirety.

[0109] In some embodiments, the AAV serotype includes, but is not limited to, AAV3-3 (SEQ ID NO: 217 in WO2005033321), AAV1 (SEQ ID NOs: 219 and 202 in WO2005033321), AAV106.1 / hu.37 (SEQ ID NO: 10 in WO2005033321), AAV114.3 / hu.40 (SEQ ID NO: 11 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 6 and 7 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 7 and 8 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 8 and 9 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 9 and 10 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 10 and 11 in WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 1 ... and 8), AAV128.3 / hu.44 (SEQ ID NO: 81 in WO 2005033321), AAV130.4 / hu.48 (SEQ ID NO: 78 in WO 2005033321), AAV145.1 / hu.53 (SEQ ID NOs: 176 and 177 in WO 2005033321), AAV145.6 / hu.56 (SEQ ID NOs: 168 and 192 in WO 2005033321), AAV16.12 / hu.11 (SEQ ID NO: 153 in WO 2005033321), and 57), AAV16.8 / hu.10 (SEQ ID NOs: 156 and 56 in WO 2005033321), AAV161.10 / hu.60 (SEQ ID NO: 170 in WO 2005033321), AAV161.6 / hu.61 (SEQ ID NO: 174 in WO 2005033321), AAV1-7 / rh.48 (SEQ ID NO: 32 in WO 2005033321), AAV1-8 / rh.49 (SEQ ID NOs: 103 and 25 in WO 2005033321), AAV2 (SEQ ID NOs: 211 and 221 in WO 2005033321), AAV2-15 / rh.62 (SEQ ID NOs: 33 and 114 in WO 2005033321), AAV2-3 / rh.61 (SEQ ID NO: 21 in WO 2005033321), AAV2-4 / rh.50 (SEQ ID NOs: 23 and 108 in WO 2005033321), AAV2-5 / rh.51 (SEQ ID NOs: 104 and 22 in WO 2005033321), AAV3.1 / hu.AAV3.1 / hu.6 (SEQ ID NOs: 5 and 84 in WO 2005033321), AAV3.1 / hu.9 (SEQ ID NOs: 155 and 58 in WO 2005033321), AAV3-11 / rh.53 (SEQ ID NOs: 186 and 176 in WO 2005033321), AAV3-3 (SEQ ID NO: 200 in WO 2005033321), AAV33.12 / hu.17 (SEQ ID NO: 4 in WO 2005033321), AAV33.4 / hu.15 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.16 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.17 (SEQ ID NO: 4 in WO 2005033321), AAV3.1 / hu.18 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.19 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.20 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.21 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.22 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.23 (SEQ ID NO: 5 in WO 2005033321), AAV3.1 / hu.24 (SEQ ID NO: 5 in WO 20050333 3321), AAV33.8 / hu.16 (SEQ ID NO: 51 in WO 2005033321), AAV3-9 / rh.52 (SEQ ID NOs: 96 and 18 in WO 2005033321), AAV4-19 / rh.55 (SEQ ID NO: 117 in WO 2005033321), AAV4-4 (SEQ ID NOs: 201 and 218 in WO 2005033321), AAV4-9 / rh.54 (SEQ ID NO: 116 ... ), AAV5 (SEQ ID NOs: 199 and 216 in WO 2005033321), AAV52.1 / hu.20 (SEQ ID NO: 63 in WO 2005033321), AAV52 / hu.19 (SEQ ID NO: 133 in WO 2005033321), AAV5-22 / rh.58 (SEQ ID NO: 27 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 105 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 110 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 111 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 112 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 113 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 114 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 115 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 116 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 117 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 118 in WO 2005033321), AAV5-3 / rh.57 (SEQ ID NO: 119 in 33321), AAV58.2 / hu.25 (SEQ ID NO: 49 in WO 2005033321), AAV6 (SEQ ID NOs: 203 and 220 in WO 2005033321), AAV7 (SEQ ID NOs: 222 and 213 in WO 2005033321), AAV7.3 / hu.7 (SEQ ID NO: 55 in WO 2005033321), AAV8 (SEQ ID NOs: 223 and 214 in WO 2005033321), AAVH-1 / hu.AAVhu.1 (SEQ ID NO: 46 in WO 2005033321), AAVH-5 / hu.3 (SEQ ID NO: 44 in WO 2005033321), AAVhu.1 (SEQ ID NO: 144 in WO 2005033321), AAVhu.10 (SEQ ID NO: 156 in WO 2005033321), AAVhu.11 (SEQ ID NO: 153 in WO 2005033321), AAVhu.12 (SEQ ID NO: 59 in WO 2005033321), AAVhu.13 (SEQ ID NO: 144 in WO 2005033321), AAVhu.14 (SEQ ID NO: 144 in WO 2005033321), AAVhu.15 (SEQ ID NO: 156 in WO 2005033321), AAVhu.16 (SEQ ID NO: 153 in WO 2005033321), AAVhu.17 (SEQ ID NO: 159 in WO 2005033321), AAVhu.18 (SEQ ID NO: 159 in WO 2005033321), AAVhu.19 (SEQ ID NO: 159 in WO 2005033321), AAVhu.20 (SEQ ID NO: 159 in WO 2005033321), AAVhu.21 (SEQ ID NO: 159 in WO 2005033321), AAVhu.22 (SEQ ID NO: 159 in WO 2005033321), AAVhu.23 (SEQ ID NO: 159 in AAVhu.14 / AAV9 (SEQ ID NO: 123 and 3 in WO 2005033321), AAVhu.15 (SEQ ID NO: 147 in WO 2005033321), AAVhu.16 (SEQ ID NO: 148 in WO 2005033321), AAVhu.17 (SEQ ID NO: 83 in WO 2005033321), AAVhu.18 (SEQ ID NO: 149 in WO 2005033321), AAVhu.19 (SEQ ID NO: 190 in WO 2005033321), AAVhu.20 (SEQ ID NO: 201 in WO 2005033321), AAVhu.21 (SEQ ID NO: 202 in WO 2005033321), AAVhu.22 (SEQ ID NO: 203 in WO 2005033321), AAVhu.23 (SEQ ID NO: 204 in WO 2005033321), AAVhu.24 (SEQ ID NO: 205 in WO 2005033321), AAVhu.25 (SEQ ID NO: 206 in WO 2005033321), AAVhu.26 (SEQ ID NO: 207 in WO 2005033321), AAVhu.27 (SEQ ID NO: 208 in WO 2005033321), AAVhu.28 (SEQ ID NO: 209 in WO 2005033321), AAVhu.29 (SEQ ID NO: 201 in WO 2005033321), AAVhu.30 (SEQ hu.19 (SEQ ID NO: 133 in WO 2005033321), AAVhu.2 (SEQ ID NO: 143 in WO 2005033321), AAVhu.20 (SEQ ID NO: 134 in WO 2005033321), AAVhu.21 (SEQ ID NO: 135 in WO 2005033321), AAVhu.22 (SEQ ID NO: 138 in WO 2005033321), AAVhu.23.2 (SEQ ID NO: 137 in WO 2005033321), AAV hu.24 (SEQ ID NO: 136 in WO 2005033321), AAVhu.25 (SEQ ID NO: 146 in WO 2005033321), AAVhu.27 (SEQ ID NO: 140 in WO 2005033321), AAVhu.29 (SEQ ID NO: 132 in WO 2005033321), AAVhu.3 (SEQ ID NO: 145 in WO 2005033321), AAVhu.31 (SEQ ID NO: 121 in WO 2005033321), AAVhu.32 (SEQ ID NO: 122 in WO 2005033321), AAVhu.34 (SEQ ID NO: 125 in WO 2005033321), AAVhu.35 (SEQ ID NO: 164 in WO 2005033321), AAVhu.37 (SEQ ID NO: 88 in WO 2005033321), AAVhu.39 (SEQ ID NO: 102 in WO 2005033321), AAVhu.4 (SEQ ID NO: 141 in WO 2005033321), AAVhu. AAVhu.40 (SEQ ID NO: 87 in WO 2005033321), AAVhu.41 (SEQ ID NO: 91 in WO 2005033321), AAVhu.42 (SEQ ID NO: 85 in WO 2005033321), AAVhu.43 (SEQ ID NO: 160 in WO 2005033321), AAVhu.44 (SEQ ID NO: 144 in WO 2005033321), AAVhu.45 (SEQ ID NO: 127 in WO 2005033321), AAVhu.46 (SEQ ID NO: 159 in WO 2005033321), AAVhu.47 (SEQ ID NO: 128 in WO 2005033321), AAVhu.48 (SEQ ID NO: 157 in WO 2005033321), AAVhu.49 (SEQ ID NO: 189 in WO 2005033321), AAVhu.51 (SEQ ID NO: 190 in WO 2005033321), AAVhu.52 (SEQ ID NO: 191 in WO 2005033321), AAVhu.5 3 (SEQ ID NO: 186 in WO 2005033321), AAVhu.54 (SEQ ID NO: 188 in WO 2005033321), AAVhu.55 (SEQ ID NO: 187 in WO 2005033321), AAVhu.56 (SEQ ID NO: 192 in WO 2005033321), AAVhu.57 (SEQ ID NO: 193 in WO 2005033321), AAVhu.58 (SEQ ID NO: 194 in WO 2005033321), AAVhu.AAVhu.6 (SEQ ID NO: 84 in WO 2005033321), AAVhu.60 (SEQ ID NO: 184 in WO 2005033321), AAVhu.61 (SEQ ID NO: 185 in WO 2005033321), AAVhu.63 (SEQ ID NO: 195 in WO 2005033321), AAVhu.64 (SEQ ID NO: 196 in WO 2005033321), AAVhu.66 (SEQ ID NO: 197 in WO 2005033321), AAVhu.67 (SEQ ID NO: 198 in WO 2005033321), AAVhu.7 (SEQ ID NO: 150 in WO 2005033321), AAVhu.8 (SEQ ID NO: 12 in WO 2005033321), AAVhu.9 (SEQ ID NO: 155 in WO 2005033321), AAVLG-10 / rh.40 (SEQ ID NO: 14 in WO 2005033321), AAVLG-4 / rh.38 (SEQ ID NO: 86 in WO 2005033321), AAVL G-4 / rh.38 (SEQ ID NO: 7 in WO 2005033321), AAVN721-8 / rh.43 (SEQ ID NO: 163 in WO 2005033321), AAVN721-8 / rh.43 (SEQ ID NO: 43 in WO 2005033321), AAVpi.1 (SEQ ID NO: 28 in WO 2005033321), AAVpi.2 (SEQ ID NO: 30 in WO 2005033321), AAVpi.3 (SEQ ID NO: 2 in WO 2005033321), 9), AAVrh.38 (SEQ ID NO: 86 in WO 2005033321), AAVrh.40 (SEQ ID NO: 92 in WO 2005033321), AAVrh.43 (SEQ ID NO: 163 in WO 2005033321), AAVrh.44 (SEQ ID NO: 34 in WO 2005033321), AAVrh.45 (SEQ ID NO: 41 in WO 2005033321), AAVrh.47 (SEQ ID NO: 38 in WO 2005033321), AAVrh.AAVrh.48 (SEQ ID NO: 115 in WO 2005033321), AAVrh.49 (SEQ ID NO: 103 in WO 2005033321), AAVrh.50 (SEQ ID NO: 108 in WO 2005033321), AAVrh.51 (SEQ ID NO: 104 in WO 2005033321), AAVrh.52 (SEQ ID NO: 104 in WO 2005033321). AAVrh.53 (SEQ ID NO: 97 in WO2005033321), AAVrh.55 (SEQ ID NO: 37 in WO2005033321), AAVrh.56 (SEQ ID NO: 152 in WO2005033321), AAVrh.57 (SEQ ID NO: 105 in WO2005033321), AAVrh.58 (SEQ ID NO: 109 in WO2005033321), AAVrh.59 (SEQ ID NO: 110 in WO2005033321), AAVrh.60 (SEQ ID NO: 111 in WO2005033321), AAVrh.61 (SEQ ID NO: 112 in WO2005033321), AAVrh.62 (SEQ ID NO: 113 in WO2005033321), AAVrh.63 (SEQ ID NO: 114 in WO2005033321), AAVrh.64 (SEQ ID NO: 115 in WO2005033321), AAVrh.65 (SEQ ID NO: 116 in WO2005033321), AAVrh.66 (SEQ ID NO: 117 in WO2005033321), AAVrh.67 (SEQ ID NO: 118 in WO2005033321), AAVrh.68 (SEQ ID NO: 119 in WO2005033321), AAVrh.69 (SEQ ID NO: 120 in WO2005033321), AAVrh.69 (SEQ ID NO: 121 in WO2005033321), AAVrh AAVrh.59 (SEQ ID NO: 42 in WO 2005033321), AAVrh.60 (SEQ ID NO: 31 in WO 2005033321), AAVrh.61 (SEQ ID NO: 107 in WO 2005033321), AAVrh.62 (SEQ ID NO: 114 in WO 2005033321), AAVrh.64 (SEQ ID NO: 99 in International Publication No. 2005033321), AAVrh.65 (SEQ ID NO: 35 in International Publication No. 2005033321), AAVrh.68 (SEQ ID NO: 16 in International Publication No. 2005033321), AAVrh.69 (SEQ ID NO: 39 in International Publication No. 2005033321), AAVrh.70 (SEQ ID NO: 20 in International Publication No. 2005033321), AAV and rh.72 (WO2005033321 SEQ ID NO: 9), or variants thereof, including but not limited to AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.25 / 42 15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh14.Non-limiting examples of variants include SEQ ID NOs: 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62, 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219, 224-236 of WO2005033321, the contents of which are herein incorporated by reference in their entirety.

[0110] In some embodiments, the AAV serotype may be or have a sequence as described in WO2015168666 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, AAVrh8R (SEQ ID NO: 9 of WO2015168666), AAVrh8R A586R mutant (SEQ ID NO: 10 of WO2015168666), AAVrh8R R533A mutant (SEQ ID NO: 11 of WO2015168666), or variants thereof.

[0111] In some embodiments, the AAV serotype may be, but is not limited to, AAVhE1.1 (SEQ ID NO: 44 of U.S. Pat. No. 9,233,131), AAVhEr1.5 (SEQ ID NO: 45 of U.S. Pat. No. 9,233,131), AAVhER1.14 (SEQ ID NO: 46 of U.S. Pat. No. 9,233,131), AAVhEr1.8 (SEQ ID NO: 47 of U.S. Pat. No. 9,233,131), AAVhEr1.16 (U.S. Pat. No. 9,233,131), AAVhEr1.18 (SEQ ID NO: 48 of U.S. Pat. No. 9,233,131), AAVhEr1.19 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.20 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.21 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.22 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.23 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.24 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.25 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.26 (S No. 31), AAVhEr1.18 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.35 (SEQ ID NO: 50 of U.S. Pat. No. 9,233,131), AAVhEr1.7 (SEQ ID NO: 51 of U.S. Pat. No. 9,233,131), AAVhEr1.36 (SEQ ID NO: 52 of U.S. Pat. No. 9,233,131), AAVhEr2.29 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr3.18 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr3.35 (SEQ ID NO: 50 of U.S. Pat. No. 9,233,131), AAVhEr3.7 (SEQ ID NO: 51 of U.S. Pat. No. 9,233,131), AAVhEr3.36 (SEQ ID NO: 52 of U.S. Pat. No. 9,233,131), AAVhEr4.29 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr5.29 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr6.29 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr7.29 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr8.29 (SEQ ID 53), AAVhEr2.4 (SEQ ID NO: 54 in U.S. Pat. No. 9,233,131), AAVhEr2.16 (SEQ ID NO: 55 in U.S. Pat. No. 9,233,131), AAVhEr2.30 (SEQ ID NO: 56 in U.S. Pat. No. 9,233,131), AAVhEr2.31 (SEQ ID NO: 58 in U.S. Pat. No. 9,233,131), AAVhEr2.36 (SEQ ID NO: 57 in U.S. Pat. No. 9,233,131), AAVhEr1 The AAV may be or have a sequence as described in U.S. Patent No. 9,233,131 (the contents of which are incorporated by reference in their entirety) such as AAVhEr3.1 (SEQ ID NO: 59 of U.S. Patent No. 9,233,131), AAV2.5T (SEQ ID NO: 42 of U.S. Patent No. 9,233,131), or a variant thereof.

[0112] In some embodiments, the AAV serotype may be, but is not limited to, AAV-PAEC (SEQ ID NO: 1 in US Patent Application Publication No. 20150376607), AAV-LK01 (SEQ ID NO: 2 in US Patent Application Publication No. 20150376607), AAV-LK02 (SEQ ID NO: 3 in US Patent Application Publication No. 20150376607), AAV-LK03 (SEQ ID NO: 4 in US Patent Application Publication No. 20150376607), AAV-LK04 (SEQ ID NO: 5 in US Patent Application Publication No. 20150376607), AAV-LK05 (US Patent Application Publication No. AAV-LK06 (SEQ ID NO: 6 in U.S. Patent Application Publication No. 20150376607), AAV-LK07 (SEQ ID NO: 8 in U.S. Patent Application Publication No. 20150376607), AAV-LK08 (SEQ ID NO: 9 in U.S. Patent Application Publication No. 20150376607), AAV-LK09 (SEQ ID NO: 10 in U.S. Patent Application Publication No. 20150376607), AAV-LK10 (SEQ ID NO: 11 in U.S. Patent Application Publication No. 20150376607), AAV-LK11 (U.S. Patent Application Publication No. 20150376607), AAV-LK12 (SEQ ID NO: 13 in U.S. Patent Application Publication No. 20150376607), AAV-LK13 (SEQ ID NO: 14 in U.S. Patent Application Publication No. 20150376607), AAV-LK14 (SEQ ID NO: 15 in U.S. Patent Application Publication No. 20150376607), AAV-LK15 (SEQ ID NO: 16 in U.S. Patent Application Publication No. 20150376607), AAV-LK16 (SEQ ID NO: 17 in U.S. Patent Application Publication No. 20150376607), AAV-LK17 (SEQ ID NO: 18 in U.S. Patent Application Publication No. 20150376607), AAV-LK18 (SEQ ID NO: 19 in U.S. Patent Application Publication No. 20150376607), AAV-LK19 (SEQ ID NO: 20 AAV-LK12 (SEQ ID NO: 12 in U.S. Patent Application Publication No. 20150376607), AAV-LK12 (SEQ ID NO: 13 in U.S. Patent Application Publication No. 20150376607), AAV-LK13 (SEQ ID NO: 14 in U.S. Patent Application Publication No. 20150376607), AAV-LK14 (SEQ ID NO: 15 in U.S. Patent Application Publication No. 20150376607), AAV-LK15 (SEQ ID NO: 16 in U.S. Patent Application Publication No. 20150376607), AAV-LK16 (SEQ ID NO: 17 in U.S. Patent Application Publication No. 20150376607), AAV-LK1 7 (SEQ ID NO: 18 in U.S. Patent Application Publication No. 20150376607), AAV-LK18 (SEQ ID NO: 19 in U.S. Patent Application Publication No. 20150376607), AAV-LK19 (SEQ ID NO: 20 in U.S. Patent Application Publication No. 20150376607), AAV-PAEC2 (SEQ ID NO: 21 in U.S. Patent Application Publication No. 20150376607), AAV-PAEC4 (SEQ ID NO: 22 in U.S. Patent Application Publication No. 20150376607), AAV-PAEC6 (SEQ ID NO: 23 in U.S. Patent Application Publication No. 20150376607),The vector may be or have a sequence as described in U.S. Patent Application Publication No. 20150376607 (the contents of which are incorporated by reference in their entirety), such as AAV-PAEC7 (SEQ ID NO: 24 of U.S. Patent Application Publication No. 20150376607), AAV-PAEC8 (SEQ ID NO: 25 of U.S. Patent Application Publication No. 20150376607), AAV-PAEC11 (SEQ ID NO: 26 of U.S. Patent Application Publication No. 20150376607), AAV-PAEC12 (SEQ ID NO: 27 of U.S. Patent Application Publication No. 20150376607), or a variant thereof.

