Compositions and methods for gene therapy

Abstract

A polynucleotide is provided comprising a nucleotide sequence encoding a biomolecule (e.g., human α-galactosidase A), a nucleotide sequence encoding a promoter (e.g., a hepatocyte-specific promoter or a hepatocyte-myocyte dual-specific promoter), or a nucleotide sequence encoding an expression cassette (e.g., a human α-galactosidase A expression cassette). Furthermore, promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant adeno-associated virus (AAV) particles and recombinant viral particles such as nonviral gene delivery systems), related pharmaceutical compositions, and methods for using them are also provided. Such compositions and methods are particularly suitable for gene therapy, and especially for lysosomal storage disorders, including Fabry disease.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the rights and priority of PCT application number PCT / CN2023 / 088738, filed on 17 April 2023. The contents of these applications are incorporated herein by reference for all purposes.

[0002] A polynucleotide is provided comprising a nucleotide sequence encoding a biomolecule (e.g., human α-galactosidase A), a nucleotide sequence encoding a promoter (e.g., a hepatocyte-specific promoter or a hepatocyte-myocyte dual-specific promoter), or a nucleotide sequence encoding an expression cassette (e.g., a human α-galactosidase A expression cassette). Furthermore, promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant adeno-associated virus (AAV) particles and recombinant viral particles such as nonviral gene delivery systems), related pharmaceutical compositions, and methods for using them are also provided. Such compositions and methods are particularly suitable for gene therapy, and especially for lysosomal storage disorders, including Fabry disease. [Background technology]

[0003] Gene therapy (including gene therapy using AAV) has the potential to be a promising treatment option for many diseases.

[0004] Lysosomal storage disorders are genetic metabolic disorders characterized by the accumulation of abnormal amounts of substrate due to lysosomal dysfunction in cells of various organs. 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) resulting from a pathogenic mutation in the GLA gene. The gradual accumulation of substrate within lysosomes leads to cellular dysfunction and multi-organ damage.

[0005] There has been a need for the development of compositions, systems, and methods for gene therapy, particularly for lysosomal storage disorders (including Fabry disease). [Overview of the Initiative]

[0006] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A comprises or is shown at SEQ ID NO:1. In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:1. In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A is shown at SEQ ID NO:1.

[0007] In one embodiment, a polynucleotide comprising a nucleotide sequence encoding a promoter is provided, wherein the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) the polynucleotide 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-myocyte dual-specific promoter. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:10. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:10.

[0008] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding human α-galactosidase A, and the nucleotide sequence encoding human α-galactosidase A comprises or is shown at SEQ ID NO:1. In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:1. In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A is shown at SEQ ID NO:1. In a particular embodiment, the promoter is a hepatocyte-specific promoter. In a particular embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a particular embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In a particular embodiment, the nucleotide sequence encoding the promoter is shown at SEQ ID NO:3. In a particular embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter includes SEQ ID NO:10. In certain embodiments, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:10. In various embodiments, the polynucleotide further includes a nucleotide sequence encoding the polyA signal.

[0009] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the biomolecule, and the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) the polynucleotide 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-myocyte dual-specific promoter. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:10. In a particular embodiment, the nucleotide sequence encoding the promoter is shown as 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 the polyA signal.

[0010] In one aspect, 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 a polynucleotide shown in SEQ ID NO:16 or SEQ ID NO:18 is provided. In certain embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO:16. In certain embodiments, the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO:16. In certain embodiments, the nucleotide sequence encoding the expression cassette comprises SEQ ID NO:18. In certain embodiments, the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO:18.

[0011] In one aspect, a promoter encoded by a nucleotide sequence, wherein the nucleotide sequence (i) comprises SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:10, or (ii) a promoter shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:10 is provided. In specific embodiments, the promoter is a hepatocyte-specific promoter. In specific embodiments, the promoter is a hepatocyte-myocyte dual-specific promoter. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:10. In certain embodiments, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:10.

[0012] In one aspect, there is provided an expression cassette comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding human α-galactosidase A, and the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:1 or is as set forth in SEQ ID NO:1. In certain embodiments, the nucleotide sequence encoding human α-galactosidase A comprises SEQ ID NO:1. In certain embodiments, the nucleotide sequence encoding human α-galactosidase A is as set forth 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-myocyte dual-specific promoter. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the promoter is as set forth in SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter is as set forth in SEQ ID NO:4. In certain embodiments, the nucleotide sequence encoding the promoter comprises SEQ ID NO:10. In certain embodiments, the nucleotide sequence encoding the promoter is as set forth in SEQ ID NO:10. In various embodiments, the expression cassette further comprises a nucleotide sequence encoding a polyA signal.

[0013] In one embodiment, an expression cassette is provided comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the biomolecule, and the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) an expression cassette 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-myocyte dual-specific promoter. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:3. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter is shown in SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the promoter comprises SEQ ID NO:10. In a particular embodiment, the nucleotide sequence encoding the promoter is shown as SEQ ID NO:10. In a specific embodiment, the biomolecule is human α-galactosidase A. In various embodiments, the expression cassette further comprises a nucleotide sequence encoding the polyA signal.

[0014] In one embodiment, an expression cassette is provided which is encoded by (i) a nucleotide sequence including SEQ ID NO:16 or SEQ ID NO:18, or (ii) a nucleotide sequence shown in SEQ ID NO:16 or SEQ ID NO:18. In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:16. In a particular embodiment, the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO:16. In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:18. In a particular embodiment, the nucleotide sequence encoding the expression cassette is shown in SEQ ID NO:18.

[0015] In one embodiment, a polynucleotide comprising a nucleotide sequence encoding the expression cassette described herein is provided.

[0016] In one embodiment, a vector comprising a polynucleotide described herein, a promoter described herein, or an expression cassette described herein is provided.

[0017] In one embodiment, recombinant viral particles are provided that include a recombinant viral genome containing an expression cassette as described herein.

[0018] In one embodiment, a recombinant adeno-associated virus (AAV) particle is provided, comprising (a) an AAV capsid and (b) a recombinant AAV genome comprising an expression cassette described herein in which an AAV terminal inverted repeat (ITR) is flanked. 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 mutant AAV9 capsid protein. In a particular embodiment, the mutant AAV9 capsid protein comprises an amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59, or 61. In a particular embodiment, the mutant AAV9 capsid protein comprises an amino acid sequence of SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58, or 60.

[0019] In one embodiment, a host cell comprising a polynucleotide described herein, a vector described herein, a recombinant viral particle described herein, or a recombinant AAV particle described herein is provided.

[0020] In one embodiment, a population of host cells stably transduced by recombinant virus particles or recombinant AAV particles described herein is provided.

[0021] In one embodiment, a pharmaceutical composition is provided comprising recombinant virus particles or recombinant AAV particles as described herein and a pharmaceutically acceptable carrier.

[0022] In one embodiment, a pharmaceutical composition is provided comprising a host cell population and a pharmaceutically acceptable carrier as described herein.

[0023] In one embodiment, a method for producing recombinant virus particles or recombinant AAV particles is provided, comprising culturing host cells as described herein.

[0024] In one embodiment, a method is provided for treating a disease or disorder in a subject as required, comprising administering to the subject recombinant virus particles, recombinant AAV particles, or pharmaceutical compositions described herein. In a particular embodiment, the disease or disorder is a lysosomal storage disorder. In a specific embodiment, the disease or disorder is Fabry disease. In a specific embodiment, the subject is a human.

[0025] Descriptive embodiment This disclosure includes the following non-limiting embodiments. 1. A polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:1 or is represented by SEQ ID NO:1. 2. The nucleotide sequence encoding human α-galactosidase A is the polynucleotide described in Embodiment 1, including SEQ ID NO:1. 3. The nucleotide sequence encoding human α-galactosidase A is the polynucleotide described in Embodiment 1, as shown in SEQ ID NO:1. 4. A polynucleotide comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) the sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10. 5. A polynucleotide comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding human α-galactosidase A, and the nucleotide sequence encoding human α-galactosidase A includes or is indicated by SEQ ID NO:1. 6. The nucleotide sequence encoding human α-galactosidase A is the polynucleotide described in Embodiment 5, including SEQ ID NO:1. 7. The nucleotide sequence encoding human α-galactosidase A is the polynucleotide described in Embodiment 5, as shown in SEQ ID NO:1. 8. The promoter is a hepatocyte-specific promoter, the polynucleotide according to any one of embodiments 4 to 7. 9. The polynucleotide according to any one of embodiments 4 to 8, wherein the promoter is a hepatocyte-myocyte dual-specific promoter. 10. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of Embodiments 4 to 9, including SEQ ID NO:3. 11. The nucleotide sequence encoding the promoter is the polynucleotide described in any one of Embodiments 4 to 8, as shown in SEQ ID NO:3. 12. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of Embodiments 4 to 9, including SEQ ID NO:4. 13. The nucleotide sequence encoding the promoter is the polynucleotide described in any one of Embodiments 4 to 9, as shown in SEQ ID NO:4. 14. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of Embodiments 4 to 9, including SEQ ID NO:10. 15. The nucleotide sequence encoding the promoter is the polynucleotide described in any one of Embodiments 4 to 9, as shown in SEQ ID NO:10. 16. A polynucleotide comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably ligated to the nucleotide sequence encoding the biomolecule, and the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) the sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10. 17. The polynucleotide according to Embodiment 16, wherein the promoter is a hepatocyte-specific promoter. 18. The polynucleotide according to Embodiment 16 or 17, wherein the promoter is a hepatocyte-myocyte dual-specific promoter. 19. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of embodiments 16 to 18, including SEQ ID NO:3. 20. The nucleotide sequence encoding the promoter is the polynucleotide shown in SEQ ID NO:3, according to Embodiment 16 or 17. 21. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of embodiments 16 to 18, including SEQ ID NO:4. 22. The nucleotide sequence encoding the promoter is the polynucleotide described in any one of embodiments 16 to 18, as shown in SEQ ID NO:4. 23. The nucleotide sequence encoding the promoter is a polynucleotide according to any one of embodiments 16 to 18, comprising SEQ ID NO: 10. 24. The nucleotide sequence encoding the promoter is the polynucleotide described in any one of embodiments 16 to 18, as shown in SEQ ID NO:10. 25. The polynucleotide according to any one of embodiments 16 to 24, wherein the biomolecule is human α-galactosidase A. 26. A polynucleotide according to any one of embodiments 5 to 25, further comprising a nucleotide sequence encoding a polyA signal. 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 is shown in SEQ ID NO:16 or SEQ ID NO:18. 28. The nucleotide sequence encoding the expression cassette is the polynucleotide described in Embodiment 27, including SEQ ID NO:16. 29. The nucleotide sequence encoding the expression cassette is the polynucleotide described in Embodiment 27, as shown in SEQ ID NO:16. 30. The nucleotide sequence encoding the expression cassette is the polynucleotide described in Embodiment 27, including SEQ ID NO:18. 31. The nucleotide sequence encoding the expression cassette is the polynucleotide described in Embodiment 27, as shown in SEQ ID NO:18. 32. (i) A promoter encoded by a nucleotide sequence including SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) a promoter encoded by the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10. 33. The promoter according to embodiment 32, which is a hepatocyte-specific promoter. 34. The promoter according to embodiment 32 or 33, which is a hepatocyte-myocyte dual-specific promoter. 35. The promoter according to any one of embodiments 32 to 34, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:3. 36. The nucleotide sequence encoding the promoter is the promoter according to Embodiment 32 or 33, as shown in SEQ ID NO:3. 37. The promoter according to any one of embodiments 32 to 34, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:4. 38. The nucleotide sequence encoding the promoter is the promoter according to any one of embodiments 32 to 34, as shown in SEQ ID NO:4. 39. The promoter according to any one of embodiments 32 to 34, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:10. 40. The nucleotide sequence encoding the promoter is the promoter according to any one of embodiments 32 to 34, as shown in SEQ ID NO:10. 41. An expression cassette comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably ligated to the nucleotide sequence encoding human α-galactosidase A, and the nucleotide sequence encoding human α-galactosidase A includes or is indicated by SEQ ID NO:1. 42. The expression cassette according to Embodiment 41, comprising the nucleotide sequence encoding human α-galactosidase A, including SEQ ID NO:1. 43. The nucleotide sequence encoding human α-galactosidase A is the expression cassette described in Embodiment 41, as shown in SEQ ID NO:1. 44. The expression cassette according to any one of embodiments 41 to 43, wherein the promoter is a hepatocyte-specific promoter. 45. The expression cassette according to any one of embodiments 41 to 44, wherein the promoter is a hepatocyte-myocyte dual-specific promoter. 46. ​​An expression cassette according to any one of embodiments 41 to 45, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:3. 47. The nucleotide sequence encoding the promoter is the expression cassette according to any one of embodiments 41 to 44, as shown in SEQ ID NO:3. 48. An expression cassette according to any one of embodiments 41 to 45, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:4. 49. The nucleotide sequence encoding the promoter is the expression cassette according to any one of embodiments 41 to 45, as shown in SEQ ID NO: 4. 50. An expression cassette according to any one of Embodiments 41 to 45, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:10. 51. The nucleotide sequence encoding the promoter is the expression cassette according to any one of embodiments 41 to 45, as shown in SEQ ID NO:10. 52. An expression cassette comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably ligated to the nucleotide sequence encoding the biomolecule, and the nucleotide sequence encoding the promoter comprises (i) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10, or (ii) SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:10. 53. The expression cassette according to Embodiment 52, wherein the promoter is a hepatocyte-specific promoter. 54. The expression cassette of Embodiment 52 or 53, wherein the promoter is a hepatocyte-myocyte dual-specific promoter. 55. An expression cassette according to any one of embodiments 52 to 54, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:3. 56. The nucleotide sequence encoding the promoter is the expression cassette according to Embodiment 52 or 53, as shown in SEQ ID NO:3. 57. An expression cassette according to any one of embodiments 52 to 54, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:4. 58. The nucleotide sequence encoding the promoter is the expression cassette according to any one of embodiments 52 to 54, as shown in SEQ ID NO:4. 59. An expression cassette according to any one of embodiments 52 to 54, wherein the nucleotide sequence encoding the promoter includes SEQ ID NO:10. 60. The nucleotide sequence encoding the promoter is the expression cassette according to any one of embodiments 52 to 54, as shown in SEQ ID NO:10. 61. The expression cassette according to any one of embodiments 52 to 60, wherein the biomolecule is human α-galactosidase A. 62. An expression cassette according to any one of embodiments 41 to 61, further comprising a nucleotide sequence encoding a polyA signal. 63. (i) an expression cassette encoded by a nucleotide sequence including SEQ ID NO:16 or SEQ ID NO:18, or (ii) an expression cassette encoded by the nucleotide sequence shown in SEQ ID NO:16 or SEQ ID NO:18. 64. The expression cassette according to Embodiment 63, wherein the nucleotide sequence encoding the expression cassette includes SEQ ID NO:16. 65. The nucleotide sequence encoding the expression cassette is the expression cassette according to Embodiment 63, as shown in SEQ ID NO:16. 66. The expression cassette according to Embodiment 63, wherein the nucleotide sequence encoding the expression cassette includes SEQ ID NO:18. 67. The nucleotide sequence encoding the expression cassette is the expression cassette according to Embodiment 63, as shown in SEQ ID NO:18. 68. A polynucleotide comprising a nucleotide sequence encoding an expression cassette according to any one of embodiments 41 to 67. 69. A vector comprising a polynucleotide according to any one of embodiments 1 to 31 and 68, a promoter according to any one of embodiments 32 to 40, or an expression cassette according to any one of embodiments 41 to 67. 70. Recombinant virus particles comprising a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette as described in any one of embodiments 41 to 67. 71. Recombinant adeno-associated virus (AAV) particle comprising (a) an AAV capsid and (b) a recombinant AAV genome comprising an expression cassette according to any one of embodiments 41 to 67 in which an AAV terminal inverted repeat sequence (ITR) is flanked. 72. Recombinant AAV particles according to Embodiment 71, which are recombinant AAV serotype 9 (rAAV9) particles. 73. Recombinant AAV particles according to Embodiment 71 or 72, wherein the AAV capsid comprises a mutant AAV9 capsid protein. 74. The recombinant AAV particle according to Embodiment 73, wherein the mutant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59, or 61. 75. The recombinant AAV particle according to Embodiment 74, wherein the mutant AAV9 capsid protein comprises the amino acid sequence SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58, or 60. 76. A host cell comprising a polynucleotide according to any one of embodiments 1 to 31 and 68, a vector according to embodiment 69, a recombinant viral particle according to embodiment 70, or a recombinant AAV particle according to any one of embodiments 71 to 75. 77. A group of host cells that are stably transduced by recombinant virus particles as described in Embodiment 70 or recombinant AAV particles as described in any one of Embodiments 71 to 75. 78. A pharmaceutical composition comprising recombinant virus particles as described in Embodiment 70 or recombinant AAV particles as described in any one of Embodiments 71 to 75, and a pharmaceutically acceptable carrier. 79. A pharmaceutical composition comprising the host cell population described in Embodiment 77 and a pharmaceutically acceptable carrier. 80. A method for producing recombinant virus particles or recombinant AAV particles, comprising culturing the host cells described in Embodiment 76. 81. A method for treating a disease or disorder in a subject as needed, comprising administering to the subject recombinant virus particles described in Embodiment 70, recombinant AAV particles described in any one of Embodiments 71 to 75, or a pharmaceutical composition described in Embodiment 78 or 79. 82. The method according to Embodiment 81, wherein the disease or disorder is a lysosomal storage disorder. 83. The method according to Embodiment 81, wherein the disease or disorder is Fabry disease. 84. The method according to any one of embodiments 81 to 83, wherein the subject is a human. [Brief explanation of the drawing]

[0026] [Figure 1] Codon optimization of human α-galactosidase A (GLA). Figure 1A: Results of ELISA assay. Figure 1B: Results of α-galactosidase A activity. Figure 1C: Western blot analysis of cell culture supernatant from HepG2 transfected with a plasmid encoding hGLA. WT: wild type; hGLA: human α-galactosidase A.

