Compositions and methods for gene therapy

Abstract

Provided herein are polynucleotides comprising a nucleotide sequence encoding a biomolecule, such as human alpha-galactosidase A, a nucleotide sequence encoding a promoter, such as a hepatocyte-specific promoter or a hepatocyte-muscle cell bispecific promoter, or a nucleotide sequence encoding an expression cassette, such as a human alpha-galactosidase A expression cassette. Also provided herein are promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant viral particles, such as recombinant adeno-associated virus (AAV) particles and non-viral gene delivery systems), related pharmaceutical compositions, and methods of using the same. Such compositions and methods are particularly suitable for gene therapy, particularly for lysosomal storage disorders, including Fabry disease.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of PCT application No. PCT / CN2023 / 088738, filed on April 17, 2023. The contents of these applications are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This document provides polynucleotides comprising nucleotide sequences encoding biomolecules such as human α-galactosidase A, nucleotide sequences encoding promoters such as hepatocyte-specific promoters or hepatocyte-muscle cell bispecific promoters, or nucleotide sequences encoding expression cassettes such as human α-galactosidase A expression cassettes. This document also provides promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant viral particles, such as recombinant adeno-associated virus (AAV) particles, and nonviral gene delivery systems), related pharmaceutical compositions, and methods of using them. Such compositions and methods are particularly suitable for gene therapy, especially for lysosomal storage diseases, including Fabry disease. Background Technology

[0003] Gene therapy (including AAV-based gene therapy) has the potential to become a promising treatment for many diseases.

[0004] Lysosomal storage disorders are inherited metabolic disorders characterized by the accumulation of abnormal amounts of substrates in cells of various organs due to lysosomal dysfunction. For example, Fabry disease (FD) is a rare X-linked metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) due to a pathogenic mutation in the GLA gene. The gradual accumulation of substrates within the lysosome leads to cellular dysfunction and multi-organ damage.

[0005] To date, there remains a need to develop compositions, systems, and methods for gene therapy, particularly for gene therapy of lysosomal storage diseases, including Fabry disease. Summary of the Invention

[0006] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1.

[0007] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, or (ii) is as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myoblast bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0008] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myotrophic lateral sclerosis (HMS) bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10. In various embodiments, the polynucleotide further comprises a nucleotide sequence encoding a polyA signal.

[0009] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myoblast bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10. In a specific embodiment, the biomolecule is human α-galactosidase A. In various embodiments, the polynucleotide further comprises a nucleotide sequence encoding a polyA signal.

[0010] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding an expression cassette, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16 or SEQ ID NO: 18, or as shown in SEQ ID NO: 16 or SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18.

[0011] In one aspect, this document provides a promoter encoded by a nucleotide sequence, said nucleotide sequence (i) comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myoblast bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0012] In one aspect, this document provides an expression cassette comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myotrophic lateral sclerosis (HMS) bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10. In various embodiments, the expression cassette further comprises a nucleotide sequence encoding a polyA signal.

[0013] In one aspect, this document provides an expression cassette comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10. In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myoblast bispecific promoter. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10. In a specific embodiment, the biomolecule is human α-galactosidase A. In various embodiments, the expression cassette also includes a nucleotide sequence encoding a polyA signal.

[0014] In one aspect, this document provides an expression cassette encoded by (i) a nucleotide sequence comprising SEQ ID NO: 16 or SEQ ID NO: 18 or (ii) a nucleotide sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 18.

[0015] In one aspect, this article provides a polynucleotide comprising a nucleotide sequence encoding the expression cassette described herein.

[0016] In one aspect, this document provides vectors comprising the polynucleotides, promoters, or expression cassettes described herein.

[0017] In one aspect, this document provides recombinant viral particles comprising a recombinant viral genome containing the expression cassette described herein.

[0018] In one aspect, this document provides a recombinant adeno-associated virus (AAV) particle comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an expression cassette described herein side-joined with an AAV terminal inverted repeat (ITR). In a specific embodiment, the recombinant AAV particle is a recombinant AAV serotype 9 (rAAV9) particle. In a specific embodiment, the AAV capsid comprises a variant AAV9 capsid protein. In some embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59, or 61. In some embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58, or 60.

[0019] In one aspect, this article provides a host cell comprising the polynucleotides described herein, the vectors described herein, the recombinant viral particles described herein, or the recombinant AAV particles described herein.

[0020] In one respect, this article provides a host cell population stably transduced by the recombinant viral particles or the recombinant AAV particles described herein.

[0021] In one aspect, this article provides a pharmaceutical composition comprising the recombinant viral particles or recombinant AAV particles described herein, and a pharmaceutically acceptable carrier.

[0022] In one aspect, this article provides a pharmaceutical composition comprising the host cell population described herein and a pharmaceutically acceptable carrier.

[0023] In one aspect, this article provides a method for producing recombinant viral particles or recombinant AAV particles, comprising culturing the host cells described herein.

[0024] In one aspect, this document provides a method for treating a disease or disorder in a subject in need, comprising administering to the subject the recombinant viral particles described herein, the recombinant AAV particles described herein, or the pharmaceutical composition described herein. In some embodiments, the disease or disorder is lysosomal storage disease. In a particular embodiment, the disease or disorder is Fabry disease. In a particular embodiment, the subject is a human.

[0025] Illustrative Implementation This disclosure includes the following non-limiting illustrative embodiments: 1. A polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1.

[0026] 2. The polynucleotide according to Embodiment 1, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1.

[0027] 3. The polynucleotide according to Embodiment 1, wherein the nucleotide sequence encoding human α-galactosidase A is shown in SEQ ID NO: 1.

[0028] 4. A polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10.

[0029] 5. A polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, said nucleotide sequence encoding the promoter being operatively linked to said nucleotide sequence encoding human α-galactosidase A, wherein said nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1.

[0030] 6. The polynucleotide according to embodiment 5, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1.

[0031] 7. The polynucleotide according to embodiment 5, wherein the nucleotide sequence encoding human α-galactosidase A is shown in SEQ ID NO: 1.

[0032] 8. The polynucleotide according to any one of embodiments 4-7, wherein the promoter is a hepatocyte-specific promoter.

[0033] 9. The polynucleotide according to any one of embodiments 4-8, wherein the promoter is a hepatocyte-myocell bispecific promoter.

[0034] 10. The polynucleotide according to any one of embodiments 4-9, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3.

[0035] 11. The polynucleotide according to any one of embodiments 4-8, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 3.

[0036] 12. The polynucleotide according to any one of embodiments 4-9, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4.

[0037] 13. The polynucleotide according to any one of embodiments 4-9, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 4.

[0038] 14. The polynucleotide according to any one of embodiments 4-9, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10.

[0039] 15. The polynucleotide according to any one of embodiments 4-9, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 10.

[0040] 16. A polynucleotide comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10.

[0041] 17. The polynucleotide according to embodiment 16, wherein the promoter is a hepatocyte-specific promoter.

[0042] 18. The polynucleotide according to embodiment 16 or 17, wherein the promoter is a hepatocyte-myoblast bispecific promoter.

[0043] 19. The polynucleotide according to any one of embodiments 16-18, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3.

[0044] 20. The polynucleotide according to embodiment 16 or 17, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 3.

[0045] 21. The polynucleotide according to any one of embodiments 16-18, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4.

[0046] 22. The polynucleotide according to any one of embodiments 16-18, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 4.

[0047] 23. The polynucleotide according to any one of embodiments 16-18, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10.

[0048] 24. The polynucleotide according to any one of embodiments 16-18, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 10.

[0049] 25. The polynucleotide according to any one of embodiments 16-24, wherein the biomolecule is human α-galactosidase A.

[0050] 26. The polynucleotide according to any one of embodiments 5-25, further comprising a nucleotide sequence encoding a polyA signal.

[0051] 27. A polynucleotide comprising a nucleotide sequence encoding an expression cassette, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16 or SEQ ID NO: 18 or as shown in SEQ ID NO: 16 or SEQ ID NO: 18.

[0052] 28. The polynucleotide according to embodiment 27, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16.

[0053] 29. The polynucleotide according to embodiment 27, wherein the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO: 16.

[0054] 30. The polynucleotide according to embodiment 27, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18.

[0055] 31. The polynucleotide according to embodiment 27, wherein the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO: 18.

[0056] 32. A promoter encoded by (i) a nucleotide sequence comprising SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10 or (ii) a nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 10.

[0057] 33. The promoter according to embodiment 32 is a hepatocyte-specific promoter.

[0058] 34. The promoter according to embodiment 32 or 33 is a hepatocyte-myocell bispecific promoter.

[0059] 35. The promoter according to any one of embodiments 32-34, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3.

[0060] 36. The promoter according to embodiment 32 or 33, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 3.

[0061] 37. The promoter according to any one of embodiments 32-34, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4.

[0062] 38. The promoter according to any one of embodiments 32-34, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 4.

[0063] 39. The promoter according to any one of embodiments 32-34, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10.

[0064] 40. The promoter according to any one of embodiments 32-34, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 10.

[0065] 41. An expression cassette comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1 or as shown in SEQ ID NO: 1.

[0066] 42. The expression cassette according to embodiment 41, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1.

[0067] 43. The expression cassette according to embodiment 41, wherein the nucleotide sequence encoding human α-galactosidase A is shown in SEQ ID NO: 1.

[0068] 44. The expression cassette according to any one of embodiments 41-43, wherein the promoter is a hepatocyte-specific promoter.

[0069] 45. The expression cassette according to any one of embodiments 41-44, wherein the promoter is a hepatocyte-myoblast bispecific promoter.

[0070] 46. ​​The expression cassette according to any one of embodiments 41-45, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3.

[0071] 47. The expression cassette according to any one of embodiments 41-44, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 3.

[0072] 48. The expression cassette according to any one of embodiments 41-45, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4.

[0073] 49. The expression cassette according to any one of embodiments 41-45, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 4.

[0074] 50. An expression cassette according to any one of embodiments 41-45, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10.

[0075] 51. The expression cassette according to any one of embodiments 41-45, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 10.

[0076] 52. An expression cassette comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter (i) comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10, or (ii) as shown in SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 10.

[0077] 53. The expression cassette according to embodiment 52, wherein the promoter is a hepatocyte-specific promoter.

[0078] 54. An expression cassette according to embodiment 52 or 53, wherein the promoter is a hepatocyte-myoblast bispecific promoter.

[0079] 55. The expression cassette according to any one of embodiments 52-54, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3.

[0080] 56. The expression cassette according to embodiment 52 or 53, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 3.

[0081] 57. An expression cassette according to any one of embodiments 52-54, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4.

[0082] 58. The expression cassette according to any one of embodiments 52-54, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 4.

[0083] 59. The expression cassette according to any one of embodiments 52-54, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10.

[0084] 60. The expression cassette according to any one of embodiments 52-54, wherein the nucleotide sequence encoding the promoter is shown in SEQ ID NO: 10.

[0085] 61. The expression cassette according to any one of embodiments 52-60, wherein the biomolecule is human α-galactosidase A.

[0086] 62. The expression cassette according to any one of embodiments 41-61 further comprises a nucleotide sequence encoding a polyA signal.

[0087] 63. An expression cassette encoded by (i) a nucleotide sequence comprising SEQ ID NO: 16 or SEQ ID NO: 18 or (ii) a nucleotide sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 18.

[0088] 64. The expression cassette according to embodiment 63, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO.16.

[0089] 65. The expression cassette according to embodiment 63, wherein the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO. 16.

[0090] 66. The expression cassette according to embodiment 63, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO.18.

[0091] 67. The expression cassette according to embodiment 63, wherein the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO. 18.

[0092] 68. A polynucleotide comprising a nucleotide sequence encoding the expression cassette of any one of embodiments 41-67.

[0093] 69. A vector comprising a polynucleotide as described in any one of embodiments 1-31 and 68, a promoter as described in any one of embodiments 32-40, or an expression cassette as described in any one of embodiments 41-67.

[0094] 70. A recombinant viral particle comprising a recombinant viral genome, said recombinant viral genome comprising the expression cassette of any one of embodiments 41-67.

[0095] 71. A recombinant adeno-associated virus (AAV) particle comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an expression cassette of any one of embodiments 41-67 with an AAV terminal inverted repeat (ITR) lateralized to it.

[0096] 72. The recombinant AAV particles according to embodiment 71 are recombinant AAV serotype 9 (rAAV9) particles.

[0097] 73. The recombinant AAV particle according to embodiment 71 or 72, wherein the AAV capsid comprises variant AAV9 capsid protein.

[0098] 74. The recombinant AAV particle according to embodiment 73, wherein the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59 or 61.

[0099] 75. The recombinant AAV particle according to embodiment 74, wherein the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58 or 60.

[0100] 76. A host cell comprising any one of embodiments 1-31 and 68, the vector of embodiment 69, the recombinant viral particle of embodiment 70, or the recombinant AAV particle of any one of embodiments 71-75.

[0101] 77. A host cell population stably transduced by the recombinant viral particles of embodiment 70 or the recombinant AAV particles of any one of embodiments 71-75.

[0102] 78. A pharmaceutical composition comprising the recombinant viral particles of embodiment 70 or the recombinant AAV particles of any one of embodiments 71-75, and a pharmaceutically acceptable carrier.

[0103] 79. A pharmaceutical composition comprising the host cell population described in embodiment 77 and a pharmaceutically acceptable carrier.

[0104] 80. A method for producing recombinant viral particles or recombinant AAV particles, comprising culturing the host cells described in embodiment 76.

[0105] 81. A method of treating a disease or disorder in a subject in need, comprising administering to the subject the recombinant viral particles of embodiment 70, the recombinant AAV particles of any one of embodiments 71-75, or the pharmaceutical composition of embodiment 78 or 79.

[0106] 82. The method according to embodiment 81, wherein the disease or disorder is lysosomal storage disease.

[0107] 83. The method according to embodiment 81, wherein the disease or disorder is Fabry disease.

[0108] 84. The method according to any one of embodiments 81-83, wherein the subject is a human. Attached Figure Description

[0109] Figure 1 Codon optimization for human α-galactosidase A (GLA). Figure 1 A: ELISA test results. Figure 1 B: Results of α-galactosidase A activity. Figure 1 C: Western blot analysis of protein blots from HepG2 cell culture supernatant transfected with a plasmid encoding hGLA. WT: Wild type; hGLA: Human α-galactosidase A.

[0110] Figure 2 In vitro assay for hepatocyte-cardiomyocyte (HC) bispecific promoters. The RLuc / FLuc ratio was used to measure the promoter strength of five promoters (HC1-HC5). RLuc: Renal luciferase; FLuc: Firefly luciferase.

[0111] Figure 3 In vitro assay of the truncated mouse muscle creatine kinase (MCK) promoter. Figure 3 A: The RLuc / FLuc ratio is used to measure the promoter strength of three versions of the MCK promoter (MCK-D1~MCK-D3). Figure 3 B and 3C: The activity of promoter HC7 was assessed using an in vivo imaging system (IVIS). One week after injection ( Figure 3 B) and three weeks ( Figure 3 C) Strong luminescent signals were observed in the liver and skeletal muscle.

[0112] Figure 4 Human GLA expression cassette-mediated α-galactosidase A expression in HepG2 ( Figure 4 A) and activity ( Figure 4 B). Figure 4 A: The α-Gal A concentrations of the negative control, box 1, box 2, box 3 and box 4 were 2 ng / mL, 2993 ng / mL, 2225 ng / mL, 1919 ng / mL and 1684 ng / mL, respectively. Figure 4 B: The α-Gal A activities of the negative control, box 1, box 2, box 3, and box 4 were 1.66 x 10⁻⁶. 6 nmol / mg / h, 1.87x10 6 nmol / mg / h, 2.00x10 6 nmol / mg / h and 2.73x10 6 nmol / mg / h.

[0113] Figure 5 Human GLA expression cassette-mediated α-galactosidase A expression in H9c2 cells ( Figure 5 A) and activity ( Figure 5 B). Figure 5 A: The α-Gal A concentrations of the negative control, box 1, box 2, box 3, and box 4 were 2 x 10⁻⁶. -1 ng / mL, 306 ng / mL, 249 ng / mL, 954 ng / mL and 1350 ng / mL. Figure 5 B: The α-Gal A activities of the negative control, box 1, box 2, box 3, and box 4 were 1.14 x 10⁻⁶. 4 nmol / mg / h, 1.45x10 4 nmol / mg / h, 1.80x10 4 nmol / mg / h and 2.57x10 4 nmol / mg / h.

[0114] Figures 6A-6J Characterization of box 1-mediated activity and efficacy in a Fabry mouse model. Figure 6A Changes in serum α-Gal A activity over time. Figure 6B Serum α-Gal A activity on day 61 (the numbers at the top of the column from left to right are: 11, 3, 1959, 25956, 80140 and 203339). Figure 6C : hGLA transgenic mRNA levels in the liver (numbers at the top of the column from left to right: 0.3200, 2.2752, 3.9636, 9.6976 and 0.0001). Figure 6DhGLA transgenic mRNA levels in the heart (numbers at the top of the column from left to right: 3.3037 x 10⁻⁶) -5 0.0005, 0.0005 and 0.0008). Figure 6E : Vector genome copies in the liver (numbers at the top of the column from left to right: 0.94, 10.88, 28.28, 79.60 and 0.02). Figure 6F : Vector genome copies in the liver (numbers at the top of the column from left to right: 0.03, 0.24, 0.87 and 1.64). Figure 6G Results of Western blot analysis of proteins. Figure 6H α-Gal A activity in the liver, heart, and kidneys. Liver data shown from left to right: 2043 (2E11 vg / kg), 48242 (2E12 vg / kg), 108653 (6E12 vg / kg), 175176 (2E13 vg / kg), 12 (GLA-KO), and 35 (wild-type). Heart data shown from left to right: 31 (2E11 vg / kg), 1186 (2E12 vg / kg), 1533 (6E12 vg / kg), 12153 (2E13 vg / kg), 26 (GLA-KO), and 10 (wild-type). The kidney data shown from left to right are: 18 (2E11 vg / kg), 217 (2E12 vg / kg), 501 (6E12 vg / kg), 1821 (2E13 vg / kg), 6 (GLA-KO), and 21 (wild type). Figure 6I Serum lyso-Gb3 levels (numbers at the top of the columns from left to right: 0.57, 147.22, 3.67, 0.76, 0.59, and 0.55). Figure 6J : Lyso-Gb3 levels in the liver, heart, and kidneys.

[0115] Figures 7A-7E Characterization of AAV-hGLA-mediated activity and efficacy in a Fabry mouse model. Figure 7A Changes in serum α-GAL A activity over time. Figure 7B : Vector genome copies in the liver, heart, and quadriceps. Figure 7C hGLA transgenic mRNA levels in the liver, heart, and quadriceps. Figure 7D Results of Western blot analysis of proteins. Figure 7E α-Gal A activity mediated by box 3 in the liver, heart, and kidneys. Figure 7FHC7-driven hGLAco1 (box 3), packaged in AVT908 capsid, was administered intravenously to male 8-week-old Gla-ko mice at a dose of 2e12 vg / kg. Eight weeks post-administration, liver, heart, and kidneys were harvested for immunohistochemical (IHC) staining. Rabbit anti-human GLA antibody (Sigma-Aldrich, catalog number HPA000237) was diluted 1:1000 and used as the primary antibody. Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (jacksonimmuno, 111-035-003) was diluted 1000-fold and used as the detection antibody. DAB was used as the substrate. Brown staining indicates positive staining for human GalA protein.

[0116] Figures 8A-8B Adult male cynomolgus monkeys were intravenously administered hGLA expression cassette 3, packaged in capsids AVT917, AVT918, or AAV9, at a dose of 2E13 vg / kg (N=1 / capsid). Plasma was collected before and after administration. Figure 8A Gal A activity in plasma was measured by a fluorescence-based 4-MU method. Figure 8A The expression of human Gal A protein in plasma was measured using ELISA.

[0117] Figure 9A Genomic biodistribution of the AAV vector in NHP. Adult male cynomolgus monkeys were administered AVT917 containing the hGLA expression cassette 3 intravenously at a dose of 2E13 vg / kg. Thirteen weeks post-administration, tissues were collected and processed for vector genome copy analysis by ddPCR. The RNase P gene was used as a reference.

[0118] Figure 9B The AVT917 and HC7 promoters mediate hGLA transgene expression in NHP. Adult male cynomolgus monkeys were intravenously administered AVT917 containing the hGLA expression cassette 3 at a dose of 2E13 vg / kg. Thirteen weeks post-administration, tissues were collected and processed for transgene mRNA level analysis by RT-qPCR. The GADPH gene was used as a reference. Data are presented as 2. -ΔCt .

[0119] Figure 9C Gal A activity in NHP tissues treated with AVT917-hGLA. Adult male cynomolgus monkeys were intravenously administered AVT917 containing hGLA expression cassette 3 at a dose of 2E13 vg / kg. Thirteen weeks after administration, tissues were collected and processed for Gal A activity analysis by a fluorescence-based 4-MU method.

[0120] Figure 10AVT917 mediates high hGLA expression in Fabry mice. Adult (16-week-old) male Fabry mice (Gla-KO) were intravenously injected with either solvent (formulation buffer) or AVT919-HC7-hGLA (box 3) at three doses (n=10 / group). Plasma was collected every two weeks for α-Gal A activity measurements. Data are presented as mean with SD. **p<0.01, ***p<0.001, ****p<0.0001, two-way repeated measures ANOVA followed by Tukey multiple comparison test.

[0121] Figure 11 AVT917 mediates hGLA mRNA expression in Fabry mice tissues. Adult (16-week-old) male Fabry mice (Gla-KO) were intravenously injected with either solvent (formulation buffer) or AVT919-HC7-hGLA (box 3) at three doses (n=10 / group). Eight weeks post-administration, major tissues were collected and processed for total RNA isolation. hGLA-specific mRNA transcripts were quantified by RT-qPCR. Mouse GAPDH (MsGAPDH) was used as a housekeeping gene. Data are presented as mean with SD. *p<0.05, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test.

[0122] Figure 12 Substrate was significantly reduced in Fabry mice treated with AVT917-HC7-hGLA. Adult (16-week-old) male Fabry mice (Gla-KO) were intravenously injected with either solvent (formulation buffer) or AVT919-HC7-hGLA (box 3) at three doses (n=10 / group). Eight weeks after administration, major tissues were collected and processed for Lyso-Gb3 quantification using LC-MS / MS. Data are presented as mean with SD. Percentages on columns represent relative Lyso-Gb3 levels, with levels in the solvent group set as 100%. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test; #p<0.0001, unpaired t-test.

[0123] Figure 13A AVT917-mediated hGLA expression in NHP. Adult male cynomolgus monkeys were administered AVT917-HC7-hGLA intravenously at doses of 2E12 vg / kg (N=3) and 6E12 vg / kg (N=2). Plasma was collected before and after administration. Plasma α-Gal A activity was measured by a fluorescence-based 4-MU assay.

[0124] Figure 13BAdult male cynomolgus monkeys were administered AVT917-HC7-hGLA intravenously at doses of 2E12 vg / kg (N=3) and 6E12 vg / kg (N=2). Plasma was collected before and after administration. Human α-Gal A protein expression in plasma was measured by ELISA.