[0113] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Pat. No. 9,163,261 (the contents of which are incorporated by reference in their entirety) such as, but not limited to, AAV-2-pre-miRNA-101 (SEQ ID NO: 1 U.S. Pat. No. 9,163,261), or a variant thereof.

[0114] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. 20150376240 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, AAV-8h (SEQ ID NO: 6 of U.S. Patent Application Publication No. 20150376240), AAV-8b (SEQ ID NO: 5 of U.S. Patent Application Publication No. 20150376240), AAV-h (SEQ ID NO: 2 of U.S. Patent Application Publication No. 20150376240), AAV-b (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150376240), or variants thereof.

[0115] In some embodiments, the AAV serotype includes, but is not limited to, AAV SM 10-2 (US Patent Application Publication No. 20160017295 SEQ ID NO: 22), AAV Shuffle 100-1 (US Patent Application Publication No. 20160017295 SEQ ID NO: 23), AAV Shuffle 100-3 (US Patent Application Publication No. 20160017295 SEQ ID NO: 24), AAV Shuffle 100-7 (US Patent Application Publication No. 20160017295 SEQ ID NO: 25), AAV Shuffle 100-8 (US Patent Application Publication No. 20160017295 SEQ ID NO: 26), AAV Shuffle 100-9 (US Patent Application Publication No. 20160017295 SEQ ID NO: 27), AAV Shuffle 100-1 (US Patent Application Publication No. 20160017295 SEQ ID NO: 28), AAV Shuffle 100-2 (US Patent Application Publication No. 20160017295 SEQ ID NO: 29), AAV Shuffle 100-3 (US Patent Application Publication No. 20160017295 SEQ ID NO: 30), AAV Shuffle 100-4 (US Patent Application Publication No. 20160017295 SEQ ID NO: 31), AAV Shuffle 100-5 (US Patent Application Publication No. 20160017295 SEQ ID NO: 32), AAV Shuffle 100-6 (US Patent Application Publication No. 20160017295 SEQ ID NO: 33), AAV Shuffle 100-7 (US Patent Application Publication No. 20160017295 SEQ ID NO: 34 shuffle10-2 (SEQ ID NO: 34 in U.S. Patent Application Publication No. 20160017295), AAV shuffle10-6 (SEQ ID NO: 35 in U.S. Patent Application Publication No. 20160017295), AAV shuffle10-8 (SEQ ID NO: 36 in U.S. Patent Application Publication No. 20160017295), AAV shuffle100-2 (SEQ ID NO: 37 in U.S. Patent Application Publication No. 20160017295), AAV The AAV SM 10-1 may be or have a sequence as described in U.S. Patent Application Publication No. 20160017295 (the contents of which are incorporated by reference in their entirety), such as SM 10-1 (SEQ ID NO: 38 of U.S. Patent Application Publication No. 20160017295), AAV SM 10-8 (SEQ ID NO: 39 of U.S. Patent Application Publication No. 20160017295), AAV SM 100-3 (SEQ ID NO: 40 of U.S. Patent Application Publication No. 20160017295), AAV SM 100-10 (SEQ ID NO: 41 of U.S. Patent Application Publication No. 20160017295), or a variant thereof.

[0116] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. 20150238550 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, BNP61 AAV (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150238550), BNP62 AAV (SEQ ID NO: 3 of U.S. Patent Application Publication No. 20150238550), BNP63 AAV (SEQ ID NO: 4 of U.S. Patent Application Publication No. 20150238550), or variants thereof.

[0117] In some embodiments, the AAV serotype includes, but is not limited to, AAVrh.50 (US Patent Application Publication No. 20150315612, SEQ ID NO: 108), AAVrh.43 (US Patent Application Publication No. 20150315612, SEQ ID NO: 163), AAVrh.62 (US Patent Application Publication No. 20150315612, SEQ ID NO: 114), AAVrh.48 (US Patent Application Publication No. 20150315612, SEQ ID NO: 115), AAVhu.19 (US Patent Application Publication No. 20150315612, SEQ ID NO: 116), AAVrh.50 (US Patent Application Publication No. 20150315612, SEQ ID NO: 117), AAVrh.51 (US Patent Application Publication No. 20150315612, SEQ ID NO: 118), AAVrh.52 (US Patent Application Publication No. 20150315612, SEQ ID NO: 119), AAVrh.53 (US Patent Application Publication No. 20150315612, SEQ ID NO: 120), AAVrh.54 (US Patent Application Publication No. 20150315612, SEQ ID NO: 121), AAVrh.55 (US Patent Application Publication No. 20150315612, SEQ ID NO: 122), AAVrh.56 (US Patent Application Publication No. 20150315612, SEQ ID NO: 123), AAVrh.57 (US Patent Application Publication No. 20150315612, SEQ ID NO: 124), AAVrh.58 (US Patent Application Publication No. 2015031561 AAVhu.11 (SEQ ID NO: 153 in U.S. Patent Application Publication No. 20150315612), AAVhu.53 (SEQ ID NO: 186 in U.S. Patent Application Publication No. 20150315612), AAV4-8 / rh.64 (SEQ ID NO: 15 in U.S. Patent Application Publication No. 20150315612), AAVLG-9 / hu.39 (SEQ ID NO: 24 in U.S. Patent Application Publication No. 20150315612), AAV54.5 / hu.23 (U.S. Patent Application Publication No. 20150315612), ...hu.53 (SEQ ID NO: 186 in U.S. Patent Application Publication No. 20150315612), AAV No. 315612), AAV54.2 / hu.22 (SEQ ID NO: 67 of U.S. Patent Application Publication No. 20150315612), AAV54.7 / hu.24 (SEQ ID NO: 66 of U.S. Patent Application Publication No. 20150315612), AAV54.1 / hu.21 (SEQ ID NO: 65 of U.S. Patent Application Publication No. 20150315612), AAV54.4R / hu.27 (SEQ ID NO: 64 of U.S. Patent Application Publication No. 20150315612), AAV46.2 / hu.28 (U.S. Patent Application Publication No. 20150 ...4R / hu.27 (SEQ ID NO: 64 of U.S. Patent Application Publication No. 20150315612), AAV46.2 / hu.28 (U.S. Patent Application Publication No. 20150315612), AAV54.4R / hu AAV46.6 / hu.29 (SEQ ID NO: 68 of U.S. Patent Application Publication No. 20150315612), AAV46.6 / hu.29 (SEQ ID NO: 69 of U.S. Patent Application Publication No. 20150315612), AAV128.1 / hu.43 (SEQ ID NO: 80 of U.S. Patent Application Publication No. 20150315612), or a variant thereof, may be or have a sequence as described in U.S. Patent Application Publication No. 20150315612 (the contents of which are incorporated by reference in their entirety).

[0118] In some embodiments, the AAV serotype may be or have a sequence as described in WO2015121501 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, truetype AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), "Eupen AAV10" (SEQ ID NO: 8 of WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501), or variants thereof.

[0119] According to the present invention, the selection or use of AAV capsid serotypes can be from various species. In one embodiment, the AAV can be avian AAV (AAAV). The AAAV serotype can be or have a sequence as described in U.S. Patent No. 9,238,800 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAAV (SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Patent No. 9,238,800), or a variant thereof.

[0120] In one embodiment, the AAV may be bovine AAV (BAAV). BAAV serotypes may be or have sequences as described in U.S. Patent No. 9,193,769 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, BAAV (SEQ ID NOS: 1 and 6 of U.S. Patent No. 9,193,769), or variants thereof. BAAV serotypes may be or have sequences as described in U.S. Patent No. 7,427,396 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, BAAV (SEQ ID NOS: 5 and 6 of U.S. Patent No. 7,427,396), or variants thereof.

[0121] In one embodiment, the AAV may be a caprine AAV. The caprine AAV serotype may be or have a sequence as described in U.S. Patent No. 7,427,396 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, a caprine AAV (SEQ ID NO: 3 of U.S. Patent No. 7,427,396), or a variant thereof.

[0122] In other embodiments, AAV may be engineered as a hybrid AAV from two or more parental serotypes.In one embodiment, this AAV may be AAV2G9, which comprises sequences from AAV2 and AAV9.The AAV2G9 AAV serotype may be or have the sequence described in U.S. Patent Application Publication No. 20160017005 (the contents of which are incorporated herein by reference in their entirety).

[0123] In one embodiment, the AAV may be a serotype generated by an AAV9 capsid library with mutations at amino acids 390-627 (VP1 numbering) as described by Pulicherla et al. (Molecular Therapy, Vol. 19, No. 6, pp. 1070-1078, 2011, the contents of which are incorporated by reference in their entirety). Serotypes and corresponding nucleotide and amino acid substitutions include, but are not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV6.3 (T1418A and T1436X; V473D and I479K), AAV9.4 (T1418C and T1436X; V473D and I479K), AAV9.5 (T1418C and T1436X; V473D and I479K), AAV9.6 (T1418C and T1436X; V473D and I479K), AAV9.7 (T1418C and T1436X; V473D and I479K), AAV9.8 (T1418C and T1436X; V473D and I479K), AAV9.9 (T1418C and T1436X; V473D and I479K), AAV9.1 ...2 (T1418C and ), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9 .13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T152 1G;W503R), AAV9.26(A1337G, A1769C;Y446C, Q590P), AAV9.33(A1667C;D556A), AAV9.34(A1534G, C1794T;N512D), AAV9.35(A1289T, T1450 A. 84C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L) , AAV9.50(A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53(G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A ;T492I, H527N), AAV.59(T1336C;Y446H), AAV9.61(A1493T;N498I), AAV9.64(C1531A, A1617T;L511I), AAV9. 65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A, ;G481R), AAV9.83 (C1402A, A1500T;P468T, E500D), AAV9.87(T1464C, T1468C;S490P), AAV9.90(A1196T;Y399F), AAV9.91(T1316G, A1583 and AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L), and AAV9.95 (T1605A; F535L).

[0124] In some embodiments, the AAV serotypes include, but are not limited to, AAVF1 / HSC1 (SEQ ID NOs: 2 and 20 in WO2016049230), AAVF2 / HSC2 (SEQ ID NOs: 3 and 21 in WO2016049230), AAVF3 / HSC3 (SEQ ID NOs: 5 and 22 in WO2016049230), AAVF4 / HSC4 (SEQ ID NOs: 6 and 23 in WO2016049230), AAVF5 / HSC5 (SEQ ID NOs: 11 and 25 in WO 2016049230), AAVF6 / HSC6 (SEQ ID NOs: 7 and 24 in WO 2016049230), AAVF7 / HSC7 (SEQ ID NOs: 8 and 27 in WO 2016049230), AAVF8 / HSC8 (SEQ ID NOs: 9 and 28 in WO 2016049230), AAVF9 / HSC9 (SEQ ID NOs: 10 and 29 in WO 2016049230), AA VF11 / HSC11 (SEQ ID NOs: 4 and 26 in WO 2016049230), AAVF12 / HSC12 (SEQ ID NOs: 12 and 30 in WO 2016049230), AAVF13 / HSC13 (SEQ ID NOs: 14 and 31 in WO 2016049230), AAVF14 / HSC14 (SEQ ID NOs: 15 and 32 in WO 2016049230), AAVF15 / HSC15 (SEQ ID NOs: 16 and 33 in WO 2016049230), The AAVF16 / HSC16 fragment may be or have a sequence as described in WO2016049230 (the contents of which are incorporated by reference in their entirety), such as AAVF16 / HSC16 (SEQ ID NOs: 16 and 33 of WO2016049230), AAVF16 / HSC16 (SEQ ID NOs: 17 and 34 of WO2016049230), AAVF17 / HSC17 (SEQ ID NOs: 13 and 35 of WO2016049230), or a variant or derivative thereof.

[0125] In some embodiments, the AAV serotypes include, but are not limited to, AAV CBr-El (SEQ ID NOs: 13 and 87 in U.S. Pat. No. 8,734,809), AAV CBr-E2 (SEQ ID NOs: 14 and 88 in U.S. Pat. No. 8,734,809), AAV CBr-E3 (SEQ ID NOs: 15 and 89 in U.S. Pat. No. 8,734,809), AAV CBr-E4 (SEQ ID NOs: 16 and 90 in U.S. Pat. No. 8,734,809), AAV CBr-E5 (SEQ ID NOs: 17 and 91 in U.S. Pat. No. 8,734,809), AAV CBr-e5 (SEQ ID NOs: 18 and 92 in U.S. Pat. No. 8,734,809), AAV CBr-E6 (SEQ ID NOs: 19 and 93 in U.S. Pat. No. 8,734,809), AAV CBr-E7 (SEQ ID NOs: 19 and 94 in U.S. Pat. No. 8,734,809), AAV CBr-E8 (SEQ ID NOs: 20 and 21 in U.S. Pat. No. 8,734,809), AAV CBr-E9 (SEQ ID NOs: 21 and 22 in U.S. Pat. No. 8,734,809), AAV CBr-E10 (SEQ ID NOs: 22 and 23 in U.S. Pat. No. 8,734,809), AAV CBr-E11 (SEQ ID NOs: 23 and 24 in U.S. Pat. No. 8,734,80 CBr-E7 (SEQ ID NOs: 20 and 94 in U.S. Patent No. 8,734,809), AAV CBr-E8 (SEQ ID NOs: 21 and 95 in U.S. Patent No. 8,734,809), AAV CLv-D1 (SEQ ID NOs: 22 and 96 in U.S. Patent No. 8,734,809), AAV CLv-D2 (SEQ ID NOs: 23 and 97 in U.S. Patent No. 8,734,809), AAV CLv-D3 (SEQ ID NOs: 24 and 98 in U.S. Patent No. 8,734,809), AAV CLv-D4 (SEQ ID NOs: 25 and 99 in U.S. Patent No. 8,734,809), AAV CLv-D5 (SEQ ID NOs: 26 and 100 in U.S. Patent No. 8,734,809), AAV CLv-D6 (SEQ ID NOs: 27 and 101 in U.S. Patent No. 8,734,809), AAV CLv-D7 (SEQ ID NOs: 28 and 102 in U.S. Patent No. 8,734,809), AAV CLv-D8 (SEQ ID NOs: 29 and 103 in U.S. Patent No. 8,734,809), AAV CLv-E1 (SEQ ID NOs: 13 and 87 in U.S. Patent No. 8,734,809), AAV CLv-R1 (SEQ ID NOs: 30 and 104 in U.S. Patent No. 8,734,809), AAV CLv-R2 (SEQ ID NOs: 31 and 105 in U.S. Patent No. 8,734,809), AAV CLv-R3 (SEQ ID NOs: 32 and 106 in U.S. Patent No. 8,734,809), AAV CLv-R4 (SEQ ID NOs: 33 and 107 in U.S. Patent No. 8,734,809), AAV CLv-R5 (SEQ ID NOs: 34 and 108 in U.S. Patent No. 8,734,809), AAV CLv-R6 (SEQ ID NOs: 35 and 109 in U.S. Patent No. 8,734,809), AAV CLv-R7 (SEQ ID NOs: 36 and 110 in U.S. Patent No. 8,734,809), AAV CLv-R8 (SEQ ID NOs: 37 and 111 in U.S. Patent No. 8,734,809), AAV CLv-R9 (SEQ ID NOs: 38 and 112 in U.S. Patent No. 8,734,809), AAV CLg-F1 (SEQ ID NOs: 39 and 113 in U.S. Patent No. 8,734,809), AAV CLg-F2 (SEQ ID NOs: 40 and 114 in U.S. Patent No. 8,734,809), AAV CLg-F3 (SEQ ID NOs: 41 and 115 in U.S. Patent No. 8,734,809), AAV CLg-F4 (SEQ ID NOs: 42 and 116 in U.S. Patent No. 8,734,809), AAVCLg-F5 (SEQ ID NOs: 43 and 117 in U.S. Patent No. 8,734,809), AAV CLg-F6 (SEQ ID NOs: 43 and 117 in U.S. Patent No. 8,734,809), AAV CLg-F7 (SEQ ID NOs: 44 and 118 in U.S. Patent No. 8,734,809), AAV CLg-F8 (SEQ ID NOs: 43 and 117 in U.S. Patent No. 8,734,809), AAV CSp-1 (SEQ ID NOs: 45 and 119 in U.S. Patent No. 8,734,809), AAV CSp-10 (SEQ ID NOs: 46 and 120 in U.S. Patent No. 8,734,809), AAV CSp-11 (SEQ ID NOs: 47 and 121 in U.S. Patent No. 8,734,809), AAV CSp-2 (SEQ ID NOs: 48 and 122 in U.S. Patent No. 8,734,809), AAV CSp-3 (SEQ ID NOs: 49 and 123 in U.S. Patent No. 8,734,809), AAV CSp-4 (SEQ ID NOs: 50 and 124 in U.S. Patent No. 8,734,809), AAV CSp-6 (SEQ ID NOs: 51 and 125 in U.S. Patent No. 8,734,809), AAV CSp-7 (SEQ ID NOs: 52 and 126 in U.S. Patent No. 8,734,809), AAV CSp-8 (SEQ ID NOs: 53 and 127 in U.S. Patent No. 8,734,809), AAV CSp-9 (SEQ ID NOs: 54 and 128 in U.S. Patent No. 8,734,809), AAV CHt-2 (SEQ ID NOs: 55 and 129 in U.S. Patent No. 8,734,809), AAV CHt-3 (SEQ ID NOs: 56 and 130 in U.S. Patent No. 8,734,809), AAV CKd-1 (SEQ ID NOs: 57 and 131 in U.S. Patent No. 8,734,809), AAV CKd-10 (SEQ ID NOs: 58 and 132 in U.S. Patent No. 8,734,809), AAV CKd-2 (SEQ ID NOs: 59 and 133 in U.S. Patent No. 8,734,809), AAV CKd-3 (SEQ ID NOs: 60 and 134 in U.S. Patent No. 8,734,809), AAV CKd-4 (SEQ ID NOs: 61 and 135 in U.S. Patent No. 8,734,809), AAV CKd-6 (SEQ ID NOs: 62 and 136 in U.S. Patent No. 8,734,809), AAV CKd-7 (SEQ ID NOs: 63 and 137 in U.S. Patent No. 8,734,809), AAV CKd-8 (SEQ ID NOs: 64 and 138 in U.S. Patent No. 8,734,809), AAVCLv-1 (SEQ ID NOs: 35 and 139 in U.S. Patent No. 8,734,809), AAV CLv-12 (SEQ ID NOs: 66 and 140 in U.S. Patent No. 8,734,809), AAV CLv-13 (SEQ ID NOs: 67 and 141 in U.S. Patent No. 8,734,809), AAV CLv-2 (SEQ ID NOs: 68 and 142 in U.S. Patent No. 8,734,809), AAV CLv-3 (SEQ ID NOs: 69 and 143 in U.S. Patent No. 8,734,809), AAV CLv-4 (SEQ ID NOs: 70 and 144 in U.S. Patent No. 8,734,809), AAV CLv-6 (SEQ ID NOs: 71 and 145 in U.S. Patent No. 8,734,809), AAV CLv-8 (SEQ ID NOs: 72 and 146 in U.S. Patent No. 8,734,809), AAV CKd-B1 (SEQ ID NOs: 73 and 147 in U.S. Patent No. 8,734,809), AAV CKd-B2 (SEQ ID NOs: 74 and 148 in U.S. Patent No. 8,734,809), AAV CKd-B3 (SEQ ID NOs: 75 and 149 in U.S. Patent No. 8,734,809), AAV CKd-B4 (SEQ ID NOs: 76 and 150 in U.S. Patent No. 8,734,809), AAV CKd-B5 (SEQ ID NOs: 77 and 151 in U.S. Patent No. 8,734,809), AAV CKd-B6 (SEQ ID NOs: 78 and 152 in U.S. Patent No. 8,734,809), AAV CKd-B7 (SEQ ID NOs: 79 and 153 in U.S. Patent No. 8,734,809), AAV CKd-B8 (SEQ ID NOs: 80 and 154 in U.S. Patent No. 8,734,809), AAV CKd-H1 (SEQ ID NOs: 81 and 155 in U.S. Patent No. 8,734,809), AAV CKd-H2 (SEQ ID NOs: 82 and 156 in U.S. Patent No. 8,734,809), AAV CKd-H3 (SEQ ID NOs: 83 and 157 in U.S. Patent No. 8,734,809), AAV CKd-H4 (SEQ ID NOs: 84 and 158 in U.S. Patent No. 8,734,809), AAV CKd-H5 (SEQ ID NOs: 85 and 159 in U.S. Patent No. 8,734,809), AAV CKd-H6 (SEQ ID NOs: 77 and 151 in U.S. Patent No. 8,734,809), AAV CHt-1 (SEQ ID NOs: 86 and 160 in U.S. Patent No. 8,734,809), AAV CLv1-1 (SEQ ID NO: 171 in U.S. Patent No. 8,734,809), AAV CLv1-2 (SEQ ID NO: 172 in U.S. Patent No. 8,734,809), AAV CLv1-3 (SEQ ID NO: 173 in U.S. Patent No. 8,734,809), AAV CLv1-4 (SEQ ID NO: 174 in U.S. Patent No. 8,734,809), AAV Clv1-7 (SEQ ID NO: 175 in U.S. Patent No. 8,734,809), AAV Clv1-8 (SEQ ID NO: 176 in U.S. Patent No. 8,734,809), AAV Clv1-9 (SEQ ID NO: 177 in U.S. Patent No. 8,734,809), AAVIt may be or have a sequence as described in U.S. Pat. No. 8,734,809 (the contents of which are incorporated by reference in their entirety), such as Clv1-10 (SEQ ID NO: 178 of U.S. Pat. No. 8,734,809), AAV.VR-355 (SEQ ID NO: 181 of U.S. Pat. No. 8,734,809), AAV.hu.48R3 (SEQ ID NO: 183 of U.S. Pat. No. 8,734,809), or a variant or derivative thereof.