[0027] [Figure 2] In vitro testing of hepatocyte-cardiomyocyte (HC) bispecific promoters. The RLuc / FLuc ratio was used to measure the promoter strength of five promoters (HC1-HC5). RLuc: reniral luciferase; FLuc: firefly luciferase.

[0028] [Figure 3]In vitro studies of truncated mouse muscle creatine kinase (MCK) promoters. Figure 3A: The RLuc / FLuc ratio was used to measure the promoter strength of three versions of the MCK promoter (MCK-D1 to MCK-D3). Figures 3B and 3C: Promoter HC7 activity was evaluated by an in vivo imaging system (IVIS). Strong luminescence signals were observed in the liver and skeletal muscle one week (Figure 3B) and three weeks (Figure 3C) after injection.

[0029] [Figure 4] Expression (Figure 4A) and activity (Figure 4B) of α-galactosidase A mediated by human GLA expression cassettes in HepG2. Figure 4A: The α-Gal A concentrations of the negative control, cassette 1, cassette 2, cassette 3, and cassette 4 were 2 ng / mL, 2993 ng / mL, 2225 ng / mL, 1919 ng / mL, and 1684 ng / mL, respectively. Figure 4B: The α-Gal A activities of the negative control, cassette 1, cassette 2, cassette 3, and cassette 4 were 1.66 x 10⁶ nmol / mg / h, 1.87 x 10⁶ nmol / mg / h, 2.00 x 10⁶ nmol / mg / h, and 2.73 x 10⁶ nmol / mg / h, respectively.

[0030] [Figure 5] Expression (Figure 5A) and activity (Figure 5B) of α-galactosidase A mediated by human GLA expression cassettes in H9c2 cells. Figure 5A: α-Gal A concentrations for negative control, cassette 1, cassette 2, cassette 3, and cassette 4 were 2 x 10⁻¹ ng / mL, 306 ng / mL, 249 ng / mL, 954 ng / mL, and 1350 ng / mL, respectively. Figure 5B: α-Gal A activity for negative control, cassette 1, cassette 2, cassette 3, and cassette 4 was 1.14 x 10⁴ nmol / mg / h, 1.45 x 10⁴ nmol / mg / h, 1.80 x 10⁴ nmol / mg / h, and 2.57 x 10⁴ nmol / mg / h, respectively.

[0031] [Figure 6A]Characterization of Cassette 1-mediated activity and effect in a mouse model of Fabry disease. Figure 6A: Time course of serum α-Gal A activity. Figure 6B: Serum α-Gal A activity on day 61 (numbers at the top of the column from left to right are 11, 3, 1959, 25956, 80140, and 203339). Figure 6C: hGLA transgene mRNA levels in the liver (numbers at the top of the column from left to right are 0.3200, 2.2752, 3.9636, 9.6976, and 0.0001). Figure 6D: hGLA transgene mRNA levels in the heart (numbers at the top of the column from left to right are 3.3037x10⁻⁵, 0.0005, 0.0005, and 0.0008). Figure 6E: Vector genome copies in the liver (the numbers at the top of the columns from left to right are 0.94, 10.88, 28.28, 79.60, and 0.02). Figure 6F: Vector genome copies in the liver (the numbers at the top of the columns from left to right are 0.03, 0.24, 0.87, and 1.64). Figure 6G: Western blot results. Figure 6H: α-Gal A activity in the liver, heart, and kidney. Liver data shown from left to right are 2043 (2E11vg / kg), 48242 (2E12vg / kg), 108653 (6E12vg / kg), 175176 (2E13vg / kg), 12 (GLA-KO), and 35 (wild type). The cardiac data, displayed from left to right, are 31 (2E11vg / kg), 1186 (2E12vg / kg), 1533 (6E12vg / kg), 12153 (2E13vg / kg), 26 (GLA-KO), and 10 (wild-type). The kidney data, displayed from left to right, are 18 (2E11vg / kg), 217 (2E12vg / kg), 501 (6E12vg / kg), 1821 (2E13vg / kg), 6 (GLA-KO), and 21 (wild-type). Figure 6I: Serum lyso-Gb3 levels (the numbers at the top of the column, from left to right, are 0.57, 147.22, 3.67, 0.76, 0.59, and 0.55). Figure 6J: Lyso-Gb3 levels in the liver, heart, and kidneys. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 6I] Same as above. [Figure 6J] Same as above.

[0032] [Figure 7A] Characterization of activity and efficacy mediated by AAV-hGLA in a Fabry disease mouse model. Figure 7A: Time course of serum α-GAL A activity. Figure 7B: Vector genome copies in liver, heart, and quadriceps muscle. Figure 7C: mRNA levels of hGLA transgenes in liver, heart, and quadriceps muscle. Figure 7D: Western blot results. Figure 7E: Cassette 3-mediated α-Gal A activity in liver, heart, and kidney. Figure 7F: HC7-driven hGLAco1 (cassette 3) packaged with capsid AVT908 was intravenously administered to 8-week-old male GLa-ko mice. The dose was 2e12vg / kg. Eight weeks after administration, liver, heart, and kidney were collected and immunohistochemical (IHC) stained. 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 indicated positive staining for human Gal A protein. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above.

[0033] [Figure 8A]hGLA expression cassette 3, packaged in capsids AVT917, AVT918, or AAV9, was administered intravenously to adult male cynomolgus monkeys at a dose of 2E13vg / kg (N=1 / capsid). Plasma samples were collected before and after administration. Figure 8A: Plasma Gal A activity was measured by fluorescence-based 4-MU method. Figure 8A: Human Gal A protein expressed in plasma was measured by ELISA. [Figure 8B] Same as above.

[0034] [Figure 9A] The biological distribution of AAV vector genomes in NHP (Nutrient-Based Hypoplasia). Adult male cynomolgus monkeys were intravenously administered AVT917 containing hGLA expression cassette 3 at a dose of 2E13vg / kg. Thirteen weeks after administration, tissue samples were collected and processed, and ddPCR-based analysis of vector genome copies was performed. The RNA polymerase P gene was used as a reference.

[0035] [Figure 9B] AVT917 and the HC7 promoter mediated hGLA transgene expression in NHP. AVT917 containing hGLA expression cassette 3 was administered intravenously to adult male cynomolgus monkeys at a dose of 2E13vg / kg. Thirteen weeks post-administration, tissue samples were collected and processed, and transgene mRNA levels were analyzed by RT-qPCR. The GADPH gene was used as a reference. Data are expressed as 2-ΔCt.

[0036] [Figure 9C] Gal A activity in NHP tissue treated with AVT917-hGLA. AVT917 containing hGLA expression cassette 3 was administered intravenously to adult male cynomolgus monkeys at a dose of 2E13vg / kg. Thirteen weeks after administration, tissue was collected and processed, and Gal A activity was analyzed using the fluorescence-based 4-MU method.

[0037] [Figure 10]AVT917 mediated high hGLA expression in Fabry disease mice. Adult (16-week-old) male Fabry disease mice (GIa-KO) were intravenously injected with either a solvent (formulation buffer) or AVT919-HC7-hGLA (cassette 3) at three different doses (n=10 / group). Plasma was collected every two weeks and used for α-Gal A activity measurement. Data are presented as mean values ​​with SD. Two-way repeated measures ANOVA was performed for **p<0.01, ***p<0.001, and ****p<0.0001, followed by Tukey multiple comparison tests.

[0038] [Figure 11] AVT917 mediated hGLA mRNA expression in the tissues of Fabry disease mice. Adult (16-week-old) male Fabry disease mice (GIa-KO) were intravenously injected with either a solvent (formulation buffer) or AVT919-HC7-hGLA (cassette 3) at three different doses (n=10 / group). Eight weeks after administration, major tissues were collected and processed, and total RNA was isolated. hGLA-specific mRNA transcripts were quantified by RT-qPCR. Mouse GAPDH (MsGAPDH) was used as a housekeeping gene. Data are presented as mean values ​​with SD. *If p<0.05, ****p<0.0001, one-way ANOVA was performed, followed by Tukey multiple comparison tests.

[0039] [Figure 12]In Fabry disease mice treated with AVT917-HC7-hGLA, the substrate level was significantly reduced. Adult (16-week-old) male Fabry disease mice (GIa-KO) were intravenously injected with either the solvent (formulation buffer) or AVT919-HC7-hGLA (cassette 3) at three different doses (n=10 / group). Eight weeks after administration, major tissues were collected and processed, and Lyso-Gb3 was quantified using LC-MS / MS. Data are presented as mean values ​​with SD. Percentages on the column indicate relative Lyso-Gb3 levels, with the level in the solvent group set to 100%. *One-way ANOVA was performed for p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, followed by Tukey's multiple comparison test. #Unpaired t-tests were performed for p<0.0001.

[0040] [Figure 13A] AVT917-mediated hGLA expression in NHP. AVT917-HC7-hGLA was administered intravenously to adult male cynomolgus monkeys at doses of 2E12vg / kg (N=3) and 6E12vg / kg (N=2). Plasma samples were collected before and after administration. Plasma α-Gal A activity was measured by fluorescence-based 4-MU method.

[0041] [Figure 13B] AVT917-HC7-hGLA was administered intravenously to adult male cynomolgus monkeys at doses of 2E12vg / kg (N=3) and 6E12vg / kg (N=2). Plasma samples were collected before and after administration. Human α-Gal A protein expressed in the plasma was measured by ELISA.

[0042] [Figure 14] Adult male cynomolgus monkeys were intravenously administered AVT917 or AVT919 containing cassette 3 (SEQ ID NO: 18) at a dose of 2E12vg / kg (N=1 / capsid). Plasma was collected and used for the measurement of human α-Gal A protein concentration by ELISA. [Modes for carrying out the invention]

[0043] This disclosure describes codon-optimized nucleotide sequences encoding human α-galactosidase A, and includes data demonstrating that these nucleotide sequences are particularly suitable for gene therapy for Fabry disease. This disclosure further describes nucleotide sequences encoding hepatocyte-specific or hepatocyte-myocyte dual-specific promoters, and includes data demonstrating that these nucleotide sequences are particularly suitable for gene therapy, particularly for lysosomal storage disorders, including Fabry disease. This disclosure further describes nucleotide sequences encoding human α-galactosidase A expression cassettes, and includes data demonstrating that these nucleotide sequences are particularly suitable for gene therapy for Fabry disease. This disclosure provides polynucleotides, promoters, expression cassettes, vectors, host cells, gene delivery systems (e.g., recombinant adeno-associated virus (AAV) particles and recombinant viral particles such as nonviral gene delivery systems), and pharmaceutical compositions related to these nucleotide sequences, as well as methods for using them.

[0044] definition The techniques and programs described or referenced herein include those commonly understood and / or used by those skilled in the art, such as Green and Sambrook, *Molecular Cloning: A Laboratory Manual*, 4th edition; 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 This includes the widely used methods described in Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010).

[0045] Unless otherwise defined herein, technical and scientific terms used herein have the meanings generally understood by those skilled in the art. The following terms are used to describe this specification.

[0046] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass naturally occurring or interveningly modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification. The definitions further include polypeptides containing one or more amino acid analogs, including, but not limited to, non-natural amino acids and other modifications known in the art.

[0047] The terms “polynucleotide” or “nucleic acid,” which may be used interchangeably herein, refer to polymers of nucleotides of any length, including DNA (including cDNA) and RNA (including mRNA). Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or synthetic reactions. Polynucleotides may be single-stranded or double-stranded. Polynucleotides may be linear or cyclic. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, “oligonucleotide” refers to a common, but not necessarily, short, synthetic polynucleotide of about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above descriptions of polynucleotides are equally and fully applicable to oligonucleotides. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of a nascent RNA transcript from 5' to 3' is called the transcription direction, the sequence region located at the 5' end of the RNA transcript on the DNA strand and having the same sequence as the 5' RNA transcript is called the “upstream sequence”, and the sequence region located at the 3' end of the RNA transcript on the DNA strand and having the same sequence as the 3' RNA transcript is called the “downstream sequence”. In certain embodiments, the polynucleotides described herein may be optimized by alternative or preferred codon use for a particular type of host cell or delivery target cell. Codon optimization can be performed by using any suitable method known in the art (e.g., using suitable software known in the art).

[0048] As used herein, “isolated polynucleotide” or “isolated nucleic acid” refers to nucleic acids, such as RNA, DNA, or mixed nucleic acids, which are typically substantially isolated from other genomic DNA sequences and proteins or complexes (e.g., ribosomes and polymerases) naturally associated with the native sequence. “Isolated” nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, “isolated” nucleic acid molecules, such as cDNA molecules, may substantially not contain other cellular material or culture media if produced by recombinant technology, or substantially not contain chemical precursors or other chemicals if chemically synthesized. The term encompasses nucleic acid sequences removed from their naturally occurring environment, recombinant or cloned DNA isolates and chemically synthesized analogs, or analogs biosynthesized in heterologous systems. A substantially pure molecule may include the isolated form of the molecule. Specifically, “isolated” nucleic acid molecules encoding polypeptides as described herein are nucleic acid molecules identified and isolated from at least one contaminated nucleic acid molecule, which is typically associated with it in its production environment.

[0049] As used herein, the term “homologousity” refers to the percentage of identity between two polynucleotides or two polypeptide parcels. Two DNA or polypeptide sequences are “substantially homologous” to each other if, in a molecule of a limited length, they exhibit sequence identity of 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 in between. As used herein, substantially homologous also refers to sequences that exhibit complete identity with a given DNA or polypeptide sequence.

[0050] As used herein, the term “identity” refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Methods for measuring identity percentage are known in the art. For example, identity percentage can be determined by directly comparing the sequence information between two molecules by comparing the sequences, counting the exact number of matches between the two comparison sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. To assist in the analysis, existing computer programs such as ALIGN, Dayhoff, MOin Atlas of Protein Sequence and Structure MODayhoff ed., 5 Suppl.3:353-358, National Biomedical Research Foundation, Washington, DC can be used, and this program is a modified version for peptide analysis of the local homology algorithm described in the paper by Smith and Waterman (Advances in Appl. Math. 2:482-489, 1981). Programs for determining nucleotide sequence identity can be obtained from the Wisconsin sequencing package version 8 (available from Genetics Computer Group, Madison, Wis), including programs such as BESTFIT, FASTA, and GAP, which also rely on the Smith and Waterman algorithms. These programs are easily used with the default parameters recommended by the manufacturer and described in the Wisconsin sequencing package 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 algorithms, along with a default scoring table and a gap penalty at six nucleotide positions.Another method for establishing the identity percentage in the context of this invention is to use the MPSRCH program package, copyrighted by Arberger University, developed by John F. Collins and Shane S. Sturrok, and published by IntelliGenetics, Inc. (Mountain View, CA). From this software package, the Smith-Waterman algorithm can be employed, and default parameters (e.g., gap start penalty 12, gap extension penalty 1, gap 6) are used in the scoring table. Based on the generated data, the "matching" value reflects the "sequence identity." Other suitable programs for calculating the identity or similarity percentage between sequences are generally known in the art; for example, another alignment program is BLAST, which is used with default parameters. For example, BLASTN and BLASTP can be used by using the default parameters: Genetic code = standard; filter = none; strand = both; cutoff value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting criterion = high score; database = non-duplicate, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these programs are publicly known in this field. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form stable double helixes between homologous regions, then digesting with a single-strand specific nuclease and determining the size of the digested fragments. Substantially homologous DNA sequences can be identified in Southern hybridization experiments under stringent conditions, for example, defined for the particular system in question. Defining appropriate hybridization conditions is within the technical scope of this field. See, for example, Sambrook et al., op. cit.; DNA cloning, op. cit.; nucleic acid hybridization, op. cit.