[0125] Figure 14 Adult male cynomolgus monkeys were intravenously administered AVT917 or AVT919 containing box 3 (SEQ ID: 18) at a dose of 2E12 vg / kg (N=1 / capsid). Plasma was collected for the measurement of human α-Gal A protein concentration by ELISA. Detailed Implementation

[0126] This disclosure describes codon-optimized nucleotide sequences encoding human α-galactosidase A and provides data indicating that these nucleotide sequences are particularly suitable for gene therapy of Fabry disease. This disclosure also describes nucleotide sequences encoding hepatocyte-specific or hepatocyte-muscle cell-specific promoters and provides data indicating that these nucleotide sequences are particularly suitable for gene therapy, especially for lysosomal storage diseases including Fabry disease. This disclosure further describes nucleotide sequences encoding human α-galactosidase A expression cassettes and provides data indicating that these nucleotide sequences are particularly suitable for gene therapy of Fabry disease. This disclosure provides polynucleotides, promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant viral particles, such as recombinant adeno-associated virus (AAV) particles, and nonviral gene delivery systems) and pharmaceutical compositions associated with these nucleotide sequences, as well as methods of using them.

[0127] 5.1 Definition The techniques and procedures described or referenced herein include those that are generally well understood by those skilled in the art and / or commonly used with conventional methods, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 th Ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY 2012); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols(Albitar ed. 2010); and Antibody Engineering The widely used method described in Vols 1 and 2 (Kontermann and Dübel eds., 2d ed. 2010).

[0128] Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terminology will be used.

[0129] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers of amino acids, either naturally occurring or modified through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification. The definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, and other modifications known in the art.

[0130] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length and includes DNA (including cDNA) and RNA (including mRNA). Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can be single-stranded or double-stranded. Polynucleotides can be linear or circular. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and their analogues. As used herein, “oligonucleotide” refers to a short synthetic polynucleotide that is typically, but not necessarily, less than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides. Unless otherwise stated, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition to a newly formed RNA transcript is referred to as the transcriptional direction; a sequence region on the DNA strand that shares the same sequence as the 5' end of the RNA transcript is referred to as the "upstream sequence"; a sequence region on the DNA strand that shares the same sequence as the 3' end of the RNA transcript is referred to as the "downstream sequence". In some embodiments, the polynucleotides described herein can be optimized by using alternative or preferred codons for a specific host cell or delivery target cell type. Codon optimization can be performed using any suitable method known in the art (e.g., using suitable software known in the art).

[0131] As used herein, “isolated polynucleotide” or “isolated nucleic acid” refers to nucleic acids, such as RNA, DNA, or a mixture of nucleic acids, which are generally substantially isolated from other genomic DNA sequences and proteins or complexes (e.g., ribosomes and polymerases), and whose natural accompaniment is a natural sequence. An “isolated” nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, an “isolated” nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term covers nucleic acid sequences that have been removed from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized through heterologous systems. A substantially pure molecule can include the isolated form of the molecule. Specifically, an “isolated” nucleic acid molecule encoding the polypeptide described herein is a nucleic acid molecule identified and isolated from at least one contaminating nucleic acid molecule that is generally associated with it in the environment in which it was produced.

[0132] As used herein, the term "homology" refers to the percentage of identity between two polynucleotides or two polypeptide motifs. Two DNA or polypeptide sequences are "substantially homologous" to each other when they exhibit at least about 50%, at least about 75%, at least about 80%–85%, at least about 90%, at least about 95%–98%, at least about 99%, or any percentage between these values ​​on a molecule of a defined length. As used herein, substantially homologous also refers to a sequence that shows complete identity with a specified DNA or polypeptide sequence.

[0133] As used herein, the term "identity" refers to the precise nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Methods for determining the percentage of identity are well known in the art. For example, the percentage of identity can be determined by directly comparing the sequence information between two molecules, such as by aligning sequences, counting the precise number of matches between two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Existing computer programs can be used to assist in the analysis, such as the local homology algorithm for peptide analysis in ALIGN, Dayhoff, MO in Atlas of Protein Sequence and Structure MO Dayhoff ed., 5 Suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, adapted from Smith and Waterman's Advances in Appl. Math. 2:482-489, 1981. Procedures for determining nucleotide sequence identity are available in Wisconsin Sequence Analysis Package Version 8 (available from Genetics Computer Group, Madison, Wis), such as the BESTFIT, FASTA, and GAP programs, which also rely on the Smith and Waterman algorithms. These programs are readily usable with the manufacturer-recommended default parameters described in the Wisconsin Sequence Analysis Packages mentioned above. For example, the percentage of identity between a particular nucleotide sequence and a reference sequence can be determined using the Smith and Waterman homology algorithm, along with a default scoring table and gap penalties at six nucleotide positions. Another approach to establishing the percentage of identity in the context of this invention is to use the MPSRCH package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and published by IntelliGenetics, Inc. (Mountain View, CA). From this package, the Smith-Waterman algorithm can be employed, with default parameters for the scoring table (e.g., a gap open penalty of 12, a gap extension penalty of 1, and 6 gaps). Based on the generated data, the "match" value reflects "sequence identity". Other suitable procedures for calculating the percentage of identity or similarity between sequences are generally known in the art; for example, another alignment procedure is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used with the following default parameters: Genetic Code = Standard; Filter = None; Strands = Both; Truncation Value = 60; Expected Value = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort By = High Score; Database = Non-Redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+Swiss Protein+Spupdate+PIR. Details of these procedures are well known in the art. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease, and determining the size of the digested fragments. Essentially homologous DNA sequences can be identified in Southern hybridization experiments under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the scope of the art. See, for example, Sambrook et al., ibid.; DNA cloning, ibid.; Nucleic acid hybridization, ibid.

[0134] As used herein, the term "vector" refers to a substance used to carry or include a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plasmids, baculovirus plasmids, sticky plasmids, constructs, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the chromosome of a host cell. Additionally, a vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes may include, for example, providing resistance to antibiotics or toxins, compensating for auxotrophic deficiencies, or supplying key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more nucleic acid molecules are to be co-expressed, the two nucleic acid molecules may be inserted into, for example, a single expression vector or a separate expression vector. The introduction of the nucleic acid molecule into the host cell can be confirmed using methods known in the art. Such methods include, for example, nucleic acid analysis, such as RNA blotting or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. The term "vector" includes cloning and expression vectors, as well as viral vectors. In some embodiments, the vector provided herein is a recombinant viral vector. In a particular embodiment, the vector provided herein is a recombinant AAV vector.

[0135] As used herein, the term "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing a nucleic acid sequence derived from AAV and one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV). In some embodiments, one or more heterologous sequences are side-joined with at least one (in some embodiments, two) AAV terminal inverted repeat (ITR) sequence. In some embodiments, such rAAV vectors can be replicated and packaged into infectious viral capsid particles, for example, when present in host cells that have been infected with a suitable helper virus (or express a suitable helper function) and express the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). rAAV vectors can be incorporated into larger polynucleotides (e.g., in chromosomes or in another vector such as plasmids used for cloning or transfection) and can be "rescued" by replication and encapsulation in the presence of AAV packaging function and suitable helper functions. rAAV vectors can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral capsid particles (especially AAV particles). The rAAV vector can be packaged into an AAV capsid to produce "recombinant adeno-associated virus capsid particles (rAAV particles)".

[0136] As used herein, the term "heterologous" in conjunction with nucleic acid sequences such as coding and control sequences refers to sequences that are not typically linked together and / or not typically associated with a particular cell. Therefore, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or linked to another nucleic acid molecule that does not coexist with that molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence flanked by a sequence not associated with that coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself does not exist in nature (e.g., a synthetic sequence with codons different from those of a natural gene).

[0137] As used herein, the term “side-joined” in relation to a sequence with other elements indicates the presence of one or more side-joined elements upstream and / or downstream of the sequence, i.e., 5' and / or 3'. The term “side-joined” is not intended to indicate that the sequence must be continuous. For example, intercalation sequences may exist between the nucleic acid encoding a transgene and the side-joined element. “Side-joined” in a sequence with two other elements (e.g., an ITR) (e.g., a transgene) indicates that one element is located at the 5' of the sequence and the other at the 3'; however, intercalation sequences may exist between them.

[0138] As used herein, the term "terminal inverted repeat" or "ITR" sequence refers to a relatively short sequence found at the opposite ends of a viral genome. "AAV terminal inverted repeat (ITR)" sequences are well known in the art and are typically approximately 145 nucleotides long and present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (designated as A, A', B, B', C, C', and D regions) that allow intrastrand base pairing to occur within this portion of the ITR.

[0139] The "coding sequence" or "encoding" of a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The transcription termination sequence may be located at the 3' position of the coding sequence.

[0140] The term "control sequence" refers to the DNA sequence necessary for the expression of an operatively linked coding sequence in a specific host organism. Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0141] As used herein, when referring to nucleic acids or amino acids, the term "operably linked" and similar phrases (e.g., genetically fused) refer to the operational linking of nucleic acid or amino acid sequences placed together in a functional relationship. For example, an operationally linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequence results in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, an operationally linked nucleic acid element results in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operationally linked peptide is a peptide in which functional domains are placed at appropriate distances from each other to confer the intended function of each domain.

[0142] As used herein, the term "expression cassette" or "nucleic acid expression cassette" refers to a nucleic acid molecule that includes one or more transcriptional control elements (e.g., but not limited to promoters, response elements, polyadenylated sequences, and introns) that direct the expression of a biomolecule in one or more desired cell types, tissues, or organs, such as directing (transgenic) gene expression. Typically, they will also contain transgenes, although the directing of endogenous gene expression in cells with inserted nucleic acid sequences is also considered.

[0143] As used herein, the term "response element (RE)" refers to a transcriptional control element capable of regulating and / or controlling gene transcription, particularly a non-coding cis-acting transcriptional control element. A response element contains at least one transcription factor binding site (TFBS). Response elements can be located upstream (e.g., in the promoter region) or downstream (e.g., in the 3' UTR) of the gene they regulate in vivo, and can be located near or further from the gene. Notably, response elements can be naturally occurring sequences or non-naturally occurring sequences.

[0144] As used in this article, the term "enhancer" refers to a nucleic acid sequence that increases the transcription rate by increasing the activity of the promoter.

[0145] As used herein, the term "promoter" in its general sense refers to a nucleotide region containing a DNA regulatory sequence derived from a gene capable of binding to RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcription promoters can include "inducible promoters" (where the expression of a polynucleotide sequence operatively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where the expression of a polynucleotide sequence operatively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0146] As used herein, the term "TATA box" refers to a DNA sequence found in the promoter region of a gene that can be bound by TATA-binding proteins and transcription factor II during DNA unwinding and binding by RNA polymerase II. TATA box sequences typically include the TATAAA sequence and usually include an additional 3' adenine nucleotide.

[0147] As used in this article, the term "transcription start site" refers to the location where transcription begins at the 5' end of a gene sequence.

[0148] As used in this article, the term "ORF (open reading frame)" refers to a series of nucleotide triplets (codons) that encode amino acids. These sequences are typically translated into peptides.

[0149] As used herein, the term "transcription factor (TF)" refers to a protein that binds to a specific DNA sequence and thereby controls the transfer (or transcription) of genetic information from DNA to RNA. TFs perform this function either by promoting (as activators) or blocking (as repressors) RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes, alone or in conjunction with other proteins in the complex. The specific DNA sequence that a TF binds to is called a response element (RE) or regulatory element. Other names include cis-elements and cis-acting transcriptional regulatory elements. Transcription factors interact with their binding sites using a combination of electrostatics (where hydrogen bonds are an exception) and van der Waals forces. Due to the nature of these chemical interactions, most transcription factors bind to DNA in a sequence-specific manner. However, not all bases at the transcription factor binding site can actually interact with the transcription factor. Furthermore, some of these interactions may be weaker than others. Therefore, many transcription factors bind not only to a single sequence but also to subsets of closely related sequences, each with a different interaction strength. For example, although the common binding site for TATA-binding proteins (TBP) is TATAAAA, TBP transcription factors can also bind similar sequences, such as TATATAT or TATATAA. Transcription factors (TFs) can be classified based on many aspects. These include the secondary, tertiary, and quaternary structures and properties of the protein-DNA binding sequence, their interaction with the DNA double helix, and their metal and other binding properties. The JASPAR database and TRANSFAC (TRANSFAC.RTM.7.0 Public 2005) are two web-based databases of transcription factors that include their experimentally proven binding sites and regulatory genes.

[0150] As used herein, the broad term “transgenic” refers to any heterologous nucleotide sequence incorporated into a vector, such as a viral vector, for example for expression in target cells, and which may be associated with an expression control sequence, such as a promoter. Those skilled in the art will understand that the expression control sequence will be selected based on its ability to promote transgenic expression in target cells. Examples of transgenics are nucleic acids encoding therapeutic peptides or detectable tags.

[0151] As used herein, the terms “AAV capsid” or “AAV capsid protein” or “AAV cap” refer to a protein or variant thereof encoded by an AAV capsid (cap) gene (e.g., VP1, VP2, and VP3). For example, the term includes, but is not limited to, capsid proteins derived from any AAV serotype, such as AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV-2 / 1, AAV 2 / 6, AAV 2 / 7, AAV 2 / 8, AAV 2 / 9, AAV LK03, AAVrh10, AAVrh74, AAV44-9, or variants thereof. The term also includes capsid proteins expressed or derived from recombinant AAVs, such as chimeric AAVs.

[0152] As used herein, the term “AAV capsid particle” or “AAV particle” includes at least one AAV capsid protein (e.g., VP1 protein, VP2 protein, VP3 protein or variants thereof) and optionally encapsulates nucleic acids from the AAV genome (including the rAAV genome) or nucleic acids derived from the AAV genome (including the rAAV genome).

[0153] The term "serotype" used for vectors or viral capsids is defined by different immunological profiles based on capsid protein sequences and capsid structure.

[0154] As used herein, with respect to viral capsids or particles, the term "chimerism" means that the capsid or particle comprises sequences from different parvoviruses, preferably different AAV serotypes, as described in U.S. Patent No. 6,491,907 to Rabinowitz et al., the disclosure of which is incorporated herein by reference in its entirety.

[0155] The term “recombinant” refers to a genetic entity that is different from what is normally found in nature. When applied to polynucleotides, genes, promoters, expression cassettes, etc., it means the product of various combinations of cloning, restriction, ligation, and / or synthetic steps, as well as other procedures that result in constructs different from those naturally occurring polynucleotides, genes, promoters, expression cassettes, etc. When used herein to describe viruses, the term “recombinant” refers to a virus that has undergone genetic alteration, for example, by adding or inserting a heterologous nucleic acid construct into the particle. For example, as used herein, the term “recombinant AAV particle” or “rAAV” refers to an AAV that has undergone genetic alteration, for example, by deletion or other mutation of the endogenous AAV gene and / or by adding or inserting a heterologous nucleic acid construct into the polynucleotide of the AAV particle.

[0156] As used in this article, "specific hybridization" refers to an antisense compound having sufficient complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids under conditions requiring specific binding, i.e., physiological conditions under which in vivo assays and therapeutic treatments are performed.

[0157] As used in this article, “strict hybridization conditions” or “strict conditions” refer to conditions under which an oligomer will hybridize with its target sequence but with a minimum number of other sequences.

[0158] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. For example, the term “transfection” is used to refer to the uptake of exogenous DNA by cells, and when exogenous DNA has been introduced into the cell membrane, the cell has been “transfected.” Many transfection techniques are well known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 th Ed., ColdSpring Harbor Laboratory (Cold Spring Harbor, NY 2012), Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous molecules into a suitable host cell. Viral transduction of cells refers to the transfer of nucleic acids, such as DNA or RNA, from viral particles into cells.

[0159] As used herein, the term "host cell" refers to a specific cell that can be transfected with nucleic acid molecules, vectors, viral particles, or non-viral gene delivery systems, as well as the progeny or potential progeny of such cells. Host cells can be bacterial cells, yeast cells, insect cells, or mammalian cells.

[0160] The term "purified" refers to the separation of a substance (compound, polynucleotide, protein, peptide, peptide composition) such that the substance of interest constitutes the majority of the sample. Typically, the purified component in a sample comprises approximately 50%, 80%-85%, 90%-99%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Techniques for purifying polynucleotides and peptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density-based sedimentation.

[0161] As used herein, the term “treatment” refers to a reduction or relief of the progression, severity, and / or duration of a disease or condition resulting from the application of one or more therapies. Treatment can be determined by assessing the presence of a reduction, relief, and / or relief of one or more symptoms associated with an underlying disorder, such that the patient observes improvement even though the underlying disorder may still be present. The term “treatment” includes controlling and alleviating disease. The term “management” refers to a beneficial effect obtained by a subject from a therapy that does not necessarily lead to a cure for the disease. “Treatment” includes: (1) preventing disease, i.e., preventing the development of disease or causing disease to occur at a lower intensity in subjects who may be exposed to or susceptible to disease but have not yet experienced or displayed symptoms of disease; (2) suppressing disease, i.e., preventing, delaying, or reversing the disease state; (3) alleviating symptoms of disease, i.e., reducing the number of symptoms experienced by the subject; and (4) reducing, preventing, or delaying the progression of disease or its symptoms. The term “prevention” refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom.

[0162] As used herein, “administration” means the act of injecting or otherwise physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject or patient (e.g., a person), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intra-articular, subretinal, intramuscular, intrathecal delivery, and / or any other physical delivery method described herein or known in the art. In certain embodiments, administration is by intravenous infusion. Therapeutic agents (e.g., pharmaceutical compositions) provided herein may be delivered systemically or to a specific tissue.

[0163] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount of a therapeutic agent (e.g., the pharmaceutical composition provided herein) sufficient to treat, diagnose, prevent, delay the onset of a given condition, disorder, or disease and / or associated symptoms, reduce and / or alleviate its severity and / or duration. These terms also cover the amount required to reduce, slow, or alleviate the progression or development of a given disease, reduce, slow, or alleviate the recurrence, development, or onset of a given disease, and / or enhance or strengthen the preventive or therapeutic effect of another therapy or serve as a bridge to another therapy. In some embodiments, "effective amount" as used herein also refers to the amount of the therapeutic agent (e.g., the pharmaceutical composition provided herein) described herein that achieves the specified result.

[0164] As used herein, the terms "subject" and "patient" are used interchangeably and refer to mammals, such as nonprimates (e.g., cattle, pigs, horses, cats, dogs, sheep, rabbits, rats, mice, etc.) or primates (e.g., monkeys and humans), such as humans. In some embodiments, the subject is a mammal diagnosed with the disease or disorder described herein, such as a human. In another embodiment, the subject is a mammal at risk of developing the disease or disorder described herein, such as a human. In a particular embodiment, the subject is a human. In a particular embodiment, the subject is an adult. In a particular embodiment, the subject is a human adolescent. In a particular embodiment, the subject is a human child.

[0165] As used herein, the term "therapy" can refer to any procedure, method, composition, formulation, and / or reagent that can be used to prevent, treat, manage, or alleviate a disease or disorder or its symptoms (e.g., the disease or disorder described herein or one or more symptoms or conditions associated with it). In some embodiments, the term "therapy" refers to gene therapy that can be used to treat, manage, prevent, or alleviate a disease or disorder or its symptoms.

[0166] As used herein, the term "gene therapy" refers to the introduction of heterologous nucleic acid molecules into one or more recipient cells, whereby the heterologous nucleic acid itself or its expression in the recipient cells affects cell function and produces a therapeutic effect in the subject. For example, the heterologous nucleic acid molecule may encode a protein that affects the function of the recipient cells.

[0167] As used herein and unless otherwise stated, the terms “about” and “approximately” should be interpreted as allowing for normal variation as judged by those skilled in the art, or meaning within an acceptable margin of error for a particular value as determined by those of ordinary skill in the art, which will depend in part on how the value is measured or determined (i.e., the limitations of the measurement system), for example, a variation within 20%, 10%, or 5% of the value, or a variation within one or more standard deviations in every practice in the art. In specific embodiments, the terms “about” and “approximately” cover the precise values ​​stated herein. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term “about”.

[0168] As used in this disclosure, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Unless the context clearly specifies otherwise, the term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably herein.

[0169] It should be understood that wherever the term "comprising" is used to describe an embodiment herein, other similar embodiments described as "consisting of" and / or "substantially consisting of" are also provided. It should also be understood that wherever the phrase "substantially consisting of" is used to describe an embodiment herein, similar embodiments described as "consisting of" are also provided.

[0170] The term "between" in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0171] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0172] The terms “or” and “and” are used interchangeably and can be understood as meaning “and / or” unless the context clearly specifies otherwise.

[0173] 5.2 Polynucleotides, promoters, and expression cassettes This disclosure describes a codon-optimized nucleotide sequence encoding human α-galactosidase A. The human α-galactosidase A encoded by the nucleotide sequence described in this disclosure may be wild-type human α-galactosidase A or a mutant (e.g., truncated) human α-galactosidase A, but preferably functional human α-galactosidase A. In a specific embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with SEQ ID NO: 1. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A is shown in SEQ ID NO: 1. In a specific embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 2. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 2. In a specific embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 20. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 20. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 20. Not wishing to be bound by any single theory, these nucleotide sequences are particularly suitable for gene therapy for Fabry disease.

[0174] This disclosure also describes nucleotide sequences encoding hepatocyte-specific or hepatocyte-muscle cell bispecific promoters. In some embodiments, the nucleotide sequence encodes a hepatocyte-specific promoter. In some embodiments, the nucleotide sequence encodes a hepatocyte-muscle cell bispecific promoter.

[0175] In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 3. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 3. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 3. Not wanting to be bound by any theory, these nucleotide sequences are particularly well-suited for directing the expression of biomolecules, such as directing (trans)gene expression, in hepatocytes (i.e., in the liver) and optionally in other cell types (e.g., myocytes and / or kidney cells), and are therefore particularly well-suited for gene therapy, especially for lysosomal storage diseases including Fabry disease.

[0176] In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 4. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 4. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 4. In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 5. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 5. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 5. In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 6. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 6. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 6. In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myoblast bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 7.In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 7. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 7. In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 8. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 8. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 8. In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 9. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 9. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 9. In a specific embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 10. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter comprises SEQ ID NO: 10. In one embodiment, the nucleotide sequence encoding a hepatocyte-specific or hepatocyte-muscle cell bispecific promoter is as shown in SEQ ID NO: 10.Not wishing to be bound by any single theory, these nucleotide sequences are particularly well-suited for directing the expression of biomolecules, such as directing (trans)gene expression, in hepatocytes (i.e., in the liver) and muscle cells (i.e., in muscles) (e.g., in cardiac muscle (e.g., atrial and / or ventricular muscle) and / or skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major)) and optionally in other cell types (e.g., kidney cells), and therefore particularly suitable for gene therapy, especially for lysosomal storage diseases including Fabry disease. In certain embodiments, muscle can be used as a production plant for biomolecules secreted from muscle cells and ultimately absorbed by cells of another tissue or organ to exert their therapeutic effects.

[0177] This disclosure further describes a nucleotide sequence encoding a human α-galactosidase A expression cassette. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 16. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises SEQ ID NO: 16. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is as shown in SEQ ID NO: 16. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 17. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises SEQ ID NO: 17. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is as shown in SEQ ID NO: 17. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 18. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises SEQ ID NO: 18. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is as shown in SEQ ID NO: 18. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 19. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette comprises SEQ ID NO: 19.In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is shown in SEQ ID NO: 19. Without wishing to be bound by any single theory, these nucleotide sequences are particularly suitable for gene therapy of Fabry disease.

[0178] This disclosure provides, in particular, polynucleotides, promoters, and expression cassettes associated with these nucleotide sequences.

[0179] 5.2.1 Polynucleotides In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 20.

[0180] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1.

[0181] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 2.

[0182] In some embodiments, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO: 20. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 20.

[0183] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0184] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0185] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0186] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0187] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0188] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0189] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0190] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0191] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0192] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0193] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 20.

[0194] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1.

[0195] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 2.

[0196] In some embodiments, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO: 20. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 20.