[0126] In some embodiments, the AAV serotypes include, but are not limited to, AAV CHt-P2 (SEQ ID NOs: 1 and 51 in WO2016065001), AAV CHt-P5 (SEQ ID NOs: 2 and 52 in WO2016065001), AAV CHt-P9 (SEQ ID NOs: 3 and 53 in WO2016065001), AAV CBr-7.1 (SEQ ID NOs: 4 and 54 in WO2016065001), AAV CBr-7.2 (SEQ ID NOs: 5 and 55 in WO2016065001), AAV CBr-7.3 (SEQ ID NOs: 6 and 56 in WO2016065001), AAV CBr-7.4 (SEQ ID NOs: 7 and 57 in WO2016065001), AAV CBr-7.5 (SEQ ID NOs: 8 and 58 in WO 2016065001), AAV CBr-7.7 (SEQ ID NOs: 9 and 59 in WO 2016065001), AAV CBr-7.8 (SEQ ID NOs: 10 and 60 in WO 2016065001), AAV CBr-7.10 (SEQ ID NOs: 11 and 61 in WO 2016065001), AAV CKd-N3 (SEQ ID NOs: 12 and 62 in WO 2016065001), AAV CKd-N4 (SEQ ID NOs: 13 and 63 in WO 2016065001), AAV CKd-N9 (SEQ ID NOs: 14 and 64 in WO 2016065001), AAV CLv-L4 (SEQ ID NOs: 15 and 65 in WO 2016065001), AAV CLv-L5 (SEQ ID NOs: 16 and 66 in WO 2016065001), AAV CLv-L6 (SEQ ID NOs: 17 and 67 in WO 2016065001), AAV CLv-K1 (SEQ ID NOs: 18 and 68 in WO 2016065001), AAV CLv-K3 (SEQ ID NOs: 19 and 69 in WO 2016065001), AAV CLv-K6 (SEQ ID NOs: 20 and 70 in WO 2016065001), AAV CLv-M1 (SEQ ID NOs: 21 and 71 in WO 2016065001), AAV CLv-M11 (SEQ ID NOs: 22 and 72 in WO 2016065001), AAV CLv-M2 (SEQ ID NOs: 23 and 73 in WO 2016065001), AAV CLv-M5 (SEQ ID NOs: 24 and 74 in WO 2016065001), AAV CLv-M6 (SEQ ID NOs: 25 and 75 in WO 2016065001), AAV CLv-M7 (SEQ ID NOs: 26 and 76 in WO 2016065001), AAV CLv-M8 (SEQ ID NOs: 27 and 77 in WO 2016065001), AAV CLv-M9 (SEQ ID NOs: 28 and 78 in WO 2016065001), AAV CHt-P1 (SEQ ID NOs: 29 and 79 in WO 2016065001), AAV CHt-P6 (SEQ ID NOs: 30 and 80 in WO 2016065001), AAV CHt-P8 (SEQ ID NOs: 31 and 81 in WO 2016065001), and AAV CHt-6.1 (SEQ ID NOs: 32 and 82 in International Publication No. 2016065001), AAV CHt-6.10 (SEQ ID NOs: 33 and 83 in International Publication No. 2016065001), AAV CHt-6.5 (SEQ ID NOs: 34 and 84 in International Publication No. 2016065001), AAV CHt-6.6 (SEQ ID NOs: 35 and 85 in International Publication No. 2016065001), AAV CHt-6.7 (SEQ ID NOs: 36 and 86 in International Publication No. 2016065001), AAV CHt-6.8 (SEQ ID NOs: 37 and 87 in International Publication No. 2016065001), AAV CSp-8.10 (SEQ ID NOs: 38 and 88 in International Publication No. 2016065001), AAV CSp-8.2 (SEQ ID NOs: 39 and 89 in WO 2016065001), AAV CSp-8.4 (SEQ ID NOs: 40 and 90 in WO 2016065001), AAV CSp-8.5 (SEQ ID NOs: 41 and 91 in WO 2016065001), AAV CSp-8.6 (SEQ ID NOs: 42 and 92 in WO 2016065001), AAV CSp-8.7 (SEQ ID NOs: 43 and 93 in WO 2016065001), AAV CSp-8.8 (SEQ ID NOs: 44 and 94 in WO 2016065001), AAV CSp-8.9 (SEQ ID NOs: 45 and 95 in WO 2016065001), AAV The AAV may be or have a sequence as described in WO2016065001 (the contents of which are incorporated by reference in their entirety), such as CBr-B7.3 (SEQ ID NOs: 46 and 96 in WO2016065001), AAV CBr-B7.4 (SEQ ID NOs: 47 and 97 in WO2016065001), AAV3B (SEQ ID NOs: 48 and 98 in WO2016065001), AAV4 (SEQ ID NOs: 49 and 99 in WO2016065001), AAV5 (SEQ ID NOs: 50 and 100 in WO2016065001), or a variant or derivative thereof.

[0127] In some embodiments, the AAV serotype may be or have modifications as described in U.S. Patent Application Publication No. 20160361439 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof.

[0128] In some embodiments, the AAV serotype may be or have mutations as described in U.S. Patent No. 9,546,112 (the contents of which are incorporated by reference herein in their entirety), including, but not limited to, at least two, but not all, of the F129L, D418E, K531E, L584F, V598A, and H642N mutations in the sequence of AAV6 (SEQ ID NO: 4 of U.S. Patent No. 9,546,112), AAV1 (SEQ ID NO: 6 of U.S. Patent No. 9,546,112), AAV2, AAV3, AAV4, AAV5, AAV7, AAV9, AAV10, or AAV11, or derivatives thereof. In yet another embodiment, the AAV serotype may be or have an AAV6 sequence (SEQ ID NO: 5 of U.S. Patent No. 9,546,112) containing the K531E mutation.

[0129] In some embodiments, the AAV serotype may be or have mutations in the AAV1 sequence as described in U.S. Patent Application Publication No. 20130224836 (the contents of which are incorporated by reference in their entirety) such as, but not limited to, at least one of the surface-exposed tyrosine residues at positions 252, 273, 445, 701, 705, and 731 of AAV1 (SEQ ID NO: 2 of U.S. Patent Application Publication No. 20130224836) substituted with another amino acid, preferably a phenylalanine residue. In one embodiment, the AAV serotype may be or have a mutation in the AAV9 sequence, such as, but not limited to, at least one of the surface-exposed tyrosine residues at positions 252, 272, 444, 500, 700, 704, and 730 of AAV2 (US Patent Publication No. 20130224836 SEQ ID NO: 4) substituted with another amino acid, preferably a phenylalanine residue. In one embodiment, the tyrosine residue at position 446 of AAV9 (US Patent Publication No. 20130224836 SEQ ID NO: 6) is substituted with a phenylalanine residue.

[0130] In some embodiments, the AAV serotype may be AAV2 or a variant thereof, as described in International Publication No. WO2016130589, which is incorporated herein by reference in its entirety. The amino acid sequence of AAV2 may include an N587A, E548A, or N708A mutation. In one embodiment, the amino acid sequence of any AAV may include a V708K mutation.

[0131] In one embodiment, the AAV may be of a serotype selected from any of those listed in Table 1. In one embodiment, the AAV may comprise a sequence of a sequence in Table 1, a fragment or variant thereof.

[0132] In one embodiment, the AAV may be encoded by a sequence, fragment or variant as set forth in Table 1.

[0133] Table 1-1

[0134] Table 1-2

[0135] Table 1-3

[0136] Table 1-4

[0137] Table 1-5

[0138] Table 1-6

[0139] Table 1-7

[0140] Table 1-8

[0141] Table 1-9

[0142] Table 1-10

[0143] Table 1-11

[0144] Table 1-12

[0145] Table 1-13

[0146] Table 1-14

[0147] Table 1-15

[0148] Table 1-16

[0149] Table 1-17

[0150] Table 1-18

[0151] Table 1-19

[0152] Table 1-20

[0153] Table 1-21

[0154] [Table 1-22]

[0155] [Table 1-23]

[0156] [Table 1-24]

[0157] [Table 1-25]

[0158] [Table 1-26]

[0159] [Table 1-27]

[0160] [Table 1-28]

[0161] [Table 1-29]

[0162] [Table 1-30] Each of the patents, applications and / or publications listed in Table 1 is hereby incorporated by reference in its entirety.

[0163] In one embodiment, the AAV serotypes include, but are not limited to, AAV9 (SEQ ID NOs: 2 and 11 in WO2015038958 or SEQ ID NOs: 127 and 126, respectively, herein), PHP.B (SEQ ID NOs: 8 and 9 in WO2015038958, SEQ ID NOs: 868 and 869 herein), G2B-13 (SEQ ID NO: 12 in WO2015038958, SEQ ID NO: 870 herein), G2B-26 (SEQ ID NO: 13 in WO2015038958, SEQ ID NO: 140 herein), G2B-36 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 141 herein), G2B-46 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 142 herein), G2B-56 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 143 herein), G2B-66 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 144 herein), G2B-76 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 145 herein), G2B-86 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 146 herein), G2B-96 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 147 herein), G2B-106 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO The targeting peptides may be or have a sequence as described in WO2015038958 (the contents of which are incorporated by reference in their entirety), such as SEQ ID NO: 13, SEQ ID NOs: 868 and 869 herein), TH1.1-32 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 871 herein), TH1.1-35 (SEQ ID NO: 15 in WO2015038958, SEQ ID NO: 872 herein), or variants thereof. Additionally, any of the targeting peptides or amino acid inserts described in WO2015038958 may be inserted into any parent AAV serotype, such as, but not limited to, AAV9 (SEQ ID NO: 126 for the DNA sequence and SEQ ID NO: 127 for the amino acid sequence). In one embodiment, the amino acid insert is inserted at amino acids 586-592 of the parent AAV (e.g., AAV9). In another embodiment, the amino acid insert is inserted at amino acids 588-589 of the parent AAV sequence.The amino acid insert may include, but is not limited to, the following amino acid sequences: TLAVPFK (SEQ ID NO: 1 in WO2015038958; SEQ ID NO: 873 herein), KFPVALT (SEQ ID NO: 3 in WO2015038958; SEQ ID NO: 874 herein), LAVPFK (SEQ ID NO: 31 in WO2015038958; SEQ ID NO: 875 herein), AVPFK (SEQ ID NO: 32 in WO2015038958; SEQ ID NO: 876 herein), LAVPFK (SEQ ID NO: 33 in WO2015038958; SEQ ID NO: 877 herein), AVPFK (SEQ ID NO: 34 in WO2015038958; SEQ ID NO: 879 herein), LAVPFK (SEQ ID NO: 35 in WO2015038958; SEQ ID NO: 878 herein), AVPFK (SEQ ID NO: 36 in WO2015038958; SEQ ID NO: 879 herein), AVPFK (SEQ ID NO: 37 in WO2015038958; SEQ ID NO: 879 herein), AVPFK (SEQ ID NO: 38 in WO2015038958; SEQ ID NO: 879 herein), AVPFK (SEQ ID NO: 39 in WO2015038958; SEQ ID NO: 40 in WO2015038958), AVPFK (SEQ ID NO: 41 in WO2015038958; SEQ ID NO: 42 in WO2015038958), AVPFK (SEQ ID NO: 43 in WO2015038 ; SEQ ID NO: 876 herein), VPFK (SEQ ID NO: 33 in WO2015038958; SEQ ID NO: 877 herein), TLAVPF (SEQ ID NO: 34 in WO2015038958; SEQ ID NO: 878 herein), TLAVP (SEQ ID NO: 35 in WO2015038958; SEQ ID NO: 879 herein), TLAV (SEQ ID NO: 36 in WO2015038958; SEQ ID NO: 880 herein) , SVSKPFL (SEQ ID NO: 28 in WO 2015038958; SEQ ID NO: 881 herein), FTLTTPK (SEQ ID NO: 29 in WO 2015038958; SEQ ID NO: 882 herein), MNATKNV (SEQ ID NO: 30 in WO 2015038958; SEQ ID NO: 883 herein), QSSQTPR (SEQ ID NO: 54 in WO 2015038958; SEQ ID NO: 884 herein), ILGTGTS (SEQ ID NO: 55 in WO2015038958; SEQ ID NO: 885 herein), TRTNPEA (SEQ ID NO: 56 in WO2015038958; SEQ ID NO: 886 herein), NGGTSSS (SEQ ID NO: 58 in WO2015038958; SEQ ID NO: 887 herein), or YTLSQGW (SEQ ID NO: 60 in WO2015038958; SEQ ID NO: 888 herein).Non-limiting examples of nucleotide sequences that may encode the amino acid insert include the following: AAGTTCCTGTGGCGTTGACT (for SEQ ID NO: 3 in WO2015038958; SEQ ID NO: 889 herein), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NOs: 24 and 49 in WO2015038958; SEQ ID NO: 890 herein), AGTGTGAGTAAGCCTTTTTTG (SEQ ID NO: 25 in WO2015038958; SEQ ID NO: 891 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 26 in WO2015038958; SEQ ID NO: 892 herein), ATGAATGCTACGAAGAATGTG (SEQ ID NO: 27 in WO2015038958; and SEQ ID NO: 893 herein), CAGTCGTCGCAGACGCCTAGG (SEQ ID NO: 48 in WO2015038958; SEQ ID NO: 894 herein), ATTCTGGGGACTGGTACTTCG (SEQ ID NOs: 50 and 52 in WO2015038958; SEQ ID NO: 895 herein), ACGCGGACTAATCCTGAGGCT (SEQ ID NO: 51 in WO2015038958; SEQ ID NO: 896 herein), AATGGGGGGACTAGTAGTTCT (SEQ ID NO: 53 in WO2015038958; SEQ ID NO: 897 herein), or TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 59 in WO2015038958; SEQ ID NO: 898 herein).

[0164] In one embodiment, an AAV serotype may be engineered to include at least one AAV capsid CD8+ T-cell epitope of AAV2, such as, but not limited to, SADNNNSEY (SEQ ID NO: 899), LIDQYLYYL (SEQ ID NO: 900), VPQYGYLTL (SEQ ID NO: 901), TTSTRTWAL (SEQ ID NO: 902), YHLNGRDSL (SEQ ID NO: 903), SQAVGRSSF (SEQ ID NO: 904), VPANPSTTF (SEQ ID NO: 905), FPQSGVLIF (SEQ ID NO: 906), YFDFNRFHCHFSPRD (SEQ ID NO: 907), VGNSSGNWHCDSTWM (SEQ ID NO: 908), QFSQAGASDIRDQSR (SEQ ID NO: 909), GASDIRQSRNWLP (SEQ ID NO: 910), and GNRQAATADVNTQGV (SEQ ID NO: 911).

[0165] In one embodiment, the AAV serotype may be engineered to include at least one AAV capsid CD8+ T cell epitope of AAV1, such as, but not limited to, LDRLMNPLI (SEQ ID NO: 912), TTSTRTWAL (SEQ ID NO: 902), and QPAKKRLNF (SEQ ID NO: 913).