[0051] As used herein, the term “vector” refers to a substance for supporting or encapsulating a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Applicable vectors include, for example, expression vectors, plasmids, bacmids, cosmids, constructs, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain a selectable sequence or marker that can be manipulated for stable integration into the chromosomes of a host cell. Vectors may also contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, compensate for nutritional deficiencies, or provide essential nutrients that are not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcriptional enhancers, transcriptional terminators, etc., known in the art. When co-expressing two or more nucleic acid molecules, the two nucleic acid molecules can be inserted, for example, into a single expression vector or a standalone expression vector. The introduction of nucleic acid molecules into host cells can be confirmed by methods known in the art. Such methods include, for example, nucleic acid analysis such as Northern blotting or mRNA polymerase chain reaction (PCR) amplification, Western blotting for gene product expression, or other appropriate analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. The term “vector” includes clone and expression vectors, as well as viral vectors. In certain embodiments, the vectors provided herein are recombinant viral vectors. In specific embodiments, the vectors provided herein are recombinant AAV vectors.

[0052] As used herein, the term “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector comprising a nucleic acid sequence from AAV and one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences). In some embodiments, one or more heterologous sequences are flanked to at least one (two in certain embodiments) AAV terminal inverted repeat sequences (ITRs). In some embodiments, such an rAAV vector may be replicated and packaged into an infectious viral capsid particle, for example, when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper function) and expresses AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). The rAAV vector may be incorporated into a relatively large polynucleotide (e.g., a chromosome or another vector, e.g., a plasmid for cloning or transfection) and may be “rescued” by replication and packaging in the presence of AAV packaging function and suitable helper function. rAAV vectors may take any of the following forms, including, but are not limited to, plasmids, linear artificial chromosomes, complexes with lipids, encapsulation in liposomes, and encapsulation in viral capsid particles (particularly AAV particles). By packaging rAAV vectors into AAV capsids, "recombinant adeno-associated virus capsid particles (rAAV particles)" can be produced.

[0053] As used herein, the term “heterogeneous,” used in conjunction with nucleic acid sequences such as coding sequences and regulatory sequences, refers to sequences that do not generally bind and / or sequences that are not generally associated with a particular cell. Therefore, the “heterogeneous” region of a nucleic acid construct or vector is a nucleic acid segment that does not exist in nature with other molecules, and is linked to or within another nucleic acid molecule. For example, the heterogeneous region of a nucleic acid construct may include a coding sequence to which sequences unrelated to the coding sequence in nature have been flanked. Another example of a heterogeneous 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 native gene).

[0054] As used herein, the term “flanking” in relation to sequences in which other elements are flanked indicates the presence of one or more flanking elements upstream and / or downstream, i.e., 5' and / or 3', of the sequence. The term “flanking” does not necessarily indicate that the sequence is continuous. For example, an intervening sequence may exist between a nucleic acid encoding a transgene and a flanking element. A sequence (e.g., a transgene) in which two other elements (e.g., ITRs) are “flanked” indicates that one element is located at 5' of the sequence and the other at 3', but an intervening sequence may exist between them.

[0055] 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 known in the art and are generally sequences of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of an ITR can exist in one of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. These outermost 125 nucleotides further contain several short self-complementary regions (designated as regions A, A', B, B', C, C', and D), which allow for intra-strand base pairing in this region of the ITR.

[0056] A "coding sequence," or a sequence that "codes" a selected polypeptide, is a nucleic acid molecule that, when controlled by appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide. The boundaries of the coding sequence are determined by the 5' (amino) terminal start codon and the 3' (carboxyl) terminal translation termination codon. The transcription termination sequence may be located at the 3' position of the coding sequence.

[0057] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a manipulably linked coding sequence in a specific host organism. Regulatory sequences suitable for prokaryotes include, for example, promoters, arbitrary operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0058] As used herein, when used to refer to nucleic acids or amino acids, the terms “operatably linked” and similar phrases (e.g., genetically fused) refer, respectively, to the operational linkage of nucleic acid sequences or amino acid sequences arranged in a functional relationship with one another. For example, operationally linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA). In some embodiments, operationally linked nucleic acid elements induce transcription of an open reading frame, ultimately producing 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 arranged at appropriate distances from each other to confer a desired function of each domain.

[0059] As used herein, the term “expression cassette” or “nucleic acid expression cassette” refers to a nucleic acid molecule comprising one or more transcriptional regulatory elements (e.g., promoters, response elements, polyadenylated sequences, and introns) that guide the expression of biomolecules in one or more desired cell types, tissues, or organs, for example, guiding (trans) gene expression. Typically, these may also include guiding the expression of endogenous genes in cells into which the expression cassette has been inserted into a nucleic acid sequence, but also include transgenes.

[0060] As used herein, the term “response element (RE)” refers to a transcriptional regulatory element, particularly a non-coding transcriptional regulatory element, that can regulate and / or control gene transcription. A response element comprises at least one transcription factor binding site (TFBS). A response element may be located upstream (e.g., in the promoter region) or downstream (e.g., in the 3'UTR) of a gene regulated in vivo, or in the vicinity or further away from the gene. A response element may be a naturally occurring sequence or a non-naturally occurring sequence.

[0061] As used herein, the term “enhancer” refers to a nucleic acid sequence that increases the transcription rate by increasing the activity of the promoter.

[0062] As used herein, the term “promoter” in its general sense refers to a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence originates from a gene that binds to RNA polymerase and initiates transcription of a downstream (3' direction) coding sequence. Transcription promoters may include “inductive promoters” (where the expression of a polynucleotide sequence manipulably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where the expression of a polynucleotide sequence manipulably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.”

[0063] As used herein, the term “TATA box” refers to a DNA sequence found in the promoter region of a gene that can bind to TATA-binding protein and transcription factor II D during the process of DNA dissociation and binding with RNA polymerase II. TATA box sequences typically include a TATAAA sequence and generally contain an additional 3' adenine nucleotide.

[0064] As used herein, the term “transcription start site” refers to the location where transcription begins at the 5' end of a gene sequence.

[0065] As used herein, the term "ORF (Open Reading Frame)" refers to a sequence of nucleotide triplets (codons) that code for an amino acid. These sequences can typically be translated into peptides.

[0066] As used herein, the term “transcription factor (TF)” refers to a protein that binds to a specific DNA sequence and thereby regulates the transfer (or transcription) of genetic information from DNA to RNA. TFs perform this function alone or in conjunction with other proteins in a complex by promoting (as an activator) or inhibiting (as a repressor) the recruitment of RNA polymerase (the enzyme that transcribes genetic information from DNA to RNA) to a specific gene. The specific DNA sequence to which a TF binds is called a response element (RE) or regulatory element. Other names include cis-elements and cis-acting transcription regulatory elements. Transcription factors interact with their binding sites through a combination of electrostatic (hydrogen bonding is 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 actually interact with the transcription factor. Also, some of these interactions may be weaker than others. Therefore, many transcription factors can bind not just one sequence, but a subset of closely related sequences, each with a different interaction strength. For example, the covalent binding site of TATA-binding protein (TBP) is TATAAAA, but TBP transcription factors may bind to similar sequences, such as TATATAT or TATAA. Transcription factors (TFs) can be classified based on many aspects. These include the protein structure, the secondary, tertiary, and quaternary structures and properties of the DNA binding sequence, the interaction with the DNA double helix, and the metal and other binding properties. The JASPAR database and TRANSFAC (TRANSFAC.RTM.7.0 Public 2005) are two network-based databases of transcription factors, including experimentally proven binding sites and regulatory genes.

[0067] As used herein, the broad term “transgene” means any heterologous nucleotide sequence incorporated into a vector, such as a viral vector, for expression in target cells, and may be associated with an expression regulatory sequence, such as a promoter. Those skilled in the art will understand that the expression regulatory sequence is selected based on its ability to promote the expression of the transgene in target cells. Examples of transgenes are nucleic acids encoding therapeutic polypeptides or detectable markers.

[0068] As used herein, the terms “AAV capsid,” “AAV capsid protein,” or “AAV cap” refer to the protein encoded by the AAV capsid protein (cap) gene (e.g., VP1, VP2, and VP3) or its variants. For example, the terms include, but are 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, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV LK03, AAVrh10, AAVrh74, AAV44-9, or their variants. The term further includes capsid proteins expressed by or derived from recombinant AAVs, such as chimeric AAVs.

[0069] As used herein, the term “AAV capsid particle” or “AAV particle” comprises at least one AAV capsid protein (e.g., VP1 protein, VP2 protein, VP3 protein or a variant thereof) and optionally encapsulates nucleic acids from an AAV genome (including rAAV genome) or nucleic acids from an AAV genome (including rAAV genome).

[0070] The term "serotype," as used in relation to vectors or viral capsids, is defined by different immunological spectra based on the capsid protein sequence and capsid structure.

[0071] As used herein, with respect to a viral capsid or particle, the term “chimera” means that the capsid or particle contains sequences derived from different parvoviruses, preferably different AAV serotypes, as described, for example, in U.S. Patent No. 6,491,907 by Rabinowitz et al., the disclosure of which is incorporated herein by whole reference.

[0072] The term “recombinant” means different from the genetic entities normally found in nature. When applied to polynucleotides, genes, promoters, expression cassettes, etc., it means the products of various combinations of cloning, restriction, ligation and / or synthesis processes, and other programs that produce constructs different from naturally occurring polynucleotides, genes, promoters, expression cassettes, etc. When used herein to describe viruses, the term “recombinant” refers to a virus that has been genetically altered, for example, by adding or inserting a heterologous nucleic acid construct into a particle. For example, as used herein, the term “recombinant AAV particle” or “rAAV” refers to an AAV that has been genetically altered, 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 polynucleotides of an AAV particle.

[0073] As used herein, “specific hybridization” means that the antisense compound has sufficient complementarity to induce the desired effect between the antisense oligonucleotide and the target nucleic acid, and exhibits minimal or no effect on the non-target nucleic acid under conditions where specific binding is required, i.e., physiological conditions under which it is measured and treated in the body.

[0074] As used herein, “stringent hybridization conditions” or “stringent conditions” means conditions under which an oligo compound hybridizes with its target sequence, but with the fewest number of other sequences.

[0075] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids. For example, the term “transfection” means the intracellular uptake of exogenous DNA, and a cell is “transfected” when exogenous DNA is introduced into the cell membrane. Many transfection techniques are known in this field. For example, Graham et al. (1973) Virology, 52:456, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 th See Ed., Cold Spring 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 types of exogenous molecules into suitable host cells. "Transduction" of a virus into a cell means transferring nucleic acids, such as DNA or RNA, from a viral particle to a cell.

[0076] As used herein, the term “host cell” refers to a particular cell that can be transfected by nucleic acid molecules, vectors, viral particles, or nonviral gene delivery systems, and to the progeny or potential progeny of such a cell. The host cell may be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0077] The term "purification" refers to the separation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) so that the substance of interest constitutes the majority of the sample in which it is located. Typically, in a sample, substantially purified components constitute 50%, 80%-85%, 90%-99% of the sample, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. Techniques for purifying polynucleotides and polypeptides of interest are known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density precipitation.

[0078] As used herein, the term “treatment” means a reduction or mitigation of the progression, severity, and / or duration of a disease or condition by the administration of one or more therapies. Treatment can be determined by assessing whether there is a reduction, mitigation, and / or mitigation of one or more symptoms associated with the latent disorder, thereby indicating that the patient may still be suffering from the latent disorder but is still showing improvement. The term “treatment” includes disease control and mitigation. The term “management” means a beneficial effect obtained by a subject from a therapy that does not necessarily contribute to a cure of the disease. “Treatment” includes (1) preventing the disease, i.e., preventing the progression of the disease, or causing the disease to develop at a lower intensity in a subject who is susceptible to the disease but has not experienced or displayed symptoms of the disease; (2) suppressing the disease, i.e., blocking the progression of the disease, preventing or delaying the progression of the disease, or improving the state of the disease; (3) mitigating the symptoms of the disease, i.e., reducing the number of symptoms experienced by the subject; and (4) reducing, preventing, or delaying the progression of the disease or its symptoms. The term "prevention" means reducing the likelihood of the onset (or recurrence) of a disease, disorder, illness, or related symptoms.

[0079] As used herein, “administration” means the act of physically delivering a substance (e.g., a pharmaceutical composition provided herein) to a subject or patient (e.g., a human) by injection or other means, for example, orally, via mucous membrane, topically, intradermally, parenterally, intravenously, intravitreously, intra-articularly, subretinally, intramuscularly, intra-sheathly, and / or via any other physical delivery method described herein or known in the art. In certain embodiments, administration is administered by intravenous infusion. The therapeutic agents (e.g., pharmaceutical compositions) provided herein may be delivered systemically or to specific tissues.

[0080] As used herein, the terms “effective dose” or “therapeutic effective dose” mean an amount of a therapeutic agent (e.g., a pharmaceutical composition provided herein) sufficient to treat, diagnose, prevent or delay the onset of a given disease, disorder or illness and / or symptoms associated therewith, and to reduce and / or mitigate its severity and / or duration. These terms further include amounts necessary to reduce, delay or mitigate the progression or advancement of a given disease, reduce, delay or mitigate the recurrence, progression or onset of a given disease, and / or to enhance or enhance the preventive or therapeutic effect of other therapies, or to serve as a bridge to other therapies. In some embodiments, “effective dose” as used herein further means an amount of a therapeutic agent described herein (e.g., a pharmaceutical composition provided herein) that achieves a specified result.

[0081] As used herein, the terms “subject” and “patient” may be used interchangeably and refer to mammals, e.g., non-primate animals (e.g., cattle, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or primates (e.g., monkeys and humans), e.g., humans. In a particular embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder described herein. In another embodiment, the subject is a mammal, e.g., a human, at risk of progression of a disease or disorder described herein. In a specific embodiment, the subject is a human. In a specific embodiment, the subject is an adult. In a specific embodiment, the subject is a human adolescent. In a specific embodiment, the subject is a human child.

[0082] As used herein, the term “therapy” may mean any operating 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., one or more symptoms or conditions associated with the disease or disorder described herein). In certain embodiments, the term “therapy” may mean gene therapy that can be used to treat, manage, prevent or alleviate a disease or disorder or one or more symptoms thereof.

[0083] As used herein, the term “gene therapy” refers to the introduction of heterologous nucleic acid molecules into one or more receptor cells, where the heterologous nucleic acid itself or its expression in the receptor cell affects the function of the cell and produces a therapeutic effect on the subject. For example, the heterologous nucleic acid molecule may encode a protein that affects the function of the receptor cell.

[0084] As used in this disclosure, unless otherwise specified, the terms “about” and “approximately” should be interpreted as meaning that a normal variation as judged by a person skilled in the art is permissible or falls within a tolerance range of a particular value as determined by a person skilled in the art, which in part depends on how the value is measured or determined (i.e., limitations of the measuring system), for example, a variation within 20%, 10%, or 5% of the value, or a variation within one or more standard deviations of each practice of the art. In specific embodiments, the terms “about” and “approximately” encompass the exact value. All numerical values ​​provided herein are modified by the term “about” unless the context specifically indicates otherwise.

[0085] As used in this disclosure, the singular “1,” “one,” and “the said” include plural nouns unless the context explicitly states otherwise. Unless the context explicitly states otherwise, the terms “1” (or “one”) and “one or more” and “at least one” may be used interchangeably herein.

[0086] In this specification, when the term “including” is used to describe embodiments, it should be understood that other similar embodiments described as “consisting of ○○” and / or “essentially consisting of ○○” are also provided. In this specification, when the phrase “substantially consisting of ○○” is used to describe embodiments, it should also be understood that similar embodiments described as “consisting of ○○” are also provided.