[0197] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0198] In various embodiments, the nucleotide sequence encoding the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0199] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0200] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0201] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0202] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0203] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0204] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0205] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0206] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0207] In various embodiments, the polynucleotide also includes one or more regulatory control elements (see Section 5.2.5). In a specific embodiment, the polynucleotide further includes a nucleotide sequence encoding a polyA signal.

[0208] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0209] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0210] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0211] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0212] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0213] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0214] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0215] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0216] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0217] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0218] Further description of the biomolecule is provided in Section 5.2.4. In a specific embodiment, the biomolecule is human α-galactosidase A (preferably functional human α-galactosidase A).

[0219] In various embodiments, the polynucleotide also includes one or more regulatory control elements (see Section 5.2.5). In a specific embodiment, the polynucleotide further includes a nucleotide sequence encoding a polyA signal.

[0220] In one aspect, this document provides a polynucleotide comprising a nucleotide sequence encoding an expression cassette, wherein the nucleotide sequence encoding the expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0221] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 16.

[0222] In some embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO: 17. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 17.

[0223] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 18.

[0224] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 19.

[0225] In one respect, this document provides polynucleotides comprising nucleotide sequences encoding the expression cassette described in Section 5.2.3.

[0226] In all aspects and embodiments, the polynucleotides described herein are purified. In all aspects and embodiments, the polynucleotides described herein are isolated. In all aspects and embodiments, the polynucleotides described herein are recombinant polynucleotides.

[0227] In some embodiments, the polynucleotides described herein are DNA polynucleotides. In specific embodiments, the polynucleotides described herein are double-stranded DNA. In specific embodiments, the polynucleotides described herein are single-stranded DNA.

[0228] Further description of polynucleotides is provided in Section 5.1.

[0229] 5.2.2 Promoter In one aspect, this document provides a promoter encoded by a nucleotide sequence comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0230] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0231] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0232] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0233] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0234] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0235] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0236] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0237] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0238] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0239] In all aspects and implementations, the promoters described herein are recombinant promoters.

[0240] Further description of promoters is provided in Sections 5.1 and 5.2.5.

[0241] 5.2.3 Expression Box In one aspect, this document provides an expression cassette comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 20.

[0242] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 1. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 1.

[0243] In some embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 2.

[0244] In some embodiments, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO: 20. In some embodiments, the nucleotide sequence encoding human α-galactosidase A is as shown in SEQ ID NO: 20.

[0245] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0246] In various embodiments, the nucleotide sequence encoding the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0247] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0248] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0249] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0250] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0251] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0252] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0253] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0254] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0255] In various embodiments, the expression cassette also includes one or more regulatory control elements (see Section 5.2.5). In a specific embodiment, the expression cassette further includes a nucleotide sequence encoding a polyA signal.

[0256] In one aspect, this document provides an expression cassette comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, the nucleotide sequence encoding the promoter being operatively linked to the nucleotide sequence encoding the biomolecule, wherein the nucleotide sequence encoding the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8% or 100% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0257] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-kidney cell bispecific promoter. In a specific embodiment, the promoter is a hepatocyte-muscle cell-kidney cell multispecific promoter.

[0258] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 3.

[0259] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 4.

[0260] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 5.

[0261] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 6. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 6.

[0262] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 7.

[0263] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 8.

[0264] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 9. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 9.

[0265] In some embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the promoter is as shown in SEQ ID NO: 10.

[0266] Further description of the biomolecule is provided in Section 5.2.4. In a specific embodiment, the biomolecule is human α-galactosidase A (preferably functional human α-galactosidase A).

[0267] In various embodiments, the expression cassette also includes one or more regulatory control elements (see Section 5.2.5). In a specific embodiment, the expression cassette further includes a nucleotide sequence encoding a polyA signal.

[0268] In one aspect, this document provides an expression cassette encoded by a nucleotide sequence comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identity with the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0269] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 16.

[0270] In some embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO: 17. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 17.

[0271] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 18. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 18.

[0272] In some embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the expression cassette is as shown in SEQ ID NO: 19.

[0273] In all aspects and implementations, the expression cassette described herein is a recombinant expression cassette.

[0274] Further description of the expression box is provided in Section 5.1.

[0275] 5.2.4 Biomolecules The biomolecules described in this disclosure can be, for example, but not limited to, polypeptides, proteins, nucleic acids (e.g., DNA or RNA), or oligonucleotides (e.g., siRNA, shRNA, miRNA, or aptamers). In specific embodiments, the biomolecule is a polypeptide or protein. In a preferred embodiment, the biomolecule is a human polypeptide or human protein. The biomolecule can be a reporter molecule, such as a reporter protein (e.g., a fluorescent protein (e.g., green fluorescent protein (GFP)), a luciferase (e.g., firefly luciferase), a β-lactamase, or a β-galactosidase (LacZ)). The biomolecule can also be a therapeutic molecule, such as a therapeutic protein. Therapeutic molecules can be used to correct or alleviate genetic defects associated with diseases or disorders. Exemplary therapeutic molecules can include, but are not limited to, enzymes, cytokines, growth factors, kinases, dominant-negative mutant proteins, antibodies and their antigen-binding fragments, interleukins, hormones, and differentiation factors.

[0276] In some embodiments, the biomolecule is a therapeutic protein. The therapeutic proteins described herein include hormones and growth and differentiation factors, including but not limited to insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), any one of the transforming growth factor α superfamily (including TGFα), activin, inhibin, or bone morphogenetic protein (BMP). Any one of 1-15, any one of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT-4 / 5, ciliary neurotrophic factor (TF), glial cell-derived neurotrophic factor (GDNF), neurturin, aggregate protein, any one of the semaphorins / collapsins family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), hepatin, noggin, sound hedgehog factor, and tyrosine hydroxylase.

[0277] Other therapeutic proteins described herein include those that regulate the immune system, including but not limited to cytokines and lymphokines such as thrombopoietin (TPO), interleukins (ILs) IL-1 to IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemoattractant proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β, and γ, stem cell factor, and flk-2 / flt3 ligand. Proteins produced by the immune system are also useful. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T-cell receptors, chimeric T-cell receptors, single-chain T-cell receptors, class I and II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful proteins also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactors (MCPs), decay accelerator factor (DAF), CR1, CF2, and CD59.

[0278] Other therapeutic proteins described herein are receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. This disclosure covers receptors for cholesterol and / or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. This disclosure also covers proteins, such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. In addition, useful proteins include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myoblasts, proteins containing ETS boxes, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZFS, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding proteins, STAT, GATA box-binding proteins (e.g., GATA-3), and the forkhead family of winged helix proteins.

[0279] Other useful biomolecules described herein include carbamoyl synthase I, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, arginase, fumaroyl acetoacetate hydrolase, phenylalanine hydroxylase, α-1 antitrypsin, glucose-6-phosphatase, bile pigmentogen deaminase, cystathionine β-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence. Other useful proteins include enzymes, such as those available for enzyme replacement therapy, which can be used for various conditions caused by enzyme activity defects. For example, enzymes containing mannose-6-phosphate can be used to treat lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)).

[0280] Other useful biomolecules described in this article include non-naturally occurring peptides, such as chimeric or hybrid peptides with non-naturally occurring amino acid sequences containing insertions, deletions, or substitutions. For example, single-chain engineered immunoglobulins can be used in certain immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce the overexpression of targets.

[0281] Other suitable biomolecules described herein include those that can be used to treat individuals with autoimmune diseases and disorders by conferring a broad protective immune response against autoimmune-related targets, including cell receptors and cells that produce “self”-directed antibodies. T-cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis. These diseases are all characterized by the binding of T-cell receptors (TCRs) to endogenous antigens and the triggering of an inflammatory cascade associated with autoimmune diseases.

[0282] Biomolecules can be used to reduce and / or modulate the expression of proteins of interest to treat hyperproliferative disorders characterized by excessive cell proliferation, such as cancer and psoriasis. Target peptides include those that are produced specifically or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include peptides encoded by oncogenes such as myb, myc, fyn and translocation genes bcr / abl, ras, src, p53, neu, trk and EGRF. In addition to oncogene products as target antigens, target peptides for anticancer therapies and protective regimens include variable regions of antibodies prepared from B-cell lymphomas and variable regions of T-cell receptors from T-cell lymphomas, which in some embodiments are also used as target antigens for autoimmune diseases. Other tumor-associated peptides can be used as target peptides, such as peptides found at higher levels in tumor cells, including peptides recognized by monoclonal antibody 17-1A and folic acid-binding peptides.

[0283] In some embodiments, the biomolecule is RNA. These RNA molecules include, but are not limited to, molecules that function through RNA interference (shRNA, RNAi), microRNA regulation (miR), catalytic RNA, antisense RNA, RNA aptamers, etc.

[0284] Biomolecules can be used to induce an immune response to a selected antigen. For example, to promote an immune response, the antigen can be expressed by a promoter disclosed herein, the vector can be adjuvanted as described herein, and / or the vector can be placed in degenerative tissues.

[0285] Examples of suitable immunogenic antigens include those selected from a variety of viral families. Ideal examples of viral families expected to elicit an immune response include the Picornaviridae family, which includes the genus *Rhinovirus*, causing approximately 50% of common cold cases; the genus *Enterovirus*, which includes poliovirus, Coxsackievirus, Echovirus, and human enteroviruses such as hepatitis A virus; and the genus *Foot-and-Mouthvirus*, which primarily causes foot and mouth diseases in non-human animals. Within the Picornaviridae family, target antigens include VP1, VP2, VP3, VP4, and VPG. Other viral families include the astroviruses and Calicviridae families. The Calicviridae family encompasses the Norwalk virus group, a major pathogen of gastroenteritis epidemics. Another ideal viral family for targeting antigens to induce an immune response in humans and non-human animals is the Clonorviridae family, which includes the genus *Avira*, including Sindbisvirus, Ross River Virus, and Venezuelan, Eastern & Western Equine Encephalitis Virus, and the genus *Russiavirus*, including rubella virus. The Flaviviridae family includes dengue fever, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Other target antigens can be generated by the Hepatitis C or Coronaviridae families, which include many non-human viruses such as infectious bronchitis virus (poultry), transmissible gastroenteritis virus (swine), hemagglutinin encephalomyelitis virus (swine), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), canine coronavirus (dog), and human respiratory coronavirus, which can cause the common cold and / or non-A, B, or C hepatitis, and include the presumed cause of severe acute respiratory syndrome (SARS). Within the Coronaviridae family, target antigens include E1 (also known as M or matrix protein), E2 (also known as S or spike protein), E3 (also known as HE or hemagglutinin esterase), glycoproteins (not all coronaviruses have this), or N (nucleocapsid). Other antigens can target the Arteriviridae and Rhabdoviridae families. The Rhabdoviridae family includes the genus *Vesicularis* (e.g., vesicular stomatitis virus) and the genus *Lyssavirus* (e.g., rabies virus). In the Rhabdoviridae family, suitable antigens can be derived from either the G or N protein. The Filoviridae family, including hemorrhagic fever viruses (such as Marburg and Ebola viruses), can be suitable sources of antigens. The Paramyxoviridae family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, mumps virus genus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, measles virus including measles and canine distemper, and pneumoviruses including respiratory syncytial virus. Influenza viruses are classified in the Orthomyxoviridae family and are suitable sources of antigens (e.g., HA protein, N1 protein). The Bunyaviridae family includes Bunyavirus genus (California encephalitis, La Crosse), Phlebovirus genus (Rift Valley fever), Hantavirus genus (puremala is a hemorrhagic fever virus), Nairovirus genus (Nairobi sheep disease), and various unclassified Bunyaviruses.The Arenaviridae family provides antigenic sources for LCM and Lassa fever viruses. Another source of antigens is the Bornavirus family. The Reoviridae family includes the genera Reovirus, Rotavirus (which causes acute gastroenteritis in children), Circovirus, and Colorado Tickvirus (Colorado tick fever, Lebombo (human), equine encephalitis, bluetongue). The Retroviridae family includes the Oncorivirinal subfamily, which covers human and veterinary diseases such as feline leukemia virus, HTLVI and HTLVII, and lentiviruses (including HIV, simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, and spumavirinal). The Papillomavirus family includes the Polyomavirus subfamily (BKU and JCU viruses) and the Papillomavirus subfamily (associated with the malignant progression of cancer or papilloma). The Adenoviridae family includes viruses that cause respiratory illness and / or enteritis (EX, AD7, ARD, OB). The Parvoviridae family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The Herpesvirus family includes the Alpha Herpesvirus subfamily, encompassing the genera *Herpes simplex virus* (HSVI, HSVII), *Varizovirus* (pseudorabies, varicella-zoster virus), and the Beta Herpesvirus subfamily, including the genera *Cytomegalovirus* (HCMV, murine cytomegalovirus), and the Gamma Herpesvirus subfamily, including the genera *Lymphoidfollicle virus*, *EBV* (Burkit lymphoma), human herpesviruses 6A, 6B, and 7, Kaposi's sarcoma-associated herpesvirus, and macaque herpesvirus (B virus), infectious rhinotracheitis virus, Marek's disease virus, and rhadinovirus. The Poxviridae family includes the subfamily Chordopoxvirinae, which encompasses the genera *Orthopoxvirus* (smallpox and cowpox), *Parapoxvirus*, *Follpoxvirus*, *Goatpoxvirus*, *Tauripoxvirus*, *Swinepoxvirus*, and *Enteropoxvirus*. The Hepatotropic DNA Viridae family includes hepatitis B virus. One unclassified virus that may be a suitable antigenic source is hepatitis D virus, hepatitis E virus, and prions. Another virus that could be an antigenic source is Nipan virus. Other viral sources may include infectious bursal disease virus in birds and porcine respiratory and reproductive syndrome virus. The Alphaviridae family includes equine arteritis virus and various encephalitis viruses.

[0286] This disclosure may also cover protein-based immunogens as biomolecules that can be used to immunize humans or non-human animals against other pathogens, including bacteria, fungi, parasitic microorganisms, or multicellular parasites that infect humans and non-human vertebrates, or those derived from cancer cells or tumor cells. Examples of bacterial pathogens include pathogenic Gram-positive cocci, including pneumococci; staphylococci (and the toxins produced therefrom, such as enterotoxin B); and streptococci. Pathogenic Gram-negative cocci include meningococci; and gonococci. Pathogenic enteric Gram-negative bacilli include Enterobacteriaceae; Pseudomonas, Acinetobacter, and Eikenella; melioidosis; Salmonella; Shigella; Haemophilus; Moraxella; Haemophilus ducreyi (which causes chancroid); Brucella (brucellosis); Tularemia (which causes tularemia); Yersinia pestis (plague) and other Yersinia (Pasteurella); Streptococcus moniliforme and Spirils; Gram-positive bacilli include Listeria; Erysipelothrix rhusiopathiae; Corynebacterium diphtheriae (diphtheria); Cholera; Bacillus anthracis (anthrax); Dunofan disease (granuloma inguinale); and Bartonella disease. Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism (Clostridium botulinum and its toxin); Clostridium perfringens and its ε-toxin; other clostridiums; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetes include syphilis; treponeosis: yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by highly pathogenic bacteria and pathogenic fungi include glanders (Melilotes); actinomycosis; nocardiacosis; cryptococcosis, blastomycosis, histoplasmosis, and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidioidomycosis, petriellidiosis, coccidioidomycosis, mycoidomycosis, and chromomycosis; and dermatophytosis. Rickettsia infections include typhus, Rocky Mountain spotted fever, Q fever (Coxella burgdorferi), and rickettsial pox. Examples of mycoplasma and chlamydia infections include: Mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydia infection. Pathogenic eukaryotes encompass pathogenic protozoa and worms and the infections arising therefrom, including: amoebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; trichomonas; Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodeiasis; trematodeiasis or fluke; and cestode / tapeworm infection.

[0287] Many of these organisms and / or the toxins they produce have been identified by the Centers for Disease Control and Prevention [(CDC), Department of Health and Human Services, U.S.] as agents with potential for biological attack. For example, some of these biological agents include Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), Smallpox meliticum (smallpox), Tularemia tularemia (tularemia), and viral hemorrhagic fevers [filoviruses (e.g., Ebola virus, Marburg virus) and arenaviruses [e.g., Lassa fever virus, Machupo virus]], all of which are currently classified as Category A agents; Coxella burgdorferi (Q fever); Brucella spp. (brucellosis), Meredithiae (meredithiae), Pseudomonas melioides (meredithiae), Ricinus ricin and its toxin (ricin), Clostridium perfringens and its toxin (ε toxin), Staphylococcus spp. and its toxin (enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats (e.g., Vibrio cholerae, Cryptosporidium microsporum), and Typhus meliticum (Richettsia). (e.g., powazekii), and viral encephalitis (alphaviruses, such as Venezuelan equine encephalitis; Eastern equine encephalitis; Western equine encephalitis); all of which are currently classified as Category B agents; as well as Nipan virus and Hantavirus, which are currently classified as Category C agents. Furthermore, other organisms classified in this way or differently may be identified and / or used for such purposes in the future. It is readily understood that the viral vectors and other constructs described herein can be used to deliver antigens from these organisms, viruses, their toxins, or other byproducts, which will prevent and / or treat infections or other adverse reactions with these biological agents.

[0288] In some embodiments, the biomolecule is a protein that is a segment of a variable region of a T cell that triggers an immune response (i.e., the elimination of cytotoxic T cells). In rheumatoid arthritis (RA), several specific variable regions of the TCRs involved in the disease have been characterized. These TCRs include V-3, V-14, V-17, and V-17. Therefore, delivery of a nucleic acid sequence encoding at least one of these polypeptides will trigger an immune response targeting T cells involved in RA. In multiple sclerosis (MS), several specific variable regions of the TCRs involved in the disease have been characterized. These TCRs include V-7 and V-10. Therefore, delivery of a transgene encoding at least one of these polypeptides will trigger an immune response targeting T cells involved in MS. In scleroderma, several specific variable regions of the TCRs involved in the disease have been characterized. These TCRs include V-6, V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28, and V-12. Therefore, delivery of a nucleic acid molecule encoding at least one of these polypeptides will trigger an immune response that targets the T cells involved in scleroderma.

[0289] In specific embodiments, the biomolecule is typically expressed in hepatocytes (e.g., typically expressed in hepatocytes at a higher level relative to other cell types). In specific embodiments, the biomolecule is typically expressed in muscle cells (e.g., typically expressed in muscle cells at a higher level relative to other cell types). In specific embodiments, the biomolecule is typically expressed in kidney cells (e.g., typically expressed in kidney cells at a higher level relative to other cell types). In specific embodiments, the biomolecule is typically expressed in both hepatocytes and muscle cells (e.g., expressed in both hepatocytes and muscle cells at a higher level relative to other cell types). In specific embodiments, the biomolecule is typically expressed in both hepatocytes and kidney cells (e.g., expressed in both hepatocytes and kidney cells at a higher level relative to other cell types). In specific embodiments, the biomolecule is typically expressed in hepatocytes, muscle cells, and kidney cells (e.g., expressed in hepatocytes, muscle cells, and kidney cells at a higher level relative to other cell types). In specific embodiments, the biomolecule functions in hepatocytes (e.g., is essential or important for the normal function of hepatocytes). In specific embodiments, the biomolecules function in muscle cells (e.g., are essential or important for the normal function of muscle cells). In specific embodiments, the biomolecules function in kidney cells (e.g., are essential or important for the normal function of kidney cells). In specific embodiments, the biomolecules function in both hepatocytes and muscle cells (e.g., are essential or important for the normal function of both hepatocytes and muscle cells). In specific embodiments, the biomolecules function in both hepatocytes and kidney cells (e.g., are essential or important for the normal function of both hepatocytes and kidney cells). In specific embodiments, the biomolecules function in hepatocytes, muscle cells, and kidney cells (e.g., are essential or important for the normal function of hepatocytes, muscle cells, and kidney cells). In specific embodiments, the muscle cells described herein are skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major muscle cells) and / or cardiomyocytes (e.g., atrial and / or ventricular muscle cells). In certain implementations, muscles can be used as a production plant for biomolecules that are secreted from muscle cells and ultimately absorbed by cells of another tissue or organ to exert their therapeutic effects.

[0290] In specific embodiments, the biomolecules described herein are associated with lysosomal storage diseases. In specific embodiments, defects in the biomolecules described herein are at least a partial cause of lysosomal storage diseases. In specific embodiments, abnormally low expression or lack of expression of the biomolecules described herein is at least a partial cause of lysosomal storage diseases.