[0166] In one embodiment, the AAV serotype may be or have a sequence as described in WO2017100671 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, AAV9 (SEQ ID NO: 45 in WO2017100671, SEQ ID NO: 1861 herein), PHP.N (SEQ ID NO: 46 in WO2017100671, SEQ ID NO: 1859 herein), PHP.S (SEQ ID NO: 47 in WO2017100671, SEQ ID NO: 1860 herein), or variants thereof. Additionally, any of the targeting peptides or amino acid inserts described in WO2017100671 may be inserted into any parent AAV serotype, such as, but not limited to, AAV9 (SEQ ID NO: 127 or SEQ ID NO: 1861). In one embodiment, the amino acid insert is inserted at amino acids 586-592 of the parent AAV (e.g., AAV9). In another embodiment, the amino acid insert is inserted at amino acids 588-589 of the parent AAV sequence. Amino acid inserts may include, but are not limited to, the following amino acid sequences: AQTLAVPFKAQ (SEQ ID NO: 1 in WO2017100671; SEQ ID NO: 2245 herein), AQSVSKPFLAQ (SEQ ID NO: 2 in WO2017100671; SEQ ID NO: 2246 herein), AQFTLTTPKAQ (SEQ ID NO: 3 in the Sequence Listing of WO2017100671; SEQ ID NO: 2247 herein), DGTLAVPF KAQ (SEQ ID NO: 4 in the Sequence Listing of WO 2017100671; SEQ ID NO: 2248 herein), ESTLAVPFKAQ (SEQ ID NO: 5 in WO 2017100671; SEQ ID NO: 2249 herein), GGTLAVPFKAQ (SEQ ID NO: 6 in WO 2017100671; SEQ ID NO: 2250 herein), AQTLATPFKAQ (SEQ ID NOs: 7 and 33 in WO 2017100671;SEQ ID NO: 2251 herein), ATTLATPFKAQ (SEQ ID NO: 8 in WO 2017100671; SEQ ID NO: 2252 herein), DGTLATPFKAQ (SEQ ID NO: 9 in WO 2017100671; SEQ ID NO: 2253 herein), GGTLATPFKAQ (SEQ ID NO: 10 in WO 2017100671; SEQ ID NO: 2254 herein), SGSLAVPFKAQ (SEQ ID NO: 11 in WO 2017100671; SEQ ID NO: 2255 herein), GGTLATPFKAQ (SEQ ID NO: 12 in WO 2017100671; SEQ ID NO: 2256 herein), GGTLATPFKAQ (SEQ ID NO: 13 in WO 2017100671; SEQ ID NO: 2257 herein), SGSLAVPFKAQ (SEQ ID NO: 14 in WO 2017100671; SEQ ID NO: 2259 herein), GGTLATPFKAQ (SEQ ID NO: 15 in WO 2017100671; SEQ ID NO: 2259 herein), GGTLATPFKAQ (SEQ ID NO: 16 in WO 2017100671; SEQ ID NO: 2259 herein), GGTLATPFKAQ (SEQ ID NO: 17 in WO 2017100671; SEQ ID NO: 2259 herein), GGTLATPFKAQ (SEQ ID NO: 18 in WO 2017100671; SEQ ID NO: 2259 herein), GGTLATPFKAQ (SEQ ID NO: 19 in WO 20171006 SEQ ID NO: 2255 in the specification), AQTLAQPFKAQ (SEQ ID NO: 12 in WO 2017100671; SEQ ID NO: 2256 herein), AQTLQQPFKAQ (SEQ ID NO: 13 in WO 2017100671; SEQ ID NO: 2257 herein), AQTLSNPFKAQ (SEQ ID NO: 14 in WO 2017100671; SEQ ID NO: 2258 herein), AQTLAVPFSNP (SEQ ID NO: 15 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 16 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 17 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 18 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 19 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 20 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 21 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 22 in WO 2017100671; SEQ ID NO: 2259 herein), AQTLAPFSNP (SEQ ID NO: 23 in WO 2017100671 SEQ ID NO: 2259 in the specification), QGTLAVPFKAQ (SEQ ID NO: 16 in WO 2017100671; SEQ ID NO: 2260 herein), NQTLAVPFKAQ (SEQ ID NO: 17 in WO 2017100671; SEQ ID NO: 2261 herein), EGSLAVPFKAQ (SEQ ID NO: 18 in WO 2017100671; SEQ ID NO: 2262 herein), SGNLAVPFKAQ (SEQ ID NO: 19 in WO 2017100671; SEQ ID NO: 2263 herein), SEQ ID NO: 2263 in the specification), EGTLAVPFKAQ (SEQ ID NO: 20 in WO 2017100671; SEQ ID NO: 2264 herein), DSTLAVPFKAQ (SEQ ID NO: 21 in Table 1 of WO 2017100671; SEQ ID NO: 2265 herein), AVTLAVPFKAQ (SEQ ID NO: 22 in WO 2017100671; SEQ ID NO: 2266 herein), AQTLSTPFKAQ (SEQ ID NO: 23 in WO 2017100671;SEQ ID NO: 2267 herein), AQTLPQPFKAQ (SEQ ID NOs: 24 and 32 in WO2017100671; SEQ ID NO: 2268 herein), AQTLSQPFKAQ (SEQ ID NO: 25 in WO2017100671; SEQ ID NO: 2269 herein), AQTLQLPFKAQ (SEQ ID NO: 26 in WO2017100671; SEQ ID NO: 2270 herein), AQTLTMPFKAQ (SEQ ID NOs: 27 ...TLQLPFKAQ (SEQ ID NO: 26 in WO2017100671; SEQ ID NO: 2270 herein), AQTLTMPFKAQ (SEQ ID NOs: 27 and 32 in WO2017100671; SEQ ID NO: 2268 herein), AQTLTMPFKAQ (SEQ ID NO: 27 in WO2017100671; SEQ ID NO: 2268 herein), AQTLTMPFKAQ (SEQ ID NO: 27 in WO2017100671; SEQ ID NO: 2268 herein), AQTLTMPFKAQ (SEQ ID NO: 34, and SEQ ID NO: 35 in the Sequence Listing of WO 2017100671; SEQ ID NO: 2271 herein), AQTLTTPFKAQ (SEQ ID NO: 28 in WO 2017100671; SEQ ID NO: 2272 herein), AQYTLSQGWAQ (SEQ ID NO: 29 in WO 2017100671; SEQ ID NO: 2273 herein), AQMNATKNVAQ (SEQ ID NO: 30 in WO 2017100671; SEQ ID NO: 2274 herein), AQVSGGHH SAQ (SEQ ID NO: 31 in WO 2017100671; SEQ ID NO: 2275 herein), AQTLTAPFKAQ (SEQ ID NO: 35 in Table 1 of WO 2017100671; SEQ ID NO: 2276 herein), AQTLSKPFKAQ (SEQ ID NO: 36 in WO 2017100671; SEQ ID NO: 2277 herein), QAVRTSL (SEQ ID NO: 37 in WO 2017100671; SEQ ID NO: 2278 herein), YTLSQGW (WO 2017100671; SEQ ID NO: 2279 herein), and YTLSKPFKAQ (WO 2017100671; SEQ ID NO: 2280 herein). SEQ ID NO: 38 in WO 2017100671; SEQ ID NO: 888 herein), LAKERLS (SEQ ID NO: 39 in WO 2017100671; SEQ ID NO: 2279 herein), TLAVPFK (SEQ ID NO: 40 in the Sequence Listing of WO 2017100671; SEQ ID NO: 873 herein), SVSKPFL (SEQ ID NO: 41 in WO 2017100671; SEQ ID NO: 881 herein), FTLTTPK (SEQ ID NO: 42 in WO 2017100671;SEQ ID NO: 882 herein), MNSTKNV (SEQ ID NO: 43 in WO 2017100671; SEQ ID NO: 2280 herein), VSGGHHS (SEQ ID NO: 44 in WO 2017100671; SEQ ID NO: 2281 herein), SAQTLAVPFKAQAQ (SEQ ID NO: 48 in WO 2017100671; SEQ ID NO: 2282 herein), SXXXLAVPFKAQAQ (SEQ ID NO: 49 in WO 2017100671, where X is any amino acid X may be any amino acid; SEQ ID NO: 2283 herein), SAQXXXVPFKAQAQ (SEQ ID NO: 50 in WO 2017100671, where X may be any amino acid; SEQ ID NO: 2284 herein), SAQTLXXXFKAQAQ (SEQ ID NO: 51 in WO 2017100671, where X may be any amino acid; SEQ ID NO: 2285 herein), SAQTLAVXXXAQAQ (SEQ ID NO: 52 in WO 2017100671, where X may be any amino acid; SEQ ID NO: 2286 herein), SAQTLAVPFXXXAQ (SEQ ID NO: 53 in WO 2017100671, where X may be any amino acid; SEQ ID NO: 2287 herein), TNHQSAQ (SEQ ID NO: 65 in WO 2017100671; SEQ ID NO: 2288 herein), AQAQTGW (SEQ ID NO: 66 in WO 2017100671; SEQ ID NO: 2289 herein), DGTLATPFK (SEQ ID NO: 2289 in WO 2017100671 SEQ ID NO: 67; SEQ ID NO: 2290 herein), DGTLATPFKXX (SEQ ID NO: 68 in WO2017100671, where X may be any amino acid; SEQ ID NO: 2291 herein), LAVPFKAQ (SEQ ID NO: 80 in WO2017100671; SEQ ID NO: 2292 herein), VPFKAQ (SEQ ID NO: 81 in WO2017100671; SEQ ID NO: 2293 herein), FKAQ (SEQ ID NO: 82 in WO2017100671;SEQ ID NO: 2294 herein), AQTLAV (SEQ ID NO: 83 in WO 2017100671; SEQ ID NO: 2295 herein), AQTLAVPF (SEQ ID NO: 84 in WO 2017100671; SEQ ID NO: 2296 herein), QAVR (SEQ ID NO: 85 in WO 2017100671; SEQ ID NO: 2297 herein), AVRT (SEQ ID NO: 86 in WO 2017100671; SEQ ID NO: 2298 herein), VRTS (SEQ ID NO: 87 in WO 2017100671; SEQ ID NO: 2299 herein), RTSL ... SEQ ID NO: 88 in WO 2017100671; SEQ ID NO: 2300 herein), QAVRT (SEQ ID NO: 89 in WO 2017100671; SEQ ID NO: 2301 herein), AVRTS (SEQ ID NO: 90 in WO 2017100671; SEQ ID NO: 2302 herein), VRTSL (SEQ ID NO: 91 in WO 2017100671; SEQ ID NO: 2303 herein), QAVRTS (SEQ ID NO: 92 in WO 2017100671; SEQ ID NO: 2304 herein), or AVRTSL (SEQ ID NO: 93 in WO 2017100671; SEQ ID NO: 2305 herein).

[0167] Non-limiting examples of nucleotide sequences that may encode the amino acid insert include the following: GATGGGACTTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 54 in WO2017100671; SEQ ID NO: 2306 herein), GATGGGACGTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 55 in WO2017100671; SEQ ID NO: 2307 herein), CAGGCGGTTAGGACGTCTTTG (SEQ ID NO: 56 in WO2017100671; SEQ ID NO: 2308 herein), CAGGTCTTCACGGACTCAGACTATCAG (SEQ ID NOs: 57 and 78 in WO2017100671; SEQ ID NO: 2309 herein), CAAGTAAAACCTCTACAAATGTGGTAAAATCG (SEQ ID NO: 58 in WO2017100671; SEQ ID NO: 2310 herein), ACT CATCGACCAATACTTGTACTATCTCTCTAGAAC (SEQ ID NO: 59 in WO 2017100671; SEQ ID NO: 2311 herein), GGAAGTATTCCTTGGTTTTGAACCCA (SEQ ID NO: 60 in WO 2017100671; SEQ ID NO: 2312 herein), GGTCGCGGTTCTTGTTTGTGGAT (SEQ ID NO: 61 in WO 2017100671; SEQ ID NO: 2312 herein), SEQ ID NO: 2313), CGACCTTGAAGCGCATGAACTCCT (SEQ ID NO: 62 in WO2017100671; SEQ ID NO: 2314 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNMNNMNNMNNMNNTTGGGCACTCTGGTGGTTTGTC (SEQ ID NO: 63 in WO2017100671, where N may be A, C, T, or G;SEQ ID NO: 2315 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCMNNMNNMNNAAAAGGCACCGCCAAAGTTTG (SEQ ID NO: 69 in WO2017100671, where N can be A, C, T, or G; SEQ ID NO: 2316 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNCACCGCCAAAGTTTGGGCACT (WO20171006 71, where N may be A, C, T, or G; SEQ ID NO: 2317 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAMNNMNNMNNCAAAGTTTGGGCACTCTGGTGG (SEQ ID NO: 71 in WO 2017100671, where N may be A, C, T, or G; SEQ ID NO: 2318 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCG CCTGTGCCTTAAAAGGCACMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG (SEQ ID NO: 72 in WO2017100671, where N can be A, C, T, or G; SEQ ID NO: 2319 herein), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NO: 74 in WO2017100671; SEQ ID NO: 890 herein), AGTGTGAGTAAGCCTTTTTTG (SEQ ID NO: 10 in WO2017100671 No. 75; SEQ ID NO: 891 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 76 in WO2017100671; SEQ ID NO: 892 herein), TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 77 in WO2017100671; SEQ ID NO: 898 herein), or CTTGCGAAGGAGCGGCTTTCG (SEQ ID NO: 79 in WO2017100671; SEQ ID NO: 2320 herein).

[0168] In one embodiment, the AAV serotypes include, but are not limited to, AAV1 (SEQ ID NO: 181 in U.S. Pat. No. 9,624,274), AAV6 (SEQ ID NO: 182 in U.S. Pat. No. 9,624,274), AAV2 (SEQ ID NO: 183 in U.S. Pat. No. 9,624,274), AAV3b (SEQ ID NO: 184 in U.S. Pat. No. 9,624,274), AAV7 (SEQ ID NO: 185 in U.S. Pat. No. 9,624,274), AAV8 (SEQ ID NO: 186 in U.S. Pat. No. 9,624,274), AAV10 (SEQ ID NO: 187 in U.S. Pat. No. 9,624,274), AAV4 (SEQ ID NO: 188 in U.S. Pat. No. 9,624,274), AAV11 (SEQ ID NO: 189 in U.S. Pat. No. 9,624,274), bAAV (SEQ ID NO: 190 in U.S. Pat. No. 9,624,274), AAV5 (SEQ ID NO: 191 in U.S. Pat. No. 9,624,274), AAV6 (SEQ ID NO: 192 in U.S. Pat. No. 9,624,274), AAV7 (SEQ ID NO: 185 in U.S. Pat. No. 9,624,274), AAV8 (SEQ ID NO: 186 in U.S. Pat. No. 9,624,274), AAV10 (SEQ ID NO: 187 in U.S. Pat. No. 9, No. 9,624,274 (the contents of which are herein incorporated by reference in their entirety), such as GPV (SEQ ID NO: 191 in U.S. Pat. No. 9,624,274; SEQ ID NO: 1862 herein), B19 (SEQ ID NO: 193 in U.S. Pat. No. 9,624,274; SEQ ID NO: 1863 herein), MVM (SEQ ID NO: 194 in U.S. Pat. No. 9,624,274; SEQ ID NO: 1864 herein), FPV (SEQ ID NO: 195 in U.S. Pat. No. 9,624,274; SEQ ID NO: 1865 herein), CPV (SEQ ID NO: 196 in U.S. Pat. No. 9,624,274; SEQ ID NO: 1866 herein), or variants thereof. Additionally, any of the structural protein inserts described in U.S. Patent No. 9,624,274 may be inserted into, but not limited to, I-453 and I-587 of any parent AAV serotype, such as, but not limited to, AAV2 (U.S. Patent No. 9,624,274 SEQ ID NO: 183). Amino acid inserts may include, but are not limited to, the following amino acid sequences: VNLTWSRASG (U.S. Patent No. 9,624,274 SEQ ID NO: 50; SEQ ID NO: 2321 herein), EFCINHRGYWVCGD (U.S. Patent No. 9,624,274 SEQ ID NO: 55;No. 9,624,274; SEQ ID NO: 2322 herein), EDGQVMDVDLS (SEQ ID NO: 85 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2323 herein), EKQRNGTLT (SEQ ID NO: 86 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2324 herein), TYQCRVTHPHLPRALMR (SEQ ID NO: 87 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2325 herein), RHSTTQPRKTKGSG (SEQ ID NO: 88 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2326 herein). 326), DSNPRGVSAYLSR (SEQ ID NO: 89 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2327 herein), TITCLWDLAPSK (SEQ ID NO: 90 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2328 herein), KTKGSGFFVF (SEQ ID NO: 91 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2329 herein), THPHLPRALMRS (SEQ ID NO: 92 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2330 herein), GETYQCRVTHPHL PRALMRSTTK (SEQ ID NO: 93 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2331 herein), LPRALMRS (SEQ ID NO: 94 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2332 herein), INHRGYWV (SEQ ID NO: 95 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2333 herein), CDAGSVRTNAPD (SEQ ID NO: 60 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2334 herein), AKAVSNLTESRSESLQS (U.S. Pat. No. 9,624,274; SEQ ID NO: 2334 herein), and AKAVSNLTESRSESLQS (U.S. Pat. No. 9,624,274; SEQ ID NO: 2334 herein). 274; SEQ ID NO:2335 herein), SLTGDEFKKVLET (U.S. Pat. No. 9,624,274; SEQ ID NO:2336 herein), REAVAYRFEED (U.S. Pat. No. 9,624,274; SEQ ID NO:2337 herein), INPEIITLDG (U.S. Pat. No. 9,624,274; SEQ ID NO:2338 herein), DISVTGAPVITATYL (U.S. Pat. No. 9,624,274; SEQ ID NO:100 herein),No. 9,624,274; SEQ ID NO: 2340 herein), PKTVSNLTESSSESVQS (SEQ ID NO: 102 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2341 herein), SLMGDEFKAVLET (SEQ ID NO: 103 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2342 herein), QHSVAYTFEED (SEQ ID NO: 104 in U.S. Pat. No. 9,624,274; SEQ ID NO: 23 43), INPEIITRDG (SEQ ID NO: 105 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2344 herein), DISLTGDPVITASYL (SEQ ID NO: 106 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2345 herein), DISLTGDPVITA (SEQ ID NO: 107 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2346 herein), DQSIDFEIDSA (SEQ ID NO: 108 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2347 herein), KNVSEDLPLPTFSPT LLGDS (SEQ ID NO: 109 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2348 herein), KNVSEDLPLPT (SEQ ID NO: 110 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2349 herein), CDSGRVRTDAPD (SEQ ID NO: 111 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2350 herein), FPEHLLVDFLQSLS (SEQ ID NO: 112 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2351 herein), DAEFRHDSG (SEQ ID NO: 113 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2352 herein), No. 9,624,274; SEQ ID NO: 2352 herein), HYAAAQWDFGNTMCQL (SEQ ID NO: 113 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2353 herein), YAAQWDFGNTMCQ (SEQ ID NO: 114 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2354 herein), RSQKEGLHYT (SEQ ID NO: 115 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2355 herein), SSRTPSDKPVAHWANPQAE (SEQ ID NO: 116 in U.S. Pat. No. 9,624,274;No. 9,624,274; SEQ ID NO: 2356 herein), SRTPSDKPVAHWANP (SEQ ID NO: 117 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2357 herein), SSRTPSDKP (SEQ ID NO: 118 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2358 herein), NADGNVDYHMNSVP (SEQ ID NO: 119 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2359 herein), DGNVDYHMNSV (U.S. Pat. No. 9,624,274 No. 9,624,274; SEQ ID NO: 2360 herein), RSFKEFLQSSLRALRQ (SEQ ID NO: 121 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2361 herein), FKEFLQSSLRA (SEQ ID NO: 122 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2362 herein), or QMWAPQWGPD (SEQ ID NO: 123 in U.S. Pat. No. 9,624,274; SEQ ID NO: 2363 herein);