[0087] The term "between ○○" used in phrases such as "between A and B" or "between A and B" means a range that includes both A and B.

[0088] In this specification, for example, the term "and / or" as used in the phrase "A and / or B" is intended to include "both A and B", "A or B", "A (alone)", and "B (alone)". Similarly, for example, the term "and / or" as used in the phrase "A, B and / or C" is intended to include each of the embodiments of "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)".

[0089] The terms "or" and "and" may be used interchangeably and may be understood to mean "and / or" unless the context explicitly states otherwise.

[0090] Polynucleotides, promoters, and expression cassettes This disclosure describes codon-optimized nucleotide sequences encoding human α-galactosidase A. The human α-galactosidase A encoded by the nucleotide sequences described in this disclosure may be wild-type human α-galactosidase A or mutant (e.g., cleaved) human α-galactosidase A, but is preferably functional human α-galactosidase A. In specific embodiments, the codon-optimized nucleotide sequences encoding human α-galactosidase A include nucleotide 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%, or 99.8% or 100% identity with SEQ ID NO:1. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:1. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A is shown as SEQ ID NO:1. In a specific embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A includes 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:2. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:2. In a specific embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A includes 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:20.In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:20. In one embodiment, the codon-optimized nucleotide sequence encoding human α-galactosidase A is shown at SEQ ID NO:20. It is undesirable to be bound by any theory, and these nucleotide sequences are particularly suitable for gene therapy for Fabry disease.

[0091] This disclosure further describes nucleotide sequences encoding hepatocyte-specific or hepatocyte-myocyte dual-specific promoters. In certain embodiments, the nucleotide sequences encode hepatocyte-specific promoters. In certain embodiments, the nucleotide sequences encode hepatocyte-myocyte dual-specific promoters.

[0092] In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:3. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:3. It is undesirable to be bound by any particular theory, and these nucleotide sequences are particularly suitable for guiding the expression of biomolecules, such as genes (transgenes), especially in hepatocytes (i.e., in the liver), and optionally in other cell types (e.g., muscle cells and / or kidney cells), making them particularly suitable for gene therapy for lysosomal storage disorders, including Fabry disease.

[0093] In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:4. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:4. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:4. In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:5. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:5. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:5. In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:6. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:6. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:6.In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:7. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:7. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:7. In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:8. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:8. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:8. In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:9. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:9. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:9.In a specific embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes 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:10. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter includes SEQ ID NO:10. In one embodiment, the nucleotide sequence encoding the hepatocyte-specific or hepatocyte-myocyte bispecific promoter is shown in SEQ ID NO:10. It is undesirable to be bound by any particular theory, and these nucleotide sequences are particularly well-suited for gene therapy, especially for lysosomal storage disorders, including Fabry disease, because they are suitable for guiding the expression of biomolecules, such as genes (transgenes), 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). 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 a therapeutic effect.

[0094] This disclosure further describes nucleotide sequences encoding human α-galactosidase A expression cassettes. In specific embodiments, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes 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:16. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes SEQ ID NO:16. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is shown in SEQ ID NO:16. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes 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:17. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes SEQ ID NO:17. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is shown in SEQ ID NO:17. In a specific embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes 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:18. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes SEQ ID NO:18. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette is shown in SEQ ID NO:18.In specific embodiments, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes 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:19. In one embodiment, the nucleotide sequence encoding the human α-galactosidase A expression cassette includes 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. It is undesirable to be bound by any theory, and these nucleotide sequences are particularly suitable for gene therapy for Fabry disease.

[0095] This disclosure provides, in particular, polynucleotides, promoters, and expression cassettes related to these nucleotide sequences.

[0096] Polynucleotides In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding human α-galactosidase A, wherein the nucleotide sequence encoding human α-galactosidase A has 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.

[0097] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:1.

[0098] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:2.

[0099] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:20.

[0100] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter has 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.

[0101] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0102] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0103] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0104] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0105] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0106] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0107] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0108] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0109] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0110] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding human α-galactosidase A, and 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.

[0111] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:1.

[0112] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:2.

[0113] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:20.

[0114] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0115] In various embodiments, the nucleotide sequence encoding the promoter includes 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.

[0116] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0117] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0118] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0119] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0120] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0121] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0122] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0123] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0124] In various embodiments, the polynucleotide further comprises one or more regulatory elements (see Section 5.2.5). In specific embodiments, the polynucleotide further comprises a nucleotide sequence encoding a polyA signal.

[0125] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the biomolecule, and 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.

[0126] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0127] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0128] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0129] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0130] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0131] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0132] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0133] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0134] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0135] Further description of the biomolecule is provided in Section 5.2.4. In a specific embodiment, the biomolecule is human α-galactosidase A (functional human α-galactosidase A is preferred).

[0136] In various embodiments, the polynucleotide further comprises one or more regulatory elements (see Section 5.2.5). In specific embodiments, the polynucleotide further comprises a nucleotide sequence encoding a polyA signal.

[0137] In one embodiment, a polynucleotide is provided comprising a nucleotide sequence encoding an expression cassette, wherein the nucleotide sequence encoding the expression cassette has 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:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

[0138] In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:16.

[0139] In certain embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:17.

[0140] In certain embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:18.

[0141] In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:19.

[0142] In one embodiment, a polynucleotide is provided that includes a nucleotide sequence encoding the expression cassette described in Section 5.2.3.

[0143] In various embodiments and designs, the polynucleotides described herein are purified. In various embodiments and designs, the polynucleotides described herein are isolated. In various embodiments and designs, the polynucleotides described herein are recombinant polynucleotides.

[0144] In certain 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.

[0145] A further explanation of polynucleotides is provided in Section 5.1.

[0146] promoter In one embodiment, a promoter is provided which is encoded by 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.

[0147] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0148] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0149] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0150] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0151] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0152] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0153] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0154] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0155] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0156] In various embodiments and designs, the promoters described herein are recombinant promoters.

[0157] Further explanation of the promoter is provided in sections 5.1 and 5.2.5.

[0158] Expression Cassette In one embodiment, an expression cassette is provided comprising a nucleotide sequence encoding human α-galactosidase A and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding human α-galactosidase A, and 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.

[0159] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:1.

[0160] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:2.

[0161] In a particular embodiment, the nucleotide sequence encoding human α-galactosidase A includes SEQ ID NO:20.

[0162] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0163] In various embodiments, the nucleotide sequence encoding the promoter includes 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.

[0164] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0165] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0166] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0167] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0168] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0169] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0170] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0171] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0172] In various embodiments, the expression cassette further comprises one or more regulatory elements (see Section 5.2.5). In specific embodiments, the expression cassette further comprises a nucleotide sequence encoding a polyA signal.

[0173] In one embodiment, an expression cassette is provided comprising a nucleotide sequence encoding a biomolecule and a nucleotide sequence encoding a promoter, wherein the nucleotide sequence encoding the promoter is operably linked to the nucleotide sequence encoding the biomolecule, and 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.

[0174] In a specific embodiment, the promoter is a hepatocyte-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-renal cell dual-specific promoter. In a specific embodiment, the promoter is a hepatocyte-myocyte-renal cell multiple-specific promoter.

[0175] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:3.

[0176] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:4.

[0177] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:5.

[0178] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:6.

[0179] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:7.

[0180] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:8.

[0181] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:9.

[0182] In a particular embodiment, the nucleotide sequence encoding the promoter includes SEQ ID NO:10.

[0183] Further description of the biomolecule is provided in Section 5.2.4. In a specific embodiment, the biomolecule is human α-galactosidase A (functional human α-galactosidase A is preferred).

[0184] In various embodiments, the expression cassette further comprises one or more regulatory elements (see Section 5.2.5). In specific embodiments, the expression cassette further comprises a nucleotide sequence encoding a polyA signal.

[0185] In one embodiment, an expression cassette is provided which is encoded by 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:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

[0186] In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:16.

[0187] In certain embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:17.

[0188] In certain embodiments, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:18.

[0189] In a particular embodiment, the nucleotide sequence encoding the expression cassette includes SEQ ID NO:19.

[0190] In various aspects and embodiments, the expression cassette described herein is a recombinant expression cassette.

[0191] Further explanation of the expression cassette is provided in Section 5.1.

[0192] Biomolecules The biomolecules described herein may, but are 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 a protein. In preferred embodiments, the biomolecule is a human polypeptide or a human protein. The biomolecule may 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 may also be a therapeutic protein, such as a therapeutic protein. Therapeutic molecules can be used to correct or improve gene deficiencies associated with disease or disorder. Exemplary therapeutic molecules 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.

[0193] In certain embodiments, the biomolecules are therapeutic proteins. The therapeutic proteins described herein include hormones and growth and differentiation factors, such as 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), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and IGF-II), and transforming growth factor α-super. —Includes, but is not limited to, any one of the families (including TGFα), activin, inhibin, or any of the bone morphogenetic proteins (BMPs) BMP1-15, any of the growth factors of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, chorionic neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), any one of the families of neuruturin, agrin, semaphorin / colapsin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0194] Other therapeutic proteins described herein include those that modulate the immune system, and these include, but are not limited to, thrombopoietin (TPO), interleukins (IL) IL-1 to IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemotactic proteins, leukemia inhibitors, granulocyte-macrophage colony-stimulating factors, Fas ligands, tumor necrosis factor α and β, interferon α, β, and γ, stem cell factors, and cytokines and lymphokines such as flk-2 / flt3 ligands. 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 class II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful proteins further include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor proteins (MCPs), pharmacoagulation factors (DAFs), CR1, CF2, and CD59.

[0195] Other therapeutic proteins described herein include receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. This disclosure encompasses 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 encompasses proteins such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors, estrogen receptors, vitamin D receptors, and other nuclear receptors. Furthermore, useful proteins include jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZFS, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-binding proteins, interferon regulator (IRF-1), Wilms tumor protein, ETS-binding proteins, STAT, GATA box-binding proteins (e.g., GATA-3), and transcription factors such as the forkhead family of winged helix proteins.

[0196] Other useful biomolecules described herein include carbamoyl synthase I, ornithine transcarbamylase, arginosuccinate synthase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, α-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminosease, cystathion β-synthase, branched chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane regulator (CFTR) sequence, and dystrophin cDNA sequence. Other useful proteins include enzymes that may be useful in enzyme replacement therapy for a variety of conditions resulting from enzyme deficiency. For example, enzymes containing mannose-6-phosphate can be used to treat lysosomal storage disorders (e.g., those in which the appropriate gene encodes β-glucuronidase (GUSB)).

[0197] Other useful biomolecules described herein include unnaturally occurring polypeptides, such as chimeric or heteropolypeptides having unnaturally occurring amino acid sequences, including insertions, deletions, or amino acid substitutions. For example, single-stranded engineered immunoglobulins may be used in patients with weakened immune function. Other types of unnaturally occurring gene sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, which may be used to reduce the overexpression of a target.

[0198] Other suitable biomolecules described herein may be useful in treating individuals suffering from autoimmune diseases and disorders by providing a broad protective immune response against autoimmune-related targets, including cell receptors and cells that produce antibodies directed toward "self." T-cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren'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. Each of these diseases is characterized by a T-cell receptor (TCR) that binds to an endogenous antigen and initiates an inflammatory cascade associated with the autoimmune disease.

[0199] Biomolecules can be used to reduce and / or regulate the expression of proteins of interest in order to treat hyperproliferative disorders characterized by excessive cell proliferation, such as cancer and psoriasis. Target polypeptides include polypeptides that are produced specifically or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, and fyn, and translocation genes such as bcr / abl, ras, src, P53, neu, trk, and EGRF. In addition to oncogene products as target antigens, target polypeptides used in anti-cancer therapeutic and protective schemes include the variable regions of antibodies prepared by B-cell lymphoma and the variable regions of T-cell receptors in T-cell lymphoma, and in some embodiments, they are also used as target antigens for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides; for example, polypeptides found at higher levels in tumor cells include polypeptides recognized by the monoclonal antibody 17-1A and folate-binding polypeptides.

[0200] In certain embodiments, the biomolecules are RNA. These RNA molecules include, but are not limited to, those that perform their functions through RNA interference (shRNA, RNAi), micro-RNA regulation (miR), catalytic RNA, antisense RNA, RNA aptamers, and others.

[0201] Biomolecules can be used to induce an immune response to a selected antigen. For example, to promote an immune response, the antigen may be expressed by a promoter disclosed herein, the vector may be adjuvanted as described herein, and / or the vector may be introduced into degenerative tissue.

[0202] Examples of suitable immunogenic antigens include those selected from a variety of viridae. Desired viridae for which an immune response is expected are the Picornaviridae, which includes the Rhinovirus genus, responsible for about 50% of common cold cases; the Enterovirus genus, which includes human enteroviruses such as poliovirus, Coxsackievirus, echovirus, and hepatitis A virus; and the Aftovirus genus, which is responsible for foot-and-mouth disease, primarily in non-human animals. Within the Picornaviridae, target antigens include VP1, VP2, VP3, VP4, and VPG. Other viridae include the Astrovirus and Caliciviridae. The Caliciviridae includes the Norwalk group viruses, which are important pathogens of epidemic gastroenteritis. Yet another viridae desirable for use in targeting antigens to induce an immune response in humans and non-human animals are the Alphavirus genus, which includes Sindbisvirus, Ross River virus, and Venezuelan, Eastern and Western Equine Encephalitis viruses, and the Rubivirus genus, which includes 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 from the Coronaviridae family, which includes hepatitis C virus, or numerous non-human viruses such as infectious bronchitis virus (poultry), porcine infectious gastroenteritis virus (swine), porcine hemagglutinin encephalomyelitis virus (swine), feline infectious peritonitis virus (cat), feline enteric coronavirus (cat), and canine coronavirus (dog), as well as human respiratory coronaviruses that can cause the common cold and / or non-A, B, or C hepatitis and are the putative cause of severe acute respiratory syndrome (SARS). Within the Coronaviridae family, target antigens include E1 (also called M or matrix protein), E2 (also called S or spike protein), E3 (also called HE or hemagglutinin-elterose), glycoprotein (not present in all coronaviruses), or N (nucleocapsid). Furthermore, other antigens may target the Arteriviridae and Rhabdoviridae families. The Rhabdoviridae family includes the Becyclovirus genus (e.g., vesicular stomatitis virus) and common lyssaviruses (e.g., rabies).Within the Rhabdoviridae family, suitable antigens can originate from either the G protein or the N protein. The Filoviridae family, which includes hemorrhagic fever viruses such as Marburg and Ebola viruses, can be a suitable source of antigens. The Paramyxoviridae family includes parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, rubravirus (including mumps virus, parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest, measles and canine distemper), and pneumovirus (including respiratory syncytial virus). Influenza viruses are classified within the Orthomyxoviridae family and are a suitable source of antigens (e.g., HA protein, N1 protein). The Bunyaviridae family includes Bunyavirus (California encephalitis, Lacrosse), Phlebovirus (Kenya hemorrhagic fever), Hantavirus (Premara is a type of hemorrhagic fever virus), Nairovirus (Nairobi sheep disease), and various unassigned Bunyaviruses. The Arenaviridae family provides a source of antigens for LCM and Lassa fever virus. Another source of antigens is the Bornavirus family. The Reoviridae family includes the genera Reovirus, Rotavirus (which causes acute gastroenteritis in children), Orbivirus, and Coltivirus (Colorado tick fever, Lebombo (human), equine encephalopathy, Bluetongue). The Retroviridae family includes feline leukemia virus, HTLV-VI and HTLV-II, and lentivirus (HIV, monkey The family includes the Oncoriviridae subfamily, which encompasses human and veterinary diseases such as immunodeficiency viruses, feline immunodeficiency virus, equine infectious anemia virus, and spumaviral. The Pavobaviridae family includes the Polyomavirinae subfamily (BKU and JCU viruses) and the Papillomavirinae subfamily (associated with the malignant progression of cancer or papillomas). The Adenoviridae family includes viruses that cause respiratory diseases and / or enteritis (EX, AD7, ARD, OB).The Parvoviridae family includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The Herpesviridae family includes the Alphaherpesvirinae subfamily, which includes the Simplexvirus genus (HSVI, HSVII) and the Varoselovirus genus (pseudorabies, herpes zoster); the Betaherpesvirinae subfamily, which includes the Cytomegalovirus genus (HCMV, muromegalovirus); and the Gammaherpesvirinae subfamily, which includes the Lymphocryptovirus genus, EBV (Burkitt lymphoma), human herpesviruses 6A, 6B, and 7, Kaposi's sarcoma-related herpesviruses, as well as cercopithecine herpesvirus (B virus), infectious rhinotracheitis, Marek's disease virus, and the Radinovirus genus. The Poxviridae family includes the subfamily Choldopoxvirinae, which encompasses the genera Orthopoxvirus (smallpox and cowpox), Parapoxvirus, Avipoxvirus, Capripoxvirus, Lepolipoxvirus, and Suipoxvirus, as well as the subfamily Entomopoxvirinae. The Hepadnaviridae family includes hepatitis B virus. One unclassified virus that can be a suitable source of antigen is hepatitis D virus, hepatitis E virus, and prions. Another virus that can be a source of antigen is Nipah virus. Other virus sources may include avian bursal disease virus and porcine reproductive and respiratory syndrome virus. The Alphaviridae family includes equine arteritis virus and various encephalitis viruses.