[0291] In specific embodiments, the biomolecule is a protein, specifically α-galactosidase A (GLA) (preferably human GLA). In specific embodiments, the biomolecule is a protein, specifically scavenger receptor class B member 2 (SCARB2) (preferably human SCARB2). In specific embodiments, the biomolecule is a protein, specifically aspartic acid glucosidase (AGA) (preferably human AGA). In specific embodiments, the biomolecule is a protein, specifically palmitoyl protein thioesterase 1 (PPT1) (preferably human PPT1). In specific embodiments, the biomolecule is a protein, specifically tripeptidyl peptidase 1 (TPP1) (preferably human TPP1). In specific embodiments, the biomolecule is a protein, specifically CLN3 lysosomal / endosomal transmembrane protein, battenin (CLN3) (preferably human CLN3). In specific embodiments, the biomolecule is a protein, specifically DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5) (preferably human DNAJC5). In specific embodiments, the biomolecule is a protein, specifically CLN5 intracellular transport protein (CLN5) (preferably human CLN5). In specific embodiments, the biomolecule is a protein, specifically CLN6 transmembrane ER protein (CLN6) (preferably human CLN6). In specific embodiments, the biomolecule is a protein containing the Major Facilitator Superfamily Domain Containing 8 (MFSD8) (preferably human MFSD8). In specific embodiments, the biomolecule is a protein, specifically CLN8 transmembrane ER and ERGIC protein (CLN8) (preferably human CLN8). In specific embodiments, the biomolecule is a protein, specifically cathepsin D (CTSD) (preferably human CTSD). In specific embodiments, the biomolecule is a protein, specifically granular protein precursor (GRN) (preferably human GRN). In specific embodiments, the biomolecule is a protein, specifically ATPase cation transporter 13A2 (ATP13A2) (preferably human ATP13A2). In a specific embodiment, the biomolecule is a protein, namely cathepsin F (CTSF) (preferably human CTSF). In a specific embodiment, the biomolecule is a protein, namely potassium channel tetramerization domain-containing protein 7 (KCTD7) (preferably human KCTD7). In a specific embodiment, the biomolecule is a protein, namely cystinosin, lysosomal cystine transporter (CTNS) (preferably human CTNS).In specific embodiments, the biomolecule is a protein, specifically lysosome-associated membrane protein 2 (LAMP2) (preferably human LAMP2). In specific embodiments, the biomolecule is a protein, specifically N-acylsphingosine hydrolase 1 (ASAH1) (preferably human ASAH1). In specific embodiments, the biomolecule is a protein, specifically α-L-fucosidase 1 (FUCA1) (preferably human FUCA1). In specific embodiments, the biomolecule is a protein, specifically cathepsin A (CTSA) (preferably human CTSA). In specific embodiments, the biomolecule is a protein, specifically β-glucocerebrosidase (GBA) (preferably human GBA). In specific embodiments, the biomolecule is a protein, specifically acidic α-glucosidase (GAA) (preferably human GAA). In specific embodiments, the biomolecule is a protein, specifically myosin VA (MYO5A) (preferably human MYO5A). In specific embodiments, the biomolecule is a protein, specifically RAB27A (Member RAS Oncogene Family, RAB27A) (preferably human RAB27A). In specific embodiments, the biomolecule is a protein, specifically lysosomal transport regulator (LYST) (preferably human LYST). In specific embodiments, the biomolecule is a protein, specifically galactosidase β1 (GLB1) (preferably human GLB1). In specific embodiments, the biomolecule is a protein, specifically hexosaminease subunit α (HEXA) (preferably human HEXA). In specific embodiments, the biomolecule is a protein, specifically hexosaminease subunit β (HEXB) (preferably human HEXB). In specific embodiments, the biomolecule is a protein, specifically ganglioside GM2 activator (GM2A) (preferably human GM2A). In a specific embodiment, the biomolecule is a protein, specifically HPS1 biogenesis of lysosomal organelles complex 3 subunit 1 (HPS1) (preferably human HPS1). In a specific embodiment, the biomolecule is a protein, specifically adaptor-associated protein complex 3 subunit β1 (AP3B1 or HPS2) (preferably human HPS2). In a specific embodiment, the biomolecule is a protein, specifically HPS3 lysosomal organelles biogenesis complex 2 subunit 1 (HPS3) (preferably human HPS3). In a specific embodiment, the biomolecule is a protein, specifically HPS4 lysosomal organelles biogenesis complex 3 subunit 2 (HPS4) (preferably human HPS4).In a specific embodiment, the biomolecule is a protein, specifically HPS5 (preferably human HPS5), which is a subunit 2 of the lysosomal organelles biogenesis complex 2. In a specific embodiment, the biomolecule is a protein, specifically HPS6 (preferably human HPS6), which is a subunit 3 of the lysosomal organelles biogenesis complex 2. In a specific embodiment, the biomolecule is a protein, specifically dystrobrevin binding protein 1 (DTNBP1 or HPS7) (preferably human HPS7). In a specific embodiment, the biomolecule is a protein, specifically lysosomal organelles biogenesis complex 1 subunit 3 (BLOC1S3 or HPS8) (preferably human HPS8). In a specific embodiment, the biomolecule is a protein, specifically lysosomal organelles biogenesis complex 1 subunit 6 (BLOC1S6 or HPS9) (preferably human HPS9). In a specific embodiment, the biomolecule is a protein, specifically galactosylceramidinase (GALC) (preferably GALC). In a specific embodiment, the biomolecule is a protein, specifically mannosidase α 2B class member 1 (MAN2B1) (preferably MAN2B1). In a specific embodiment, the biomolecule is a protein, specifically mannosidase β (MANBA) (preferably MANBA). In a specific embodiment, the biomolecule is a protein, specifically arylsulfatase A (ARSA) (preferably ARSA). In a specific embodiment, the biomolecule is a protein, specifically N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably GNPTAB). In a specific embodiment, the biomolecule is a protein, specifically N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably GNPTAB). In a specific embodiment, the biomolecule is a protein, specifically N-acetylglucosamine-1-phosphotransferase subunit γ (GNPTG) (preferably GNPTG). In a specific embodiment, the biomolecule is a protein, specifically a mucolipin TRP cation channel 1 (MCOLN1) (preferably MCOLLN1). In a specific embodiment, the biomolecule is a protein, specifically α-L-iduronidase (IDUA) (preferably IDUA). In a specific embodiment, the biomolecule is a protein, specifically iduronate 2-sulfatase (IDS) (preferably IDS). In a specific embodiment, the biomolecule is a protein, specifically N-sulfamethoxyglucosylhydrogenase (SGSH) (preferably SGSH).In specific embodiments, the biomolecule is a protein, specifically N-acetyl-α-glucosidase (NAGLU) (preferably NAGLU). In specific embodiments, the biomolecule is a protein, specifically heparin-α-glucosamine N-acetyltransferase (HGSNAT) (preferably HGSNAT). In specific embodiments, the biomolecule is a protein, specifically glucosamine (N-acetyl)-6-sulfatase (GNS) (preferably GNS). In specific embodiments, the biomolecule is a protein, specifically galactosamine (N-acetyl)-6-sulfatase (GALNS) (preferably GALNS). In specific embodiments, the biomolecule is a protein, specifically galactosidase β1 (GLB1) (preferably GLB1). In specific embodiments, the biomolecule is a protein, specifically arylsulfatase B (ARSB) (preferably ARSB). In specific embodiments, the biomolecule is a protein, specifically β-glucuronidase (GUSB) (preferably GUSB). In specific embodiments, the biomolecule is a protein, specifically hyaluronidase 1 (HYAL1) (preferably HYAL1). In specific embodiments, the biomolecule is a protein, specifically sulfatase modifying factor 1 (SUMF1) (preferably SUMF1). In specific embodiments, the biomolecule is a protein, specifically sphingomyelin phosphodiesterase 1 (SMPD1) (preferably SMPD1). In specific embodiments, the biomolecule is a protein, specifically NPC intracellular cholesterol transporter 1 (NPC1) (preferably NPC1). In specific embodiments, the biomolecule is a protein, specifically NPC intracellular cholesterol transporter 2 (NPC2) (preferably NPC2). In specific embodiments, the biomolecule is a protein, specifically solute carrier family 17 member 5 (SLC17A5) (preferably SLC17A5). In specific embodiments, the biomolecule is a protein, specifically α-N-acetylgalactosaminease (NAGA) (preferably NAGA). In specific embodiments, the biomolecule is a protein, specifically neuraminidase 1 (NEU1) (preferably NEU1). In a specific embodiment, the biomolecule is a protein, namely lipase A, lysosomal acid form (LIPA) (preferably human LIPA). In a specific embodiment, the biomolecule is a protein, namely motor neuron survival (SMN) protein (preferably human SMN protein). In a specific embodiment, the biomolecule is a protein, namely dystrophin (preferably human dystrophin). In a specific embodiment, the biomolecule is a protein, namely phenylalanine hydroxylase (PAH) (preferably human PAH).In a specific embodiment, the biomolecule is a protein, specifically coagulation factor VIII (FVIII) (preferably human FVIII). In another specific embodiment, the biomolecule is a protein, specifically coagulation factor IX (FIX) (preferably human FIX). In a preferred embodiment, the biomolecule is a functional protein (e.g., a wild-type protein).

[0292] 5.2.5 Adjustment and control elements Regulatory control elements include expression control elements that are sequential to the nucleotide sequence encoding the biomolecule of interest and expression control elements that act in a trans or cis but at a distance to control the expression of the biomolecule of interest.

[0293] Expression control elements include, but are not limited to, appropriate transcription initiation, termination, promoter and enhancer sequences; polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; secretion signals; and nuclear localization sequences.

[0294] In various embodiments, the regulatory control element is a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a natural promoter encoding a nucleotide sequence of a biomolecule. In some embodiments, the promoter is a tissue-specific promoter. In a preferred embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-specific promoter. In a specific embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-myocell (i.e., muscle) bispecific promoter. In a specific embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-kidney bispecific promoter. In a specific embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-myocell (i.e., muscle)-kidney multispecific promoter. The muscle may be cardiac muscle (e.g., atrial and / or ventricular muscle) and / or skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major).

[0295] Further descriptions of the regulating control elements are provided in Sections 5.1 and 5.3.

[0296] 5.3 Vectors, gene delivery systems, and viral particles This disclosure also provides vectors and gene delivery systems associated with the above-mentioned nucleotide sequences, polynucleotides, promoters and / or expression cassettes.

[0297] In one aspect, this document provides vectors containing the polynucleotides described herein (e.g., the polynucleotides described in Section 5.2.1).

[0298] In one aspect, this document provides a carrier containing the promoters described herein (e.g., the promoters described in Section 5.2.2).

[0299] In one aspect, this document provides a carrier that includes the expression box described herein (e.g., the expression box described in Section 5.2.3).

[0300] In various embodiments, the vector described herein is a plasmid. In various embodiments, the vector described herein is an expression vector.

[0301] In various embodiments, the vector described herein is a recombinant viral vector. In various embodiments, the vector described herein is a recombinant AAV vector.

[0302] Further description of the carrier is provided below this section and in Section 5.1.

[0303] The gene delivery systems described in this article include recombinant viral particles, such as recombinant AAV particles, and non-viral gene delivery systems.

[0304] Viral particles consist of a viral genome encapsulated in a protein coat called a capsid. An "AAV particle" refers to an AAV virus composed of at least one AAV capsid protein and a packaged AAV genome. The AAV genome is a linear single-stranded DNA molecule containing terminal inverted repeat (ITR) sequences at the 5' and 3' ends. ITRs act as the origin of DNA replication and as packaging signals for the viral genome.

[0305] In one aspect, this document provides recombinant viral particles containing the polynucleotides described herein (e.g., the polynucleotides described in Section 5.2.1).

[0306] In one aspect, this document provides recombinant viral particles comprising a recombinant viral genome containing an expression cassette described herein (e.g., the expression cassette described in Section 5.2.3).

[0307] In one aspect, this document provides recombinant viral particles comprising the vectors described herein (e.g., the vectors described in this section).

[0308] In one aspect, this document provides recombinant AAV particles comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an expression cassette (e.g., the expression cassette described in Section 5.2.3) with AAV terminal inverted repeat (ITR) side-mounted.

[0309] Numerous virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well-known in the art and is illustrated, for example, in Green and Sambrook, *Molecular Cloning: A Laboratory Manual*, 4. th As described in Ed., Cold SpringHarbor Laboratory (Cold Spring Harbor, NY 2012) and other virology and molecular biology handbooks.

[0310] In some embodiments, the viral vectors or viral particles provided herein are derived from adenoviruses. Exemplary vectors are based on or derived from HAd5, ChAd3, HAd26, HAd6, AdCH3NSmut, HAd35, ChAd63, HAd4, and rcAd26. Recombinant adenovirus vectors can be constructed according to methods known in the art. See, for example, O'Connor et al. Virology , 217(1):11-22(1996); Hardy et al., Journal of Virology, 73(9):7835-7841 (1999); Hardy et al., Journal of Virology , 71(3):1842-1849(1997). In some embodiments, third-generation adenovirus vectors (also known as “high-capacity adenovirus vectors” (HCAds), helper-dependent, or “Gutless” adenovirus vectors) may be used herein to deliver longer sequences. In some embodiments, the nucleic acid construct of interest is cloned into an adenovirus vector containing only the ITR and packaging signal. Helper adenovirus vectors may be co-transfected into HEK cells to generate adenovirus particles. See Lee et al., Genes and Diseases ,4(2):43-63 (2007).

[0311] In some embodiments, the viral vectors or viral particles provided herein are derived from lentiviruses. Exemplary vectors are based on or derived from HIV-1, HIV-2, SIVSM, SIVAGM, EIAV, FIV, VNV, CAEV, or BIV. Lentiviral vectors can be produced according to methods known in the art, such as Chribs, etc. BMC Biotechnology , 13:98 (2003); Merten et al., Mol Ther Methods Clin Dev., 13(3):16017(2016); Durand and Cimarelli, Viruses As described in , 3:132-159 (2011). In some embodiments, this document uses a third-generation self-inactivating lentiviral vector.

[0312] In some embodiments, the viral vectors or viral particles provided herein are derived from herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof. Exemplary vectors are based on or derived from HSV-1, HSV-2, CMV, VZV, EBV, and KSHV. HSV-based vectors can be constructed according to methods known in the art, such as those described in U.S. Patents 7,078,029, 6,261,552, 5,998,174, 5,879,934, 5,849,572, 5,849,571, 5,837,532, 5,804,413, and 5,658,724, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, the entire contents of which are incorporated herein by reference.

[0313] In some embodiments, the HSV-based vectors provided herein are amplicon vectors. In other embodiments, the HSV-based vectors provided herein are replication-defective vectors. In other embodiments, the HSV-based vectors provided herein are replication-competent vectors.

[0314] Amplicones are plasmid-derived vectors engineered to contain an HSV DNA replication origin (ori) and an HSV cleavage-packaging recognition sequence (pac). When amplicons are transfected into mammalian cells with HSV helper function, they replicate, forming head-to-tail linked multiply, which are then packaged into viral particles. Currently, there are two main methods for producing amplicon particles: one based on defective helper HSV infection, and the other based on transfection of the HSV-1 gene, such as a set of overlapping adhesive plasmids lacking pac or BAC-HSV-1 lacking both pac and ICP27. In some embodiments, the amplicon used herein can accommodate large fragments of foreign DNA (e.g., up to 152 kb), including multiple copies of transgenes (e.g., up to 15 copies), and is non-toxic.

[0315] In some embodiments, the HSV-based vectors used herein lack at least one essential HSV gene, and the HSV-based vectors may also contain one or more deletions of non-essential genes. In some embodiments, the HSV-based vectors are replication-deficient. Most replication-deficient HSV-based vectors contain deletions to remove one or more immediate early, early, or late HSV genes to prevent replication. In other embodiments, the HSV-based vectors lack immediate early genes selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47, and combinations thereof. In specific embodiments, the HSV-based vectors are all deficient for ICP0, ICP4, ICP22, ICP27, and ICP47. Exemplary replication-deficient vectors include NV-1020 (HSV-1), RAV9395 (HSV-2), AD-472 (HSV-2), NS-gEnull (HSV-1), and ImmunoVEX (HSV2). Exemplary replication-defective vectors include dl5-29 (HSV-2), dl5-29-41L (HSV-1), DISC-dH (HSV-1 and HSV-2), CJ9gD (HSV-1), TOH-OVA (HSV-1), d106 (HSV-1), d81 (HSV-1), HSV-SIV d106 (HSV-1), and d106 (HSV-1).

[0316] Replication-deficient HSV-based vectors are typically produced in complement cell lines that provide appropriate levels of gene function absent in the replication-deficient HSV-based vectors but required for viral replication, in order to generate a high-titer stock of viral vectors. Exemplary cell lines complement at least one of the replication-deficient HSV-based vectors not present, and in some embodiments, all replication-essential gene functions. For example, ICP0, ICP4, ICP22, ICP27, and ICP47-deficient HSV-based vectors can be complemented by the human osteosarcoma cell line U2OS. Cell lines can also complement non-essential genes (e.g., UL55) that reduce growth or replication efficiency when missing. Complement cell lines can complement deficiencies in at least one replication-essential gene function encoded by an early region, an immediate early region, a late region, a viral packaging region, a viral-associated region, or a combination thereof, including all HSV functions (e.g., to enable HSV amplicons comprising minimal HSV sequences (e.g., only terminal inverted repeat sequences and packaging signals or only ITR and HSV promoters) to replicate). In some embodiments, the cell line is further characterized by containing a complementary gene in a non-overlapping manner with the HSV-based vector, which minimizes and virtually eliminates the possibility of recombination between the HSV-based vector genome and the cellular DNA. Therefore, the presence of replicable HSV is minimized if not avoided in the vector library, thus making it suitable for certain therapeutic purposes, particularly gene therapy. The construction of the complementary cell line involves standard molecular biology and cell culture techniques well-known in the art.

[0317] In some embodiments, the viral vectors or viral particles provided herein are derived from adeno-associated virus (AAV). A more detailed description related to AAV is provided in Section 5.3.1 below.

[0318] The nucleotide sequences, promoters, and expression cassettes described herein can be cloned into viral vectors using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selection markers.

[0319] In one embodiment, the viral vector genome contains at least one element that enhances transgene target specificity and expression (see, for example, 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), such as an intron or a synthetic intron with a modified sequence derived from a mammalian genome. Non-limiting examples of introns include MVM (67-97 bps), F.IX truncated intron 1 (300 bps), β-globulin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobulin splice acceptor (500 bps), SV40 late splice donor / splicing acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps). In one embodiment, the intron may be 100-500 nucleotides in length. The length of the introns can be 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.

[0320] In some embodiments, the viral vector further contains a selection marker gene or a reporter gene to select cells expressing the protein from a host cell population transfected by the viral vector. Both the selection marker and the reporter gene may be side-mounted with appropriate regulatory sequences to enable expression in the host cells. For example, the viral vector may contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate nucleic acid sequence expression.

[0321] The non-viral gene delivery systems described herein include, but are not limited to, lipid-based gene delivery systems (e.g., liposome-based gene delivery systems), peptide-based gene delivery systems, inorganic gene delivery systems, and polymer delivery systems. Non-limiting exemplary non-viral gene delivery systems are described, for example, in Sung and Kim, 2019, Biomater Res 23:8; Zu and Gao, 2021, AAPS J 23:78; and Lan et al., 2022, Mol Cancer 21:71; each of which is incorporated herein by reference in its entirety.

[0322] In one aspect, this document provides a nonviral gene delivery system comprising the polynucleotides described herein (e.g., the polynucleotides described in Section 5.2.1).

[0323] In one aspect, this document provides a nonviral gene delivery system comprising a polynucleotide containing a nucleotide sequence encoding an expression cassette described herein (e.g., the expression cassette described in Section 5.2.3).

[0324] In one aspect, this article provides a nonviral gene delivery system comprising the vectors described herein (e.g., the vectors described in this section).

[0325] 5.3.1 AAV carrier and AAV particles Any AAV serotype or variant thereof may be used in this disclosure. AAV serotypes may include, but are not limited to, AAV1 (Genbank Accession No. NC_002077.1; HC000057.1), AAV2 (Genbank Accession No. NC_001401.2, JC527779.1), AAV2i8 (Asokan, A., 2010, Discov. Med. 9:399), AAV3 (Genbank Accession No. NC_001729.1), AAV3-B (Genbank Accession No. AF028705.1), AAV4 (Genbank Accession No. NC_001829.1), AAV5 (Genbank Accession No. NC_006152.1; JC527780.1), and AAV6 (Genbank Accession No. NC_002077.1). AF028704.1; JC527781.1), AAV7 (Genbank Accession No. NC_006260.1; JC527782.1), AAV8 (Genbank Accession No. NC_006261.1; JC527783.1), AAV9 (Genbank Accession No. AX753250.1; JC527784.1), AAV10 (Genbank Accession No. AY631965.1), AAVrh10 (Genbank Accession No. AY243015.1), AAV11 (Genbank Accession No. AY631966.1), AAV12 (Genbank Accession No. AY631966.1) NoDQ813647.1), AAV13 (Genbank Accession No EU285562.1), AAV LK03, AAVrh74, AAV DJ (WuZ, et al., J Virol. 80:11393–7 (2006)), AAVAnc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127 (Zin, E. et al., Cell. Rep. 12:1056 (2016)), AAV_go.1 (Arbetum, AE et al., J. Virol. 79:15238 (2005)), AAVhu.37, AAVrh8, AAVrh8R and AAVrh.8 (Wang et al., Mol. Ther. 18:119-125 (2010) or its variants.)

[0326] AAV variants include, but are not limited to, AAV1 variants (e.g., AAV containing AAV1 variant capsid protein), AAV2 variants (e.g., AAV containing AAV2 variant capsid protein), AAV3 variants (e.g., AAV containing AAV3 variant capsid protein), AAV3-B variants (e.g., AAV containing AAV3-B variant capsid protein), AAV4 variants (e.g., AAV containing AAV4 variant capsid protein), AAV5 variants (e.g., AAV containing AAV5 variant capsid protein), AAV6 variants (e.g., AAV containing AAV6 variant capsid protein), AAV7 variants (e.g., AAV7 variant capsid protein), and AAV8 variants (e.g., AAV containing AAV8 variant capsid protein), AAVrh8, AAVrh8R (e.g., AAV containing AAVrh8 or AAVrh8R variant capsid protein), AAV9 variant (e.g., AAV containing AAV9 variant capsid protein), AAV10 variant (e.g., AAV containing AAV10 variant capsid protein), AAVrh10 variant (e.g., AAV containing AAVrh10 variant capsid protein), AAV11 variant (e.g., AAV containing AAV11 variant capsid protein), AAV12 variant (e.g., AAV containing AAV12 variant capsid protein), AAV13 variant (e.g., AAV containing AAV13 variant capsid protein), AAV LK03 variant (e.g., AAV containing AAV LK03 variant capsid protein), and AAVrh74 variant (e.g., AAV containing AAVrh74 variant capsid protein).

[0327] The recombinant AAV (rAAV) vectors used in this disclosure can be constructed according to known techniques. In some embodiments, the rAAV vector is constructed to include components operatively linked in the transcriptional direction. Control elements may include the recombinant promoter, response element, transcription initiation region, and transcription termination region provided herein. Control elements may be selected based on the cell of interest. In some embodiments, the resulting rAAV vector construct containing operatively linked components has functional AAV ITR sequences flanked (5' and 3'). In some embodiments, the resulting rAAV vector may have a genome structured as (5'AAV ITR)-(promoter)-transfer of interest)-(3'AAV ITR).

[0328] In some embodiments, the promoter described herein is operatively linked to at least one additional regulatory sequence. In some embodiments, the regulatory sequence may include, for example, an enhancer sequence, such as an upstream enhancer sequence (USE), an RNA processing signal, such as a splicing signal, a polyadenylation signal sequence, a sequence stabilizing cytoplasmic mRNA, a post-transcriptional regulatory element (PRE), and / or a microRNA (miRNA) target sequence. In some embodiments, the regulatory sequence may include sequences that enhance translation efficiency (e.g., Kozak sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion. In some embodiments, the included regulatory sequence promotes in vivo transcription or expression of a transgene operatively linked to the promoter described herein. The regulatory sequence may include a control sequence typically associated with the transgene of interest or optionally a heterologous control sequence.

[0329] In some embodiments, the regulatory sequence includes a regulatory control element. In some embodiments, the regulatory control element is the 5' of the transgene (i.e., present in the 5' untranslated region; 5'UTR). In other embodiments, the regulatory control element is the 3' of the transgene (i.e., present in the 3' untranslated region; 3'UTR). In some embodiments, the regulatory sequence includes more than one regulatory control element, for example, it may include two, three, four, or five control elements. When the regulatory sequence includes more than one control element, each control element may independently be the 5', 3', flanking, or internal part of the promoter described herein.

[0330] In some embodiments, the viral vector may contain at least one polyadenylation (polyA) signal sequence, which is well known in the art. Where a polyadenylation sequence is present, it is typically located between the 3' end of the transgene and the 5' end of the 3' ITR. In some embodiments, the viral vector also contains an upstream enhancer sequence of polyA at the 5' end of the polyA signal sequence. In some cases, the regulatory sequence is a sequence that improves translation efficiency, such as a Kozak sequence.

[0331] In some embodiments, the viral vector contains introns. In some embodiments, the introns are present within the transgene. In some embodiments, the introns are the 5' or 3' end of the transgene. In some embodiments, the introns are flanked by the 5' or 3' end of the transgene. In some embodiments, the nucleic acid construct contains two introns. In some embodiments, one intron is the 5' end of the transgene, and the other intron is the 3' end of the transgene. In some embodiments, one intron is flanked by the 5' end of the transgene, and the second intron is flanked by the 3' end of the transgene. In some embodiments, the introns are SV40 introns, such as the 5' UTR SV40 intron.

[0332] AAV ITR sequences known in the art can be used in the rAAV vectors of the present invention. In some embodiments, the AAV ITR used in the vectors of the present invention has a wild-type nucleotide sequence. In other embodiments, the AAV ITR sequence used in the vectors of the present invention is not a wild-type sequence, but contains, for example, nucleotide insertions, deletions, or substitutions. The AAV ITRs provided herein can be derived from any AAV serotype, including but not limited to AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or variants thereof.