[0169] In one embodiment, the AAV serotype may be or have a sequence as described in U.S. Pat. No. 9,475,845, the contents of which are incorporated by reference in their entirety, including, but not limited to, an AAV capsid protein comprising one or more amino acid modifications at amino acid positions 585-590 of the native AAV2 capsid protein. Further modifications may include, but are not limited to, the following amino acid sequences: RGNRQA (SEQ ID NO: 3 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2364 herein), SSSTDP (SEQ ID NO: 4 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2365 herein), SSNTAP (SEQ ID NO: 5 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2366 herein), SNSNLP (SEQ ID NO: 6 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2367 herein), SSTTAP (SEQ ID NO: 7 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2368 herein), AANTAA (SEQ ID NO: 8 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2369 herein), QQNTAP (SEQ ID NO: 9 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2370 herein), SAQAQA (SEQ ID NO: 10 in U.S. Pat. No. 9,475,845; SEQ ID NO: 11 herein), SAQAQA (SEQ ID NO: 12 in U.S. Pat. No. 9,475,845; SEQ ID NO: 13 herein), SAQAQA (SEQ ID NO: 14 in U.S. Pat. No. 9,475,845; SEQ ID NO: 15 herein), SAQAQA (SEQ ID NO: 16 in U.S. Pat. No. 9,475,845; SEQ ID NO: 16 herein), SAQAQA (SEQ ID NO No. 9,475,845; SEQ ID NO: 2371 herein), QANTGP (SEQ ID NO: 11 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2372 herein), NATTAP (SEQ ID NO: 12 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2373 herein), SSTAGP (SEQ ID NOs: 13 and 20 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2374 herein), QQNTAA (SEQ ID NO: 14 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2375 herein), PSTAGP (SEQ ID NO: 15 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2376 herein), NQNTAP (SEQ ID NO: 16 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2377 herein), QAANAP (SEQ ID NO: 17 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2378 herein), SIVGLP (SEQ ID NO: 18 in U.S. Pat. No. 9,475,845;No. 9,475,845; SEQ ID NO: 2379 herein), AASTAA (SEQ ID NOs: 19 and 27 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2380 herein), SQNTTA (SEQ ID NO: 21 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2381 herein), QQDTAP (SEQ ID NO: 22 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2382 herein), QTNTGP (SEQ ID NO: 23 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2383 herein), QTNGAP (SEQ ID NO: 24 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2384 herein), QQNAAP (SEQ ID NO: 25 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2385 herein), or AANTQA (SEQ ID NO: 26 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2386 herein). In one embodiment, the amino acid modification is a substitution at amino acid positions 262-265 of the native AAV2 capsid protein, or at the corresponding positions of a capsid protein of another AAV that has a targeting sequence, including, but not limited to, the following amino acid sequences: NGRAHA (SEQ ID NO: 38 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2387 herein), QPEHSST (SEQ ID NOs: 39 and 50 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2388 herein), VNTANST (SEQ ID NO: 40 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2389 herein), HGPMQKS (SEQ ID NO: 41 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2390 herein), PHKPPLA (SEQ ID NO: 42 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2391 herein), PHKPPLA (SEQ ID NO: 43 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2392 herein), PHKPPLA (SEQ ID NO: 44 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2393 herein), PHKPPLA (SEQ ID NO: 45 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2394 herein), PHKPPLA (SEQ ID NO: 46 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2395 herein), PHKPPLA (SEQ ID NO: 47 in U.S. Pat. No. 9,4 SEQ ID NO: 42 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2391 herein), IKNNEMW (SEQ ID NO: 43 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2392 herein), RNLDTPM (SEQ ID NO: 44 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2393 herein), VDSHRQS (SEQ ID NO: 45 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2394 herein), YDSKTKT (SEQ ID NO: 46 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2395 herein), SQLPHQK (SEQ ID NO: 47 in U.S. Pat. No. 9,475,845;SEQ ID NO: 2396 herein), STMQQNT (SEQ ID NO: 48 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2397 herein), TERYMTQ (SEQ ID NO: 49 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2398 herein), DASLSTS (SEQ ID NO: 51 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2399 herein), DLPNKKT (SEQ ID NO: 52 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2400 herein), DLTAARL (U.S. Pat. No. 9,475,845 No. 9,475,845; SEQ ID NO: 2401 herein), EPHQFNY (SEQ ID NO: 54 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2402 herein), EPQSNHT (SEQ ID NO: 55 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2403 herein), MSSWPSQ (SEQ ID NO: 56 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2404 herein), NPKHNAT (SEQ ID NO: 57 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2405 herein), PDGMRTT (U.S. Pat. No. 9,475,845; SEQ ID NO: 2406 herein), SEQ ID NO: 58 in U.S. Patent No. 9475845; SEQ ID NO: 2406 herein), PNNNKTT (SEQ ID NO: 59 in U.S. Patent No. 9475845; SEQ ID NO: 2407 herein), QSTTHDS (SEQ ID NO: 60 in U.S. Patent No. 9475845; SEQ ID NO: 2408 herein), TGSKQKQ (SEQ ID NO: 61 in U.S. Patent No. 9475845; SEQ ID NO: 2409 herein), SLKHQAL (SEQ ID NO: 62 in U.S. Patent No. 9475845; SEQ ID NO: 2410 herein) , SPIDGEQ (SEQ ID NO: 63 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2411 herein), WIFPWIQL (SEQ ID NOs: 64 and 112 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2412 herein), CDCRGDCFC (SEQ ID NO: 65 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2413 herein), CNGRC (SEQ ID NO: 66 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2414 herein), CPRECES (SEQ ID NO: 67 in U.S. Pat. No. 9,475,845;SEQ ID NO: 2415 herein), CTTHWGFTLC (SEQ ID NOs: 68 and 123 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2416 herein), CGRRAGGSC (SEQ ID NO: 69 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2417 herein), CKGGRAKDC (SEQ ID NO: 70 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2418 herein), CVPELGHEC (SEQ ID NOs: 71 and 115 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2419 herein), CR RETAWAK (SEQ ID NO: 72 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2420 herein), VSWFSHRYSPFAVS (SEQ ID NO: 73 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2421 herein), GYRDGYAGPILYN (SEQ ID NO: 74 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2422 herein), XXXYXXX (SEQ ID NO: 75 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2423 herein), YXNW (SEQ ID NO: 76 in U.S. Pat. No. 9,475,845 ; SEQ ID NO: 2424 herein), RPLPPLP (SEQ ID NO: 77 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2425 herein), APPLPPR (SEQ ID NO: 78 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2426 herein), DVFYPYPYASGS (SEQ ID NO: 79 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2427 herein), MYWYPY (SEQ ID NO: 80 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2428 herein), DITWDQLWDLMK ​​(U.S. Pat. No. No. 9,475,845; SEQ ID NO: 2429 herein), CWDDXWLC (SEQ ID NO: 82 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2430 herein), EWCEYLGGYLRCYA (SEQ ID NO: 83 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2431 herein), YXCXXGPXTWXCXP (SEQ ID NO: 84 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2432 herein), IEGPTLRQWLAARA (SEQ ID NO: 85 in U.S. Pat. No. 9,475,845;SEQ ID NO: 2433 herein), LWXXX (SEQ ID NO: 86 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2434 herein), XFXXYLW (SEQ ID NO: 87 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2435 herein), SSIISHFRWGLCD (SEQ ID NO: 88 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2436 herein), MSRPACPPNDKYE (SEQ ID NO: 89 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2437 herein), CLRSGRGC (U.S. Pat. No. 9475,845; SEQ ID NO: 2438 herein), No. 5845; SEQ ID NO: 2438 herein), CHWMFSPWC (SEQ ID NO: 91 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2439 herein), WXXF (SEQ ID NO: 92 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2440 herein), CSSRLDAC (SEQ ID NO: 93 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2441 herein), CLPVASC (SEQ ID NO: 94 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2442 herein), CGFECVRQCPER C (SEQ ID NO: 95 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2443 herein), CVALCREACGEGC (SEQ ID NO: 96 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2444 herein), SWCEPGWCR (SEQ ID NO: 97 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2445 herein), YSGKWGW (SEQ ID NO: 98 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2446 herein), GLSGGRS (SEQ ID NO: 99 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2447 herein), No. 2447), LMLPRAD (SEQ ID NO: 100 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2448 herein), CSCFRDVCC (SEQ ID NO: 101 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2449 herein), CRDVVSVIC (SEQ ID NO: 102 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2450 herein), MARSGL (SEQ ID NO: 103 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2451 herein), MARAKE (SEQ ID NO: 104 in U.S. Pat. No. 9,475,845;SEQ ID NO:2452 herein), MSRTMS (SEQ ID NO:105 in U.S. Pat. No. 9,475,845; SEQ ID NO:2453 herein), KCCYSL (SEQ ID NO:106 in U.S. Pat. No. 9,475,845; SEQ ID NO:2454 herein), MYWGDSHWLQYWYE (SEQ ID NO:107 in U.S. Pat. No. 9,475,845; SEQ ID NO:2455 herein), MQLPLAT (SEQ ID NO:108 in U.S. Pat. No. 9,475,845; SEQ ID NO:2456 herein), EWLS (SEQ ID NO:109 in U.S. Pat. No. 9,475,845; SEQ ID NO:2457 herein; ), SNEW (SEQ ID NO: 110 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2458 herein), TNYL (SEQ ID NO: 111 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2459 herein), WDLAWMFRLPVG (SEQ ID NO: 113 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2460 herein), CTVALPGGYVRVC (SEQ ID NO: 114 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2461 herein), CVAYCIEHHCWTC (U.S. Pat. No. 9,475,845 No. 9,475,845; SEQ ID NO: 2462 herein), CVFAHNYDYLVC (SEQ ID NO: 117 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2463 herein), CVFTSNYAFC (SEQ ID NO: 118 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2464 herein), VHSPNKK (SEQ ID NO: 119 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2465 herein), CRGDGWC (SEQ ID NO: 120 in U.S. Pat. No. 9,475,845; SEQ ID NO: 246 6), XRGCDX (SEQ ID NO: 121 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2467 herein), PXXX (SEQ ID NO: 122 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2468 herein), SGKGPRQITAL (SEQ ID NO: 124 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2469 herein), AAAAAAAAAXXXXX (SEQ ID NO: 125 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2470 herein), VYMSPF (SEQ ID NO: 126 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2471 herein), VYMSPF (SEQ ID NO: 127 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2472 herein), VYMSPF (SEQ ID NO: 128 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2473 herein), VYMSPF (SEQ ID NO: 129 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2474 herein), VYMSPF (SEQ ID NO: 129 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2475 herein), VYMSPF (SEQ ID NO: 129 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2476 herein), VYMSPF (SEQ ID NO: 129 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2477 SEQ ID NO: 126; SEQ ID NO: 2471 herein), ATWLPPR (SEQ ID NO: 127 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2472 herein), HTMYYHHYQHHL (SEQ ID NO: 128 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2473 herein), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 129 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2474 herein), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 130 in U.S. Pat. No. 9,475,845;No. 9,475,845; SEQ ID NO: 2475 herein), CKGQCDRFKGLPWEC (SEQ ID NO: 131 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2476 herein), SGRSA (SEQ ID NO: 132 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2477 herein), WGFP (SEQ ID NO: 133 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2478 herein), AEPMPHSLNFSQYLWYT (SEQ ID NO: 134 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2479 herein), WAYXSP (U.S. Pat. No. 9,475,845; SEQ ID NO: 2479 herein), No. 475845 SEQ ID NO: 135; SEQ ID NO: 2480 herein), IELLQAR (U.S. Pat. No. 9,475,845 SEQ ID NO: 136; SEQ ID NO: 2481 herein), AYTKCSRQWRTCMTTH (U.S. Pat. No. 9,475,845 SEQ ID NO: 137; SEQ ID NO: 2482 herein), PQNSKIPGPTFLDPH (U.S. Pat. No. 9,475,845 SEQ ID NO: 138; SEQ ID NO: 2483 herein), SMEPALPDWWWKMFK (U.S. Pat. No. 9,475,845 SEQ ID NO: 139; No. 9,475,845; SEQ ID NO: 2485 herein), ANTPCGPYTHDCPVKR (SEQ ID NO: 140 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2485 herein), TACHQHVRMVRP (SEQ ID NO: 141 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2486 herein), VPWMEPAYQRFL (SEQ ID NO: 142 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2487 herein), DPRATPGS (SEQ ID NO: 143 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2488 herein), FRPNRA QDYNTN (SEQ ID NO: 144 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2489 herein), CTKNSYLMC (SEQ ID NO: 145 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2490 herein), CXXTXXXGXGC (SEQ ID NO: 146 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2491 herein), CPIEDRPMC (SEQ ID NO: 147 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2492 herein), HEWSYLAPYPWF (SEQ ID NO: 148 in U.S. Pat. No. 9,475,845;No. 9,475,845; SEQ ID NO: 2493 herein), MCPKHPLGC (SEQ ID NO: 149 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2494 herein), RMWPSSTVNLSAGRR (SEQ ID NO: 150 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2495 herein), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 151 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2496 herein), KSREHVNNSACPSKRITAAL (SEQ ID NO: 152 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2497 herein), EGFR (SEQ ID NO: 153 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2498 herein), AGLGVR (SEQ ID NO: 154 in U.S. Pat. No. 9,475,845; SEQ ID NO: 2499 herein), GTRQGHTMRLGVSDG (U.S. Pat. No. No. 9,475,845; SEQ ID NO: 2500 herein), IAGLAATPGWSHWLAL (U.S. Pat. No. 9,475,845; SEQ ID NO: 2501 herein), SMSIARL (U.S. Pat. No. 9,475,845; SEQ ID NO: 2502 herein), HTFEPGV (U.S. Pat. No. 9,475,845; SEQ ID NO: 158 herein), NTSLKRISNKRIRRK (U.S. Pat. No. 9,475,845; SEQ ID NO: 2504 herein), LRIKRKRRKRKKTRK (U.S. Pat. No. 9,475,845; SEQ ID NO: 2505 herein), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH, and GTV.

[0170] In one embodiment, the AAV serotype may be or have a sequence as described in US Patent Publication No. 20160369298 (the contents of which are herein incorporated by reference in their entirety), such as, but not limited to, a site-directed mutant capsid protein of AAV2 (SEQ ID NO: 97 of US Patent Publication No. 20160369298; SEQ ID NO: 2506 herein) or a variant thereof, wherein the specific site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.

[0171] Additionally, any of the mutant sequences described in US Patent Application Publication No. 20160369298 may be mutated using any of the following sequences, including, but not limited to, SDSGASN (SEQ ID NO: 1 and SEQ ID NO: 231 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2507 herein), SPSGASN (SEQ ID NO: 2 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2508 herein), SHSGASN (SEQ ID NO: 3 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2509 herein), SR SGASN (SEQ ID NO: 4 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2510 herein), SKSGASN (SEQ ID NO: 5 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2511 herein), SNSGASN (SEQ ID NO: 6 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2512 herein), SGSGASN (SEQ ID NO: 7 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2513 herein), SASGASN (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2514 herein), SEQ ID NOs: 8, 175, and 221 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2514 herein), SESGTSN (SEQ ID NO: 9 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2515 herein), STTGGSN (SEQ ID NO: 10 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2516 herein), SSAGSTN (SEQ ID NO: 11 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2517 herein), NNDSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2518 herein), and NNDSQA (U.S. Patent Application Publication No. 20160369298). 69298; SEQ ID NO:2518 herein), NNRNQA (SEQ ID NO:13 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2519 herein), NNNKQA (SEQ ID NO:14 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2520 herein), NAKRQA (SEQ ID NO:15 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2521 herein), NDEHQA (SEQ ID NO:16 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2522 herein), NTSQKA (SEQ ID NO: 17 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2523 herein), YYLSRTNTPSGTDTQSRLVFSQAGA (SEQ ID NO: 18 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2524 herein), YYLSRTNTDSGTETQSGLDFSQAGA (SEQ ID NO: 19 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2525 herein), YYLSRTNTESGTPTQ SALEFSQAGA (SEQ ID NO: 20 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2526 herein), YYLSRTNTHSGTHTQSPLHFSQAGA (SEQ ID NO: 21 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2527 herein), YYLSRTNTSSGTITISHLIFSQAGA (SEQ ID NO: 22 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2528 herein), YYLSRTNTRSGIMTKSSLMFSQAGA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2529 herein), YYLSRTNTHSGTHTQSPLHFSQAGA (SEQ ID NO: 23 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2529 herein), YYLSRTNTRSGIMTKSSLMFSQAGA (U ... YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 23 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2529 herein), YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 24 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2530 herein), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 25 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2531 herein), YYLSRTNAASGHATHSDLKFSQPGA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2532 herein), YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 24 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2533 herein), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 25 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2534 herein), YYLSRTNAASGHATHSDLKFSQPGA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2535 herein), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 25 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2536 herein), YYLSRTNAASGHATHSDLKFSQPGA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2537 herein), YYLSRTNDGSGPVTPSK 298; SEQ ID NO: 2532 herein), YYLSRTNGQAGSLTMSELGFSQVGA (SEQ ID NO: 27 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2533 herein), YYLSRTNSTGGNQTTSQLLFSQLSA (SEQ ID NO: 28 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2534 herein), YFLSRTNNNTGLNTNSTLNFSQGRA (SEQ ID NO: 29 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2535 herein), SKTGADNNNSEYSWTG (SEQ ID NO: 30 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2536 herein), SKTDADNNNSEYSWTG (SEQ ID NO: 31 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2537 herein), SKTEADNNNSEYSWTG (SEQ ID NO: 32 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2538 herein), SKTPADNNNSEYSWT G (SEQ ID NO: 33 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2539 herein), SKTHADNNNSEYSWTG (SEQ ID NO: 34 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2540 herein), SKTQADNNNSEYSWTG (SEQ ID NO: 35 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2541 herein), SKTIADNNNSEYSWTG (SEQ ID NO: 36 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2542 herein), SKTMADNNNSEYSWTG (SEQ ID NO: 37 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2543 herein), SKTRADNNNSEYSWTG (SEQ ID NO: 38 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2544 herein), SKTNADNNNSEYSWTG (SEQ ID NO: 39 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2545 herein), SKTVGRNNNSEYSWT G (SEQ ID NO: 40 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2546 herein), SKTADRNNNSEYSWTG (SEQ ID NO: 41 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2547 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 42 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2548 herein), SKPTTGNNNSDYSWPG (SEQ ID NO: 43 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2549 herein), STQKNENNNSNYSWPG (SEQ ID NO: 44 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2550 herein), HKDDEGKF (SEQ ID NO: 45 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2551 herein), HKDDNRKF (SEQ ID NO: 46 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2552 herein), HKDDTNKF (SEQ ID NO: 46 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2553 herein), SEQ ID NO: 47 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2553 herein), HEDSDKNF (SEQ ID NO: 48 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2554 herein), HRDGADSF (SEQ ID NO: 49 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2555 herein), HGDNKSRF (SEQ ID NO: 50 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2556 herein), KQGSEKTNVDFEEV (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2557 herein), KQGSEKTNVDFEEV (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2559 ... KQGSEKTNVDSEEV (SEQ ID NO: 51 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2557 herein), KQGSEKTNVDSEEV (SEQ ID NO: 52 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2558 herein), KQGSEKTNVDVEEV (SEQ ID NO: 53 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2559 herein), KQGSDKTNVDDAGV (SEQ ID NO: 54 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2560 herein), KQGSSK TNVDPREV (SEQ ID NO: 55 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2561 herein), KQGSRKTNVDHKQV (SEQ ID NO: 56 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2562 herein), KQGSKGGNVDTNRV (SEQ ID NO: 57 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2563 herein), KQGSGEANVDNGDV (SEQ ID NO: 58 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2564 herein), KQDAAADNIDYDHV (SEQ ID NO: 59 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2565 herein), KQSGTRSNAAASSV (SEQ ID NO: 60 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2566 herein), KENTNTNDTELTNV (SEQ ID NO: 61 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2567 herein), QRGNNVAATADVNT (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2568 herein), KQDAAADNIDYDHV (SEQ ID NO: 59 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2568 herein), KQSGTRSNAAASSV (SEQ ID NO: 60 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2569 herein), KENTNTNDTELTNV (SEQ ID NO: 61 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2569 herein), QRGNNVAATADVNT (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2569 herein), KQSGTRSNAAASSV (SEQ ID NO: 62 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2569 herein), KQSGTRSNAAASSV (SEQ ID NO: 63 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2 69298; SEQ ID NO: 2568 herein), QRGNNEAATADVNT (SEQ ID NO: 63 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2569 herein), QRGNNPAATADVNT (SEQ ID NO: 64 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2570 herein), QRGNNHAATADVNT (SEQ ID NO: 65 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2571 herein), QEENNIAATPGVNT (SEQ ID NO: 66 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2572 herein), QPPNNMAATHEVNT (SEQ ID NO: 67 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2573 herein), QHHNNSAATTIVNT (SEQ ID NO: 68 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2574 herein), QTTNNRAAFNMVET (SEQ ID NO: 69 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2575 herein) ), QKKNNNAASKKVAT (SEQ ID NO: 70 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2576 herein), QGGNNKAADDAVKT (SEQ ID NO: 71 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2577 herein), QAAKGGAADDAVKT (SEQ ID NO: 72 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2578 herein), QDDRAAAANESVDT (SEQ ID NO: 73 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2579 herein), QQQHDDAAYQRVHT (SEQ ID NO: 74 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2580 herein), QSSSSLAAVSTVQT (SEQ ID NO: 75 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2581 herein), QNNQTTAAIRNVTT (SEQ ID NO: 76 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2582 herein), NYNKKSDNVDFT (SEQ ID NO: 77 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2583 herein), NYNKKSENVDFT (SEQ ID NO: 77 in U.S. Patent Application Publication No. 20160369298; 8; SEQ ID NO: 2584 herein), NYNKKSLNVDFT (SEQ ID NO: 79 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2585 herein), NYNKKSPNVDFT (SEQ ID NO: 80 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2586 herein), NYSKKSHCVDFT (SEQ ID NO: 81 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2587 herein), NYRKTIYVDFT (SEQ ID NO: 100 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 101 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 102 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 103 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 104 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 105 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 106 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 107 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 108 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 109 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 109 in U. SEQ ID NO: 82; SEQ ID NO: 2588 herein), NYKEKKDVHFT (SEQ ID NO: 83 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2589 herein), NYGHRAIVQFT (SEQ ID NO: 84 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2590 herein), NYANHQFVVCT (SEQ ID NO: 85 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2591 herein), NYDDDPTGVLLT (SEQ ID NO: 86 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2592 herein), NYDDDPTGVLLT (SEQ ID NO: 87 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2593 herein), NYDDDPTGVLLT (SEQ ID NO: 88 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2594 herein), NYDDDPTGVLLT (SEQ ID NO: 89 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2595 herein), NYDDDPTGVLLT (SEQ ID NO: 89 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2596 herein), NYDDDPTGVLLT (SEQ ID NO: 89 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2597 herein), NYDDDPTGVLLT (SEQ ID NO: 89 in U.S. Patent SEQ ID NO: 86 in the specification; SEQ ID NO: 2592 herein), NYDDPTGVLLT (SEQ ID NO: 87 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2593 herein), NFEQQNSVEWT (SEQ ID NO: 88 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2594 herein), SQSGASN (SEQ ID NO: 89 and SEQ ID NO: 241 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2595 herein), NNGSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2596 herein), NNGSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2597 herein), NNGSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2599 herein), NNGSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2598 herein), NNGSQA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 259 ... No. 8, SEQ ID NO: 90; SEQ ID NO: 2596 herein), YYLSRTNTPSGTTTWSRLQFSQAGA (SEQ ID NO: 91 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2597 herein), SKTSADNNNSEYSWTG (SEQ ID NO: 92 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2598 herein), HKDDEEKF (SEQ ID NOs: 93, 209, 214, 219, 224, 234, 239, and 244 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2599 herein), KQGSEKTNVDIEEV (SEQ ID NO: 94 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2600 herein), QRGNNQAATADVNT (SEQ ID NO: 95 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2601 herein), NYNKKSVNVDFT (SEQ ID NO: 96 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2602 herein), SQSGASNYNTPSGTTTQSRLQFSTS ADNNNSEYSWTGATKYH (SEQ ID NO: 106 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2603 herein), SASGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 107 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2604 herein), SQSGASNYNTPSGTTTQSRLQFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 1 in U.S. Patent Application Publication No. 20160369298 08; SEQ ID NO: 2605 herein), SASGASNYNTPSGTTTQSRLQFSTSADNNNSEFSWPGATTYH (SEQ ID NO: 109 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2606 herein), SQSGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 110 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2607 herein), SASGASNYNTPSGSLTQSSLGFSTDGE NNNSDFSWTGATKYH (SEQ ID NO: 111 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2608 herein), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSDFSWTGATKYH (SEQ ID NO: 112 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2609 herein), SGAGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 113 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2610 herein), SGAGASN (SEQ ID NO: 176 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2611 herein), NSEGGSLTQSSLGFS (SEQ ID NOs: 177, 185, 193, and 202 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2612 herein), TDGENNNSDFS (SEQ ID NO: 178 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2613 herein), SEFSWPGATT (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2614 herein), SEQ ID NO: 179 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2614 herein), TSADNNNSDFSWT (SEQ ID NO: 180 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2615 herein), SQSGASNY (SEQ ID NOs: 181, 187, and 198 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2616 herein), NTPSGTTTQSRLQFS (SEQ ID NOs: 182, 188, 191, and 199 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2616 herein), SEQ ID NO: 2617), TSADNNNSEYSWTGATKYH (SEQ ID NO: 183 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2618 herein), SASGASNF (SEQ ID NO: 184 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2619 herein), TDGENNNSDFSWTGATKYH (SEQ ID NOs: 186, 189, 194, 197, and 203 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2620 herein), SASGASNY (U.S. Patent Application SEQ ID NO:190 and SEQ ID NO:195 in US Patent Application Publication No. 20160369298; SEQ ID NO:2621 herein), TSADNNNSEFSWPGATTYH (SEQ ID NO:192 in US Patent Application Publication No. 20160369298; SEQ ID NO:2622 herein), NTPSGSLTQSSLGFS (SEQ ID NO:196 in US Patent Application Publication No. 20160369298; SEQ ID NO:2623 herein), TSADNNNSDFSWTGATKYH (SEQ ID NO:200 in US Patent Application Publication No. 20160369298;SEQ ID NO: 2624 herein), SGAGASNF (SEQ ID NO: 201 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2625 herein), CTCCAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACACAA (SEQ ID NO: 204 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2626 herein), CTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2627 herein), GAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2628 herein), GAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAA (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2629 ... 298; SEQ ID NO: 2627 herein), SAAGASN (SEQ ID NO: 206 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2628 herein), YFLSRTNTESGSTTQSTLRFSQAG (SEQ ID NO: 207 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2629 herein), SKTSADNNNSDFS (SEQ ID NOs: 208, 228, and 253 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2630 herein), KQGSEKTDVDIDKV (SEQ ID NO: 210 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2631 herein), STAGASN (SEQ ID NO: 211 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2632 herein), YFLSRTNTTSGIETQSTLRFSQAG (SEQ ID NO: 212 and SEQ ID NO: 247 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2633 herein), SKTDGENNNSDFS (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2634 herein), No. 8; SEQ ID NO: 2634 herein), KQGAAADDVEIDGV (SEQ ID NO: 215 and SEQ ID NO: 250 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2635 herein), SEAGASN (SEQ ID NO: 216 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2636 herein), YYLSRTNTPSGTTTQSRLQFSQAG (SEQ ID NOs: 217, 232, and 242 in U.S. Patent Application Publication No. 20160369298;SEQ ID NO: 2637 herein), SKTSADNNNSEYS (SEQ ID NOs: 218, 233, 238, and 243 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2638 herein), KQGSEKTNVDIEKV (SEQ ID NOs: 220, 225, and 245 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2639 herein), YFLSRTNDASGSDTKSTLLFSQAG (SEQ ID NO: 222 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2640 herein), ), STTPSENNNSEYS (SEQ ID NO: 223 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2641 herein), SAAGATN (SEQ ID NO: 226 and SEQ ID NO: 251 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2642 herein), YFLSRTNGEAGSATLSELRFSQAG (SEQ ID NO: 227 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2643 herein), HGDDADRF (SEQ ID NO: 2644 in US Patent Application Publication No. 20160369298), SEQ ID NO:229 and SEQ ID NO:254; SEQ ID NO:2644 herein), KQGAEKSDVEVDRV (SEQ ID NO:230 and SEQ ID NO:255 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2645 herein), KQDSGGDNIDIDQV (SEQ ID NO:235 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2646 herein), SDAGASN (SEQ ID NO:236 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO:2647 herein), YFLSRTNTEGGHD TQSTLRFSQAG (SEQ ID NO: 237 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2648 herein), KEDGGGSDVAIDEV (SEQ ID NO: 240 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2649 herein), SNAGASN (SEQ ID NO: 246 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2650 herein), and YFLSRTNGEAGSATLSELRFSQPG (SEQ ID NO: 252 in U.S. Patent Application Publication No. 20160369298;Non-limiting examples of nucleotide sequences that can encode the amino acid mutation site include the following: AGCVVMDCAGGARSCASCAAC (SEQ ID NO: 97 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2652 herein), AACRACRRSMRSMAGGCA (SEQ ID NO: 98 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2653 herein), CACRRGGACR; RCRMSRRSARSTTT (SEQ ID NO: 99 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2654 herein), TATTTCTTGAGCAGAACAAACRVCVVSRSCGGAMNCVHSACGMHSTCAVVSCTTVDSTTTTCTCAGSBCRGSGCG (SEQ ID NO: 100 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2655 herein), TCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCGTGGMMAGG A (SEQ ID NO: 101 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2656 herein), AAGSAARCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTC (SEQ ID NO: 102 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2657 herein), CAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACA (SEQ ID NO: 103 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2658 herein) , AACTWCRVSVASMVSVHSDDTGTGSWSTKSACT (SEQ ID NO: 104 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2659 herein), TTGTTGAACATCACCACGTGACGCACGTTC (SEQ ID NO: 256 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2660 herein), TCCCCGTGGTTCTACTACATAATGTGGCCG (SEQ ID NO: 257 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2660 herein). SEQ ID NO: 2661 in US Patent Application Publication No. 20160369298), TTCCACACTCCGTTTTGGATAATGTTGAAC (SEQ ID NO: 258 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2662 herein), AGGGACATCCCCAGCTCCATGCTGTGGTCG (SEQ ID NO: 259 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2663 herein), AGGGACAACCCCTCCGACTCGCCCTAATCC (SEQ ID NO: 260 in US Patent Application Publication No. 20160369298;SEQ ID NO: 2664 herein), TCCTAGTAGAAGACACCCTCTCACTGCCCG (SEQ ID NO: 261 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2665 herein), AGTACCATGTACACCCACTCTCCCAGTGCC (SEQ ID NO: 262 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2666 herein), ATATGGACGTTCATGCTGATCACCATACCG (U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2667 herein), SEQ ID NO: 263 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2667 herein), AGCAGGAGCTCCTTGGCCTCAGCGTGCGAG (SEQ ID NO: 264 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2668 herein), ACAAGCAGCTTCACTATGACAACCACTGAC (SEQ ID NO: 265 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2669 herein), CAGCCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGAGAGTCTCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCCTGGMMAGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCCGGACCAGCTATGGCAAGCCACRRGGACRRCRMSRRSARSTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGSAARRCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTCATGATT ACAGACGAAGAGGAGATCTGGAC (SEQ ID NO: 266 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2670 herein), TGGGACAATGGCGGTCGTCTCTCAGAGTTKTKKT (SEQ ID NO: 267 in US Patent Application Publication No. 20160369298; SEQ ID NO: 2671 herein), AGAGGACCKKTCCTCGATGGTTCATGGTGGAGTTA (SEQ ID NO: 268 in US Patent Application Publication No. 20160369298;CCACTTAGGGCCTGGTCGATACCGTTCGGTG (SEQ ID NO: 269 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2673 herein), and TCTCGCCCCAAGAGTAGAAACCCTTCSTTYYG (SEQ ID NO: 270 in U.S. Patent Application Publication No. 20160369298; SEQ ID NO: 2674 herein);