[0203] This disclosure may also include immunogens based on proteins from cancer cells or tumor cells that are useful for immunizing humans or non-human animals against other pathogens, including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates as biomolecules, or immunogens based on proteins from cancer cells or tumor cells. Examples of bacterial pathogens include pathogenic Gram-positive cocci, such as Streptococcus pneumoniae; Staphylococcus (and the toxins they produce, e.g., enterotoxin B); and Streptococcus. Pathogenic Gram-negative cocci include Neisseria meningitidis; Neisseria gonorrhoeae. Pathogenic enteric Gram-negative bacilli include the Enterobacteriaceae family; Pseudomonas, Acinetobacteria, and Eikenella; meidomygia; Salmonella; Shigella; Haemophilus; Moraxella; Haemophilus ducreyi (causes chancroid), Brucella species (brucellosis); Francisella tularensis (causes tularemia); Yersinia pestis (plague) and other Yersinia species (Pasteurella); Streptobacillus moniliformis and Spirillum species; Gram-positive bacilli include Listeria monocytogenes. This category includes monocytogenes; erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); Donovan disease (inguinal granuloma); and bartonellosis. Diseases caused by pathogenic anaerobic bacteria include tetanus; botulism (Clostridium botulinum and its toxin); Clostridium perfringens and its ε-toxin; other Clostridium species; tuberculosis; leprosy; and other mycobacteria.Pathogenic spirochete diseases include syphilis; treponemic infections; strawberry cystitis, pinta and endemic syphilis; and leptospirosis. Other infections caused by more pathogenic bacteria and pathogenic fungi include glanders (Burkholderia mallei); actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis and mucormycosis; sporotrichosis; paracoccidioidomycosis, petriellidiosis, torulopsiosis, mycomatosis and chromomycosis; and dermatophytosis. Rickettsial infections include typhus, Rocky Mountain fever, Q fever (Coxiella burnetti) and rickettsialpox. Examples of mycoplasma and chlamydia infections include mycoplasma pneumonia; lymphogranuloma venereum; psittacosis; and perinatal chlamydia infection. Pathogenic eukaryotes include pathogenic protozoa and helminths, and the infections they cause include amoebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis; giardiasis; trichinellosis; filariasis; schistosomiasis; nematodeosis; trematode / flukes; and cestode / tapeworm infections.

[0204] Many of these organisms and / or toxins produced by them have been identified by the Centers for Disease Control (CDC), the U.S. Department of Health and Human Services, as pathogens with potential for use in biological attack. For example, some of these biological agents include Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague), smallpox, Francisella tularensis (tularemia), and viral hemorrhagic fevers [filoviruses (e.g., Ebola, Marburg) and arenaviruses [e.g., Lassa, Machupo]] (all of these now belong to Category A agents); Coxiella burnetti (Q fever); Brucella (brucellosis), Burkholderia mallei (glanders), Burkholderia pseudomallei (meidomygia), and Ricinus (carambola). This includes *Clostridium communis* and its toxin (lysine toxin), *Clostridium perfringens* and its toxin (epsilon toxin), *Staphylococcus* and their toxins (enterotoxin B), *Chlamydia psittaci* (psittacosis), threats to water safety (e.g., *Vibrio cholerae*, *Crytosporidium parvum*), *Typhus* (*Richettsia powazekki*), and viral encephalitis (alphaviruses, e.g., Venezuelan horse encephalitis; Eastern horse encephalitis; Western horse encephalitis); (all currently belonging to Category B agents); as well as Nippan virus and Hantavirus (currently classified as Category C agents). In addition, other organisms classified in this way or differently may be identified and / or used for such purposes in the future.The viral vectors and other constructs described herein are useful for delivering antigens from these organisms, viruses, their toxins, or other by-products, thereby it will be readily understood that they can prevent and / or treat infection or other adverse reactions associated with these biological agents.

[0205] In certain embodiments, the biomolecule is a protein that is a segment of the T cell variable region that triggers an immune response (i.e., eliminates cytotoxic T cells). In rheumatoid arthritis (RA), several specific variable regions of the TCR 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 leads to an immune response targeting T cells involved in RA. In multiple sclerosis (MS), several specific variable regions of the TCR 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 leads to an immune response targeting T cells involved in MS. In scleroderma, several specific variable regions of the TCR 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 nucleic acid molecules encoding at least one of these polypeptides elicits an immune response targeting T cells involved in scleroderma.

[0206] In specific embodiments, the biomolecule is typically expressed in hepatocytes (for example, typically expressed at a higher level in hepatocytes than in other cells). In specific embodiments, the biomolecule is typically expressed in muscle cells (for example, typically expressed at a higher level in muscle cells than in other cells). In specific embodiments, the biomolecule is typically expressed in kidney cells (for example, typically expressed at a higher level in kidney cells than in other cells). In specific embodiments, the biomolecule is typically expressed in both hepatocytes and muscle cells (for example, expressed at a higher level in both hepatocytes and muscle cells than in other cells). In specific embodiments, the biomolecule is typically expressed in both hepatocytes and kidney cells (for example, expressed at a higher level in both hepatocytes and kidney cells than in other cells). In specific embodiments, the biomolecule is typically expressed in hepatocytes, muscle cells, and kidney cells (for example, expressed at a higher level in both hepatocytes, muscle cells, and kidney cells than in other cells). In specific embodiments, the biomolecule acts in hepatocytes (for example, necessary or important for the normal function of hepatocytes). In specific embodiments, the biomolecule acts in muscle cells (for example, necessary or important for the normal function of muscle cells). In specific embodiments, the biomolecule acts in kidney cells (for example, necessary or important for the normal function of kidney cells). In specific embodiments, the biomolecule acts in both hepatocytes and muscle cells (for example, necessary or important for the normal function of both hepatocytes and muscle cells). In specific embodiments, the biomolecule acts in both hepatocytes and kidney cells (for example, necessary or important for the normal function of both hepatocytes and kidney cells). In specific embodiments, the biomolecule acts in hepatocytes, muscle cells and kidney cells (for example, necessary 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 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 therapeutic effects.

[0207] In specific embodiments, the biomolecules described herein are associated with lysosomal storage disorders. In specific embodiments, defects in the biomolecules described herein are at least partially responsible for lysosomal storage disorders. In specific embodiments, abnormal low or deficient expression of the biomolecules described herein is at least partially responsible for lysosomal storage disorders.

[0208] In a specific embodiment, the biomolecule is a protein that is α-galactosidase A (GLA) (preferably human GLA). In a specific embodiment, the biomolecule is a protein that is scavenger receptor class B member 2 (SCARB2) (preferably human SCARB2). In a specific embodiment, the biomolecule is a protein that is aspartylglucosaminidase (AGA) (preferably human AGA). In a specific embodiment, the biomolecule is a protein that is palmitoylthioesterase 1 (PPT1) (preferably human PPT1). In a specific embodiment, the biomolecule is a protein that is tripeptidyl peptidase 1 (TPP1) (preferably human TPP1). In a specific embodiment, the biomolecule is a protein that is CLN3 lysosome / endosomal transmembrane protein battenin (CLN3) (preferably human CLN3). In specific embodiments, the biomolecule is a protein that is DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5) (preferably human DNAJC5). In specific embodiments, the biomolecule is a protein that is CLN5 intracellular transport protein (CLN5) (preferably human CLN5). In specific embodiments, the biomolecule is a protein that is CLN6 transmembrane ER protein (CLN6) (preferably human CLN6). In specific embodiments, the biomolecule is a protein that contains Major Facilator Superfamily Domain Containing 8 (MFSD8) (preferably human MFSD8). In specific embodiments, the biomolecule is a protein that is CLN8 transmembrane ER and ERGIC protein (CLN8) (preferably human CLN8). In specific embodiments, the biomolecule is a protein that is cathepsin D (CTSD) (preferably human CTSD). In a specific embodiment, the biomolecule is a protein that is a granulin precursor (GRN) (preferably human GRN).In a specific embodiment, the biomolecule is a protein that is ATPase cation transporter 13A2 (ATP13A2) (preferably human ATP13A2). In a specific embodiment, the biomolecule is a protein that is cathepsin F (CTSF) (preferably human CTSF). In a specific embodiment, the biomolecule is a protein that is potassium channel tetramerization domain containing 7 (Potassium Channel Tetramerization Domain Containing 7, KCTD7) (preferably human KCTD7). In a specific embodiment, the biomolecule is a protein that is cystinosin lysosomal cystine transporter (CTNS) (preferably human CTNS). In a specific embodiment, the biomolecule is a protein that is lysosome-associated membrane protein 2 (LAMP2) (preferably human LAMP2). In a specific embodiment, the biomolecule is a protein that is N-acyl sphingosinamide hydrolase 1 (ASAH 1) (preferably human ASAH 1). In a specific embodiment, the biomolecule is a protein that is α-L-fucosidase 1 (FUCA1) (preferably human FUCA1). In a specific embodiment, the biomolecule is a protein that is cathepsin A (CTSA) (preferably human CTSA). In a specific embodiment, the biomolecule is a protein that is β-glucocerebrosidase (GBA) (preferably human GBA). In a specific embodiment, the biomolecule is a protein that is acid α-glucosidase (GAA) (preferably human GAA). In a specific embodiment, the biomolecule is a protein that is myosin VA (MYO5A) (preferably human MYO5A). In a specific embodiment, the biomolecule is a protein that is RAB27A (RAB27A, Member of RAS Oncogene Family) (preferably human RAB27A). In a specific embodiment, the biomolecule is a protein that is lysosome transport regulator (LYST) (preferably human LYST).In a specific embodiment, the biomolecule is a protein that is galactosidase β1 (GLB1) (preferably human GLB1). In a specific embodiment, the biomolecule is a protein that is hexosaminidase subunit α (HEXA) (preferably human HEXA). In a specific embodiment, the biomolecule is a protein that is hexosaminidase subunit β (HEXB) (preferably human HEXB). In a specific embodiment, the biomolecule is a protein that is ganglioside GM2 activator (GM2A) (preferably human GM2A). In a specific embodiment, the biomolecule is a protein that is HPS1 biogenesis of lysosomal organelles complex 3 subunit 1 (HPS1) (preferably human HPS1). In a specific embodiment, the biomolecule is a protein that is adapter-related protein complex 3 subunit β1 (AP3B1 or HPS2) (preferably human HPS2). In a specific embodiment, the biomolecule is a protein that is HPS3 lysosome organelle complex biosynthesis subunit 21 (HPS3) (preferably human HPS3). In a specific embodiment, the biomolecule is a protein that is HPS4 lysosome organelle complex biosynthesis subunit 32 (HPS4) (preferably human HPS4). In a specific embodiment, the biomolecule is a protein that is HPS5 lysosome organelle complex biosynthesis subunit 22 (HPS5) (preferably human HPS5). In a specific embodiment, the biomolecule is a protein that is HPS6 lysosome organelle complex biosynthesis subunit 23 (HPS6) (preferably human HPS6). In a specific embodiment, the biomolecule is a protein that is dystrobrevin binding protein 1 (DTNBP1 or HPS7) (preferably human HPS7).In a specific embodiment, the biomolecule is a protein that is the biogenesis of lysosomal organelles complex 1 subunit 3 (BLOC1S3 or HPS8) (preferably human HPS8). In a specific embodiment, the biomolecule is a protein that is the biogenesis of lysosomal organelles complex 1 subunit 6 (BLOC1S6 or HPS9) (preferably human HPS9). In a specific embodiment, the biomolecule is a protein that is galactosylceramidase (GALC) (preferably human GALC). In a specific embodiment, the biomolecule is a protein that is mannosidase α class 2B member 1 (MAN2B1) (preferably human MAN2B1). In a specific embodiment, the biomolecule is a protein that is mannosidase β (MANBA) (preferably human MANBA). In a specific embodiment, the biomolecule is a protein that is allylsulfatase A (ARSA) (preferably human ARSA). In a specific embodiment, the biomolecule is a protein comprising N-acetylglucosamine-1-phosphate transferase subunits α and β (GNPTAB) (preferably human GNPTAB). In a specific embodiment, the biomolecule is a protein comprising N-acetylglucosamine-1-phosphate transferase subunits α and β (GNPTAB) (preferably human GNPTAB). In a specific embodiment, the biomolecule is a protein comprising N-acetylglucosamine-1-phosphate transferase subunit γ (GNPTG) (preferably human GNPTG). In a specific embodiment, the biomolecule is a protein comprising mucolipin TRP cation channel 1 (MCOLN1) (preferably human MCOLN1). In a specific embodiment, the biomolecule is a protein comprising α-L-iduronidase (IDUA) (preferably human IDUA). In a specific embodiment, the biomolecule is a protein comprising iduronic acid 2-sulfatase (IDS) (preferably human IDS).In a specific embodiment, the biomolecule is a protein that is N-sulfoglucosamine sulfohydrolase (SGSH) (preferably human SGSH). In a specific embodiment, the biomolecule is a protein that is N-acetyl-α-glucosaminidase (NAGLU) (preferably human NAGLU). In a specific embodiment, the biomolecule is a protein that is heparan-α-glucosamide N-acetyltransferase (HGSNAT) (preferably human HGSNAT). In a specific embodiment, the biomolecule is a protein that is glucosamine (N-acetyl)-6-sulfatase (GNS) (preferably human GNS). In a specific embodiment, the biomolecule is a protein that is galactosamine (N-acetyl)-6-sulfatase (GALNS) (preferably human GALNS). In a specific embodiment, the biomolecule is a protein that is galactosidase β1 (GLB1) (preferably human GLB1). In a specific embodiment, the biomolecule is a protein that is allyl sulfatase B (ARSB) (preferably human ARSB). In a specific embodiment, the biomolecule is a protein that is β-glucuronidase (GUSB) (preferably human GUSB). In a specific embodiment, the biomolecule is a protein that is hyaluronidase 1 (HYAL1) (preferably human HYAL1). In a specific embodiment, the biomolecule is a protein that is sulfatase modifier 1 (SUMF1) (preferably human SUMF1). In a specific embodiment, the biomolecule is a protein that is sphingomyelin phosphodiesterase 1 (SMPD1) (preferably human SMPD1). In a specific embodiment, the biomolecule is a protein that is NPC intracellular cholesterol transporter 1 (NPC1) (preferably human NPC1). In a specific embodiment, the biomolecule is a protein that is NPC intracellular cholesterol transporter 2 (NPC2) (preferably human NPC2). In a specific embodiment, the biomolecule is a protein that is member 5 (SLC17A5) of the solute carrier family 17 (preferably human SLC17A5).In a specific embodiment, the biomolecule is a protein that is α-N-acetylgalactosaminidase (NAGA) (preferably human NAGA). In a specific embodiment, the biomolecule is a protein that is neuraminidase 1 (NEU1) (preferably human NEU1). In a specific embodiment, the biomolecule is a protein that is lipase A, lysosomal acid (LIPA) (preferably human LIPA). In a specific embodiment, the biomolecule is a protein that is motor neuron survival (SMN) protein (preferably human SMN protein). In a specific embodiment, the biomolecule is microdist. The biomolecule is a protein that is rofin (preferably human microdystrophin). In a specific embodiment, the biomolecule is a protein that is phenylalanine hydroxylase (PAH) (preferably human PAH). In a specific embodiment, the biomolecule is a protein that is coagulation factor VIII (FVIII) (preferably human FVIII). In a specific embodiment, the biomolecule is a protein that is coagulation factor IX (FIX) (preferably human FIX). In a preferred embodiment, the biomolecule is a functional protein (e.g., wild-type protein).