[0333] In some embodiments, the rAAV carrier includes an ITR from AAV1. In some embodiments, the rAAV carrier includes an ITR from AAV2. In some embodiments, the rAAV carrier includes an ITR from AAV2i8. In some embodiments, the rAAV carrier includes an ITR from AAV3. In some embodiments, the rAAV carrier includes an ITR from AAV3-B. In some embodiments, the rAAV carrier includes an ITR from AAV4. In some embodiments, the rAAV carrier includes an ITR from AAV5. In some embodiments, the rAAV carrier includes an ITR from AAV6. In some embodiments, the rAAV carrier includes an ITR from AAV7. In some embodiments, the rAAV carrier includes an ITR from AAV8. In some embodiments, the rAAV carrier includes an ITR from AAVrh8. In some embodiments, the rAAV carrier includes an ITR from AAVrh8R. In some embodiments, the rAAV carrier includes an ITR from AAV9. In some embodiments, the rAAV carrier includes an ITR from AAV10. In some embodiments, the rAAV carrier includes an ITR from AAVrh10. In some embodiments, the rAAV carrier includes an ITR from AAV11. In some embodiments, the rAAV carrier includes an ITR from AAV12. In some embodiments, the rAAV carrier includes an ITR from AAV13. In some embodiments, the rAAV carrier includes an ITR from AAV-DJ. In some embodiments, the rAAV carrier includes an ITR from AAV LK03. In some embodiments, the rAAV carrier includes an ITR from AAVrh74.

[0334] In some embodiments, the 5' and 3' ITRs of the nucleotide sequences side-joined in the rAAV vector provided herein are identical and derived from the same AAV serotype. In other embodiments, the 5' and 3' ITRs of the nucleotide sequences side-joined in the rAAV vector provided herein are different and / or derived from different AAV serotypes.

[0335] In some implementations, an rAAV vector containing an expression cassette of interest with an AAV ITR attached to it can be constructed by directly inserting the cassette of interest into the AAV genome, for example, by inserting it into a cut-off AAV open reading frame, and certain portions of the AAV genome can be optionally deleted, as described in WO 1993 / 003769; Kotin (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J.Exp. Med. 179:1867-1875.

[0336] In other implementations, the AAV ITR is excised from the AAV genome or from an AAV vector containing such an ITR, and then fused with the 5' and 3' of the expression cassette of interest present in another vector using standard ligation techniques.

[0337] In some embodiments, the rAAV vectors provided herein contain a recombinant self-complementary genome. rAAV containing a self-complementary genome can typically rapidly form a double-stranded DNA molecule via its partially complemented sequences (e.g., complementing the coding and non-coding strands of the transgene). More specifically, in some embodiments, the rAAV vectors provided herein contain an rAAV genome comprising a first heteropolynucleotide sequence (e.g., the coding strand of a therapeutic transgene) and a second heteropolynucleotide sequence (e.g., the non-coding or antisense strand of a therapeutic transgene), and the first heteropolynucleotide sequence can form intrastrand base pairs with the second heteropolynucleotide sequence. In some embodiments, the first and second heteropolynucleotide sequences are linked by sequences that promote intrastrand base pairing (e.g., hairpin DNA structures). In some embodiments, the first and second heteropolynucleotide sequences are linked by a mutated ITR such that the rep protein does not cleave the viral genome at the mutated ITR. rAAV vectors containing self-complementary genomes can be prepared using methods known in the art, for example, as described in U.S. Patent Nos. 7,125,717; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457.

[0338] In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 5 kilobases (kb). In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 4.5 kb. In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 4.0 kb. In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 3.5 kb. In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 3.0 kb. In some embodiments, the recombinant viral genome in the rAAV vector provided herein is less than about 2.5 kb.

[0339] The capsid protein can be derived from the same serotype as the ITR or a derivative thereof. The capsid can also be a different serotype than the ITR. For example, in some embodiments, the AAV particle comprises an AAV2 ITR and an AAV6 capsid (AAV 2 / 6), an AAV2 ITR and an AAV7 capsid (AAV 2 / 7), an AAV2 ITR and an AAV8 capsid (AAV 2 / 8), or an AAV2 ITR and an AAV9 capsid (AAV 2 / 9).

[0340] Naturally occurring AAV capsids contain AAV VP1, VP2, and VP3 capsid proteins, each encoded by a splice variant of the AAV cap gene. Typically, AAV particles contain three proteins, VP1, VP2, and VP3, where VP2 and VP3 are truncated forms of VP1 and therefore contain sequences also found in VP1. Generally, the amino acid sequence of VP1 defines the capsid serotype. Therefore, for example, if the VP1 capsid protein encodes the AAV2 VP1 protein, then the AAV will be serotype AAV2, and if the VP1 capsid protein encodes the AAV9 VP1 protein, then the AAV will be serotype AAV9.

[0341] Typically, AAV capsid proteins contain multiple variable regions (VRs) and constant regions located between the variable regions. The "GH loop" refers to a loop sequence with β-chain G and β-chain H attached to the inner β-barrel of the AAV capsid protein, and contains variable regions VR IV to VRVIII.

[0342] The sequences of the VP2 and VP3 capsid proteins, as well as the positions of various domains (e.g., variable regions and GH loops), can be readily and routinely determined by a person skilled in the art from a given VP1 sequence using suitable methods known in the art, such as comparing or aligning the VP1 sequence with an annotated VP1 sequence (preferably an annotated VP1 sequence from a closely related AAV species) using a suitable algorithm. Preferably, such an annotated VP1 sequence is an annotated wild-type AAV VP1 sequence.

[0343] In some embodiments, the AAV capsid proteins (e.g., VP1, VP2, and / or VP3) in the rAAV particles of the present invention are not naturally occurring capsid proteins. In some embodiments, the AAV capsid proteins (e.g., VP1, VP2, and / or VP3) are derived from naturally occurring capsid proteins.

[0344] In some embodiments, the AAV capsid protein is VP1. In other embodiments, the AAV capsid protein is VP2. In other embodiments, the AAV capsid protein is VP3. In some embodiments, the rAAV particle comprises VP1, VP2, and / or VP3 capsid proteins. In other embodiments, the rAAV particle comprises VP1, VP2, and VP3 capsid proteins. In some embodiments, the rAAV particle comprises VP1, VP2, and / or VP3 capsid proteins, wherein the capsid proteins of the rAAV particle have the same serotype. In other embodiments, the rAAV particle comprises VP1, VP2, and VP3 capsid proteins, wherein the capsid proteins of the AAV particle have the same serotype.

[0345] In some respects, the capsid protein is a variant capsid protein. Compared to the corresponding reference capsid protein (e.g., the naturally occurring parental capsid protein, i.e., the capsid protein from which it originates), the variant capsid protein may contain one or more mutations, such as amino acid substitutions, amino acid deletions, and heteropeptide insertions. In some embodiments, the amino acid sequence of the AAV capsid protein is identical to that of the wild-type, reference, or parental AAV capsid protein, except for 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, or 30 amino acid residues, for example, except for substitutions of 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, or 30 amino acid residues. In some embodiments, the capsid protein or AAV particle described herein may be a chimeric capsid protein or AAV particle, each comprising the protein sequences of two or more AAV serum type capsid proteins or particles.

[0346] In various embodiments, the variant AAV capsid protein described herein is the variant AAV VP1 capsid protein. Variant AAV capsid proteins, which are VP2 and VP3 counterparts of the VP1 capsid protein described herein, are also described herein. Fragments of such variant AAV capsid proteins (e.g., fragments of VP1, VP2, or VP3 capsid proteins) are also described herein, including but not limited to variable regions (e.g., VR IV and VR VIII), GH loops, and functional fragments that substantially retain the biological activity and associated tissue tropism of the corresponding capsid protein. In some embodiments, the fragment length of the VP1, VP2, or VP3 capsid protein described herein is at least 7 amino acids. In some embodiments, the fragment length of the VP1, VP2, or VP3 capsid protein described herein is at least 8 amino acids. In some embodiments, the fragment length of the VP1, VP2, or VP3 capsid protein described herein is at least 9 amino acids. In some embodiments, the fragment length of the VP1, VP2, or VP3 capsid protein described herein is at least 10 amino acids. In some embodiments, the fragment length of the VP1, VP2, or VP3 capsid protein described herein is at least 20 amino acids. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 30 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 40 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 50 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 100 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 200 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 300 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 400 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are at least 500 amino acids long. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 7-10 amino acids in length. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 10-50 amino acids in length. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 50-100 amino acids in length. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 100-200 amino acids in length. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 200-300 amino acids in length.In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 300-400 amino acids in length. In some embodiments, the VP1, VP2, or VP3 capsid protein fragments described herein are 400-500 amino acids in length.

[0347] In some embodiments, the capsid proteins in the rAAV particles provided herein are derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, and AAV44-9 capsid proteins. In specific embodiments, the capsid proteins in the rAAV particles provided herein have amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% identical to the amino acid sequences of the capsid proteins AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, and AAV44-9.

[0348] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which comprise VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV1.

[0349] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which comprise VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV2.

[0350] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV2i8.

[0351] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV3.

[0352] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV3-B.

[0353] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV4.

[0354] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV5.

[0355] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV6.

[0356] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV7.

[0357] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV8.

[0358] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAVrh8.

[0359] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAVrh8R.

[0360] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV9.

[0361] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV10.

[0362] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAVrh10.

[0363] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV11.

[0364] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV12.

[0365] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV13.

[0366] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV-DJ.

[0367] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV LK03.

[0368] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAVrh74.

[0369] In some embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins, which contain VP1, VP2, and / or VP3 capsid protein sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with any VP1, VP2, or VP3 amino acid sequence of AAV44-9.

[0370] In some specific embodiments, the rAAV particles provided herein contain an AAV VP1 capsid protein, which contains an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identical to the VP2 capsid protein counterparts and / or the VP3 capsid protein counterparts of SEQ ID NO: 21, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.

[0371] In some specific embodiments, the rAAV particles provided herein contain AAV VP1 capsid protein, which contains the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141, its VP2 capsid protein counterpart and / or its VP3 capsid protein counterpart.

[0372] In a specific embodiment, the rAAV particles provided herein are recombinant AAV serotype 9 (rAAV9) particles. In a specific embodiment, the rAAV particles provided herein contain the AAV9 capsid protein. In a particular embodiment, the AAV capsid of the rAAV particles provided herein contains a variant of the AAV9 capsid protein.

[0373] In various embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 25, 27, 29, 31, 33, 35, 36, 38, 40, 42, 46, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, or 135.

[0374] In various embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 24, 26, 28, 30, 32, 34, 37, 39, 41, 43, 45, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, or 134.

[0375] In various embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59, or 61.

[0376] In various embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58, or 60.

[0377] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 25. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 24.

[0378] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 27. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 26.

[0379] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 29. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 28.

[0380] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 31. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 30.

[0381] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 33. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 32.

[0382] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the variant AAV9 capsid protein provided herein comprises both the amino acid sequence of SEQ ID NO: 35 and the amino acid sequence of SEQ ID NO: 36. In a specific embodiment, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 34.

[0383] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 38. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 37.

[0384] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 40. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 39.

[0385] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 42. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 41.

[0386] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 44. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 43.

[0387] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the variant AAV9 capsid protein provided herein comprises both the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 47. In a specific embodiment, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 45.

[0388] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 49. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 48.

[0389] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 51. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 50.

[0390] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 53. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 52.

[0391] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 55. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 54.

[0392] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 57. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 56.

[0393] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 59. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 58.

[0394] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 61. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 60.

[0395] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 63. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 62.

[0396] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 65. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 64.

[0397] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 67. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 66.

[0398] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 69. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 68.

[0399] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 71. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 70.

[0400] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 73. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 72.

[0401] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 75. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 74.

[0402] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 77. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 76.

[0403] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 79. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 78.

[0404] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 81. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 80.

[0405] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 83. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 82.

[0406] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 85. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 84.

[0407] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 87. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 86.

[0408] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 89. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 88.

[0409] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 91. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 90.

[0410] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 93. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 92.

[0411] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 95. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 94.

[0412] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 97. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 96.

[0413] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 99. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 98.

[0414] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 101. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 100.

[0415] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 103. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 102.

[0416] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 105. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 104.

[0417] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 107. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 106.

[0418] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 109. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 108.

[0419] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 111. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 110.

[0420] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 113. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 112.

[0421] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 115. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 114.

[0422] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 117. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 116.

[0423] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 119. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 118.

[0424] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 121. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 120.

[0425] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 123. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 122.

[0426] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 125. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 124.

[0427] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 127. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 126.

[0428] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 129. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 128.

[0429] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 131. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 130.

[0430] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 133. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 132.

[0431] In some embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 135. In specific embodiments, the variant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 134.

[0432] In some embodiments, the variant AAV9 capsid protein provided herein comprises the VP2 capsid protein counterpart of the VP1 capsid protein described herein.

[0433] In some embodiments, the variant AAV9 capsid protein provided herein comprises the VP3 capsid protein counterpart of the VP1 capsid protein described herein.

[0434] In some embodiments, the variant AAV9 capsid protein provided herein comprises a fragment (e.g., a functional fragment) of the VP1 capsid protein described herein.

[0435] In various embodiments, the recombinant AAV particles described herein exhibit an increased tendency toward one or more tissues relative to wild-type AAV particles. These one or more tissues may be, for example, the liver, muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or cardiac muscles (e.g., atrial and / or ventricular muscles)), kidneys, brain (e.g., midbrain, mid-hindbrain, forebrain, and / or hindbrain), lungs, and / or spleen.

[0436] In some implementations, the recombinant AAV particles described herein exhibit an increased tendency to act on the liver compared to wild-type AAV particles.

[0437] In some embodiments, the recombinant AAV particles described herein exhibit an increased tendency to act on muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or cardiac muscles (e.g., atrial and / or ventricular muscles)) relative to wild-type AAV particles.

[0438] In some implementations, the recombinant AAV particles described herein exhibit an increasing tendency to benefit the kidneys compared to wild-type AAV particles.

[0439] In some implementations, the recombinant AAV particles described herein exhibit an increased tendency for both liver and muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or myocardium (e.g., atrial and / or ventricular myocardium)) relative to wild-type AAV particles.

[0440] In some implementations, the recombinant AAV particles described herein tend to increase activity in both the liver and kidneys compared to wild-type AAV particles.

[0441] In some embodiments, the recombinant AAV particles described herein exhibit an increased tendency to affect the liver, muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or myocardium (e.g., atrial and / or ventricular myocardium)) and kidneys, relative to wild-type AAV particles.

[0442] In various embodiments, the recombinant AAV particles described herein exhibit a reduced tendency to act on one or more tissues compared to wild-type AAV particles. One or more tissues may be, for example, muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or cardiac muscles (e.g., atrial and / or ventricular muscles)), kidneys, brain (e.g., midbrain, mid-hindbrain, forebrain, and / or hindbrain), lungs, and / or spleen.

[0443] In various embodiments, the recombinant AAV particles described herein have the tendency to increase one or more tissues as described above and the tendency to decrease another or more tissues as described above.

[0444] In various embodiments, the tissue tropism described herein is measured after systemic administration of the recombinant AAV particles to the subject. In some embodiments, the tissue tropism described herein is measured after intravenous administration of the recombinant AAV particles to the subject.

[0445] In a preferred embodiment, the recombinant AAV particles described herein are non-naturally occurring AAV particles. In some embodiments, the recombinant AAV particles described herein are isolated. In some embodiments, the recombinant AAV particles described herein are purified.

[0446] 5.4 Cells In one aspect, this document provides host cells containing the polynucleotides described herein (e.g., the polynucleotides described in Section 5.2.1).

[0447] In one aspect, this document provides host cells comprising the vectors described herein (e.g., the vectors described in Section 5.3).

[0448] In some embodiments, the host cell also contains a nucleotide sequence encoding the AAV rep gene in a polynucleotide or vector that is the same as or different from the polynucleotide or vector described herein (e.g., the polynucleotide described in Section 5.2.1 or the vector described in Section 5.3). In some embodiments, the host cell also contains a nucleotide sequence encoding the AAV cap gene in a polynucleotide or vector that is the same as or different from the polynucleotide or vector described herein (e.g., the polynucleotide described in Section 5.2.1 or the vector described in Section 5.3). In some embodiments, the host cell also includes helper functions, such as one or more helper plasmids and / or one or more helper viruses (see Section 5.5 for helper functions).

[0449] In one aspect, this document provides a host cell containing the recombinant viral particles described herein (e.g., the recombinant viral particles described in Section 5.3).

[0450] In one respect, this document provides host cells for producing the recombinant viral particles described herein (e.g., the recombinant viral particles described in Section 5.3).

[0451] In one aspect, this document provides host cells comprising the recombinant AAV particles described herein (e.g., the recombinant AAV particles described in Section 5.3).

[0452] In one aspect, this document provides host cells for producing the recombinant AAV particles described herein (e.g., the recombinant AAV particles described in Section 5.3).

[0453] In one aspect, this document provides a host cell comprising a non-viral delivery system as described herein (e.g., the non-viral delivery system described in Section 5.3).

[0454] In one aspect, this document provides host cells for producing the non-viral delivery systems described herein (e.g., the non-viral delivery systems described in Section 5.3).

[0455] In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is an in vitro host cell. In some embodiments, the host cell is an in vivo host cell.

[0456] As used herein, the term "host" refers to a cell carrying polynucleotides, vectors, viral particles (e.g., rAAV particles), or a non-viral gene delivery system (e.g., cells from insects, animals (including humans and non-human animals), yeast, and bacteria). This disclosure is not intended to be limited to any particular type of host cell. In fact, any suitable cell is expected to be used as a host herein. Host cells may be in the form of or derived from single cells, populations, cultures (such as liquid cultures or cultures on solid substrates), cell lines, organisms, or parts thereof.

[0457] Host cells can be used to amplify, replicate, or produce the polynucleotides, vectors, recombinant viral particles (e.g., rAAV particles), nonviral gene delivery systems, or biomolecules described herein. Suitable host cells for this purpose may be, for example, bacterial cells, yeast cells, insect cells, or mammalian cells (e.g., human cells or non-human mammalian cells). Non-limiting exemplary insect cells that can be used as host cells include Ao38, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, and Hz2E5. Non-limiting exemplary mammalian cells that can be used as host cells include HEK293, HEK293-T, HeLa, WEHI, 10T1 / 2, MDCK, W138, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, COS1, BSC 1, BSC 40, BMT 10, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, MRC-5, primary fibroblasts, hepatocytes, and myoblasts. In some embodiments, the host cell is a human cell. In a specific embodiment, the human cell is autologous to the subject to be treated (e.g., a human patient). In a specific embodiment, the human cell is allogeneic to the subject to be treated (e.g., a human patient).

[0458] The host cells themselves can also be used as a therapeutic agent. Host cells suitable for this purpose are preferably derived from the same species as the subject. Therefore, if the subject to be treated is a human patient, the host cells suitable for use as the therapeutic agent itself are preferably human cells. In a specific embodiment, the human cells are autologous to the subject to be treated with human cells (e.g., a human patient). In a specific embodiment, the human cells are allogeneic to the subject to be treated with human cells (e.g., a human patient).

[0459] In one aspect, this document provides a host cell population stably transduced by recombinant viral particles (e.g., recombinant AAV particles) as described herein. In another aspect, this document provides a host cell population stably transduced by a non-viral gene delivery system as described herein. In a specific embodiment, the host cell population is an in vitro host cell population. In a specific embodiment, the host cell population is an in vitro host cell population. In a specific embodiment, the host cell population is an in vivo host cell population. In a specific embodiment, the host cell population is a human host cell population. In a specific embodiment, the host cell population is a human in vitro host cell population. In a specific embodiment, the host cell population is a human in vitro host cell population.

[0460] In some implementations, such host cell populations can be used to generate populations of recombinant viral particles (e.g., recombinant AAV particles), which are used as therapeutic agents and administered to subjects in need (e.g., human patients).

[0461] In some implementations, such host cell populations can be used to generate nonviral gene delivery system molecules that are used as therapeutic agents and administered to subjects in need (e.g., human patients).

[0462] In some implementations, such host cell populations can be used to generate biomolecule populations as described in Section 5.2.4, which are used as therapeutic agents and administered to subjects in need (e.g., human patients).

[0463] In some embodiments, such host cell populations can be used as the therapeutic agent itself and administered to a subject in need (e.g., a human patient). In specific embodiments, the host cell population is autologous to the subject (e.g., a human patient) to be treated with the host cell population. In specific embodiments, the host cell population is allogeneic to the subject (e.g., a human patient) to be treated with the host cell population.

[0464] 5.5 Production Methods On the other hand, this document provides a method for producing the recombinant viral particles described herein, such as the recombinant AAV particles described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3).

[0465] On the other hand, this document provides methods for producing the non-viral gene delivery system described herein (e.g., the non-viral gene delivery system described in Section 5.3).

[0466] This document also describes methods for producing the polynucleotides described herein (e.g., the polynucleotides described in Section 5.2.1), the promoters described herein (e.g., the promoters described in Section 5.2.2), the expression cassettes described herein (e.g., the expression cassettes described in Section 5.2.3), the vectors described herein (e.g., the vectors described in Section 5.3), or the host cells described herein (e.g., the host cells described in Section 5.4).

[0467] The polynucleotides, promoters, expression cassettes, vectors, host cells, recombinant viral particles (e.g., recombinant AAV particles), and nonviral gene delivery systems disclosed herein can be produced by any suitable method known in the art, including recombinant production, genetic engineering, molecular cloning, chemical synthesis, and other synthetic methods. Such production methods are within the knowledge of those skilled in the art and are not intended to limit the invention.

[0468] The polynucleotides, promoters, expression cassettes, and / or vectors disclosed herein can be produced using any suitable genetic engineering and protein production techniques known in the art, including but not limited to cloning, restriction endonuclease digestion, ligation, transformation, plasmid purification, DNA sequencing, chemical synthesis, in vitro translation, and in vivo expression, such as Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4. th Described in Ed., Cold Spring Harbor Laboratory (ColdSpring Harbor, NY 2012).

[0469] Any suitable method can be used to enable host cells to carry the polynucleotides, vectors, recombinant viral particles (e.g., recombinant AAV particles) or non-viral gene delivery systems disclosed herein, including but not limited to transfection, electroporation, transduction, liposome delivery, membrane fusion technology, high-speed DNA-coated particles, viral infection, and protoplast fusion.

[0470] Recombinant viral particles (such as recombinant AAV particles) can be produced by host cells, which allows for the production and replication of recombinant viral particles (such as recombinant AAV particles).Methods for producing recombinant viral particles including recombinant AAV particles are well known in the art and are illustrated in, for example, Adeno-Associated Virus: Methods and Protocols (Methods in Molecular Biology, 280), ed. Snyder and Moullier, Humana Press, NJ (2011); Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Biology, 1937); ed. Manfredsson and Benskey, Humana Press, NJ (2019); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); US Patent Nos. US5064764, US5756283, US6194191, US6204059, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6566118, US6943019, US6953690, US7022519, US7238526, US7291498, US7491508 and US8137948; and The disclosures are described in international patent application publication numbers WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2018022608, WO2019217513, WO2019222132, WO2019222136 and WO2020232044, the entire contents of which are incorporated herein by reference.

[0471] The rAAV particles described herein can be produced using any suitable method known in the art. For example, host cells (e.g., mammalian cells) can be engineered to stably express the necessary components for AAV particle production. This can be achieved by integrating a plasmid (or multiple plasmids) containing the AAV rep and cap genes and selection markers (e.g., antibiotic resistance genes such as neomycin or ampicillin) into the cell genome. The cells can be, for example, insect or mammalian cells, and can then be co-infected with a helper virus (e.g., an adenovirus or baculovirus providing the helper function) and an rAAV vector containing 5' and 3' AAV ITRs. The use of selectable markers allows for large-scale production of rAAV. As another non-limiting example, adenovirus or baculovirus, instead of plasmids, can be used to introduce the rep and cap genes into packaging cells. As yet another non-limiting example, viral vectors containing 5' and 3' AAV ITRs, as well as the rep and cap genes, can be stably integrated into the DNA of the production cell, and the helper function can be provided by wild-type adenovirus for rAAV production.

[0472] Helper viruses used for AAV replication are viruses that allow AAV to be replicated and packaged by host cells. Helper viruses provide auxiliary functions that allow AAV replication. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass many different subgroups, although subgroup C, type 5 adenovirus (Ad5), is the most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and are available from collections such as the ATCC. Viruses of the Herpesviridae family also available from collections such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions used for AAV replication include E1A, E1B, E2A, VA, and E4orf6 functions.