[0172] In some embodiments, the AAV serotype may comprise an ocular cell targeting peptide as described in WO2016134375 (the contents of which are incorporated by reference in their entirety), such as, but not limited to, SEQ ID NO: 9 and SEQ ID NO: 10 of WO2016134375. Additionally, any of the ocular cell targeting peptides or amino acids described in WO2016134375 may be inserted into any parent AAV serotype, such as, but not limited to, AAV2 (SEQ ID NO: 8 of WO2016134375; SEQ ID NO: 2675 herein), or AAV9 (SEQ ID NO: 11 of WO2016134375; SEQ ID NO: 2676 herein). In some embodiments, modifications such as insertions are made at positions P34 to A35, T138 to A139, A139 to P140, G453 to T454, N587 to R588, and / or R588 to Q589 of the AAV2 protein. In certain embodiments, insertions are made at positions D384, G385, 1560, T561, N562, E563, E564, E565, N704, and / or Y705 of the AAV9 protein. The ocular cell targeting peptide may be, but is not limited to, any of the following amino acid sequences: GSTPPPM (SEQ ID NO: 1 in WO2016134375; SEQ ID NO: 2677 herein), or GETRAPL (SEQ ID NO: 4 in WO2016134375; SEQ ID NO: 2678 herein).

[0173] In some embodiments, the AAV serotypes may be modified as described in U.S. Patent Application Publication No. 20170145405, the contents of which are incorporated by reference in their entirety. AAV serotypes include modified AAV2 (e.g., Y444F, Y500F, Y730F, and / or S662V modifications), modified AAV3 (e.g., Y705F, Y731F, and / or T492V modifications), and modified AAV6 (e.g., S663V and / or T492V modifications).

[0174] In some embodiments, the AAV serotype may be modified as described in International Publication No. WO2017083722, the contents of which are incorporated herein by reference in their entirety. AAV serotypes may include AAV1 (Y705+731F+T492V), AAV2 (Y444+500+730F+T491V), AAV3 (Y705+731F), AAV5, AAV5 (Y436+693+719F), AAV6 (VP3 variant Y705F / Y731F / T492V), AAV8 (Y733F), AAV9, AAV9 (VP3 variant Y731F), and AAV10 (Y733F).

[0175] In some embodiments, the AAV serotype may comprise an engineered epitope comprising amino acids SPAKFA (SEQ ID NO: 24 in WO2017015102; SEQ ID NO: 2679 herein) or NKDKLN (SEQ ID NO: 2 in WO2017015102; SEQ ID NO: 2680 herein), as described in WO2017015102 (the contents of which are incorporated by reference in their entirety). The epitope may be inserted in the region of amino acids 665-670 based on the numbering of the VP1 capsid of AAV8 (SEQ ID NO: 3 in WO2017015102) and / or in the region of residues 664-668 of AAV3B (SEQ ID NO: 3).

[0176] In one embodiment, the AAV serotype is selected from the group consisting of, but not limited to, one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500, 528-534, 547-552, 588-597, 709-710, and 716-722 of AAV1, or AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26, AAV27, AAV28, AAV29, AAV30, AAV31, AAV32, AAV33, AAV34, AAV35, AAV36, AAV37, AAV38, AAV39, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV46, AAV47, AAV48, AAV49, AAV50, AAV51, AAV52, AAV53, AAV54, AAV55, AAV56, AAV57, AAV58, AAV59, AAV60, AAV61, AAV62, AAV63, AAV64, AAV65, AAV66, AAV67, AAV68, AAV69, AAV70, AAV71, AAV72, AAV73, AAV74, AAV The AAV variants may be or have the sequences as described in WO2017058892 (the contents of which are incorporated by reference herein in their entirety), such as AAV variants having capsid proteins that may contain substitutions in the equivalent amino acid residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. The amino acid substitutions may be, but are not limited to, any of the amino acid sequences described in WO2017058892.In one embodiment, the AAV comprises the following residues: 256L, 258K, 259Q, 261S, 263A, 264S, 265T, 266G, 272H, 385S, 386Q, S472R, V473D, N500E of AAV1 (SEQ ID NO: 1 of WO2017058892). any combination of 547S, 709A, 710N, 716D, 717N, 718N, 720L, A456T, Q457T, N458Q, K459S, T492S, K493A, S586R, S587G, S588N, T589R and / or 722T, 244N, 246Q, 248R, 249E, 250I, 251K, 252S, 253G, 254S, 255V, 256D, 263Y, 377E, 378N, 45 any combination of 3L, 456R, 532Q, 533P, 535N, 536P, 537G, 538T, 539T, 540A, 541T, 542Y, 543L, 546N, 653V, 654P, 656S, 697Q, 698F, 704D, 705S, 706T, 707G, 708E, 709Y and / or 710R, 248R, 316V, 317Q, 318D, 319S, 443N, 530N, 531S, 532Q of AAV5 (SEQ ID NO: 5 in WO2017058892) any combination of 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D, 706T, 708E, 709Y and / or 710R, any combination of 264S, 266G, 269N, 272H, 457Q, 588S and / or 589I of AAV6 (SEQ ID NO: 6 in WO2017058892), AAV8 ( The amino acid substitutions may be any combination of 457T, 459N, 496G, 499N, 500N, 589Q, 590N and / or 592A in AAV9 (SEQ ID NO: 8 in WO2017058892), or any combination of 451I, 452N, 453G, 454S, 455G, 456Q, 457N and / or 458Q in AAV9 (SEQ ID NO: 9 in WO2017058892).

[0177] In some embodiments, the AAV may comprise a sequence of amino acids at VP1 positions 155, 156, and 157 or VP2 positions 17, 18, 19, and 20, as described in WO2017066764, the contents of which are incorporated by reference in their entirety. The amino acid sequences may include, but are not limited to, NSS, SXS, SSY, NXS, NSY, SXY, and NXY, where N, X, and Y are independently non-serine or non-threonine amino acids, and the AAV may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the AAV may contain at least one amino acid deletion at position 156, 157 or 158 of VP1 or position 19, 20 or 21 of VP2, and the AAV may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

[0178] In one embodiment, peptides for inclusion in AAV serotypes may be identified using the method described by Hui et al. (Molecular Therapy-Methods & Clinical Development, 2015, Vol. 2, p. 15029, doi:10.1038 / mtm.2015.29; the contents of which are incorporated by reference herein in their entirety). As a non-limiting example, the procedure includes isolating human splenocytes, restimulating the splenocytes in vitro with individual peptides spanning the amino acid sequence of the AAV capsid protein, IFN-γ ELISpot with the individual peptides used for the in vitro restimulation, determining the HLA restriction of the 15mers identified by the IFN-γ ELISpot by bioinformatics analysis, identifying candidate reactive 9mer epitopes for a given HLA allele, synthesizing the candidate 9mers, a second IFN-γ ELISpot screening of splenocytes from subjects bearing HLA alleles predicted to bind the identified AAV epitopes, determining AAV capsid-reactive CD8+ T cell epitopes, and determining the frequency of subjects reactive to a given AAV epitope.

[0179] In one embodiment, the AAV may be a serotype generated by Cre-recombination-based AAV targeted evolution (CREATE) as described by Deverman et al. (Nature Biotechnology, Vol. 34, No. 2, pp. 204-209, 2016), the contents of which are incorporated herein by reference in their entirety. In one embodiment, the AAV serotype thus generated has improved CNS transduction and / or neuronal and astrocyte tropism when compared to other AAV serotypes. As non-limiting examples, the AAV serotypes may be PHP.B, PHP.B2, PHP.B3, PHP.A, G2A12, or G2A15. In one embodiment, these AAV serotypes may be derivatives of AAV9 (SEQ ID NOs: 126 and 127) with a 7-amino acid insert at amino acids 588-589. Non-limiting examples of these 7 amino acid inserts include TLAVPFK (SEQ ID NO: 873), SVSKPFL (SEQ ID NO: 1249), FTLTTPK (SEQ ID NO: 882), YTLSQGW (SEQ ID NO: 888), QAVRTSL (SEQ ID NO: 914) and / or LAKERLS (SEQ ID NO: 915).

[0180] In one embodiment, the AAV serotype may be as described in Jackson et al. (Frontiers in Molecular Neuroscience, vol. 9, p. 154, 2016), the contents of which are incorporated by reference herein in their entirety. In some embodiments, the AAV serotype is PHP.B or AAV9. In some embodiments, the AAV serotype is paired with a synapsin promoter to enhance neuronal transduction when compared to using a more ubiquitous promoter (i.e., CBA or CMV).

[0181] In one embodiment, peptides for inclusion in an AAV serotype may be identified by isolating human splenocytes, restimulating the splenocytes in vitro with individual peptides spanning the amino acid sequence of the AAV capsid protein, performing an IFN-γ ELISpot with the individual peptides used for the in vitro restimulation, determining the given allele restriction of the 15mers identified by the IFN-γ ELISpot by bioinformatics analysis, identifying candidate reactive 9mer epitopes for the given allele, synthesizing the candidate 9mers, performing a second IFN-γ ELISpot screening of splenocytes from subjects bearing the specific allele to which the identified AAV epitope is predicted to bind, determining AAV capsid-reactive CD8+ T cell epitopes, and determining the frequency of subjects reactive to the given AAV epitope.

[0182] The AAV particles that contain the regulatory polynucleotides that encode siRNA molecules can be prepared or derived from various serotypes of AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 and AAV-DJ.In some cases, different AAV serotypes can be mixed together or with other types of viruses to create chimeric AAV vectors.As a non-limiting example, AAV particles are derived from AAV9 serotype.

[0183] viral genome In one embodiment, as shown in, the AAV particle comprises a viral genome with a payload region.

[0184] In one embodiment, the viral genome may comprise components as shown in Figure 1. A payload region 110 is located within the viral genome 100. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. A promoter region 130 may be present between the 5' ITR 120 and the payload region 110. In one embodiment, the payload region may comprise at least one regulatory polynucleotide.

[0185] In one embodiment, the viral genome 100 may include components as shown in Figure 2. A payload region 110 is located within the viral genome 100. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. A promoter region 130 may be present between the 5' ITR 120 and the payload region 110. An intron region 140 may be present between the promoter region 130 and the payload region 110. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0186] In one embodiment, viral genome 100 may include components as shown in Figure 3. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of viral genome 100. An enhancer region 150, a promoter region 130, an intron region 140, and a payload region 110 may be present within viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0187] In one embodiment, viral genome 100 may include components as shown in Figure 4. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of viral genome 100. An enhancer region 150, a promoter region 130, an intron region 140, a payload region 110, and a polyadenylation signal sequence region 160 may be present within viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0188] In one embodiment, viral genome 100 may include components as shown in Figure 5. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of viral genome 100. At least one MCS region 170, an enhancer region 150, a promoter region 130, an intron region 140, a payload region 110, and a polyadenylation signal sequence region 160 may be present within viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0189] In one embodiment, the viral genome 100 may include components as shown in Figure 6. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. At least one MCS region 170, an enhancer region 150, a promoter region 130, at least one exon region 180, at least one intron region 140, a payload region 110, and a polyadenylation signal sequence region 160 may be present within the viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0190] In one embodiment, the viral genome 100 may include components as shown in Figures 7 and 8. There may be at least one promoter region 130 and a payload region 110 within the viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0191] In one embodiment, viral genome 100 may include components as shown in Figure 9. At least one promoter region 130, payload region 110, and polyadenylation signal sequence region 160 may be present in viral genome 100. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0192] Viral genome size In one embodiment, the viral genome comprising the payload described herein may be a single-stranded or double-stranded viral genome. The size of the viral genome may be small, medium, large, or maximum size. In addition, the viral genome may include a promoter and a polyA tail.