[0209] Control element The regulatory element includes an expression regulatory element that is continuous with the nucleotide sequence encoding the biomolecule of interest, and an expression regulatory element that acts in trans or cis but is located at a distance to control the expression of the biomolecule of interest.

[0210] Expression regulatory elements include, but are not limited to, appropriate transcription start, termination, promoter, and enhancer sequences; polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; secretory signals; and nuclear localization sequences.

[0211] In various embodiments, the regulatory control element is a promoter. In a particular embodiment, the promoter is a constitutive promoter. In a particular embodiment, the promoter is an inductive promoter. In a particular embodiment, the promoter is a native promoter of a nucleotide sequence encoding a biomolecule. In a particular embodiment, 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)-myocyte (i.e., muscle) dual-specific promoter. In a specific embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-kidney dual-specific promoter. In a specific embodiment, the tissue-specific promoter is a hepatocyte (i.e., liver)-myocyte (i.e., muscle)-kidney multiple-specific promoter. The muscle may be cardiac muscle (e.g., atrial muscle and / or ventricular muscle) and / or skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major).

[0212] Further explanation of the regulating control elements is provided in Sections 5.1 and 5.3.

[0213] Vectors, gene delivery systems, and viral particles This disclosure further provides vectors and gene delivery systems related to the above-mentioned nucleotide sequences, polynucleotides, promoters, and / or expression cassettes.

[0214] In one embodiment, a vector comprising a polynucleotide described herein (for example, a polynucleotide described in Section 5.2.1) is provided.

[0215] In one embodiment, a vector is provided that includes a promoter as described herein (for example, the promoter described in Section 5.2.2).

[0216] In one embodiment, a vector is provided that includes an expression cassette described herein (for example, an expression cassette described in Section 5.2.3).

[0217] In various embodiments, the vectors described herein are plasmids. In various embodiments, the vectors described herein are expression vectors.

[0218] In various embodiments, the vectors described herein are recombinant viral vectors. In various embodiments, the vectors described herein are recombinant AAV vectors.

[0219] Further explanation of vectors is provided below in this section and in Section 5.1.

[0220] The gene delivery systems provided herein include, for example, recombinant AAV particles and recombinant viral particles such as nonviral gene delivery systems.

[0221] A viral particle consists of a viral genome enclosed within a protein shell called a capsid. An "AAV particle" refers to an AAV virus consisting of at least one AAV capsid protein and the packaged AAV genome. The AAV genome is a linear, single-stranded DNA molecule containing terminal inversion repeats (ITRs) at the 5' and 3' ends of the viral genome. ITRs act as a cis-acting origin for DNA replication and as a packaging signal for the viral genome.

[0222] In one embodiment, recombinant viral particles comprising the polynucleotides described herein (for example, the polynucleotides described in Section 5.2.1) are provided.

[0223] In one embodiment, recombinant viral particles are provided that include a recombinant viral genome containing an expression cassette described herein (for example, an expression cassette described in Section 5.2.3).

[0224] In one aspect, recombinant virus particles are provided that contain a vector as described herein (e.g., a vector as described in this section).

[0225] In one aspect, recombinant AAV particles are provided that contain (a) an AAV capsid and (b) a recombinant AAV genome that contains an expression cassette as described herein (e.g., an expression cassette as described in Section 5.2.3) flanked by AAV inverted terminal repeats (ITRs).

[0226] Many viral systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4 th Ed ., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y. 2012), as well as in other virology and molecular biology manuals.

[0227] In certain 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. The helper adenovirus vector can be co-transfected into HEK cells to generate adenovirus particles. See Lee et al., Genes and Diseases, 4(2):43-63 (2007).

[0228] In certain 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 those described in Chribs et al., BMC Biotechnology, 13:98 (2003); Merten et al., Mol Ther Methods Clin Dev., 13(3):160-17 (2016); Durand and Cimarelli, Viruses, 3:132-159 (2011). In some embodiments, herein, third-generation self-inactivating lentiviral vectors are used.

[0229] In certain 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 type 1 virus (HSV-1), herpes simplex type 2 virus (HSV-2), or any derivatives thereof. Exemplary vectors are based on or derived from HSV-1, HSV-2, CMV, VZV, EBV and KSHV. For example, they can be constructed according to methods known in the art, such as those described in U.S. Patent Nos. 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, which are hereby incorporated by reference in their entirety.

[0230] 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.

[0231] Amplicons are plasmid-derived vectors engineered to contain HSV DNA replication origins (oris) and HSV cleavage and packaging recognition sequences (pacs). When amplicons are transfected into mammalian cells with HSV helper function, they replicate, form head-tail multimers, and package into viral particles. Currently, there are two main methods used for producing amplicon particles: one is based on infection with a deficient helper HSV, and the other is based on transfection of the HSV-1 gene, such as a pair of pac deletion overlap cosmids or BAC-HSV-1 with both pac deletion and ICP27 deletion. In some embodiments, the amplicons used herein are capable of accommodating large fragments of non-toxic exogenous DNA (e.g., up to 152 kb) containing multiple copies (e.g., up to 15 copies) of the transgene.

[0232] In some embodiments, the HSV-based vectors used herein are deficient in at least one essential HSV gene, and the HSV-based vectors may further include deletions of one or more non-essential genes. In some embodiments, the HSV-based vectors are replication-deficient. Most replication-deficient HSV-based vectors include deletions to remove one or more immediate early, early, or late HSV genes to prevent replication. In other embodiments, the HSV-based vectors are deficient in an immediate early gene selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47 and combinations thereof. In specific embodiments, the HSV-based vectors are deficient in all of ICP0, ICP4, ICP22, ICP27, and ICP47. Exemplary replication-competent vectors include NV-1020 (HSV-1), RAV9395 (HSV-2), AD-472 (HSV-2), NS-gEnull (HSV-1), and ImmunoVEX (HSV-2). Exemplary replication-defect type 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).

[0233] Replication-deficient HSV-based vectors are typically produced in complementary cell lines that provide adequate levels of gene function necessary for viral replication, even if it is absent in the replication-deficient HSV-based vector, thereby generating high-titer viral vector stocks. Exemplary cell lines complement at least one replication-essential gene function absent in the replication-deficient HSV-based vector, and in some embodiments, complement all replication-essential gene functions. For example, an HSV-based vector lacking ICP0, ICP4, ICP22, ICP27, and ICP47 can be complemented by the human osteosarcoma cell line U2OS. Cell lines can also complement non-essential genes (e.g., UL55) whose deficiency reduces growth or replication efficiency. Complementary cell lines can complement defects in at least one replication-essential gene function encoded by early regions, immediate early regions, late regions, viral packaging regions, virus-associated regions, or combinations thereof, and include all HSV functions (e.g., enabling the proliferation of HSV amplicons containing minimal HSV sequences (e.g., terminal inverted repeats and packaging signals only, or ITR and HSV promoter only)). In some embodiments, a further feature of the cell system is the inclusion of complementary genes that do not overlap with the HSV-based vector, which minimizes, and virtually eliminates, the possibility of the HSV-based vector genome being recombined with cellular DNA. Therefore, the presence of HSVs capable of replication, which must be avoided in the vector library, is minimized, making it suitable for certain therapeutic purposes, particularly gene therapy. The construction of the complementary cell system relates to standard molecular biology and cell culture techniques known in this field.

[0234] In certain embodiments, the viral vectors or viral particles provided herein are derived from adeno-associated viruses (AAVs). A more detailed description of AAVs is provided in Section 5.3.1 below.

[0235] The nucleotide sequences, promoters, and expression cassettes described herein can be cloned into viral vectors using any known molecular cloning method in the art, which may include, for example, the use of restriction endonuclease sites and one or more selection markers.

[0236] In one embodiment, the viral vector genome includes at least one element that enhances the specificity and expression of the transgene target (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), such as an intron or a synthetic intron having a modified sequence from a mammalian genome. Non-limiting examples of introns include MVM (67-97 bps), F.IX cleavage 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 / splice acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps). In one embodiment, the length of the intron may be 100 to 500 nucleotides. The length of the intron may 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.

[0237] In some embodiments, the viral vector further includes a selection marker gene or a reporter gene to select cells expressing a protein from a population of host cells transfected with the viral vector. Both the selection marker and reporter genes may have appropriate regulatory sequences flanked to enable expression in host cells. For example, the viral vector may include transcription and translation terminators, start sequences, and promoters to regulate the expression of nucleic acid sequences.

[0238] The nonviral gene delivery systems provided 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-exclusive exemplary nonviral gene delivery systems are described, for example, in Sung and Kim, 2019, Biomator Res 23:8; Zu and Gao, 2021, AAPS J 23:78; and Lan et al., 2022, Mol Cancer 21:71, the entirety of which is incorporated herein.

[0239] In one embodiment, a nonviral gene delivery system comprising a polynucleotide as described herein (for example, a polynucleotide as described in Section 5.2.1) is provided.

[0240] In one embodiment, a nonviral gene delivery system is provided that includes a polynucleotide comprising a nucleotide sequence encoding an expression cassette described herein (for example, an expression cassette described in Section 5.2.3).

[0241] In one embodiment, a nonviral gene delivery system comprising a vector described herein (for example, a vector described in this section) is provided.

[0242] AAV vectors and AAV particles Any AAV serotype or variant thereof may be used in this disclosure.AAV serotypes are 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), AAV6(Genbank Accession No.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 DQ813647.1), AAV13(Genbank Accession No EU285562.1), AAV LK03, AAVrh74, AAV DJ (Wu Z et al., J This may include, but is not limited to, 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 AAV rh.8 (Wang et al., Mol.Ther.18:119-125(2010), or their variants.

[0243] AAV variants include AAV1 variants (e.g., AAVs containing AAV1 variant capsid protein), AAV2 variants (e.g., AAVs containing AAV2 variant capsid protein), AAV3 variants (e.g., AAVs containing AAV3 variant capsid protein), AAV3-B variants (e.g., AAVs containing AAV3-B variant capsid protein), AAV4 variants (e.g., AAVs containing AAV4 variant capsid protein), AAV5 variants (e.g., AAVs containing AAV5 variant capsid protein), AAV6 variants (e.g., AAVs containing AAV6 variant capsid protein), AAV7 variants (e.g., AAVs containing AAV7 variant capsid protein), AAV8 variants (e.g., This includes, but is not limited to, AAVs containing AAV8 mutant capsid protein, AAVrh8, AAVrh8R (e.g., AAVs containing AAVrh8 or AAVrh8R mutant capsid protein), AAV9 mutants (e.g., AAVs containing AAV9 mutant capsid protein), AAV10 mutants (e.g., AAVs containing AAV10 mutant capsid protein), AAVrh10 mutants (e.g., AAVs containing AAVrh10 mutant capsid protein), AAV11 mutants (e.g., AAVs containing AAV11 mutant capsid protein), AAV12 mutants (e.g., AAVs containing AAV12 mutant capsid protein), AAV13 mutants (e.g., AAVs containing AAV13 mutant capsid protein), AAV LK03 mutants (e.g., AAVs containing AAV LK03 mutant capsid protein), and AAVrh74 mutants (e.g., AAVs containing AAVrh74 mutant capsid protein).

[0244] 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 that are operablely linked in the transcription direction. The regulatory elements may include recombinant promoters, response elements, transcription start regions, and transcription end regions provided herein. The regulatory elements can be selected based on the cells of interest. In some embodiments, a functional AAV ITR sequence is flanked (5' and 3') to the resulting rAAV vector construct containing operablely linked components. In some embodiments, the resulting rAAV vector may have a genome whose structure is (5'AAV ITR)-(promoter)-(transgene of interest)-(3'AAV ITR).

[0245] In certain embodiments, the promoter described herein is operably ligated to at least one other regulatory sequence. In certain 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, or a sequence that stabilizes cytoplasmic mRNA, a post-transcriptional regulatory element (PRE), and / or a microRNA (miRNA) target sequence. In certain embodiments, the regulatory sequence may include a sequence that enhances translation efficiency (e.g., a Kozak sequence), a sequence that enhances protein stability, and / or a sequence that enhances protein processing and / or secretion. In some embodiments, the included regulatory sequences promote the transcription or expression of a transgene operably ligated to the promoter described herein in vivo. The regulatory sequence may typically include a regulatory sequence related to the transgene of interest or, optionally, a heterologous regulatory sequence.

[0246] In certain embodiments, the regulatory array includes a regulatory control element. In certain embodiments, the regulatory control element is located at 5' of the transgene (i.e., in the 5' untranslated region; 5'UTR). In other embodiments, the regulatory control element is located at 3' of the transgene (i.e., in the 3' untranslated region; 3'UTR). In certain embodiments, the regulatory array includes one or more regulatory control elements, for example, two, three, four, or five control elements. If the regulatory array includes multiple control elements, each control element may independently be located in the 5', 3', flanking portion, or inside the promoter described herein.

[0247] In certain embodiments, the viral vector may include at least one polyadenylation (polyA) signaling sequence known in the art. If 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 certain embodiments, the viral vector further includes a polyA upstream enhancer sequence at 5' of the polyA signaling sequence. In some cases, the regulatory sequence is a sequence that improves translation efficiency, such as a Kozak sequence.

[0248] In certain embodiments, the viral vector includes an intron. In certain embodiments, the intron is located within a transgene. In certain embodiments, the intron is at the 5' or 3' end of the transgene. In certain embodiments, the intron is flanked at the 5' or 3' end of the transgene. In certain embodiments, the nucleic acid construct includes two introns. In some embodiments, one intron is at the 5' end of the transgene and the other intron is at the 3' end of the transgene. In certain embodiments, the first intron is flanked at the 5' end of the transgene and the second intron is flanked at the 3' end of the transgene. In certain embodiments, the intron is an SV40 intron, for example, a 5'UTR SV40 intron.

[0249] AAV ITR sequences known in the art can be used in the rAAV vectors of the present invention. In some embodiments, the AAV ITRs used in the vectors of the present invention have a wild-type nucleotide sequence. In other embodiments, the AAV ITR sequences used in the vectors of the present invention are not wild-type sequences, but include, for example, nucleotide insertions, deletions or substitutions. The AAV ITRs provided herein may be derived from any AAV serotype, and any such AAV serotype includes, but is not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9 or variants thereof.

[0250] In some embodiments, the rAAV vector includes an ITR derived from AAV1. In some embodiments, the rAAV vector includes an ITR derived from AAV2. In some embodiments, the rAAV vector includes an ITR derived from AAV2i8. In some embodiments, the rAAV vector includes an ITR derived from AAV3. In some embodiments, the rAAV vector includes an ITR derived from AAV3-B. In some embodiments, the rAAV vector includes an ITR derived from AAV4. In some embodiments, the rAAV vector includes an ITR derived from AAV5. In some embodiments, the rAAV vector includes an ITR derived from AAV6. In some embodiments, the rAAV vector includes an ITR derived from AAV7. In some embodiments, the rAAV vector includes an ITR derived from AAV8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8R. In some embodiments, the rAAV vector includes an ITR derived from AAV9. In some embodiments, the rAAV vector includes an ITR derived from AAV10. In some embodiments, the rAAV vector includes an ITR derived from AAVrh10. In some embodiments, the rAAV vector includes an ITR derived from AAV11. In some embodiments, the rAAV vector includes an ITR derived from AAV12. In some embodiments, the rAAV vector includes an ITR derived from AAV13. In some embodiments, the rAAV vector includes an ITR derived from AAV-DJ. In some embodiments, the rAAV vector includes an ITR derived from AAV LK03. In some embodiments, the rAAV vector includes an ITR derived from AAVrh74.

[0251] In some embodiments, the 5' and 3' ITRs flanked in the nucleotide sequences of the rAAV vectors provided herein are the same and derived from the same AAV serotype. In other embodiments, the 5' and 3' ITRs flanked in the nucleotide sequences of the rAAV vectors provided herein are different and / or derived from different AAV serotypes.

[0252] In some embodiments, an rAAV vector containing an expression cassette of interest with a flanked AAV ITR may be constructed by directly inserting the cassette of interest into the AAV genome, for example, into an excised AAV open reading frame, and some portions of the AAV genome may be selectively deleted, as described in WO1993 / 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.

[0253] In another embodiment, AAV ITRs are excised from an AAV genome or an AAV vector containing such ITRs and fused to the 5' and 3' of an expression cassette of interest present in another vector using standard ligation techniques.