[0473] If the ratio of infectious AAV particles to infectious helper virus particles is at least about 10²:1; at least about 10⁴:1; at least about 10⁶:1; or at least about 10⁸:1, then the AAV formulation is considered substantially free of helper virus. The formulation may also be free of an equal amount of helper virus proteins (i.e., proteins present due to this level of helper virus if the aforementioned helper virus particle impurities are present in a disrupted form). Viral and / or cellular protein contamination can typically be observed due to the presence of Coomassie staining bands on the SDS gel (e.g., the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2, and VP3).

[0474] In some embodiments, host cells containing the aforementioned rAAV vector are enabled to provide AAV helper functions to replicate and encapsulate transgenes encoding proteins of interest flanked by AAV ITRs, thereby producing rAAV particles. AAV helper functions are typically AAV-derived coding sequences that can be expressed to provide AAV gene products, which in turn act trans-act on productive AAV replication. AAV helper functions are used herein to complement essential AAV functions missing in the rAAV vector. In some embodiments, AAV helper functions include one or both of the primary AAV ORF (i.e., the rep and cap coding regions) or their functional homologs.

[0475] AAV helper functions can be introduced into host cells by transfecting them with an AAV helper construct before or simultaneously with transfection of the rAAV vector. For example, an AAV helper construct can be used to provide at least transient expression of the AAV rep and / or cap genes to compensate for the loss of AAV function necessary for productive AAV infection. Typically, AAV helpers lack AAV ITRs and cannot replicate or package themselves. AAV helpers can be in the form of, for example, plasmids, phages, transposons, sticky plasmids, viruses, or virions.

[0476] In some embodiments, the host cell may also provide or be provided with non-AAV-derived functions or "accessory functions" to produce rAAV particles. Accessory functions are non-AAV-derived viral and / or AAV-dependent cellular functions, such as non-AAV proteins and RNA required for AAV replication, including those involved in the activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. In some embodiments, virus-based accessory functions may be derived from known helper viruses.

[0477] In some embodiments, as a result of infecting host cells with helper viruses and / or helper functional vectors, recombinant AAV particles are produced, and the produced rAAV particles are infectious replication-defective viruses and include an AAV protein shell encapsulating heterologous nucleotide sequences of interest of AAV ITR at both ends.

[0478] rAAV particles can be purified from host cells using purification methods known in the art, such as chromatography, CsCl gradients, and other methods, such as those described in U.S. Patent Nos. 6,989,264 and 8,137,948 and WO 2010 / 148143. In some embodiments, known methods, such as heating, can be used to inactivate residual helper viruses.

[0479] 5.6 Pharmaceutical Compositions In one aspect, this document provides a pharmaceutical composition comprising recombinant viral particles described herein, such as recombinant AAV particles described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3), and a pharmaceutically acceptable carrier.

[0480] In one aspect, this document provides a pharmaceutical composition comprising a nonviral gene delivery system described herein (e.g., the nonviral gene delivery system described in Section 5.3) and a pharmaceutically acceptable carrier.

[0481] In one aspect, this document provides a pharmaceutical composition comprising a host cell population stably transduced by recombinant viral particles as described herein, such as recombinant AAV particles as described herein (e.g., a host cell population stably transduced by recombinant viral particles, such as recombinant AAV particles, as described in Section 5.4), and a pharmaceutically acceptable carrier.

[0482] In one aspect, this document provides a pharmaceutical composition comprising a host cell population stably transduced by a nonviral gene delivery system as described herein (e.g., a host cell population stably transduced by a nonviral gene delivery system as described in Section 5.4), and a pharmaceutically acceptable vector.

[0483] In some embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 8 vg / ml up to 1x10 20 Within the range of vg / ml. In specific embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 9 vg / ml up to 1x10 19 Within the range of vg / ml. In specific embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 10 vg / ml up to 1x10 18 Within the range of vg / ml. In specific embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 11 vg / ml up to 1x10 17 Within the range of vg / ml. In specific embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 12 vg / ml up to 1x10 16Within the range of vg / ml. In specific embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 13 vg / ml up to 1x10 15 Within the range of vg / ml. In some embodiments, the concentration of recombinant viral particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 8 vg / ml up to 1x10 15 Within the range of vg / ml.

[0484] In some embodiments, the concentration of host cells in the pharmaceutical composition described herein can be 1 x 10⁻⁶. 2 cells / ml to 1x10 12 Within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein can be 1×10⁻⁶. 3 cells / ml to 1×10 11 Within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein can be 1×10⁻⁶. 4 cells / ml to 1×10 10 Within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein can be 1 x 102 5 cells / ml to 1x10 9 Within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein can be 1×10⁻⁶. 6 cells / ml to 1×10 8 Within the range of cells / ml.

[0485] In some embodiments, the pharmaceutical composition described herein is injected in a volume of about 0.1 ml to about 20 ml. In some embodiments, the pharmaceutical composition described herein is injected in a volume of about 1 ml to about 10 ml.

[0486] Generally, a drug is pharmaceutically acceptable and acceptable for veterinary and human use when the agent (such as excipients or carriers) is safe, non-toxic, and not biologically or otherwise undesirable.

[0487] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients to provide advantageous properties for storage and / or administration to a subject to treat a disease or disorder. In some embodiments, the pharmaceutical compositions described herein comprise one or more buffers, such as disodium hydrogen phosphate and / or sodium dihydrogen phosphate monohydrate. In some embodiments, the pharmaceutical compositions described herein comprise one or more isotonic agents, such as sodium chloride. In some embodiments, the pharmaceutical compositions described herein comprise one or more fillers, such as mannitol, sucrose, dextran, lactose, trehalose, and / or povidone (PVP K24). In some embodiments, the pharmaceutical compositions described herein comprise one or more surfactants, such as polysorbate 80, polysorbate 20, sodium lauryl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, and / or poloxamer 188.

[0488] Preferably, the pharmaceutical compositions described herein are stable and can be stored for extended periods without unacceptable changes in quality, potency, or purity, for example, at temperatures below -60°C, about -20°C, about 2°C to 8°C, and / or at room temperature.

[0489] Preferably, the pharmaceutical compositions described herein are sterile and stable under the conditions of manufacture and storage. The pharmaceutical compositions described herein can be formulated as solutions, microemulsions, liposomes, lyophilized compositions, or other ordered structures suitable for high drug concentrations.

[0490] In some embodiments, the pharmaceutical compositions described herein are formulated for administration to a subject via a route of administration. Non-limiting examples of possible routes of administration include direct delivery to the target organ, orally, by inhalation, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrathecally, intrapancreatically, intraperitoneally, intratumorally, and other parenteral administration routes. In specific embodiments, the pharmaceutical compositions described herein are formulated for systemic administration to a subject. In specific embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration to a subject.

[0491] In some embodiments, the pharmaceutical compositions provided herein contain viral particles, nonviral gene delivery systems, or host cell populations as described herein in an amount effective in treating or preventing a disease or disorder (e.g., as a therapeutically or preventively effective amount). In some embodiments, therapeutic or preventative efficacy is monitored by periodically evaluating the treated subjects.

[0492] 5.7 Methods and Applications In one aspect, this document provides a method for treating a disease or disorder in a subject in need, comprising administering to the subject (preferably a therapeutically effective amount) a recombinant viral particle as described herein, such as a recombinant AAV particle as described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3).

[0493] In one aspect, this document provides a method for treating a disease or disorder in a subject in need, comprising administering to the subject (preferably a therapeutically effective amount) a nonviral gene delivery system as described herein (e.g., the nonviral gene delivery system described in Section 5.3).

[0494] In one aspect, this document provides a method for treating a disease or disorder in a subject in need, comprising administering to the subject (preferably a therapeutically effective amount) a pharmaceutical composition described herein (e.g., the pharmaceutical composition described in Section 5.6).

[0495] In one aspect, this document provides a method for delivering biomolecules to one or more ex vivo target cells, comprising transducing the one or more target cells with recombinant viral particles described herein, such as recombinant AAV particles described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3).

[0496] In one aspect, this document provides a method for delivering biomolecules to one or more ex vivo target cells, including transducing the one or more target cells using a nonviral gene delivery system described herein (e.g., the nonviral gene delivery system described in Section 5.3).

[0497] In one aspect, this document provides a method for delivering biomolecules to one or more in vitro target cells, comprising transducing the one or more target cells with recombinant viral particles described herein, such as recombinant AAV particles described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3).

[0498] In one aspect, this document provides a method for delivering biomolecules to one or more in vitro target cells, including transducing the one or more target cells using a nonviral gene delivery system described herein (e.g., the nonviral gene delivery system described in Section 5.3).

[0499] In one aspect, this document provides a method for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering to the subject recombinant viral particles described herein, such as recombinant AAV particles described herein (e.g., recombinant viral particles, such as the recombinant AAV particles described in Section 5.3).

[0500] In one aspect, this article provides a method for delivering biomolecules to one or more in vivo target cells in a subject, the method comprising administering to the subject a nonviral gene delivery system described herein (e.g., the nonviral gene delivery system described in Section 5.3).

[0501] On the other hand, this document provides a method for delivering a biomolecule to one or more target cells in vivo in a subject, including administering to the subject a pharmaceutical composition described herein (e.g., the pharmaceutical composition described in Section 5.6).

[0502] Further description of biomolecules is provided in Section 5.2.4.

[0503] In some embodiments, the one or more target cells are one or more hepatocytes. In some embodiments, the one or more target cells are one or more muscle cells. In some embodiments, the one or more target cells are one or more kidney cells. In some embodiments, the one or more target cells are one or more hepatocytes and one or more muscle cells. In some embodiments, the one or more target cells are one or more hepatocytes and one or more kidney cells. In some embodiments, the one or more target cells are one or more hepatocytes, one or more muscle cells, and one or more kidney cells. In specific embodiments, the one or more muscle cells described herein are one or more skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or one or more cardiomyocytes (e.g., atrial and / or ventricular muscle cells). In certain embodiments, muscle can be used as a production factory for biomolecules secreted from muscle cells and ultimately absorbed by cells of another tissue or organ to exert their therapeutic effects.

[0504] The route of administration or delivery, and the amount of recombinant viral particles (e.g., recombinant AAV particles), nonviral gene delivery systems, or pharmaceutical compositions to be administered to the subject, can be determined based on the nature of the disease or disorder, the subject's condition, and the physician's knowledge. Non-limiting examples of possible routes of administration or delivery include direct delivery to the target organ, oral, inhalation, intravenous, intramuscular, subcutaneous, intradermal, intranasal, intrathecal, intrapancreatic, intraperitoneal, intratumoral, and other parenteral administration routes. In specific embodiments, the recombinant viral particles (e.g., recombinant AAV particles), nonviral gene delivery systems, or pharmaceutical compositions described herein are administered or delivered systemically. In specific embodiments, the recombinant viral particles (e.g., recombinant AAV particles), nonviral gene delivery systems, or pharmaceutical compositions described herein are administered or delivered intravenously.

[0505] In some implementations, the method uses 1x10 8 Up to 1x1017 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome (vg). In some embodiments, the method is carried out at 1 x 10 9 Up to 1x10 16 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome (vg). In some embodiments, the method is carried out at 1 x 10 10 Up to 1x10 15 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome (vg). In some embodiments, the method is carried out at 1 x 10 11 Up to 1x10 14 The dose of the vector genome (vg) is administered to the subject as described herein, consisting of recombinant viral particles (e.g., recombinant AAV particles).

[0506] In some implementations, the method uses 1x10 8 Up to 1x10 17 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome / kg (vg / kg). In some embodiments, the method is carried out at 1x10 9 Up to 1x10 16 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome / kg (vg / kg). In some embodiments, the method is carried out at 1x10 10 Up to 1x10 15 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to the subject at a dose of vector genome / kg (vg / kg). In some embodiments, the method is carried out at 1x10 11 Up to 1x10 14 The recombinant viral particles (e.g., recombinant AAV particles) described herein are administered to subjects at a dose of vector genome / kg (vg / kg).

[0507] The delivery methods and treatments described herein can be used to treat a variety of diseases or disorders, including but not limited to genetic disorders (such as lysosomal storage diseases), cancers (such as carcinomas, sarcomas, leukemias, lymphomas, germ cell tumors, and blastomas), autoimmune diseases, and infectious diseases.

[0508] In a specific embodiment, the disease or disorder is caused at least by liver dysfunction. In a specific embodiment, the disease or disorder is caused at least by dysfunction of muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) and / or cardiac muscles (e.g., atrial and / or ventricular muscles)). In a specific embodiment, the disease or disorder is caused at least by kidney dysfunction. In a specific embodiment, the disease or disorder is caused by at least liver dysfunction and muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major) dysfunction). In a specific embodiment, the disease or disorder is caused by at least liver dysfunction and kidney dysfunction. In a specific embodiment, the disease or disorder is caused by at least liver dysfunction, muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major)) dysfunction, and kidney dysfunction.

[0509] In specific embodiments, the disease or disorder is associated with hepatocytes. In specific embodiments, the disease or disorder is associated with muscle cells (e.g., skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major cells) and / or cardiomyocytes (e.g., atrial and / or ventricular cells)). In specific embodiments, the disease or disorder is associated with kidney cells. In specific embodiments, the disease or disorder is associated with both hepatocytes and muscle cells (e.g., skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major cells) and / or cardiomyocytes (e.g., atrial and / or ventricular cells)). In specific embodiments, the disease or disorder is associated with both hepatocytes and kidney cells. In a specific embodiment, the disease or disorder is associated with hepatocytes, muscle cells (e.g., skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major cells) and / or cardiomyocytes (e.g., atrial and / or ventricular cells)) and kidney cells.

[0510] In some implementations, the disease or disorder is lysosomal storage disease.

[0511] In a specific embodiment, the disease or disorder is Fabry disease. In a specific embodiment, the disease or disorder is action myoclonus-renal failure syndrome. In a specific embodiment, the disease or disorder is aspartic glucosamineuria. In a specific embodiment, the disease or disorder is CLN1 disease or neuronal ceroid lipofuscin deposition syndrome 1. In a specific embodiment, the disease or disorder is CLN2 disease or Jansky–Bielschowsky disease. In a specific embodiment, the disease or disorder is CLN3 disease. In a specific embodiment, the disease or disorder is CLN4 disease. In a specific embodiment, the disease or disorder is CLN5 disease. In a specific embodiment, the disease or disorder is CLN6 disease. In a specific embodiment, the disease or disorder is CLN7 disease. In a specific embodiment, the disease or disorder is CLN8 disease. In a specific embodiment, the disease or disorder is CLN10 disease. In a specific embodiment, the disease or disorder is CLN11 disease. In a specific embodiment, the disease or disorder is CLN12 disease. In a specific embodiment, the disease or disorder is CLN13 disease. In a specific embodiment, the disease or disorder is CLN14 disease. In a specific embodiment, the disease or disorder is cystine storage disease. In a specific embodiment, the disease or disorder is Danon disease. In a specific embodiment, the disease or disorder is Farber lipogranulomatosis. In a specific embodiment, the disease or disorder is fucoside storage disease. In a specific embodiment, the disease or disorder is galactosialidin storage disease. In a specific embodiment, the disease or disorder is Gaucher disease. In a specific embodiment, the disease or disorder is glycogen storage disease type 2 (GSD II) or Pompe disease. In a specific embodiment, the disease or disorder is Griseli syndrome 1 or Elejalde syndrome. In a specific embodiment, the disease or disorder is Griseli syndrome 2. In a specific embodiment, the disease or disorder is Chédiak-Higashi syndrome. In a specific embodiment, the disease or disorder is GM1 ganglioside storage disease. In a specific embodiment, the disease or disorder is GM2 ganglioside storage disease (Tay-Sachs disease). In a specific embodiment, the disease or disorder is Sandhoff's disease, a GM2 ganglioside storage disorder. In a specific embodiment, the disease or disorder is GM2 activator deficiency in GM2 ganglioside storage disorder. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 1. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 2. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 3.In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 4. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 5. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 6. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 7. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 8. In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 9. In a specific embodiment, the disease or disorder is Krabby's disease. In a specific embodiment, the disease or disorder is α-mannosinolate storage disease. In a specific embodiment, the disease or disorder is β-mannosinolate storage disease. In a specific embodiment, the disease or disorder is metachromatic leukodystrophy. In a specific embodiment, the disease or disorder is mucolipidemia II α / β or I cell disease. In a specific embodiment, the disease or disorder is mucolipidemia III α / β or pseudo-Hurler's multiple malnutrition. In a specific embodiment, the disease or disorder is mucolipidemia III γ, variant pseudo-Hurler's multiple malnutrition. In a specific embodiment, the disease or disorder is mucolipidemia IV. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type I (MPS I) or Hurler's syndrome. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type II (MPS II) or Hunter's syndrome. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIA (MPS IIIA) or Sanfilippo syndrome A. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo syndrome B. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIC (MPS IIIC) or Sanfilippo syndrome C. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIID (MPS IIID) or Sanfilippo syndrome D. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome A. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IVB (MPS IVB) or Morquio syndrome B. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VII (MPS VII) or Sly disease.In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IX (MPS IX). In a specific embodiment, the disease or disorder is multiple sulfatase deficiency. In a specific embodiment, the disease or disorder is Niemann-Pick disease types A and B. In a specific embodiment, the disease or disorder is Niemann-Pick disease type C1. In a specific embodiment, the disease or disorder is Niemann-Pick disease type C2. In a specific embodiment, the disease or disorder is Sala disease. In a specific embodiment, the disease or disorder is Schindler disease or Kanzaki disease. In a specific embodiment, the disease or disorder is sialic acid deposition type I or cherry red spot myoclonic syndrome. In a specific embodiment, the disease or disorder is Wolman disease and cholesterol ester storage disease. In a specific embodiment, the disease or disorder is spinal muscular atrophy. In a specific embodiment, the disease or disorder is Duchenne muscular dystrophy (DMD). In a specific embodiment, the disease or disorder is phenylalanine hydroxylase deficiency. In a specific embodiment, the disease or disorder is hemophilia A. In a specific implementation, the disease or disorder is hemophilia B.