[0193] In one embodiment, the viral genome containing the payload described herein may be a small single-stranded viral genome. The small single-stranded viral genome may be 2.7 to 3.5 kb in size, such as approximately 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size. As a non-limiting example, the small single-stranded viral genome may be 3.2 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0194] In one embodiment, the viral genome containing the payload described herein may be a small double-stranded viral genome. The small double-stranded viral genome may be 1.3 to 1.7 kb in size, such as approximately 1.3, 1.4, 1.5, 1.6, and 1.7 kb in size. As a non-limiting example, the small double-stranded viral genome may be 1.6 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0195] In one embodiment, the viral genome containing the payload described herein may be a medium-sized single-stranded viral genome. The medium-sized single-stranded viral genome may be 3.6 to 4.3 kb in size, such as approximately 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, and 4.3 kb in size. As a non-limiting example, the medium-sized single-stranded viral genome may be 4.0 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0196] In one embodiment, the viral genome containing the payload described herein may be a medium-sized double-stranded viral genome. The medium-sized double-stranded viral genome may be 1.8 to 2.1 kb in size, such as approximately 1.8, 1.9, 2.0, and 2.1 kb in size. As a non-limiting example, the medium-sized double-stranded viral genome may be 2.0 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0197] In one embodiment, the viral genome containing the payload described herein may be a large single-stranded viral genome. The large single-stranded viral genome may be 4.4 to 6.0 kb in size, such as approximately 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0 kb in size. As a non-limiting example, the large single-stranded viral genome may be 4.7 kb in size. As another non-limiting example, the large single-stranded viral genome may be 4.8 kb in size. As yet another non-limiting example, the large single-stranded viral genome may be 6.0 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0198] In one embodiment, the viral genome containing the payload described herein may be a large, double-stranded viral genome. The large, double-stranded vector genome may be 2.2 to 3.0 kb in size, such as approximately 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 kb in size. As a non-limiting example, the large, double-stranded viral genome may be 2.4 kb in size. In addition, the viral genome may include a promoter and a poly(A) tail.

[0199] Viral genome components: Inverted terminal repeats (ITRs) The AAV particles of the present invention comprise a viral genome having at least one ITR region and a payload region. In one embodiment, the viral genome has two ITRs. These two ITRs flank the payload region at the 5' and 3' ends. The ITRs serve as replication origins containing recognition sites for replication. The ITRs contain sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the viral genome of the present invention can comprise naturally occurring or recombinantly derived polynucleotide sequences.

[0200] The ITRs may be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1 or derivatives thereof. The ITRs may be of a different serotype from the capsid. In one embodiment, the AAV particle has two or more ITRs. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In one embodiment, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one, or both ITRs having the same serotype as the capsid. In one embodiment, both ITRs in the viral genome of the AAV particle are AAV2 ITRs.

[0201] Independently, each ITR may be about 100 to about 150 nucleotides in length. The ITRs may be about 100 to 105, 106 to 110, 111 to 115, 116 to 120, 121 to 125, 126 to 130, 131 to 135, 136 to 140, 141 to 145, or 146 to 150 nucleotides in length. In one embodiment, the ITRs are 140 to 142 nucleotides in length. Non-limiting examples of ITR lengths are 102, 140, 141, 142, and 145 nucleotides, and those with at least 95% identity thereto.

[0202] In one embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located near the 5' end of the flip ITR in an expression vector. In another embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located near the 3' end of the flip ITR in an expression vector. In yet another embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located near the 5' end of the flop ITR in an expression vector. In yet another embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located near the 3' end of the flop ITR in an expression vector. In one embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located between the 5' end of the flip ITR and the 3' end of the flop ITR in an expression vector. In one embodiment, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located between the 3' end of the flip ITR and the 5' end of the flip ITR (e.g., between the 5' end of the flip ITR and the 3' end of the flop ITR, or midway between the 3' end of the flop ITR and the 5' end of the flip ITR) in an expression vector. As a non-limiting example, the AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides downstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the expression vector. As a non-limiting example, the AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides upstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the expression vector.As another non-limiting example, the AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the expression vector. As another non-limiting example, the AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, 15 to 30, 20 to 25, 20 to 30, or 25 to 30 upstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in the expression vector. As a non-limiting example, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of nucleotides upstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in an expression vector. As another non-limiting example, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of nucleotides downstream from the 5' or 3' end of an ITR (e.g., a flip or flop ITR) in an expression vector.

[0203] Viral genome components: promoters In one embodiment, the payload region of the viral genome comprises at least one element that enhances transgene target specificity and expression (see, e.g., Powell et al., "Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy," 2015, the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of elements that enhance transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (polyA) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0204] Those skilled in the art will recognize that expression of a polypeptide of the invention in a target cell may require a specific promoter, including, but not limited to, a species-specific, inducible, tissue-specific, or cell cycle-specific promoter (Parr et al., Nat. Med. 3:1145-9 (1997); the contents of which are incorporated herein by reference in their entirety).

[0205] In one embodiment, a promoter is considered effective if it drives expression of a polypeptide encoded in the payload region of the viral genome of an AAV particle. In one embodiment, the promoter is a promoter that is considered effective to drive expression of the regulatory polynucleotide.

[0206] In one embodiment, a promoter is a promoter that is considered effective if it drives expression in the cells that are targeted. In one embodiment, the promoter drives expression of the payload in the target tissue for a period of time, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days ... The period may be days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Expression may be 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0207] In one embodiment, the promoter is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, or more than 65 years.

[0208] The promoter may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated.

[0209] Promoters that drive or enhance expression in most tissues include, but are not limited to, human elongation factor 1 α-subunit (EF1α), cytomegalovirus (CMV) immediate-early enhancer and / or promoter, chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Expression can be restricted to particular cell types using tissue-specific expression elements, such as, but not limited to, a muscle-specific promoter, a B-cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0210] Non-limiting examples of muscle-specific promoters include the mammalian muscle creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the mammalian troponin I (TNNI2) promoter, and the mammalian skeletal alpha-actin (ASKA) promoter (see, e.g., U.S. Patent Application Publication No. 20110212529, the contents of which are incorporated by reference herein in their entirety).

[0211] Non-limiting examples of neuronal tissue-specific expression elements include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG binding protein (MeCP2), Ca 2+Examples of tissue-specific expression elements include the calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the myelin basic protein (MBP) promoter.

[0212] In one embodiment, the promoter may be less than 1 kb. In some embodiments, the amino acid sequence may be 0, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides in length. The promoter may have a length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800.

[0213] In one embodiment, the promoter may be a combination of two or more elements of the same or different initiation or parent promoters, such as, but not limited to, CMV and CBA. Each element may be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1 In some embodiments, the amino acid sequence may have a length of 0, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800. Each component may have a length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. In one embodiment, the promoter is a combination of a 382 nucleotide CMV enhancer sequence and a 260 nucleotide CBA promoter sequence.

[0214] In one embodiment, the viral genome comprises a ubiquitous promoter, non-limiting examples of which include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0215] Yu et al. (Molecular Pain, 2011, Vol. 7, p. 63; the contents of which are incorporated herein by reference in their entireties) used lentiviral vectors to evaluate eGFP expression under the CAG, EFIα, PGK, and UBC promoters in rat DRG cells and primary DRG cells. They found that UBC showed weaker expression than the other three promoters, with only 10–12% glial cell expression observed for all promoters. Soderblom et al. (E. Neuro, 2015; the contents of which are incorporated herein by reference in their entireties) evaluated eGFP expression in the motor cortex after injection of AAV8 containing the CMV and UBC promoters and AAV2 containing the CMV promoter. Intranasal administration of plasmids containing the UBC or EFIα promoters demonstrated sustained airway expression that was higher than that achieved by the CMV promoter (see, e.g., Gill et al., Gene Therapy, 2001, vol. 8, pp. 1539-1546, the contents of which are incorporated herein by reference in their entirety). Husain et al. (Gene Therapy, 2009; the contents of which are incorporated herein by reference in their entirety) evaluated an HβH construct containing the hGUSB promoter, the HSV-1 LAT promoter, and the NSE promoter and found that this HβH construct exhibited weaker expression than NSE in mouse brain. Passini and Wolfe (J. Virol., 2001, pp. 12382-12392, the contents of which are incorporated herein by reference in their entireties) evaluated the long-term effects of HβH vectors after intracerebroventricular injection in neonatal mice and found sustained expression for at least one year.Xu et al. (Gene Therapy, 2001, Vol. 8, pp. 1323-1332; the contents of which are incorporated herein by reference in their entireties) found lower expression in all brain regions when using the NFL and NFH promoters compared to CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8kb), and NSE(1.8kb+wpre). Xu et al. found that promoter activity, in descending order, was NSE(1.8kb), EF, NSE(0.3kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650-nucleotide promoter, and NFH is a 920-nucleotide promoter. Both are absent in the liver, but NFH is abundant in proprioceptive neurons, the brain, and the spinal cord, and NFH is present in the heart. Scn8a is a 470-nucleotide promoter expressed throughout the DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, the cortex, the thalamus, and the hypothalamus (see, e.g., Drews et al., "Identification of evolutionary conserved, functional noncoding elements in the promoter region of the sodium channel gene SCN8A," Mamm Genome (2007) 18:723-731; and Raymond et al., "Expression of alternatively spliced ​​sodium channel α-subunit genes."). Alternatively, see "Alternatively Spliced ​​Sodium Channel α-subunit Genes," Journal of Biological Chemistry, 2004, 279(44), pp. 46234-46241, the contents of each of which are incorporated herein by reference in their entirety.

[0216] Any of the promoters taught by Yu, Soderblom, Gill, Husain, Passini, Xu, Drews, or Raymond, supra, can be used in the present invention.

[0217] In one embodiment, the promoter is not cell-specific. In one embodiment, the promoter is the ubiquitin c (UBC) promoter. The UBC promoter can have a size of 300-350 nucleotides. As a non-limiting example, the UBC promoter is 332 nucleotides.

[0218] In one embodiment, the promoter is a β-glucuronidase (GUSB) promoter. The GUSB promoter can have a size of 350 to 400 nucleotides. As a non-limiting example, the GUSB promoter is 378 nucleotides.

[0219] In one embodiment, the promoter is a neurofilament light chain (NFL) promoter. The NFL promoter can be 600-700 nucleotides in size. As a non-limiting example, the NFL promoter is 650 nucleotides. As a non-limiting example, the construct can be AAV-promoter-CMV / globin intron-regulatory polynucleotide-RBG, where the AAV can be self-complementary and the AAV can be a DJ serotype.

[0220] In one embodiment, the promoter is a neurofilament heavy chain (NFH) promoter. The NFH promoter can be 900-950 nucleotides in size. As a non-limiting example, the NFH promoter is 920 nucleotides. As a non-limiting example, the construct can be AAV-promoter-CMV / globin intron-regulatory polynucleotide-RBG, where the AAV can be self-complementary and the AAV can be of the DJ serotype.

[0221] In one embodiment, the promoter is the scn8a promoter. The scn8a promoter can be 450-500 nucleotides in size. As a non-limiting example, the scn8a promoter is 470 nucleotides. As a non-limiting example, the construct can be AAV-promoter-CMV / globin intron-regulatory polynucleotide-RBG, where the AAV can be self-complementary and the AAV can be of the DJ serotype.

[0222] In one embodiment, the viral genome comprises a Pol III promoter. In one embodiment, the viral genome comprises a P1 promoter. In one embodiment, the viral genome comprises the FXN promoter.

[0223] In one embodiment, the promoter is a phosphoglycerate kinase 1 (PGK) promoter. In one embodiment, the promoter is the chicken β-actin (CBA) promoter.

[0224] In one embodiment, the promoter is the CAG promoter, a construct containing the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin (CBA) promoter.

[0225] In one embodiment, the promoter is a cytomegalovirus (CMV) promoter. In one embodiment, the viral genome comprises a Pol III promoter, eg, a Pol III type 3 promoter.

[0226] In one embodiment, the promoter comprises a U3, U6, U7, 7SK, H1, or MRP, EBER, seleno-cysteine ​​tRNA, 7SL, adenovirus VA-1, or telomerase gene promoter.

[0227] In one embodiment, the viral genome comprises an H1 promoter. In one embodiment, the viral genome comprises a U6 promoter. In one embodiment, the promoter is a liver or skeletal muscle promoter. Non-limiting examples of liver promoters include human alpha-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG). Non-limiting examples of skeletal muscle promoters include desmin, MCK, or synthetic C5-12.

[0228] In one embodiment, the promoter is an RNA pol III promoter. As a non-limiting example, the RNA pol III promoter is U6. As a non-limiting example, the RNA pol III promoter is H1.

[0229] In one embodiment, the promoter is an RNA Pol II promoter, including, for example, a truncated RNA Pol II promoter. In one embodiment, the viral genome comprises two promoters. As a non-limiting example, the promoters are the EF1α promoter and the CMV promoter.

[0230] In one embodiment, the viral genome comprises an enhancer element, a promoter, and / or a 5'UTR intron. The enhancer element, also referred to herein as an "enhancer," may be, but is not limited to, a CMV enhancer, the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE, synapsin, MeCP2, and GFAP promoter, and the 5'UTR / intron may be, but is not limited to, an SV40 and CBA-MVM promoter. As non-limiting examples, enhancers, promoters and / or introns used in combination may be: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV enhancer, CBA promoter, SV40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) synapsin promoter; (8) MeCP2 promoter, (9) GFAP promoter, (10) H1 promoter; and (11) U6 promoter.

[0231] In one embodiment, the viral genome comprises an engineered promoter. In another embodiment, the viral genome comprises the promoter of a naturally expressed protein.

[0232] Viral genome components: untranslated regions (UTRs) By definition, the wild-type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally, the 5' UTR begins at the transcription start site and ends at the start codon, and the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0233] Features typically found in abundantly expressed genes of specific target organs can be engineered into UTRs to enhance stability and protein production. As a non-limiting example, the 5'UTR from mRNA normally expressed in the liver (e.g., albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used in the viral genome of the AAV particles of the present invention to enhance expression in hepatocyte cell lines or the liver.

[0234] Without wishing to be bound by theory, wild-type 5' untranslated regions (UTRs) contain features that play a role in translation initiation. The Kozak sequence is widely known to be involved in the process by which the ribosome initiates translation of many genes and is typically contained in 5' UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another "G."

[0235] In one embodiment, the 5'UTR of the viral genome comprises a Kozak sequence. In one embodiment, the 5'UTR of the viral genome does not contain a Kozak sequence. Without wishing to be bound by theory, wild-type 3'UTRs are known to have adenosine and uridine stretches embedded therein. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al., 1995, the contents of which are incorporated herein by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of the AUUUA motif within the U-rich region; Class II AREs, such as, but not limited to, GM-CSF and TNF-α, have two or more overlapping UUAUUUA(U / A)(U / A) nonamers; Class III AREs, such as, but not limited to, c-Jun and myogenin, contain several dispersed copies of the AUUUA motif within the U-rich region. ARES are less well defined. These U-rich regions do not contain the AUUUA motif. Most proteins that bind to AREs are known to destabilize messengers, but members of the ELAV family, most notably HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thus message stabilization in vivo.

[0236] The introduction, removal or modification of 3'UTR AU-rich element (ARE) can be used to adjust the stability of polynucleotide.When manipulating a specific polynucleotide, such as the payload region of a viral genome, one or more copies of ARE can be introduced to make the polynucleotide less stable, thereby reducing translation and the resulting protein production.Similarly, ARE can be identified and removed or mutated to increase intracellular stability, thus increasing the translation and production of the resulting protein.

[0237] In one embodiment, the 3'UTR of the viral genome may contain an oligo(dT) sequence for templated addition of a polyA tail. In one embodiment, the viral genome may contain at least one miRNA seed, binding site, or complete sequence. MicroRNAs (or miRNAs or miRs) are 19-25 nucleotide non-coding RNAs that bind to nucleic acid target sites and downregulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. The microRNA sequence includes the "seed" region, i.e., the sequence of positions 2-8 of the mature microRNA. This sequence has perfect Watson-Crick complementarity to the miRNA target sequence of the nucleic acid.

[0238] In one embodiment, the viral genome may be engineered to include, alter or remove at least one miRNA binding site, sequence or seed region. Any UTR from any gene known in the art can be incorporated into the viral genome of an AAV particle. These UTRs or parts thereof can be placed in the same orientation as the gene from which they were selected, or their orientation or position can be changed. In one embodiment, the UTR used in the viral genome of an AAV particle can be inverted, shortened, extended, or made with one or more other 5'UTRs or 3'UTRs known in the art. As used herein, the term "modified" when referring to a UTR means that the UTR is changed in some way relative to the reference sequence. For example, the 3' or 5'UTR can be changed relative to the wild-type or natural UTR by changing its orientation or position as taught above, or by inserting additional nucleotides, deleting nucleotides, exchanging nucleotides, or rearranging nucleotides.

[0239] In one embodiment, the viral genome of the AAV particle comprises at least one artificial UTR that is not a variant of a wild-type UTR. In one embodiment, the viral genome of the AAV particle comprises UTRs whose proteins are selected from a family of transcripts that share a common function, structure, feature, or characteristic.

[0240] Viral genome components: Polyadenylation sequences In one embodiment, the viral genome of an AAV particle of the present invention comprises at least one polyadenylation sequence. The viral genome of an AAV particle may comprise a polyadenylation sequence between the 3' end of the payload coding sequence and the 5' end of the 3' ITR.

[0241] In one embodiment, the polyadenylation sequence or "poly A sequence" may range from absent to about 500 nucleotides in length. Polyadenylation sequences include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 18 0, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 1 99, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288 , 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319 , 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 35 0, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 1, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 4 12, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443 and 500 nucleotides in length.

[0242] In one embodiment, the polyadenylation sequence is 50 to 100 nucleotides in length. In one embodiment, the polyadenylation sequence is 50 to 150 nucleotides in length. In one embodiment, the polyadenylation sequence is 50 to 160 nucleotides in length.

[0243] In one embodiment, the polyadenylation sequence is 50 to 200 nucleotides in length. In one embodiment, the polyadenylation sequence is 60 to 100 nucleotides in length. In one embodiment, the polyadenylation sequence is 60 to 150 nucleotides in length.

[0244] In one embodiment, the polyadenylation sequence is 60 to 160 nucleotides in length. In one embodiment, the polyadenylation sequence is 60 to 200 nucleotides in length. In one embodiment, the polyadenylation sequence is 70 to 100 nucleotides in length.

[0245] In one embodiment, the polyadenylation sequence is 70 to 150 nucleotides in length. In one embodiment, the polyadenylation sequence is 70 to 160 nucleotides in length. In one embodiment, the polyadenylation sequence is 70 to 200 nucleotides in length.

[0246] In one embodiment, the polyadenylation sequence is 80-100 nucleotides in length. In one embodiment, the polyadenylation sequence is 80 to 150 nucleotides in length. In one embodiment, the polyadenylation sequence is 80 to 160 nucleotides in length.

[0247] In one embodiment, the polyadenylation sequence is 80 to 200 nucleotides in length. In one embodiment, the polyadenylation sequence is 90-100 nucleotides in length. In one embodiment, the polyadenylation sequence is 90 to 150 nucleotides in length.

[0248] In one embodiment, the polyadenylation sequence is 90 to 160 nucleotides in length. In one embodiment, the polyadenylation sequence is 90 to 200 nucleotides in length. In one embodiment, AAV particles comprise a nucleic acid sequence encoding an siRNA molecule, which may be located upstream of a polyadenylation sequence in an expression vector.Furthermore, AAV particles comprise a nucleic acid sequence encoding an siRNA molecule, which may be located downstream of a promoter in an expression vector, such as, but not limited to, a CMV, U6, CAG, CBA, or CBA promoter with an SV40 intron or a human β-globin intron.As a non-limiting example, AAV particles comprise a nucleic acid sequence encoding an siRNA molecule, which may be located downstream of a promoter and / or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more than 30 nucleotides upstream of a polyadenylation sequence in an expression vector. As another non-limiting example, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream from the promoter and / or upstream from the polyadenylation sequence in an expression vector. As a non-limiting example, an AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of nucleotides downstream from the promoter and / or upstream from the polyadenylation sequence in an expression vector. As another non-limiting example, the AAV particle comprises a nucleic acid sequence encoding an siRNA molecule that may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the expression vector downstream from the promoter and / or upstream of the polyadenylation sequence.