[0254] In certain embodiments, the rAAV vectors provided herein include a recombinant autocomplementary genome. rAAVs containing an autocomplementary genome can typically rapidly form a double-stranded DNA molecule by some of their complementary sequences (e.g., coding and non-coding strands of a complementary gene). More specifically, in some embodiments, the rAAV vectors provided herein include an rAAV genome comprising a first heterologous polynucleotide sequence (e.g., a therapeutic transgene coding strand) and a second heterologous polynucleotide sequence (e.g., a non-coding or antisense strand of a therapeutic transgene), wherein the first heterologous polynucleotide sequence can form intra-strand base pairs with the second polynucleotide sequence. In some embodiments, the first and second heterologous polynucleotide sequences are linked by a sequence that promotes intra-strand base pairing (e.g., a hairpin DNA structure). In some embodiments, the first and second heterologous polynucleotide sequences are linked by a mutated ITR to prevent the rep protein from cleaving the viral genome at the mutated ITR. rAAV vectors containing a self-complementary genome can be manufactured 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.

[0255] In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 5 kilobases (kb). In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 4.5 kb. In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 4.0 kb. In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 3.5 kb. In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 3.0 kb. In some embodiments, the size of the recombinant viral genome in the rAAV vector provided herein is less than about 2.5 kb.

[0256] The capsid protein may be derived from the same serotype as the ITR or a derivative thereof. The capsid may be from a different serotype than the ITR. For example, in certain embodiments, the AAV particles include AAV2 ITR and AAV6 capsid (AAV2 / 6), AAV2 ITR and AAV7 capsid (AAV2 / 7), AAV2 ITR and AAV8 capsid (AAV2 / 8), or AAV2 ITR and AAV9 capsid (AAV2 / 9).

[0257] Naturally occurring AAV capsids contain AAV VP1, VP2, and VP3 capsid proteins, each encoded by a splicing variant of the AAV cap gene. Typically, AAV particles contain three proteins, VP1, VP2, and VP3, and since VP2 and VP3 are cleavage forms of VP1, they also contain sequences found in VP1. Usually, the amino acid sequence of VP1 defines the serotype of the capsid. For example, if the VP1 capsid protein encodes the AAV2 VP1 protein, the AAV is of the AAV2 serotype, but if the VP1 capsid protein encodes the AAV9 VP1 protein, the AAV is of the AAV9 serotype.

[0258] Generally, AAV capsid proteins contain multiple variable regions (VRs) and constant regions located between them. The "GH loop" is a ring sequence in which β-strands G and β-strand H are flanked within the internal β-barrel of the AAV capsid protein, and it contains variable regions VR IV to VR VIII.

[0259] The sequences of the VP2 and VP3 capsid proteins and the positions of various domains (e.g., the variable region and the GH loop) can be easily and conventionally determined from a given VP1 sequence by appropriate methods known to those skilled in the art (e.g., by comparing or aligning the VP1 sequence with an annotated VP1 sequence (preferably an annotated VP1 sequence from a closely related AAV species) using an appropriate algorithm). Preferably, such annotated VP1 sequence is an annotated wild-type AAV VP1 sequence.

[0260] 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.

[0261] In some embodiments, the AAV capsid protein is the VP1 capsid protein. In other embodiments, the AAV capsid protein is the VP2 capsid protein. In other embodiments, the AAV capsid protein is the VP3 capsid protein. In some embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and / or the VP3 capsid protein. In other embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein. In some embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and / or the VP3 capsid protein, and the capsid proteins of the rAAV particles have the same serotype. In other embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein, and the capsid proteins of the AAV particles have the same serotype.

[0262] In certain embodiments, the capsid protein is a mutant capsid protein. Compared to the corresponding reference capsid protein (e.g., a naturally occurring parental capsid protein, i.e., the capsid protein of which it originates), the mutant capsid protein may contain one or more mutations, such as amino acid substitutions, amino acid deletions, and heterologous peptide insertions. In some embodiments, the amino acid sequence of the AAV capsid protein is the same as that of the wild-type, reference, or parent 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 residue substitutions, for example, 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 residue substitutions. In some embodiments, the capsid proteins or AAV particles described herein may be chimeric capsid proteins or AAV particles each comprising protein sequences of two or more AAV serotype capsid proteins or particles.

[0263] In various embodiments, the mutant AAV capsid proteins described herein are the capsid proteins of mutant AAV VP1. Furthermore, mutant AAV capsid proteins that are the VP2 and VP3 counterparts of the VP1 capsid protein described herein are described. Furthermore, fragments of such mutant AAV capsid proteins (e.g., fragments of the VP1, VP2, or VP3 capsid proteins) are described, which include, but are not limited to, variable regions (e.g., VR IV and VR VIII), GH loops, and functional fragments that substantially retain the corresponding capsid proteins and their associated tissue-targeting properties. In certain embodiments, the length of the fragments of the VP1, VP2, or VP3 capsid proteins described herein is at least 7 amino acids. In certain embodiments, the length of the fragments of the VP1, VP2, or VP3 capsid proteins described herein is at least 8 amino acids. In certain embodiments, the length of the fragments of the VP1, VP2, or VP3 capsid proteins described herein is at least 9 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 10 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 20 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 30 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 40 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 50 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 100 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 200 amino acids.In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 300 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 400 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is at least 500 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 7 to 10 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 10 to 50 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 50 to 100 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 100 to 200 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 200 to 300 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 300 to 400 amino acids. In certain embodiments, the length of the VP1, VP2, or VP3 capsid protein fragment described herein is 400 to 500 amino acids.

[0264] 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 are AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV It has 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% identical to the amino acid sequences of LK03, AAVrh74, and AAV44-9 capsid proteins.

[0265] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0266] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0267] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0268] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0269] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0270] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0271] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0272] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0273] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0274] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0275] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0276] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0277] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0278] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0279] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0280] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0281] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0282] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0283] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0284] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0285] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0286] In certain embodiments, the AAV particles provided herein include VP1, VP2, and / or VP3 capsid proteins comprising 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.

[0287] In some specific embodiments, the rAAV particles provided herein include an AAV VP1 capsid protein having an amino acid 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% sequence identity with 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.

[0288] In some specific embodiments, the rAAV particles provided herein include an AAV VP1 capsid protein comprising the amino acid sequence 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.

[0289] In specific embodiments, the rAAV particles provided herein are recombinant AAV serotype 9 (rAAV9) particles. In specific embodiments, the rAAV particles provided herein contain an AAV9 capsid protein. In specific embodiments, the AAV capsid of the rAAV particles provided herein contains a mutant AAV9 capsid protein.

[0290] In various embodiments, the mutant AAV9 capsid protein contains the amino acid sequence 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.

[0291] In various embodiments, the mutant AAV9 capsid protein contains the amino acid sequence 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.

[0292] In various embodiments, the mutant AAV9 capsid protein contains the amino acid sequence SEQ ID NO: 31, 38, 49, 51, 53, 55, 57, 59, or 61.

[0293] In various embodiments, the mutant AAV9 capsid protein contains the amino acid sequence SEQ ID NO: 30, 37, 48, 50, 52, 54, 56, 58, or 60.

[0294] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:25. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:24.

[0295] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:27. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:26.

[0296] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:29. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:28.

[0297] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:31. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:30.

[0298] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:33. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:32.

[0299] In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO:35. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO:36. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO:35 and also includes the amino acid sequence of SEQ ID NO:36. In specific embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO:34.

[0300] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:38. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:37.

[0301] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 40. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 39.

[0302] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:42. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:41.

[0303] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:44. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:43.

[0304] In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 46. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 47. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 46 and also includes the amino acid sequence of SEQ ID NO: 47. In specific embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 45.

[0305] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:49. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:48.

[0306] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 51. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 50.

[0307] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 53. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 52.

[0308] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 55. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 54.

[0309] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 57. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 56.

[0310] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 59. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 58.

[0311] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:61. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:60.

[0312] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:63. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:62.

[0313] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:65. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:64.

[0314] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:67. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:66.

[0315] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:69. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:68.

[0316] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:71. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:70.

[0317] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:73. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:72.

[0318] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:75. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:74.

[0319] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:77. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:76.

[0320] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:79. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:78.

[0321] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:81. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:80.

[0322] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:83. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:82.

[0323] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:85. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:84.

[0324] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:87. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:86.

[0325] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:89. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:88.

[0326] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:91. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:90.

[0327] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:93. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:92.

[0328] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:95. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:94.

[0329] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:97. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:96.

[0330] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:99. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:98.

[0331] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 101. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 100.

[0332] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:103. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:102.

[0333] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:105. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:104.

[0334] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:107. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:106.

[0335] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:109. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:108.

[0336] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:111. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:110.

[0337] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:113. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:112.

[0338] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:115. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:114.

[0339] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:117. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:116.

[0340] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:119. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:118.

[0341] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:121. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:120.

[0342] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:123. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:122.

[0343] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:125. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:124.

[0344] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:127. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:126.

[0345] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:129. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:128.

[0346] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:131. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:130.

[0347] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:133. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:132.

[0348] In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:135. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:134.

[0349] In certain embodiments, the mutant AAV9 capsid protein provided herein includes the VP2 capsid protein counterpart of the VP1 capsid protein described herein.

[0350] In certain embodiments, the mutant AAV9 capsid protein provided herein includes the VP3 capsid protein counterpart of the VP1 capsid protein described herein.

[0351] In certain embodiments, the mutant AAV9 capsid protein provided herein comprises a fragment (e.g., a functional fragment) of the VP1 capsid protein described herein.

[0352] In various embodiments, the recombinant AAV particles described herein have enhanced tropism to one or more tissues compared to wild-type AAV particles. The one or more tissues may be, for example, the liver, muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or cardiac muscle (e.g., atrial and / or ventricular muscle)), kidney, brain (e.g., mesoforebrain, mesopostracheal, forebrain and / or hindbrain), lung and / or spleen.

[0353] In certain embodiments, the recombinant AAV particles described herein have improved hepatic tropism compared to wild-type AAV particles.

[0354] In certain embodiments, the recombinant AAV particles described herein have enhanced tropism to muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or cardiac muscle (e.g., atrial muscle and / or ventricular muscle)) compared to wild-type AAV particles.

[0355] In certain embodiments, the recombinant AAV particles described herein have improved renal tropism compared to wild-type AAV particles.

[0356] In certain embodiments, the recombinant AAV particles described herein have enhanced tropism to both the liver and 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)) compared to wild-type AAV particles.

[0357] In certain embodiments, the recombinant AAV particles described herein have improved tropism to both the liver and kidneys compared to wild-type AAV particles.

[0358] In certain embodiments, the recombinant AAV particles described herein have enhanced tropism to 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 muscle (e.g., atrial and / or ventricular muscles)) and kidneys compared to wild-type AAV particles.

[0359] In various embodiments, the recombinant AAV particles described herein have reduced tropism to one or more tissues compared to wild-type AAV particles. The one or more tissues may be, for example, muscle (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or cardiac muscle (e.g., atrial muscle and / or ventricular muscle)), kidney, brain (e.g., mesoforebrain, mesopostracheal, forebrain and / or hindbrain), lung and / or spleen.

[0360] In various embodiments, the recombinant AAV particles described herein have enhanced tropism to one or more tissues as described above, and reduced tropism to one or more other tissues as described above.

[0361] In various embodiments, recombinant AAV particles are administered systemically to a subject, and then tropism to the tissues described herein is measured. In specific embodiments, recombinant AAV particles are administered intravenously to a subject, and then tropism to the tissues described herein is measured.

[0362] In a preferred embodiment, the recombinant AAV particles described herein are AAV particles that do not exist in nature. In a particular embodiment, the recombinant AAV particles described herein are isolated. In a particular embodiment, the recombinant AAV particles described herein are purified.

[0363] cell In one embodiment, a host cell containing a polynucleotide described herein (for example, a polynucleotide described in Section 5.2.1) is provided.

[0364] In one embodiment, a host cell containing a vector described herein (for example, a vector described in Section 5.3) is provided.

[0365] In certain embodiments, the host cell further comprises a nucleotide sequence encoding the AAV rep gene in a polynucleotide or vector identical or different from those described herein (e.g., the polynucleotide described in Section 5.2.1 or the vector described in Section 5.3). In certain embodiments, the host cell further comprises a nucleotide sequence encoding the AAV cap gene in a polynucleotide or vector identical or different from those described herein (e.g., the polynucleotide described in Section 5.2.1 or the vector described in Section 5.3). In certain embodiments, the host cell further comprises a helper function, e.g., one or more helper plasmids and / or one or more helper viruses (see Section 5.5 for helper functions).

[0366] In one embodiment, a host cell containing recombinant virus particles as described herein (for example, recombinant virus particles as described in Section 5.3) is provided.

[0367] In one embodiment, a host cell is provided that produces recombinant virus particles as described herein (for example, recombinant virus particles as described in Section 5.3).

[0368] In one embodiment, a host cell containing recombinant AAV particles as described herein (for example, recombinant AAV particles as described in Section 5.3) is provided.

[0369] In another embodiment, a host cell is provided that produces recombinant AAV particles as described herein (for example, recombinant AAV particles as described in Section 5.3).

[0370] In one embodiment, a host cell is provided that includes a nonviral delivery system described herein (for example, a nonviral delivery system described in Section 5.3).

[0371] In one embodiment, a host cell is provided that produces a nonviral delivery system described herein (for example, a nonviral delivery system described in Section 5.3).

[0372] In some embodiments, the host cell is an ex vivo 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.

[0373] As used herein, the term “host” means a cell containing a polynucleotide, vector, viral particle (e.g., rAAV particle) or nonviral gene delivery system (e.g., a cell derived from insects, animals (including humans and non-human animals), yeast, bacteria, etc.). This disclosure is not intended to be limited to any particular type of host cell. In practice, any suitable cell is expected to be used as a host herein. A host cell may be a single cell, a group, a culture (e.g., a liquid culture or a culture on a solid substrate), a cell line, a living organism or part thereof, or derived therefrom.

[0374] Host cells can be used for amplification, replication, or production of polynucleotides, vectors, recombinant viral particles (e.g., rAAV particles), nonviral gene delivery systems, or biomolecules as 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-limited 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, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, MRC-5, primary fibroblasts, hepatocytes, and myoblasts. In certain embodiments, the host cells are human cells. In specific embodiments, the human cells are autologous to the subject being treated (e.g., a human patient). In specific embodiments, the human cells are allogeneic to the subject being treated (e.g., a human patient).

[0375] The host cells themselves can also be used as a therapeutic agent. For this purpose, it is preferable that the host cells are of the same species derived from the subject. Therefore, if the subject to be treated is a human patient, it is preferable that the host cells suitably used as the therapeutic agent itself are human cells. In a specific embodiment, the human cells are autologous to the subject to be treated by the human cells (e.g., a human patient). In a specific embodiment, the human cells are allogeneic to the subject to be treated by the human cells (e.g., a human patient).

[0376] In one embodiment, a population of host cells that are stably transduced by recombinant virus particles (e.g., recombinant AAV particles) described herein is provided. In another embodiment, a population of host cells that are stably transduced by a nonviral gene delivery system described herein is provided. In a specific embodiment, the population of host cells is an ex vivo host cell population. In a specific embodiment, the population of host cells is an in vitro host cell population. In a specific embodiment, the population of host cells is an in vivo host cell population. In a specific embodiment, the population of host cells is a human host cell population. In a specific embodiment, the population of host cells is a human ex vivo host cell population. In a specific embodiment, the population of host cells is a human in vitro host cell population.

[0377] In certain embodiments, such host cell populations may be used to generate a population of recombinant virus particles (e.g., recombinant AAV particles) that are used as therapeutic agents and administered to subjects in need (e.g., human patients).

[0378] In certain embodiments, such host cell populations may be used to generate a group of nonviral gene delivery system molecules that are used as therapeutic agents and administered to the required subject (e.g., a human patient).

[0379] In certain embodiments, such host cell populations may be used to generate the biomolecules described in Section 5.2.4, which are used as therapeutic agents and administered to the required subjects (e.g., human patients).

[0380] In certain embodiments, such a population of host cells may be used as the therapeutic agent itself and administered to the subject in need (e.g., a human patient). In specific embodiments, the population of host cells is autologous to the subject to be treated by the population of host cells (e.g., a human patient). In specific embodiments, the population of host cells is allogeneic to the subject to be treated by the population of host cells (e.g., a human patient).