[0512] In a specific embodiment, the disease or disorder is Fabry disease, and the biomolecule is a protein, α-galactosidase A (GLA) (preferably human GLA). In a specific embodiment, the disease or disorder is action myoclonus-renal failure syndrome, and the biomolecule is a protein, scavenger receptor class B member 2 (SCARB2) (preferably human SCARB2). In a specific embodiment, the disease or disorder is aspartylglucosamineuria, and the biomolecule is a protein, aspartate glucosidase (AGA) (preferably human AGA). In a specific embodiment, the disease or disorder is CLN1 disease or neuronal ceroid lipofuscin deposition syndrome 1, and the biomolecule is a protein, palmitoyl protein thioesterase 1 (PPT1) (preferably human PPT1). In a specific embodiment, the disease or disorder is CLN2 disease or Jansky–Bielschowsky disease, and the biomolecule is a protein, specifically tripeptidyl peptidase 1 (TPP1) (preferably human TPP1). In a specific embodiment, the disease or disorder is CLN3 disease, and the biomolecule is a protein, specifically CLN3 lysosomal / endosomal transmembrane protein, battenin (CLN3) (preferably human CLN3). In a specific embodiment, the disease or disorder is CLN4 disease, and the biomolecule is a protein, specifically C5 (DNAJC5), a member of the DnaJ heat shock protein family (Hsp40) (preferably human DNAJC5). In a specific embodiment, the disease or disorder is CLN5 disease, and the biomolecule is a protein, specifically CLN5 intracellular transport protein (CLN5) (preferably human CLN5). In a specific embodiment, the disease or disorder is CLN6 disease, and the biomolecule is a protein, specifically CLN6 transmembrane ER protein (CLN6) (preferably human CLN6). In a specific embodiment, the disease or disorder is a CLN7 disease, and the biomolecule is a protein containing the major promoting factor superfamily domain 8 (MFSD8) (preferably human MFSD8). In a specific embodiment, the disease or disorder is a CLN8 disease, and the biomolecule is a protein, specifically the CLN8 transmembrane ER and ERGIC protein (CLN8) (preferably human CLN8). In a specific embodiment, the disease or disorder is a CLN10 disease, and the biomolecule is a protein, specifically cathepsin D (CTSD) (preferably human CTSD).In a specific embodiment, the disease or disorder is CLN11, and the biomolecule is a protein, specifically a granular protein precursor (GRN) (preferably human GRN). In a specific embodiment, the disease or disorder is CLN12, and the biomolecule is a protein, specifically ATPase cation transporting 13A2 (ATP13A2) (preferably human ATP13A2). In a specific embodiment, the disease or disorder is CLN13, and the biomolecule is a protein, specifically cathepsin F (CTSF) (preferably human CTSF). In a specific embodiment, the disease or disorder is CLN14, and the biomolecule is a protein, specifically potassium channel tetramer domain protein 7 (KCTD7) (preferably human KCTD7). In a specific embodiment, the disease or disorder is cystine storage disease, and the biomolecule is a protein, specifically cystinosin, lysosomal cystine transporter (CTNS) (preferably human CTNS). In a specific embodiment, the disease or disorder is Danon's disease, and the biomolecule is a protein, specifically lysosome-associated membrane protein 2 (LAMP2) (preferably human LAMP2). In a specific embodiment, the disease or disorder is Farber's lipogranulomatosis, and the biomolecule is a protein, specifically N-acylsphingosine amide hydrolase 1 (ASAH1) (preferably human ASAH1). In a specific embodiment, the disease or disorder is fucosidosis, and the biomolecule is a protein, specifically α-L-fucosidase 1 (FUCA1) (preferably human FUCA1). In a specific embodiment, the disease or disorder is galactosialidosis, and the biomolecule is a protein, specifically cathepsin A (CTSA) (preferably human CTSA). In a specific embodiment, the disease or disorder is Gaucher's disease, and the biomolecule is a protein, specifically β-glucocerebrosidase (GBA) (preferably human GBA). In a specific embodiment, the disease or disorder is glycogen storage disease type 2 (GSD II) or Pompe disease, and the biomolecule is a protein, which is acid α-glucosidase (GAA) (preferably human GAA). In a specific embodiment, the disease or disorder is Griscelli syndrome 1 or Elejalde syndrome, and the biomolecule is a protein, which is myosin VA (MYO5A) (preferably human MYO5A).In a specific embodiment, the disease or disorder is Griselly syndrome 2, and the biomolecule is a protein, RAB27A (RAB27A), a member of the RAS oncogene family (preferably human RAB27A). In a specific embodiment, the disease or disorder is Chédiak-Higashi syndrome, and the biomolecule is a protein, lysosomal transport regulator (LYST) (preferably human LYST). In a specific embodiment, the disease or disorder is GM1 gangliosidosis, and the biomolecule is a protein, galactosidase β1 (GLB1) (preferably human GLB1). In a specific embodiment, the disease or disorder is GM2 gangliosidosis (Tay-Sachs disease), and the biomolecule is a protein, hexosamine subunit α (HEXA) (preferably human HEXA). In a specific embodiment, the disease or disorder is GM2 gangliosidosis (Sandhoff disease), and the biomolecule is a protein, hexosamine subunit β (HEXB) (preferably human HEXB). In a specific embodiment, the disease or disorder is GM2 ganglioside deposition syndrome with GM2 activator deficiency, and the biomolecule is a protein, namely ganglioside GM2 activator (GM2A) (preferably human GM2A). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 1, and the biomolecule is a protein, namely HPS1 lysosomal organelle biogenesis complex 3 subunit 1 (HPS1) (preferably human HPS1). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 2, and the biomolecule is a protein, namely adaptor factor-associated protein complex 3 subunit β1 (AP3B1 or HPS2) (preferably human HPS2). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 3, and the biomolecule is a protein, namely HPS3 lysosomal organelle biogenesis complex 2 subunit 1 (HPS3) (preferably human HPS3). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 4, and the biomolecule is a protein, namely HPS4 lysosomal organelle biogenesis complex 3 subunit 2 (HPS4) (preferably human HPS4). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 5, and the biomolecule is a protein, namely HPS5 lysosomal organelle biogenesis complex 2 subunit 2 (HPS5) (preferably human HPS5).In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 6, and the biomolecule is a protein, specifically HPS6 lysosomal organelle biogenesis complex 2 subunit 3 (HPS6) (preferably human HPS6). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 7, and the biomolecule is dystrophic short protein-binding protein 1 (DTNBP1 or HPS7) (preferably human HPS7). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 8, and the biomolecule is a protein, specifically lysosomal organelle biogenesis complex 1 subunit 3 (BLOC1S3 or HPS8) (preferably human HPS8). In a specific embodiment, the disease or disorder is Hermansky-Pudlak syndrome type 9, and the biomolecule is a protein, specifically lysosomal organelle biogenesis complex 1 subunit 6 (BLOC1S6 or HPS9) (preferably human HPS9). In a specific embodiment, the disease or disorder is Krabby's disease, and the biomolecule is a protein, which is galactosylceramidinase (GALC) (preferably human GALC). In a specific embodiment, the disease or disorder is α-mannosinolate storage disease, and the biomolecule is a protein, which is mannosidase α 2B class member 1 (MAN2B1) (preferably human MAN2B1). In a specific embodiment, the disease or disorder is β-mannosinolate storage disease, and the biomolecule is a protein, which is mannosidase β (MANBA) (preferably human MANBA). In a specific embodiment, the disease or disorder is metachromatic leukodystrophy, and the biomolecule is a protein, which is arylsulfatase A (ARSA) (preferably human ARSA). In a specific embodiment, the disease or disorder is mucolipidemia II α / β or I-cell disease, and the biomolecule is a protein, which is N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably human GNPTAB). In a specific embodiment, the disease or disorder is mucolipidemia III α / β or pseudo-Hurler's multiple malnutrition, and the biomolecule is a protein, specifically N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably human GNPTAB). In a specific embodiment, the disease or disorder is mucolipidemia III γ, variant pseudo-Hurler's multiple malnutrition, and the biomolecule is a protein, specifically N-acetylglucosamine-1-phosphotransferase subunit γ (GNPTG) (preferably human GNPTG).In a specific embodiment, the disease or disorder is mucolipidemia IV, and the biomolecule is a protein, namely mucolipin TRP cation channel 1 (MCOLN1) (preferably human MCOLLN1). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type I (MPS I) or Hurler syndrome, and the biomolecule is a protein, namely α-L-iduronidase (IDUA) (preferably human IDUA). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type II (MPS II) or Hunter syndrome, and the biomolecule is a protein, namely iduronate 2-sulfatase (IDS) (preferably human IDS). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIA (MPSIIIA) or Sanfilippo syndrome A, and the biomolecule is a protein, namely N-sulfamethoxamylsulfahydrogenase (SGSH) (preferably human SGSH). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo syndrome B, and the biomolecule is a protein, namely N-acetyl-α-glucosidase (NAGLU) (preferably human NAGLU). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIC (MPS IIIC) or Sanfilippo syndrome C, and the biomolecule is a protein, namely heparan-α-glucosamine N-acetyltransferase (HGSNAT) (preferably human HGSNAT). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIID (MPS IIID) or Sanfilippo syndrome D, and the biomolecule is a protein, namely glucosamine (N-acetyl)-6-sulfatase (GNS) (preferably human GNS). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome A, and the biomolecule is a protein, which is galactosamine (N-acetyl)-6-sulfatase (GALNS) (preferably human GALNS). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IVB (MPS IVB) or Morquio syndrome B, and the biomolecule is a protein, which is galactosidase β1 (GLB1) (preferably human GLB1). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome, and the biomolecule is a protein, which is arylsulfatase B (ARSB) (preferably human ARSB).In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VII (MPS VII) or Sly disease, and the biomolecule is a protein, namely β-glucuronidase (GUSB) (preferably human GUSB). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IX (MPSIX), and the biomolecule is a protein, namely hyaluronidase 1 (HYAL1) (preferably human HYAL1). In a specific embodiment, the disease or disorder is multiple sulfatase deficiency, and the biomolecule is a protein, namely sulfatase modifying factor 1 (SUMF1) (preferably human SUMF1). In a specific embodiment, the disease or disorder is Niemann-Pick disease type A and B, and the biomolecule is a protein, namely sphingomyelin phosphodiesterase 1 (SMPD1) (preferably human SMPD1). In a specific embodiment, the disease or disorder is Niemann-Pick disease type C1, and the biomolecule is a protein, namely NPC intracellular cholesterol transporter 1 (NPC1) (preferably human NPC1). In a specific embodiment, the disease or disorder is Niemann-Pick disease C2, and the biomolecule is a protein, namely NPC intracellular cholesterol transporter 2 (NPC2) (preferably human NPC2). In a specific embodiment, the disease or disorder is Sala disease, and the biomolecule is a protein, namely solute carrier family 17 member 5 (SLC17A5) (preferably SLC17A5). In a specific embodiment, the disease or disorder is Schindler disease or Kanzaki disease, and the biomolecule is a protein, namely α-N-acetylgalactosaminease (NAGA) (preferably human NAGA). In a specific embodiment, the disease or disorder is sialic acid deposition type I or cherry rash myoclonic syndrome, and the biomolecule is a protein, namely neuraminidase 1 (NEU1) (preferably human NEU1). In a specific embodiment, the disease or disorder is Wolman disease and cholesterol ester storage disease, and the biomolecule is a protein, namely lipase A, lysosomal acid form (LIPA) (preferably human LIPA). In a specific embodiment, the disease or disorder is spinal muscular atrophy, and the biomolecule is a protein, specifically a motor neuron survival (SMN) protein (preferably human SMN protein). In a specific embodiment, the disease or disorder is Duchenne muscular dystrophy (DMD), and the biomolecule is a protein, specifically dystrophin (preferably human dystrophin). In a specific embodiment, the disease or disorder is phenylalanine hydroxylase deficiency, and the biomolecule is a protein, specifically phenylalanine hydroxylase (PAH) (preferably human PAH).In a specific embodiment, the disease or disorder is hemophilia A, and the biomolecule is a protein, specifically coagulation factor VIII (FVIII) (preferably human FVIII). In a specific embodiment, the disease or disorder is hemophilia B, and the biomolecule is a protein, specifically coagulation factor IX (FIX) (preferably human FIX). In a preferred embodiment, the biomolecule is a functional protein (e.g., a wild-type protein).

[0513] In some embodiments, the method is used to treat hereditary hemochromatosis (HH), major symptoms of iron overload, Wilson's disease, genetic symptoms of copper overload, or α1-antitrypsin deficiency. In some embodiments, the biomolecule is human α1-antitrypsin (accession number: P01009.3), HFE protein (accession number NP_000401.1 or Q30201), or hepatin ATP7B (accession number P35670.4), or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0514] In some embodiments, the method is used for the treatment of hypercholesterolemia. In some embodiments, the biomolecule is human phenylalanine hydroxylase (accession number: P00439.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0515] In some embodiments, the method is used for the treatment of type 1 tyrosinemia. In some embodiments, the biomolecule is human fumaroyl acetoacetate hydrolase (accession number: P16930.2) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95 or 95.

[0516] In some embodiments, the method is used for the treatment of type 2 tyrosinemia. In some embodiments, the biomolecule is a human tyrosine aminotransferase (accession number: P17735.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0517] In some embodiments, the method is used for the treatment of homocystinuria and hyperhomocysteinemia. In some embodiments, the biomolecule is human methyltetrahydrofolate reductase (accession number: P42898.3) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0518] In some embodiments, the method is used for the treatment of hyperlipidemia and hypercholesterolemia. In some embodiments, the biomolecule is human mid-chain acyl-CoA dehydrogenase (accession number: P11310.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0519] In some embodiments, the method is used for the treatment of galactosemia. In some embodiments, the biomolecule is human galactose-1-phosphate uridine transferase (accession number: P07902.3) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95 or 95.

[0520] In some embodiments, the method is used for the treatment of Lesch-Nyhan syndrome. In some embodiments, the biomolecule is human hypoxanthine phosphoribosyltransferase (accession number: P00492.2) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0521] In some embodiments, the method is used for the treatment of Gaucher disease. In some embodiments, the biomolecule is a human brain lipase (accession number: P07602.2, accession number: P04062.3) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95 or 95.

[0522] In some embodiments, the method is used for the treatment of Tay-Sachs disease. In some embodiments, the biomolecule is human β-hexosamine enzyme A (accession number: P06865.2) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0523] In some embodiments, the method is used for the treatment of Fabry disease. In some embodiments, the biomolecule is human α-galactosidase (accession number: P06280.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0524] In some embodiments, the method is used for the treatment of Hunter syndrome. In some embodiments, the biomolecule is human iduronate sulfatase (accession number: P22304.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0525] In some embodiments, the method is used for the treatment of glycogen storage disease type Ia. In some embodiments, the biomolecule is human glucose-6-phosphatase (accession number: P35575.2) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0526] In some embodiments, the method is used for the treatment of ammonia metabolism. In some embodiments, the biomolecule is human ornithine carbamoyltransferase (accession number: P00480.3) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95 or 95.

[0527] In some embodiments, the method is used for the treatment of phenylketonuria. In some embodiments, the biomolecule is a human low-density lipoprotein receptor (accession number: P01130.1) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95, or 95.

[0528] In some embodiments, the method is used for the treatment of propionic acidemia. In some embodiments, the biomolecule is human propionyl-CoA carboxylase, PCCA and / or PCCB (accession numbers: P05166.3β, NP_000273.2α, NP_001121164.1α) or a variant having sequence identity or similarity greater than 50, 60, 70, 80, 90, 95 or 95.

[0529] 5.8 Measurement 5.8.1 Analysis of mRNA levels Changes in gene levels or expression can be measured using a variety of methods known in the art.

[0530] For example, a variety of methods for detecting or quantifying mRNA levels are known in the art. Exemplary methods include, but are not limited to, northern blot, ribonuclease protection assays, PCR-based methods, etc. The mRNA sequence of a gene can be used to prepare probes that are at least partially complementary to the mRNA sequence. The probes can then be used to detect mRNA in a sample using any suitable assay, such as PCR-based methods, RNA blot, dipstick assays, etc.

[0531] The assay method can vary depending on the type of mRNA information required. Exemplary methods include, but are not limited to, RNA blotting and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantify the amount of mRNA in a sample.

[0532] The presence of mRNA in a sample can be determined using any suitable assay platform. For example, the assay can take the form of a measuring rod, membrane, chip, disk, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The assay system can have a solid support on which the nucleic acid corresponding to the mRNA is attached. The solid support can include, for example, plastic, silicon, metal, resin, glass, membrane, particles, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The assay components can be prepared and packaged together as a kit for detecting mRNA.

[0533] If desired, nucleic acids can be labeled to prepare labeled mRNA clusters. Typically, samples can be labeled using methods well-known in the art, such as DNA ligases, terminal transferases, or by labeling the RNA backbone. See, for example, Ausubel et al., Short Protocols in Molecular Biology (Wiley & Sons, 3rd ed. 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY, 3rd ed. 2001). In some embodiments, samples are labeled with fluorescent markers. Exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes (e.g., fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), and rhodamine dyes (e.g., rhodamine 110 (R110), N,N,N',N'). -Tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6)), anthocyanin dyes (e.g., Cy3, Cy5, and Cy7), Alexa dyes (e.g., Alexa-fluor-555), coumarin, diethylaminocoumarin, umbelliferone, benzylimine dyes (e.g., Hoechst 33258), phenanthridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethystine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazine (Ctriazinyl), eosin dyes, tetramethylrhodamine, lissamine, naphthylfluorescein, etc.

[0534] A typical mRNA assay may include the following steps: (1) obtaining a surface-bound subject probe; (2) hybridizing a population of mRNAs to the surface-bound probe under conditions sufficient to provide specific binding; (3) washing after hybridization to remove nucleic acids that have not specifically bound to the surface-bound probe; and (4) detecting the hybridized mRNA. The reagents used in each of these steps and the conditions under which they are used may vary depending on the specific application.

[0535] Hybridization can be performed under suitable hybridization conditions, the stringency of which can be varied as needed. Typical conditions are sufficient to produce probe / target complexes between complementary binding members, i.e., on a solid surface between the subject probe and the complementary mRNA bound to the surface in the sample. In some implementations, stringent hybridization conditions may be employed.

[0536] Hybridization is typically performed under strict hybridization conditions. Standard hybridization techniques (e.g., under conditions sufficient to provide specific binding of the target mRNA in the sample to the probe) are described in Kallioniemi et al., Science, 258:818-821 (1992) and International Patent Application Publication No. WO 93 / 18186. Several guidelines for general techniques are available, for example, Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993). For a description of techniques suitable for in situ hybridization, see Gall et al., Meth. Enzymol. 1981, 21:470-480; Angerer et al., Genetic Engineering: Principles and Methods, Vol 7, pgs 43-65 (Plenum Press, New York, Setlow and Hollaender, eds. 1985). The selection of appropriate conditions (including temperature, salt concentration, polynucleotide concentration, hybridization time, and the stringency of washing conditions) will depend on the experimental design, including the sample source, the type (identity) of the trapping agent, the expected degree of complementarity, etc., and can be determined based on routine experiments performed by those skilled in the art.

[0537] Ordinary technicians will readily recognize that conditions with similar stringency can be provided using alternative but comparable hybridization and washing conditions.

[0538] Following the mRNA hybridization procedure, surface-bound polynucleotides are typically washed to remove unbound nucleic acids. Washing can be performed using any convenient washing protocol, where the washing conditions are usually stringent, as described above. Hybridization of the target mRNA with the probe is then detected using standard techniques.

[0539] Other methods, such as PCR-based methods, can also be used to detect gene expression. Examples of PCR methods can be found in U.S. Patent No. 6,927,024, the entire contents of which are incorporated herein by reference. Examples of RT-PCR methods can be found in U.S. Patent No. 7,122,799, the entire contents of which are incorporated herein by reference. A method for fluorescent in situ PCR is described in U.S. Patent No. 7,186,507, the entire contents of which are incorporated herein by reference.

[0540] In some implementations, quantitative reverse transcription PCR (qRT-PCR) can be used to detect and quantify both RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). Quantitative results obtained by qRT-PCR are generally more informative than qualitative data. Therefore, in some implementations, qRT-PCR-based assays can be used to measure mRNA levels during cell-based assays. qRT-PCR methods can also be used to monitor patient treatment. Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7,101,663, the entire contents of which are incorporated herein by reference.

[0541] Unlike conventional reverse transcriptase PCR and agarose gel analysis, qRT-PCR provides quantitative results. Another advantage of qRT-PCR is its relative ease of use. Instruments for qRT-PCR (e.g., Applied Biosystems 7500) are commercially available, as are reagents (e.g., TaqMan® Sequence Detection Chemistry). For example, the TaqMan® Gene Expression Assay can be used according to the manufacturer's instructions. These kits are pre-formulated gene expression assays for the rapid and reliable detection and quantification of human, mouse, and rat mRNA transcripts. To determine the number of cycles in which the fluorescence signal associated with the accumulation of a particular amplicon crosses a threshold (called CT), data can be analyzed, for example, using the 7500 Real-Time PCR system sequence detection software relative to a comparative CT quantification method. Using this method, the output is expressed as a fold change in expression level. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set above baseline but low enough to ensure it is within the exponential growth region of the amplification curve.

[0542] In other implementations, the target RNA can be detected or quantified by next-generation sequencing (NGS).

[0543] 5.8.2 Analysis of protein levels Changes in protein expression levels can be assessed by measuring the levels of the protein of interest. Protein levels can be assessed or quantified in a variety of ways known in the art, such as immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS), LC-MS (liquid chromatography-mass spectrometry), and other methods. Antibodies against the target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or prepared using conventional monoclonal or polyclonal antibody production methods known in the art. Antibodies for detecting proteins of interest in mice, rats, monkeys, and humans are commercially available. In the case of MassSpec, protein levels can be measured using labeled or unlabeled methods.

[0544] 5.8.3 In vivo analysis In vivo assays can be used to evaluate the therapeutic efficacy of recombinant viral particles, including the recombinant AAV particles of this disclosure, non-viral gene delivery systems, or pharmaceutical compositions.

[0545] In some implementations, motor function is measured by the animal's righting and openfield performance. In some implementations, respiration is measured by whole-body plethysmograph, invasive resistance, and compliance measurement.

[0546] In some implementations, overall survival (OS) and disease-free survival (DFS) are measured by observing the weight and health status of the animals twice daily.

[0547] The tests can be performed in normal animals or experimental disease models. For administration to animals, the oligonucleotides can be formulated in pharmaceutically acceptable diluents such as phosphate-buffered saline. Administration includes parenteral routes, such as intraperitoneal, intravenous, and subcutaneous. The dosage and frequency of administration of the recombinant viral particles of this disclosure (including recombinant AAV particles, non-viral gene delivery systems, or pharmaceutical compositions) are calculated within the capabilities of those skilled in the art and depend on factors such as the route of administration and animal weight. After treatment with the recombinant viral particles of this disclosure (including recombinant AAV particles, non-viral gene delivery systems, or pharmaceutical compositions) for a period of time, RNA or proteins can be isolated from tissues of interest (including liver, muscle, kidney, spleen, CNS tissue, or CSF), and changes in the expression of the protein of interest can be measured, for example, using NGS.

[0548] 5.9 Reagent Kits and Products Kits, unit doses, and articles thereof comprising any of the compositions described herein are also provided. In some embodiments, a kit is provided containing any of the pharmaceutical compositions described herein, and preferably, instructions for use are provided.

[0549] The kit of this application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film (Mylar) or plastic bags), etc. The kit may optionally include additional components, such as buffer solutions and explanatory information. Therefore, this application also provides articles of manufacture, including vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0550] The manufactured article may include a container and a label or packaging appendix on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials such as glass or plastic. Typically, the container contains a composition that effectively treats the disease or disorder described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The label or packaging appendix indicates that the composition is intended to treat a specific condition in an individual. The label or packaging appendix will further include instructions for administering the composition to the individual. The label may indicate instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-purpose vial that allows for repeated administration (e.g., 2-6 doses) of the reconstituted formulation. The packaging appendix refers to the instructions for use typically included in the commercial packaging of a therapeutic product, which contains information about indications, uses, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Additionally, the manufactured article may include a second container containing a pharmaceutically acceptable buffer solution, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. From a commercial and user perspective, other desired materials may also be included, including additional buffers, diluents, filters, needles, and syringes.

[0551] The kit or manufactured product may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies (such as hospital pharmacies and dispensing pharmacies).

[0552] sequence Table 1. Sequence List

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585] Example Certain embodiments provided herein are illustrated by the following non-limiting examples, which describe the design and testing of various human α-galactosidase A coding sequences, promoters, expression cassettes, and recombinant adeno-associated virus (AAV) particles, demonstrating their particular suitability for gene therapy, especially for lysosomal storage diseases including Fabry disease.

[0586] Example 1: Correction of substrate levels in a mouse model of Fabry disease by intravenous administration of AAV-GLA 1. Codon optimization for human α-galactosidase A (GLA).

[0587] Two codon-optimized versions of the wild-type (WT) human GLA coding sequence (hGLAco1 and hGLAco2, with nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively) were synthesized and cloned into the expression cassette backbone along with the chicken-β actin (CB) promoter and bovine growth hormone (bGH) poly A sequence. HepG2 cells were seeded into 48-well plates (1e5 cells / well). Approximately 20 hours after seeding, 500 ng of plasmids expressing hGLA or GFP (negative control, Neg.ctrl) were transfected using Lipofectamine 3000. 72 hours after transfection, the supernatant was collected and centrifuged at 2,000 rpm for 10 min. Samples for GLA were diluted 100 or 1000-fold, and undiluted samples from the GFP group were used for ELISA using a human α-galactosidase AELISA kit (Cat. No. SEK12078). Figure 1 A). Dilute the sample 10-fold to determine α-galactosidase A activity using kit (ab239716). Figure 1 B). Using anti-α-galactosidase polyclonal antibody (1:1000, PA5-27349, Thermo Fisher), 10 μL of sample, either pretreated or untreated with PNGase F, was loaded for Western blot analysis. Figure 1 C). The pattern following PNGase F treatment also demonstrated appropriate glycosylation of α-galactosidase A expressed from HepG2 cells. hGLAco1 showed 2-fold higher protein expression than wild-type GLA. Therefore, hGLAco1 (SEQ ID NO.1) was selected for further investigation.

[0588] 2. Hepatocyte-cardiomyocyte (HC) bispecific promoter in in vitro testing.

[0589] Five promoters (HC1-HC5, with nucleotide sequences of SEQ ID NO: 4-8 respectively) containing a fixed 59-bp hepatocyte promoter (SEQ ID NO. 3) linked to various cardiomyocyte-specific transcription factor binding sites (C1-C5) were cloned upstream of the René luciferase (RLuc) reporter gene. Another reporter gene driven by the HSV-TK promoter, firefly luciferase (FLuc), was cloned on the same construct and used as an internal control. Rat cardiomyocyte cell lines H9c2 (ATCC) and HepG2 were seeded in 96-well plates. One day after seeding, plasmids containing promoters HC1-HC5 were transfected using Lipofectamine 3000. Two days after transfection, luminescent quantification was performed using a dual-luciferase assay kit (Cat. No. E1500, Promega) and a SpectraMax i3x Multi-Mode Microplate Reader with a SpectraMax Injector Cartridge, following the manufacturer's instructions. The RLuc / FLuc ratio was used to measure the intensity of the HC promoter ( Figure 2 The RLuc plasmid driven by the SV40 promoter and the FLuc plasmid driven by the HSV-TK promoter were used as positive controls (Pos.Ctrl). The HC1 promoter (SEQ ID NO.4) showed strong gene expression in both HepG2 and H9c2. Figure 2 It was selected for in vivo studies.

[0590] 3. The truncated mouse muscle creatine kinase (MCK) promoter in in vitro testing.

[0591] Three versions of the MCK promoter (MCK-D1~MCK-D3, with nucleotide sequences of SEQ ID NO: 11-13 respectively) with 50-bp, 55-bp, and 105-bp truncated versions from the CK8e promoter (SEQ ID NO: 14) were cloned upstream of the René luciferase (RLuc) reporter gene. C2C12 myoblasts were seeded in 96-well plates. One day after seeding, plasmids containing the MCK promoters were transfected using Lipofectamine 3000. Two days after transfection, luminescent quantification was performed using a dual-luciferase assay kit (Cat. No. E1500, Promega) and a SpectraMax i3xMulti-Mode Microplate Reader with a SpectraMax Injector Cartridge. The RLuc / FLuc ratio was used to measure promoter intensity (…). Figure 3A). RLuc plasmids driven by the CK8e promoter and FLuc plasmids driven by the HSV-TK promoter were used as controls. Compared with the CK8e promoter, 50-bp, 55-bp, and 105-bp truncation did not lead to a decrease in activity. Figure 3 A). The MCK-D3 promoter (SEQ ID NO. 13) was selected for further in vivo studies. A synthetic promoter HC7 (SEQ ID NO. 10) was constructed by combining a 59-bp hepatocyte promoter and the MCK-D3 promoter. The activity of promoter HC7 was assessed using an in vivo imaging system (IVIS). Six-week-old male BALB / c mice were intravenously (tail vein) injected with AAV (1E11vg / mouse) containing a firefly luciferase transgene driven by the HC7 promoter. One week after injection ( Figure 3 B) and three weeks ( Figure 3 (C) Strong luminescent signals were observed in the liver and skeletal muscle.