[0249] In one embodiment, the AAV particle comprises a rabbit globin polyadenylation (polyA) signal sequence. In one embodiment, the AAV particle comprises a human growth hormone polyadenylation (polyA) signal sequence.

[0250] Viral genome components: introns In one embodiment, the payload region comprises at least one element for enhancing expression, such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splice donor / IgG splice acceptor (230 bp).

[0251] In one embodiment, an intron or intron portion may be 100 to 500 nucleotides in length. The intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. The intron may have a length of 80 to 100, 80 to 120, 80 to 140, 80 to 160, 80 to 180, 80 to 200, 80 to 250, 80 to 300, 80 to 350, 80 to 400, 80 to 450, 80 to 500, 200 to 300, 200 to 400, 200 to 500, 300 to 400, 300 to 500, or 400 to 500.

[0252] In one embodiment, the AAV viral genome may include a promoter such as, but not limited to, CMV or U6. As a non-limiting example, the promoter of the AAV comprising the nucleic acid sequence of the siRNA molecule of the present invention is a CMV promoter. As another non-limiting example, the promoter of the AAV comprising the nucleic acid sequence of the siRNA molecule of the present invention is a U6 promoter.

[0253] In one embodiment, the AAV viral genome may comprise a CMV promoter. In one embodiment, the AAV viral genome may comprise a U6 promoter. In one embodiment, the AAV viral genome may comprise a CMV and U6 promoter.

[0254] In one embodiment, the AAV viral genome may comprise a Pol III promoter. In one embodiment, the AAV viral genome may comprise a Pol III type 3 promoter.

[0255] In one embodiment, the AAV viral genome may comprise an H1 promoter. In one embodiment, the AAV viral genome may comprise a U6 promoter. In one embodiment, the AAV viral genome may comprise a CBA promoter.

[0256] In one embodiment, the coding siRNA molecule can be located downstream of a promoter in an expression vector, such as but not limited to, CMV, U6, H1, CBA, CAG, or CBA promoter with intron, such as SV40 or other known in the art.In addition, the coding siRNA molecule can also be located upstream of a polyadenylation sequence in an expression vector.As a non-limiting example, the coding siRNA molecule can be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more than 30 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in an expression vector. As another non-limiting example, the encoded siRNA molecule may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream from the promoter and / or upstream from the polyadenylation sequence in the expression vector. As a non-limiting example, the encoded siRNA molecule may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of the nucleotides downstream from the promoter and / or upstream from the polyadenylation sequence in the expression vector. As another non-limiting example, the encoding siRNA molecule may be located in the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the expression vector downstream from the promoter and / or upstream of the polyadenylation sequence.

[0257] Viral genome components: filler sequences In one embodiment, the viral genome comprises one or more filler sequences. In one embodiment, the viral genome includes one or more filler sequences to optimize the length of the viral genome for packaging. As a non-limiting example, the viral genome includes at least one filler sequence to optimize the length of the viral genome to approximately 2.3 kb. As a non-limiting example, the viral genome includes at least one filler sequence to optimize the length of the viral genome to approximately 4.6 kb.

[0258] In one embodiment, the viral genome comprises one or more filler sequences to reduce the likelihood that the hairpin structure of the vector genome (e.g., a regulatory polynucleotide described herein) may be read as an inverted terminal repeat (ITR) during expression and / or packaging. As a non-limiting example, the viral genome comprises at least one filler sequence to bring the length of the viral genome to approximately 2.3 kb. As a non-limiting example, the viral genome comprises at least one filler sequence to bring the length of the viral genome to approximately 4.6 kb.

[0259] In one embodiment, the viral genome is a single-stranded (ss) viral genome, including but not limited to, 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1. The vector genome may include one or more filler sequences having a length of about 0.1 kb to 3.8 kb, such as 9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, or 3.8 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 3.1 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 2.7 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 0.8 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 0.4 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.8 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.4 kb.

[0260] In one embodiment, the viral genome is a self-complementary (sc) viral genome and includes one or more filler sequences having a length of about 0.1 kb to 1.5 kb, such as, but not limited to, 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, or 1.5 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 0.8 kb. As a non-limiting example, the total length of the filler sequences in the vector genome is 0.4 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.8 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.4 kb.

[0261] In one embodiment, the viral genome comprises any portion of the filler sequence. The viral genome may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the filler sequence.

[0262] In one embodiment, the viral genome is a single-stranded (ss) viral genome and includes one or more filler sequences to make the length of the viral genome approximately 4.6 kb. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 3' of the 5' ITR sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 5' of the promoter sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 3' of the polyadenylation signal sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 5' of the 3' ITR sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located between two intron sequences. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located within an intron sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 3' of the 5' ITR sequence and a second filler sequence located 3' of the polyadenylation signal sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 5' of the promoter sequence and a second filler sequence located 3' of the polyadenylation signal sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 3' of the 5' ITR sequence and a second filler sequence located 5' of the 5' ITR sequence.

[0263] In one embodiment, the viral genome is a self-complementary (sc) viral genome and includes one or more filler sequences to bring the length of the viral genome to approximately 2.3 kb. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 3' of the 5' ITR sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 5' of the promoter sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 3' of the polyadenylation signal sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located 5' of the 3' ITR sequence. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located between two intron sequences. As a non-limiting example, the viral genome includes at least one filler sequence, the filler sequence being located within an intron sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 3' of the 5' ITR sequence and a second filler sequence located 3' of the polyadenylation signal sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 5' of the promoter sequence and a second filler sequence located 3' of the polyadenylation signal sequence. As a non-limiting example, a viral genome comprises two filler sequences, a first filler sequence located 3' of the 5' ITR sequence and a second filler sequence located 5' of the 5' ITR sequence.

[0264] In one embodiment, the viral genome may include one or more filler sequences between one of many regions of the viral genome. In one embodiment, the filler region may be located before a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region. In one embodiment, the filler region may be located after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region. In one embodiment, the filler region may be located before or after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region.

[0265] In one embodiment, the viral genome may include one or more filler sequences that bifurcate at least one region of the viral genome. The bifurcated region of the viral genome may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the region 5' of the filler sequence region. As a non-limiting example, the filler sequence may bifurcate at least one region such that 10% of the region is located 5' of the filler sequence and 90% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 20% of the region is located 5' of the filler sequence and 80% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 30% of the region is located 5' of the filler sequence and 70% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 40% of the region is located 5' of the filler sequence and 60% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 50% of the region is located 5' of the filler sequence and 50% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 60% of the region is located 5' of the filler sequence and 40% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 70% of the region is located 5' of the filler sequence and 30% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 80% of the region is located 5' of the filler sequence and 20% of the region is located 3' of the filler sequence. As a non-limiting example, the filler sequence may bisect at least one region such that 90% of the region is located 5' of the filler sequence and 10% of the region is located 3' of the filler sequence.

[0266] In one embodiment, the viral genome comprises a filler sequence after the 5' ITR. In one embodiment, the viral genome comprises a filler sequence after the promoter region. In one embodiment, the viral genome comprises a filler sequence after the payload region. In one embodiment, the viral genome comprises a filler sequence after the intron region. In one embodiment, the viral genome comprises a filler sequence after the enhancer region. In one embodiment, the viral genome comprises a filler sequence after the polyadenylation signal sequence region. In one embodiment, the viral genome comprises a filler sequence after the MCS region. In one embodiment, the viral genome comprises a filler sequence after the exon region.

[0267] In one embodiment, the viral genome comprises a filler sequence before the promoter region. In one embodiment, the viral genome comprises a filler sequence before the payload region. In one embodiment, the viral genome comprises a filler sequence before the intron region. In one embodiment, the viral genome comprises a filler sequence before the enhancer region. In one embodiment, the viral genome comprises a filler sequence before the polyadenylation signal sequence region. In one embodiment, the viral genome comprises a filler sequence before the MCS region. In one embodiment, the viral genome comprises a filler sequence before the exon region.

[0268] In one embodiment, the viral genome comprises a filler sequence before the 3' ITR. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and a promoter region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and a payload region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and an intron region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and an enhancer region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and a polyadenylation signal sequence region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, a 5'ITR and an MCS region.

[0269] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, the 5' ITR and an exon region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and a payload region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and an intron region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and an enhancer region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and a polyadenylation signal sequence region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and an MCS region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and an exon region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a promoter region and a 3' ITR.

[0270] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a payload region and an intron region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a payload region and an enhancer region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a payload region and a polyadenylation signal sequence region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a payload region and an MCS region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a payload region and an exon region.

[0271] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, the payload region and the 3' ITR. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an intron region and an enhancer region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an intron region and a polyadenylation signal sequence region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an intron region and an MCS region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an intron region and an exon region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an intron region and a 3' ITR. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an enhancer region and a polyadenylation signal sequence region. In one embodiment, a filler sequence may be located between two regions, such as, but not limited to, an enhancer region and an MCS region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, an enhancer region and an exon region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, an enhancer region and a 3'ITR.

[0272] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a polyadenylation signal sequence region and an MCS region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a polyadenylation signal sequence region and an exon region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, a polyadenylation signal sequence region and a 3' ITR.

[0273] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, the MCS region and an exon region. In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, the MCS region and the 3' ITR.

[0274] In one embodiment, the filler sequence may be located between two regions, such as, but not limited to, an exon region and a 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the intron region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the intron region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the promoter region, and the second of which may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the promoter region, and the second of which may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the promoter region, and the second of which may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the promoter region, and the second of which may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the promoter region, and the second filler sequence may be located between the exon region and the 3' ITR.

[0275] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the intron region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the intron region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the payload region, and the second of which may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the payload region, and the second of which may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the payload region, and the second of which may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the payload region, and the second of which may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the payload region, and the second filler sequence may be located between the exon region and the 3' ITR.

[0276] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the intron region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the intron region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the intron region, and the second filler sequence may be located between the exon region and the 3' ITR.

[0277] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the intron region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the intron region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the enhancer region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the enhancer region, and the second of which may be located between the exon region and the 3' ITR.

[0278] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, a first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and a second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, a first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and a second filler sequence may be located between the payload region and the intron region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the polyadenylation signal sequence region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the intron region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the polyadenylation signal sequence region, and the second of which may be located between the exon region and the 3' ITR.

[0279] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the payload region and the enhancer region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the intron region and the 3' ITR.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the MCS region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the MCS region and the 3' ITR.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the MCS region, and the second of which may be located between the exon region and the 3' ITR.

[0280] In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the promoter region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the payload region and the enhancer region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the intron region and the 3' ITR.In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the 5' ITR and the exon region, and the second of which may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first filler sequence may be located between the 5' ITR and the exon region, and the second filler sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two filler sequences, a first filler sequence may be located between the 5' ITR and the exon region, and a second filler sequence may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, a first filler sequence may be located between the 5' ITR and the exon region, and a second filler sequence may be located between the exon region and the 3' ITR.

[0281] In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the payload region, and the second filler sequence may be located between the MCS region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, the first of which may be located between the promoter region and the payload region, and the second of which may be located between the exon region and the 3' ITR.

[0282] In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the payload region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the intron region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the enhancer region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the MCS region.In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the polyadenylation signal sequence region and the 3' ITR. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter region and the intron region, and the second filler sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two filler sequences, where the first filler sequence may be located between the promoter regio...

Claims

1. An adeno-associated virus (AAV) genome comprising a nucleic acid sequence located between two inverted terminal repeats (ITRs), which, when expressed, inhibits or suppresses expression of a first target gene and a second target gene in a cell; The nucleic acid sequence, in 5' to 3' order, is (i) (a) a first 5' flanking region, a nucleotide sequence encoding a first sense strand sequence, a first loop region, a nucleotide sequence encoding a first antisense strand sequence, and a first 3' flanking region; or (b) a first 5' flanking region, a nucleotide sequence encoding a first antisense strand sequence, a first loop region, a nucleotide sequence encoding a first sense strand sequence, and a first 3' flanking region; and (ii) (a) a second 5' flanking region, a nucleotide sequence encoding a second sense strand sequence, a second loop region, a nucleotide sequence encoding a second antisense strand sequence, and a second 3' flanking region; or (b) a second 5' flanking region, a nucleotide sequence encoding a second antisense strand sequence, a second loop region, a nucleotide sequence encoding a second sense strand sequence, and a second 3' flanking region. Including, the first sense strand sequence and the first antisense strand sequence share a region of complementarity of at least 12 nucleotides in length, and the first antisense strand is complementary to the mRNA of the first target gene; the second sense strand sequence and the second antisense strand sequence share a region of complementarity of at least 12 nucleotides in length, and the second antisense strand is complementary to the mRNA of the second target gene; the first 5' flanking region comprises the nucleotide sequence of any one of SEQ ID NOs: 1503-1509, 1692 or 1782, and the second 5' flanking region comprises the nucleotide sequence of any one of SEQ ID NOs: 1503-1509, 1692 or 1782; The first loop region comprises the nucleotide sequence of any one of SEQ ID NOs: 1510-1517 or 1693-1694, and the second loop region comprises the nucleotide sequence of any one of SEQ ID NOs: 1510-1517 or 1693-1694; and the first 3' flanking region comprises the nucleotide sequence of any one of SEQ ID NOs: 1518-1522, 1695 or 1783, and the second 3' flanking region comprises the nucleotide sequence of any one of SEQ ID NOs: 1518-1522, 1695 or 1783; AAV genome.

2. The AAV genome of claim 1 , wherein the first target gene is the same as the second target gene.

3. The AAV genome of claim 1 , wherein the first target gene is not the same as the second target gene. Claim 4: (i) the first 5' flanking region comprises the nucleotide sequence of SEQ ID NO:1504; the first loop region comprises the nucleotide sequence of SEQ ID NO:1511; and the first 3' flanking region comprises the nucleotide sequence of SEQ ID NO:1518; and the second 5' flanking region comprises the nucleotide sequence of SEQ ID NO:1505; the second loop region comprises the nucleotide sequence of SEQ ID NO:1513; and the second 3' flanking region comprises the nucleotide sequence of SEQ ID NO:1520; or (ii) the first 5' flanking region comprises the nucleotide sequence of SEQ ID NO:1505; the first loop region comprises the nucleotide sequence of SEQ ID NO:1513; and the first 3' flanking region comprises the nucleotide sequence of SEQ ID NO:1520; and the second 5' flanking region comprises the nucleotide sequence of SEQ ID NO:1504; the second loop region comprises the nucleotide sequence of SEQ ID NO:1511; and the second 3' flanking region comprises the nucleotide sequence of SEQ ID NO:1518. The AAV genome according to any one of claims 1 to 3.

5. The AAV genome of any one of claims 1 to 4, wherein the first target gene is huntingtin (HTT).

6. The AAV genome of any one of claims 1 to 5, wherein the second target gene is HTT.

7. The AAV genome of any one of claims 1 to 4 or 6, wherein the first target gene is SOD1.

8. The AAV genome of any one of claims 1 to 5 or 7, wherein the second target gene is SOD1.

9. (i) one or both of the first sense strand sequence and the first antisense strand sequence comprises a 3' overhang of at least one nucleotide or at least two nucleotides; (ii) one or both of the second sense strand sequence and the second antisense strand sequence comprises a 3' overhang of at least one nucleotide or at least two nucleotides; (iii) each sense strand sequence and antisense strand sequence is independently 19 to 24 nucleotides in length or 19 to 21 nucleotides in length; and / or (iv) each sense strand sequence and antisense strand sequence is independently 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides in length; The AAV genome according to any one of claims 1 to 8.

10. (i) a first promoter 5' to the first 5' flanking region; or (ii) further comprising a first promoter 5' to the first 5' flanking region and a second promoter 5' to the second 5' flanking region; The AAV genome according to any one of claims 1 to 9.

11. The AAV genome of claim 10, wherein the first promoter, the second promoter, or both are ubiquitous promoters.

12. (i) the first promoter and / or the second promoter is a CBA promoter, a CMV promoter, a PGK promoter, an H1 promoter, a UBC promoter, a GUSB promoter, an NSE promoter, a synapsin promoter, a MeCP2 promoter, or a GFAP promoter; or (ii) the first promoter is a CBA promoter and the second promoter is an H1 promoter; The AAV genome of claim 10 or 11.

13. (i) an intron; (ii) a filler sequence; and / or (iii) a polyadenylation (polyA) sequence; The AAV genome of any one of claims 1 to 12, further comprising:

14. An adeno-associated virus (AAV) particle comprising the AAV genome of any one of claims 1 to 13 and an AAV capsid protein.

15. The AAV particle of claim 14, wherein the AAV capsid protein is an AAV9 capsid protein or an AAV5 capsid protein.

16. The AAV genome according to any one of claims 1 to 13 or the AAV particle according to claim 14 or 15. cell.

17. 17. The cell of claim 16, which is a mammalian cell, a HEK293 cell, an insect cell, an Sf9 cell, a cell of the central nervous system, a neuron, a medium spiny neuron, a motor neuron, or an astrocyte.

18. A pharmaceutical composition comprising the AAV particles of claim 14 or 15 and a pharmaceutically acceptable excipient.

19. An AAV particle comprising an AAV genome according to any one of claims 1 to 13, or a composition comprising the AAV particle according to claim 14 or 15, or a pharmaceutical composition according to claim 18, for use in inhibiting expression of a target gene in a cell.

20. (i) the target gene is expressed in neural cells, tissues, or organs; (ii) the target gene is HTT or SOD1; (iii) the cell is a medium spiny neuron, a cortical neuron, a motor neuron, or an astrocyte; and / or (iv) the cell is in a subject; 20. A composition for use according to claim 19.

21. 21. The composition for use according to claim 20, wherein the subject is suffering from a disease of the central nervous system (CNS).

22. An AAV particle comprising an AAV genome according to any one of claims 1 to 13, or a composition comprising the AAV particle of claim 14 or 15, or a pharmaceutical composition according to claim 18, for use in treating a disease of the central nervous system (CNS).

23. 23. The composition for use according to claim 21 or 22, wherein the CNS disease is Huntington's disease (HD).

24. 23. The composition for use according to claim 21 or 22, wherein the CNS disease is amyotrophic lateral sclerosis (ALS).

25. 25. The composition for use according to any one of claims 19 to 24, wherein the composition is formulated for intravenous administration, intracisternal injection, intravascular administration, intraventricular administration, or a combination thereof.

26. Use of an AAV particle comprising an AAV genome according to any one of claims 1 to 13, an AAV particle according to claim 14 or 15, or a pharmaceutical composition according to claim 18 in the manufacture of a medicament for inhibiting expression of a target gene in a cell.

27. (i) the target gene is expressed in neural cells, tissues, or organs; (ii) the target gene is HTT or SOD1; (iii) the cell is a medium spiny neuron, a cortical neuron, a motor neuron, or an astrocyte; and / or (iv) the cell is in a subject; 27. The use according to claim 26.

28. 28. The use of claim 27, wherein the subject is suffering from a disease of the central nervous system (CNS).

29. Use of an AAV particle comprising an AAV genome according to any one of claims 1 to 13, an AAV particle according to claim 14 or 15, or a pharmaceutical composition according to claim 18, in the manufacture of a medicament for the treatment of a disease of the central nervous system (CNS).

30. 30. The use according to claim 28 or 29, wherein the CNS disease is Huntington's disease (HD).

31. 30. The use according to claim 28 or 29, wherein the CNS disease is amyotrophic lateral sclerosis (ALS).

32. The use of any one of claims 26 to 31, wherein the AAV particles or pharmaceutical composition are formulated for intravenous administration, intracisternal injection, intravascular administration, intraventricular administration, or a combination thereof.

33. 14. An in vitro method for producing adeno-associated virus (AAV) particles, the method comprising the steps of: supplying a cell with a polynucleotide comprising the AAV genome of any one of claims 1 to 13, at least one polynucleotide encoding an AAV rep gene, and at least one polynucleotide encoding an AAV cap gene; and recovering AAV particles from the cell, wherein the cell is a bacterial cell, a mammalian cell, or an insect cell.

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