[0592] 4. Human GLA expression cassette mediates the expression and activity of α-galactosidase A in HepG2.

[0593] Four constructs (Table 2) with different promoters or GLA variants were cloned into a self-complementary adeno-associated virus (scAAV) vector backbone. HepG2 cells were seeded into 48-well plates (1e5 cells / well). One day after seeding, 500 ng of plasmid containing the hGLA cassette (Table 2) or GFP (negative control, Neg.ctrl) was transfected using Lipofectamine 3000. Promoter HC6 (SEQ ID NO: 9) was constructed by combining a 59-bp hepatocyte promoter with a truncated and modified human MCK promoter (SEQ ID NO: 15). Three days after transfection, the supernatant was collected and centrifuged at 2,000 rpm for 10 min. Samples for GLA were diluted 100 or 1000 times, and undiluted samples from the GFP group were used for ELISA using a human α-galactosidase A ELISA kit (Cat. No. SEK12078). Figure 4 A). Dilute the sample 10-fold to determine α-galactosidase A activity using kit (ab239716). Figure 4 B). thGLAco1: A truncated human α-galactosidase A with a two-amino acid deletion at the C-terminus (nucleotide sequence shown in SEQ ID NO: 20). All constructs showed high α-galactosidase A protein expression and activity in HepG2.

[0594] Table 2. Human GLA expression cassette constructs.

[0595]

[0596] 5. Human GLA expression cassette mediates the expression and activity of α-galactosidase A in H9c2 cells.

[0597] Four constructs (boxes 1-4) were transfected into H9c2 cells using Lipofectamine 3000. A plasmid expressing GFP was used as a negative control. Three days post-transfection, the supernatant was collected for human α-galactosidase A ELISA. Figure 5 A) and activity ( Figure 5 B) Analysis. Construct cassette 3 showed 3.1-fold and 3.8-fold expression of cassettes 1 and 2, respectively. Figure 5 A). The loss of the last two amino acids resulted in a 1.4-fold increase in α-galactosidase A activity in vitro. Figure 5 B).

[0598] 6. Characterize box 1-mediated activity and efficacy in a Fabry mouse model.

[0599] Expression cassette 1 was packaged into scAAV9. scAAV9 was produced via triple plasmid transfection and purified by iodixanol gradient ultracentrifugation. Vector genomic titer was measured by ddPCR, and purity was confirmed by SDS-PAGE. The scAAV9 vector was filtered through a 0.22 μm filter and adjusted to approximately 1E13 vg / mL in phosphate-buffered saline (PBS) with 0.001% Pluronic F68 (formulation buffer). Four groups of adult male GLA-KO mice (JAX stock #003535) were intravenously injected intravenously via tail vein at four doses (2E11 vg / kg, 2E12 vg / kg, 6E12 vg / kg, 2E13 vg / kg) containing expression cassette 1. Two groups of GLA-KO and wild-type mice were used as controls, injected intravenously with formulation buffer. Serum was collected every two weeks using K2-EDTA tubes for α-galactosidase A activity analysis. Stable expression of α-Gal A in serum persisted until the end of the mouse study. Figure 6A On day 61, compared with wild-type mice treated with buffer, GLA-KO mice treated with scAAV9-box 1 showed 178-fold, 2360-fold, 7285-fold, and 18485-fold human α-Gal A activity in serum at doses of 2E11 vg / kg, 2E12 vg / kg, 6E12 vg / kg, and 2E13 vg / kg, respectively. Figure 6BAt the endpoint, mice were sacrificed and perfused with cold PBS. Total RNA was isolated from the liver and heart. Total RNA treated with DNase I was used for reverse transcription (RT). Transformed cDNA was used for quantitative real-time PCR using hGLAco1-specific primers / probes. Mouse GAPDH primers / probes were used as a normalized reference. Clearly, hGLA transgenic mRNA levels showed a dose-dependent effect in the liver. Figure 6C Compared with the low-dose group (2E11 vg / kg), the high-dose groups (2E12 vg / kg, 6E12 vg / kg, 2E13 vg / kg) showed higher levels of hGLA transgenic mRNA in the heart. Figure 6D Genomic DNA was isolated from the liver and heart using the DNeasy Blood & Tissue Kit (Qiagen). AAV vector genome copies in those tissues were measured using droplet digital PCR (ddPCR) with hGLAco1 and mouse TFRC primers / probes. Vector genome copies in the liver ( Figure 6E ) and heart ( Figure 6F The results all showed a dose-dependent approach. Approximately 15 mg of liver sample was lysed with RIPA buffer. The supernatant after centrifugation was treated with or without PNGase F. For Western blotting, 2 μg of protein / sample was loaded and anti-α-galactosidase polyclonal antibody (1:2000, PA5-27349, Thermo Fisher) was used. After treatment with scAAV9-box 1, Western blotting results showed strong human α-Gal A protein expression and appropriate glycosylation in hepatocytes ( Figure 6G Human α-Gal A protein was not detected in buffer-treated GLA-KO and wild-type mice. Lysate samples from the liver, heart, and kidney were used for α-Gal A activity assays. Figure 6H Compared with buffer-treated wild-type mice, GLA-KO mice receiving the lowest dose (2E11 vg / kg) of scAAV9-box 1 showed 58-fold, 3-fold, and 0.8-fold α-Gal A activity in the liver, heart, and kidney, respectively. GLA-KO mice treated with 2E12 vg / kg of scAAV9-box 1 showed 1378-fold, 119-fold, and 10-fold α-Gal A activity in the liver, heart, and kidney, respectively. GLA-KO mice treated with 6E12 vg / kg of scAAV9-box 1 showed 3104-fold, 153-fold, and 24-fold α-Gal A activity in the liver, heart, and kidney, respectively. GLA-KO mice treated with 2E13 vg / kg of scAAV9-box 1 showed 5005-fold, 1215-fold, and 87-fold α-Gal A activity in the liver, heart, and kidney, respectively.

[0600] Since lyso-Gb3 is a biomarker for Fabry disease, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantify substrate levels. Compared with solvent-controlled GLA-KO mice, all AAV-treated GLA-KO mice showed significantly reduced serum lyso-Gb3 levels. Figure 6I Treatment with 2E11 vg / kg of scAAV9-box 1 reduced lyso-Gb3 to 2.5% of that in GLA-KO control mice. Treatment with three higher doses (2E12, 6E12, and 2E13 vg / kg) completely restored serum lyso-Gb3 levels to normal. Figure 6I Treatment with 2E12 vg / kg scAAV9-box 1 resulted in complete restoration of lyso-Gb3 levels to normal in the liver, heart, and kidneys. Figure 6J ).

[0601] 7. Characterize the AAV-hGLA-mediated activity and efficacy in a Fabry mouse model.

[0602] Two expression cassettes (cassette 1 and cassette 3) contained the AAV9-derived liver-muscle tropism variant AVT908. AAV production, purification, and characterization were similar to those described above. For each hGLA expression cassette, four groups of adult male GLA-KO mice (JAX stock #003535) were intravenously injected via tail vein at two doses (6E11 vg / kg, 2E12 vg / kg) (n=5 / group). Serum was collected every two weeks using K2-EDTA tubes for α-galactosidase A activity measurement. Figure 7A Eight weeks after treatment, serum α-Gal A activity in cassette 3 was 134% of that in cassette 1. Similar hGAL transgene mRNA levels were observed in the liver for both expression cassettes. Similar or even lower vector genome copies were observed in muscle tissue. Figure 7B In the case of ), box 3 showed significantly higher levels of hGLA transgenic mRNA in the heart and quadriceps compared to box 1. Figure 7C Consistently, for both doses, box 3 showed 84-fold activity in the heart compared to box 1. RT-qPCR data confirmed the activity of the hepatocyte-muscle bispecific promoter HC7 in both the liver and muscle. Since the mice were fully perfused with PBS prior to tissue collection, the α-Gal A protein detected in the heart originated from cardiomyocytes and / or was taken up by them. By Western blotting, high doses of box 3 resulted in higher levels of α-Gal A protein in the heart. Figure 7DBox 1, administered at a dose of 2E12 vg / kg, also showed the presence of α-Gal A protein in the heart. Mouse GAPDH was used as a control. Liver, heart, and kidney tissue lysates were used for α-Gal A activity measurements. All tissues showed high activity after AAV-hGLA treatment. Box 3, administered at a dose of 2E12 vg / kg, showed 274%, 632%, and 189% of the α-Gal A activity in the liver, heart, and kidney, respectively, compared to the 6E11 vg / kg dose. Figure 7E Compared with buffer-treated GLA-KO mice, robust and widely expressed human α-Gal A protein was detected in the liver and heart of GLA-KO mice treated with 2E12 vg / kg cassette 3 in the context of AAV by immunostaining. Figure 7F The presence of human α-Gal A protein expressed by AAV was also observed in the kidneys. Figure 7F ).

[0603] Example 2: Correction of substrate levels in a mouse model of Fabry disease by intravenous administration of AAV-GLA Fabry disease (FD) is a rare X-linked metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) due to a pathogenic mutation in the GLA gene. The gradual accumulation of substrates within the lysosome leads to cellular dysfunction and multi-organ damage. Current enzyme replacement therapy is an effective treatment for FD; however, the lifelong infusions every two weeks are a significant burden on patients. To provide a single dose with durable effects, this study aimed to develop a gene therapy for FD based on a recombinant adeno-associated virus (rAAV) vector. Codon-optimized human GLA (hGLA) cDNA with minimal CpG was generated using two algorithms. A series of synthetic liver-affinity promoters were evaluated in vitro. An optimized transgenic expression cassette was then selected and packaged into AAV9 (scAAV9-hGLA). A single intravenous injection of scAAV9-hGLA into 10-week-old α-Gal A KO male mice resulted in dose-dependent α-Gal A activity in serum, liver, heart, and kidneys. Stable expression of α-Gal A in serum persisted until the end of the 8-week mouse study. Compared with wild-type mice treated with solvent control (buffer), mice receiving the lowest dose (2 × 10⁻⁶) showed significantly better serum expression. 11 GLA-KO mice (vg / kg) showed α-Gal A activities of 178 × 10⁻⁶, 58 × 10⁻⁶, 3 × 10⁻⁶, and 0.8 × 10⁻⁶ in serum, liver, heart, and kidney, respectively. Using 2 × 10⁻⁶ vg / kg of α-Gal A in serum, liver, heart, and kidney, respectively... 12 GLA-KO mice treated with scAAV9-hGLA at vg / kg showed α-Gal A activity of 2360×, 1378×, 119×, and 10×. The highest dose (2×10⁻⁶) was [not specified in the original text]. 13(vg / kg) resulted in serum α-Gal A activity as high as 18,485×. Since lyso-Gb3 is a biomarker for FD, substrate levels were quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Compared to solvent-controlled GLA-KO mice, all AAV-treated GLA-KO mice showed significantly reduced serum lyso-Gb3 levels. 2×10 11 Treatment with scAAV9-hGLA at a dose of vg / kg reduced lyso-Gb3 to 2.5% of that in GLA-KO control mice. Three higher doses (2 × 10⁻⁶) further reduced lyso-Gb3 levels. 12 6×10 12 and 2×10 13 Treatment with a dose of (vg / kg) completely restored lyso-Gb3 levels to normal in serum, liver, heart, and kidneys. Codon-optimized hGLA transgenic mRNA levels correlated with AAV vector genomic copies in the liver and showed a dose-dependent effect. Western blotting confirmed robust expression and appropriate glycosylation of hepatic-derived human α-Gal A protein. Immunostaining confirmed the presence of α-Gal A in the liver, heart, and kidneys. Histopathological examination showed no toxicity observed at any dose. Finally, this study demonstrated that the effective dose can be further minimized using a novel, internally developed hepatomyotactic capsid AVT908 and promoter. The data support the development of low-dose AAV-GLA gene therapy for Fabry disease.

[0604] Example 3: Robust hGLA Expression in NHP Mediated by Engineered AAV Capsid Human GLA transgenic expression cassette 3 (SEQ ID NO: 18) was packaged in capsids AAV9 (SEQ ID NO: 21), AVT917 (SEQ ID NO: 56), and AVT918 (SEQ ID NO: 58). All three AAVs were produced in HEK293 cells via triple transfection. After purification by affinity chromatography and filtration through a 0.22 μm filter, the vector genomic titer was determined by droplet digital PCR (ddPCR) using hGLA primers / probes. Purity was analyzed by SDS-PAGE. AAV products with endotoxin levels below 1 EU / mL were used for non-human primate (NHP) studies.

[0605] Three adult (>3 years old) male cynomolgus monkeys without pre-existing neutralizing antibodies against AAV9 were used to compare different AAV capsid-mediated in vivo hGLA expression.

[0606] Each animal was administered intravenously with a single AAV expressing hGLA at a dose of 2E13 vg / kg, without the use of immunosuppressants.

[0607] Animals injected with AVT918 containing the hGLA expression cassette 3 were sacrificed 8 weeks after administration. Plasma, liver, heart, kidney, and skeletal muscle were harvested and processed for α-Gal A activity measurements.

[0608] Animals injected with AVT917 containing the hGLA expression cassette 3 were sacrificed 13 weeks post-administration. Plasma, liver, heart, kidney, and skeletal muscle were harvested for α-Gal A activity measurements. Total genomic DNA was extracted from major tissues using the DNeasy Blood & Tissue Kit (Qiagen, 69506). AAV vector genome copies in tissues were measured by ddPCR (Bio-Rad, QX200) using transgene-specific primers / probes (forward primer: 5'-GGCAGCTTTGGCTACTATGA-3', reverse primer: 5'-GTCACAGTAGCAGCCATCAA-3', probe: 5'-FAM-TTGATGCCCAGACCTTTGCTGACT-BHQ1-3'). RNase P primers / probes (Thermo Fisher Scientific, 4403326) were used as a reference. To quantify transgene mRNA levels in tissues, total RNA was isolated using the TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, 9767). cDNA was generated using the PrimeScript™ RT Kit (Takara, RR047B) with a gDNA Eraser. hGLA primers / probes were used for transgene-specific transcripts. Rhesus monkey GAPDH (forward: 5'-GCACCGTGAAGGCTGAGAAC-3', reverse: 5'-GGATCTCGCTCCTGGAAGATG-3', probe: 5'-HEX-CTCGTCATCAATGGAAGCCCCATCA-BHQ1-3') was used as a housekeeping gene. Two [presumably referring to a specific gene or formula] were used. -ΔCt The relative transgenic mRNA level was calculated using this method.

[0609] Animals were released after 26 weeks of monitoring plasma α-Gal A activity and then injected with AAV9 containing hGLA expression cassette 3. Plasma α-Gal A activity and human α-Gal A protein levels in these animals remained stable from week 6 onwards (triangle symbol in Figure 8). The 26-week long-term data indicate that the HC7 promoter (SEQ ID NO: 10) has stable activity in NHP under AAV conditions.

[0610] Plasma and serum samples were collected at different time points. α-Gal A activity in plasma was measured using a fluorescence-based enzyme activity assay. Briefly, 4-methylumbelliferone (4-MU) (Sigma-Aldrich, M1381-100G) was dissolved in DMSO and serially diluted as a standard. 4-MU-α-D-galactopyranoside (4-MU-α-Gal) (Cayman Chemical, 16551) was used as a substrate. N-acetylgalactosamine (Sigma-Aldrich, A2795-500MG), an α-galactosidase B inhibitor, was also added to the samples. After incubation at 37°C for 2 hours with shaking at 100 rpm in black 96-well plates (Corning, 3603), 0.1 mL of 0.5 M glycine-NaOH buffer (pH 10.5) was added to each well to stop the reaction. Fluorescence intensity (Ex / Em = 365 / 450 nm) was measured using an endpoint setting. α-Gal A activity was calculated based on the 4MU standard curve and expressed as nmol / h / mL plasma or nmol / h / mg protein.

[0611] All three capsids (AAV9, AVT917, and AVT918) mediated high α-GalA activity levels in NHP plasma after intravenous administration. Figure 8A AVT917 mediated the highest α-Gal A activity in plasma, showing a 1,778-fold increase over baseline (Table).

[0612] Table 3. Plasma α-Gal A activity (nmol / h / mL) in NHP treated with AAV-hGLA.

[0613]

[0614] / : Not applicable; ND: Not measured.

[0615] All three capsids (AAV9, AVT917, and AVT918) mediated specific human α-Gal A protein expression in NHP plasma after intravenous administration. Figure 8B Based on the concentration of human α-Gal A protein in plasma, AVT917 showed the highest GLA expression (Table).

[0616] Table 4. AAV-mediated hGLA expression in NHP after intravenous administration (ng / mL).

[0617]

[0618] / : Not applicable; ND: Not measured.

[0619] The AAV vector genome (vg) copies in NHP tissues treated with AVT917 were measured by ddPCR. The vector genome copies in the left lobe of the liver, right lobe of the liver, heart, kidney, triceps, quadriceps, and gastrocnemius muscles were 990, 1126, 8.6, 0.7, 1.2, 1.6, and 2.0 vg / dpg, respectively. The vector genome copies in the central nervous system (CNS) tissues were less than 1 vg / dpg. Figure 9A The levels and tissue specificity of transgenic hGLAm RNA driven by the HC7 promoter (SEQ ID NO: 10) were assessed by RT-qPCR. The HC7 promoter showed robust activity in the liver, followed by the heart and skeletal muscle. Figure 9B Weak promoter activity was observed in kidney and CNS tissues. High hGLA transgene expression and high levels of α-Gal A activity mediated by the AVT917 capsid and HC7 promoter were monitored in plasma. Thirteen weeks after administration of AVT917 containing HC7-hGLA expression cassette 3, major tissues were harvested and treated for α-Gal A activity. Increases in α-Gal A activity of approximately 80×, 54×, 1.6×, 197×, 68×, and 138× were observed in the heart, liver, kidney, gastrocnemius, quadriceps, and triceps muscles, respectively, compared to tissues from non-AAV-hGLA-treated animals (Neg Ctrl). Figure 9C ).

[0620] Example 4: The efficacy of AVT917 capsid containing HC7-hGLA mediated in Fabry mice.

[0621] Human GLA transgenic expression cassette 3 (SEQ ID NO: 18) was packaged in the capsid AVT917 (SEQ ID NO: 56) and produced in HEK293 cells via triple transfection. The purified AAV preparation was designated AVT917-HC7-hGLA. To explore the minimum effective dose, adult (~16 weeks old) male Fabry mice (Gla-KO) were intravenously injected with three doses (2E11 vg / kg, 6E11 vg / kg, 2E12 vg / kg, n=10 / group) of either the solvent (formulation buffer) or AVT919-HC7-hGLA (cassette 3). Plasma was collected every two weeks for α-Gal A activity measurements. Compared with the solvent group, all mice treated with AVT917-HC7-hGLA showed supraphysiological α-Gal A activity in plasma during the 8-week study period. Figure 10 A). A dose-dependent increase in α-Gal A activity was observed in the AVT917-HC7-hGLA-treated group ( Figure 10A). Eight weeks after treatment, the mean plasma α-Gal A activities in the solvent, low-dose (2E11 vg / kg), medium-dose (6E11 vg / kg), and high-dose (2E13 vg / kg) groups were 1.37 nmol / h / mL, 2336.19 nmol / h / mL, 6140.66 nmol / h / mL, and 18189.11 nmol / h / mL, respectively, corresponding to increases of 1×, 1710×, 4495×, and 13314× in mean plasma α-Gal A activity in solvent-treated Fabry mice.

[0622] The levels of transgenic hGLAm RNA in tissues of Fabry mice treated with AVT917-HC7-hGLA were assessed by RT-qPCR. Firstly, hGLA transcripts were found in the liver and heart (…). Figure 11 A, B). Weak hGLA mRNA levels were observed in the quadriceps and gastrocnemius skeletal muscles ( Figure 11 D, E). Negligible transgene expression was observed in the kidneys and spleen. Figure 11 C, F). These data support the in vivo hepatocyte-myocellular bispecific activity of the HC7 promoter.

[0623] Eight weeks after a single intravenous administration of AVT917-HC...

Claims

1. A polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:

1.

2. An expression cassette comprising a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A as claimed in claim 1 and a polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operatively linked to the nucleotide sequence encoding human α-galactosidase A.

3. The expression cassette according to claim 2, wherein the nucleotide sequence encoding the promoter comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

10.

4. The polynucleotide according to claim 2 or 3, wherein the promoter is a hepatocyte-specific promoter.

5. The polynucleotide according to claim 2 or 3, wherein the promoter is a hepatocyte-myoblast bispecific promoter.

6. The expression cassette according to claims 2-5, further comprising a nucleotide sequence encoding a polyA signal.

7. The expression cassette of claim 6, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16 or SEQ ID NO:

18.

8. A polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence (i) encoding the promoter comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

10.

9. An expression cassette comprising a polynucleotide comprising a nucleotide sequence encoding a promoter as described in claim 8 and a polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding the promoter is operatively linked to the nucleotide sequence encoding human α-galactosidase A.

10. The expression cassette of claim 9, wherein the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:

1.

11. The expression cassette according to claim 9, wherein the promoter is a hepatocyte-specific promoter.

12. The expression cassette according to claim 9, wherein the promoter is a hepatocyte-myoblast bispecific promoter.

13. The expression cassette according to any one of claims 9-12, further comprising a nucleotide sequence encoding a polyA signal.

14. The expression cassette of claim 13, wherein the nucleotide sequence encoding the expression cassette comprises SEQ ID NO: 16 or SEQ ID NO:

18.

15. A vector comprising the polynucleotide of claim 1 or 8, or the expression cassette of any one of claims 2-7 or 9-14.

16. A recombinant viral particle comprising a recombinant viral genome, said recombinant viral genome comprising an expression cassette according to any one of claims 2-7 or 9-14.

17. A recombinant adeno-associated virus (AAV) particle comprising: (a) an AAV capsid; and (b) a recombinant AAV genome comprising an expression cassette of any one of claims 2-7 or 9-14 with an AAV terminal inverted repeat (ITR) lateralized to it.

18. The recombinant AAV particles according to claim 17, wherein the recombinant AAV serotype 9 (rAAV9) particles are recombinant AAV serotype 9 particles.

19. The recombinant AAV particle of claim 17 or 18, wherein the AAV capsid comprises a variant AAV9 capsid protein.

20. The recombinant AAV particle of claim 19, wherein the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59 or 61.

21. The recombinant AAV particle of claim 20, wherein the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58 or 60.

22. A host cell comprising the polynucleotide of claim 1 or 8, the vector of claim 15, the recombinant viral particle of claim 16, or the recombinant AAV particle of any one of claims 17-21.

23. A host cell population stably transduced by the recombinant viral particles of claim 16 or the recombinant AAV particles of any one of claims 17-21.

24. A pharmaceutical composition comprising the recombinant viral particles of claim 16 or the recombinant AAV particles of any one of claims 17-21, the host cell population of claim 23, and a pharmaceutically acceptable carrier.

25. A method for producing recombinant viral particles or recombinant AAV particles, comprising culturing the host cells of claim 22.

26. A method of treating a disease or disorder in a subject in need, comprising administering to the subject the recombinant viral particles of claim 16, the recombinant AAV particles of any one of claims 17-21, or the pharmaceutical composition of claim 24.

27. The method of claim 26, wherein the disease or disorder is lysosomal storage disease or Fabry disease.

28. The method of claim 26, wherein the subject is a human being.

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