[0381] Production method In another embodiment, a method for producing recombinant virus particles as described herein, for example, recombinant AAV particles as described herein (e.g., recombinant virus particles, for example, recombinant AAV particles as described in Section 5.3) is provided.

[0382] In another embodiment, a method for producing a nonviral gene delivery system described herein (for example, the nonviral gene delivery system described in Section 5.3) is provided.

[0383] Furthermore, this specification describes methods for producing polynucleotides described herein (e.g., polynucleotides described in Section 5.2.1), promoters described herein (e.g., promoters described in Section 5.2.2), expression cassettes described herein (e.g., expression cassettes described in Section 5.2.3), vectors described herein (e.g., vectors described in Section 5.3), or host cells described herein (e.g., host cells described in Section 5.4).

[0384] The polynucleotides, promoters, expression cassettes, vectors, host cells, recombinant viral particles (e.g., recombinant AAV particles), and nonviral gene delivery systems of this disclosure 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 scope of the knowledge of those skilled in the art and do not limit the present invention.

[0385] The generation of the polynucleotides, promoters, expression cassettes and / or vectors of this disclosure can be carried out using any suitable genetic engineering and protein production techniques known in the art, including but not limited to cloning, restriction enzyme digestion, ligation, transformation, plasmid purification, DNA sequencing, chemosynthesis, in vitro translation and in vivo expression, as described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY 2012).

[0386] Host cells can be made to contain the polynucleotides, vectors, recombinant viral particles (e.g., recombinant AAV particles) or nonviral gene delivery systems of this disclosure by any suitable method, which includes, but is not limited to, transfection, electroporation, transduction, liposome delivery, membrane fusion techniques, high-speed DNA coated pellets, viral infection, and protoplast fusion.

[0387] Recombinant viral particles (e.g., recombinant AAV particles) can be produced from host cells that allow the production and replication of recombinant viral particles (e.g., recombinant AAV particles). Methods for producing recombinant virus particles, including recombinant AAV particles, are well known in the art, for example, in *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); USPatent numbers US5064764, US5756283, US6194191, US6204059, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6566118, US6943019, US6953690, US7022519, US7238526, US7291498, US7491508 and US8137948; and international patent application publication number WO1 This information is described in 996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2018022608, WO2019217513, WO2019222132, WO2019222136 and WO2020232044, and its disclosures are incorporated herein by reference in their entirety.

[0388] The rAAV particles described herein can be produced by any suitable method known in the art. For example, a host cell (e.g., a mammalian cell) can be engineered to stably express the essential components for AAV particle production. This can be achieved by incorporating a plasmid (or multiple plasmids) containing the AAV rep and cap genes and a selection marker (e.g., an antibiotic (e.g., neomycin or ampicillin) resistance gene) into the cell genome. The cell may be, for example, an insect or mammalian cell and may be co-infected with a helper virus (e.g., an adenovirus or baculovirus that provides helper function) and an rAAV vector containing 5' and 3' AAV ITRs. The use of a selection marker enables mass production of rAAV. Another non-limiting example is that, instead of a plasmid, an adenovirus or baculovirus may be used to introduce the rep and cap genes into the packaging cell. As another non-limiting example, viral vectors containing 5' and 3' AAV ITRs, along with rep and cap genes, can both be stably integrated into the DNA of producing cells, and helper functions can be provided by wild-type adenovirus to produce rAAV.

[0389] AAV helper viruses are viruses that enable AAV to be replicated and packaged by host cells. Helper viruses provide helper functions that allow AAV replication. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as cowpox. Adenoviruses encompass many different subgroups, but the most commonly used is adenovirus type 5 (Ad5) of subgroup C. Many adenoviruses of human, non-human mammalian, and avian origin are known and can be obtained from storage institutions such as ATCC. Herpesviridae viruses that can be obtained from depositary centers such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and herpesvirus (PRV). Examples of adenovirus helper functions for AAV replication include E1A, E1B, E2A, VA, and E4 or E6 functions.

[0390] 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, the AAV preparation is considered substantially helper virus-free. The preparation does not need to contain an equal amount of helper virus protein (i.e., the protein present by this level of helper virus if the above-mentioned helper virus particle impurities are present in a disrupted form). Viral and / or cellular protein contamination is usually observed by 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).

[0391] In certain embodiments, host cells containing the rAAV vector can produce rAAV particles by providing an AAV helper function in which the AAV ITR provides an AAV helper function for replicating and encapsulating a transgene encoding a protein of interest that has been flanked. The AAV helper function is typically an AAV-derived coding sequence which can be expressed to provide an AAV gene product which is further used in trans-action for productive AAV replication. In this specification, the AAV helper function is used to compensate for a required AAV function that is lost in the rAAV vector. In some embodiments, the AAV helper function includes one or both of the major AAV ORFs (i.e., rep and cap coding regions) or their functional homologs.

[0392] AAV helper function can be introduced into host cells by transfecting them with an AAV helper construct before or simultaneously with transfection with an rAAV vector. For example, an AAV helper construct can be used to compensate for the loss of AAV function necessary for productive AAV infection by providing transient expression of at least the AAV rep and / or cap genes. Typically, AAV helper constructs are lacking in AAV ITRs and cannot replicate or package. AAV helper constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions.

[0393] In certain embodiments, host cells may provide, or could be provided with, non-AAV-derived functions or “helper functions” for the production of rAAV particles. Helper functions are cellular functions on which non-AAV-derived viruses and / or AAVs depend for replication, and include, for example, non-AAV proteins and RNAs necessary for AAV replication, which are involved in activating AAV gene transcription, splicing step-specific AAV mRNA, AAV DNA replication, synthesis of Cap expression products, and packaging of AAV capsids. In some embodiments, virus-based helper functions may be derived from known helper viruses.

[0394] In some embodiments, recombinant AAV particles are produced as a result of infecting host cells with a helper virus and / or a helper functional vector, the produced rAAV particles being infectious replication-deficient viruses and comprising an AAV protein shell encapsulating a heterologous nucleotide sequence of interest with AAV ITRs flanked at both ends.

[0395] rAAV particles can be purified from host cells using purification methods known in the art, such as chromatography, CsCl gradients, and other methods described, for example, U.S. Patents 6,989,264 and 8,137,948, and WO 2010 / 148143. In some embodiments, known methods such as inactivating residual helper viruses by heating may be used.

[0396] Pharmaceutical composition In one embodiment, a pharmaceutical composition is provided comprising recombinant virus particles as described herein, for example recombinant AAV particles as described herein (e.g., recombinant virus particles, for example, recombinant AAV particles as described in Section 5.3), and a pharmaceutically acceptable carrier.

[0397] In one embodiment, a pharmaceutical composition is provided comprising a nonviral gene delivery system described herein (for example, a nonviral gene delivery system described in Section 5.3) and a pharmaceutically acceptable carrier.

[0398] In one embodiment, a pharmaceutical composition is provided comprising a host cell population as described herein, stably transduced by recombinant virus particles as described herein, for example, recombinant AAV particles as described herein (for example, a host cell population stably transduced by recombinant virus particles, for example, recombinant AAV particles as described in Section 5.4), and a pharmaceutically acceptable carrier.

[0399] In one aspect, there is provided a pharmaceutical composition comprising a group of host cells described herein (e.g., a group of host cells stably transduced by the non-viral gene delivery system described in Section 5.4) stably transduced by the non-viral gene delivery system described herein, and a pharmaceutically acceptable carrier.

[0400] In certain embodiments, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 8 vg / ml to 1×10 20 vg / ml. In a specific embodiment, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 9 vg / ml to 1×10 19 vg / ml. In a specific embodiment, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 10 vg / ml to 1×10 18 [[ID=十六]]vg / ml. In a specific embodiment, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 11 vg / ml to 1×10 17 vg / ml. In a specific embodiment, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 12 vg / ml to 1×10 16 vg / ml. In a specific embodiment, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 13 vg / ml to 1×10 15 vg / ml. In certain embodiments, the concentration of recombinant virus particles (e.g., recombinant AAV particles) in the pharmaceutical composition described herein may be in the range of 1×10 8 vg / ml to 1×10 15 vg / ml.

[0401] In a particular embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1 × 10⁻⁶ 2 Cells / ml to ~1 x 10 12 It may be within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1 × 10⁻⁶ 3 From cells / ml to 1 × 10 11 It may be within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1 × 10⁻⁶ 4 From cells / ml to 1 × 10 10 It may be within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1 × 10⁻⁶ 5 From cells / ml to 1 × 10 9 It may be within the range of cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1 × 10⁻⁶ 6 From cells / ml to 1 × 10 8 It may be within the range of cells / ml.

[0402] In certain embodiments, the pharmaceutical compositions described herein are injected in volumes ranging from about 0.1 ml to about 20 ml. In certain embodiments, the pharmaceutical compositions described herein are injected in volumes ranging from about 1 ml to about 10 ml.

[0403] Generally, if a drug (e.g., an excipient or carrier) is safe, non-toxic, and not undesirable from a biological or other standpoint, then the drug is pharmaceutically acceptable and suitable for both veterinary and human pharmaceutical uses.

[0404] In certain embodiments, the pharmaceutical compositions described herein include one or more pharmaceutically acceptable excipients to impart properties to the composition that are advantageous for preservation and / or administration to a subject for the treatment of a disease or disorder. In certain embodiments, the pharmaceutical compositions described herein include one or more buffers, such as disodium hydrogen phosphate and / or sodium dihydrogen phosphate monohydrate. In certain embodiments, the pharmaceutical compositions described herein include one or more isotonic agents, such as sodium chloride. In certain embodiments, the pharmaceutical compositions described herein include one or more fillers, such as mannitol, sucrose, dextran, lactose, trehalose and / or povidone (PVP K24). In certain embodiments, the pharmaceutical compositions described herein include one or more surfactants, such as polysorbate 80, polysorbate 20, sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407 and / or poloxamer 188.

[0405] Preferably, the pharmaceutical compositions described herein are stable and can be stored for long periods of time at, for example, below -60°C, about -20°C, about 2°C to 8°C and / or room temperature without unacceptable changes in quality, potency, or purity.

[0406] Preferably, the pharmaceutical compositions described herein are sterile and stable under manufacturing and storage conditions. The pharmaceutical compositions described herein may be prepared as solutions, microemulsions, liposomes, lyophilized compositions, or other ordered structures suitable for application to high-concentration drugs.

[0407] In specific embodiments, the pharmaceutical compositions described herein are prepared according to the route of administration to a subject. Non-limiting examples of usable routes of administration include direct delivery to a target organ, orally, by inhalation, intravenously, intramuscularly, subcutaneously, intradermally, nasally, intrathecally, intrapancreatally, intraperitoneally, intratumorally, and other parenteral routes. In specific embodiments, the pharmaceutical compositions described herein are prepared for systemic administration to a subject. In specific embodiments, the pharmaceutical compositions described herein are prepared for intravenous administration to a subject.

[0408] In some embodiments, the pharmaceutical compositions provided herein contain the viral particles, the nonviral gene delivery system, or the host cell population described herein in an amount (e.g., a therapeutic or preventive dose) that effectively treats or prevents a disease or disorder. In some embodiments, the therapeutic or preventive effect is monitored by periodically evaluating the treated subject.

[0409] Method and Use In one embodiment, a method is provided for treating a disease or disorder in a subject in need of treatment, comprising administering to the subject (preferably in a therapeutically effective amount) recombinant virus particles as described herein, for example recombinant AAV particles as described herein (e.g., recombinant virus particles, for example, recombinant AAV particles as described in Section 5.3).

[0410] In one embodiment, a method is provided for treating a disease or disorder in a subject requiring treatment, comprising administering to the subject (preferably in a therapeutically effective amount) a nonviral gene delivery system described herein (e.g., a nonviral gene delivery system described in Section 5.3).

[0411] In one embodiment, a method is provided for treating a disease or disorder in a subject, comprising administering to the subject (preferably in a therapeutically effective amount) of a pharmaceutical composition described herein (for example, a pharmaceutical composition described in Section 5.6).

[0412] In one embodiment, a method is provided for delivering a biomolecule to one or more ex vivo target cells, comprising transducing one or more target cells with recombinant viral particles as described herein, for example, recombinant AAV particles as described herein (e.g., recombinant viral particles, for example, recombinant AAV particles as described in Section 5.3).

[0413] In one embodiment, a method is provided for delivering a biomolecule to one or more ex vivo target cells, comprising transducing the one or more target cells by a nonviral gene delivery system described herein (for example, a nonviral gene delivery system described in Section 5.3).

[0414] In one embodiment, a method is provided for delivering a biomolecule to one or more in vitro target cells, comprising transducing one or more target cells with recombinant virus particles as described herein, for example recombinant AAV particles as described herein (e.g., recombinant virus particles, for example, recombinant AAV particles as described in Section 5.3).

[0415] One embodiment provides a method for delivering a biomolecule to one or more in vitro target cells, comprising transducing one or more target cells using a nonviral gene delivery system described herein (for example, a nonviral gene delivery system described in Section 5.3).

[0416] In one embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells of a subject, comprising administering recombinant viral particles as described herein, for example, recombinant AAV particles as described herein (e.g., recombinant viral particles, for example, recombinant AAV particles as described in Section 5.3), to the subject.

[0417] In another embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells of a subject, comprising administering a nonviral gene delivery system described herein (for example, a nonviral gene delivery system described in Section 5.3) to the subject.

[0418] In another embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells of a subject, comprising administering a pharmaceutical composition described herein (for example, a pharmaceutical composition described in Section 5.6) to the subject.

[0419] Further explanation of biomolecules is provided in Section 5.2.4.

[0420] In a particular embodiment, the one or more target cells are one or more hepatocytes. In a particular embodiment, the one or more target cells are one or more muscle cells. In a particular embodiment, the one or more target cells are one or more renal cells. In a particular embodiment, the one or more target cells are one or more hepatocytes and one or more muscle cells. In a particular embodiment, the one or more target cells are one or more hepatocytes and one or more renal cells. In a particular embodiment, the one or more target cells are one or more hepatocytes, one or more muscle cells and / or one or more renal cells. In a specific embodiment, 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 myocytes). In certain embodiments, muscle can be used as a production factory for biomolecules secreted from muscle cells that are ultimately absorbed by cells of another tissue or organ to exert therapeutic effects.

[0421] The route of administration or delivery, and the dosage of recombinant viral particles (e.g., recombinant AAV particles), nonviral gene delivery systems, or pharmaceutical compositions administered to a subject, may be determined based on the nature of the disease or disorder, the subject's condition, and the physician's knowledge. Non-limiting examples of usable routes of administration or delivery include direct delivery to target organs, orally, by inhalation, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrathecally, intrapancreatally, intraperitoneally, intratumorally, and other parenteral 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.

[0422] In a particular embodiment, the method involves distributing recombinant virus particles (e.g., recombinant AAV particles) as described herein, in a 1 × 10⁻⁶ mixture. 8 From 1 x 10 17 The subject is administered a dose of the vector genome (vg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) as described herein to 1 × 10⁻⁶ 9 From 1 x 10 16 The subject is administered a dose of the vector genome (vg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) as described herein to 1 × 10⁻⁶ 10 From 1 x 10 15 The subject is administered a dose of the vector genome (vg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) as described herein to 1 × 10⁻⁶ 11 From 1 x 10 14 The vector genome (vg) is administered to the subjects at the specified dose.

[0423] In a particular embodiment, the method involves distributing recombinant virus particles (e.g., recombinant AAV particles) as described herein, in a 1 × 10⁻⁶ mixture. 8 From 1 x 10 17The subject is administered a dose of vector genome / kg (vg / kg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) described herein to 1 × 10⁻⁶ 9 From 1 x 10 16 The subject is administered a dose of vector genome / kg (vg / kg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) described herein to 1 × 10⁻⁶ 10 From 1 x 10 15 The subject is administered a dose of vector genome / kg (vg / kg). In a particular embodiment, the method involves administering recombinant virus particles (e.g., recombinant AAV particles) described herein to 1 × 10⁻⁶ 11 From 1 x 10 14 The vector genome is administered to the subject at a dose of 1 / kg (vg / kg).

[0424] The delivery and treatment methods described herein can be used to treat a variety of diseases or disorders, including, but not limited to, genetic disorders (e.g., lysosomal storage disorders), cancers (e.g., carcinomas, sarcomas, leukemias, lymphomas, germ cell tumors and blastomas), autoimmune diseases and infectious diseases.

[0425] In specific embodiments, the disease or disorder is caused by at least liver dysfunction. In specific embodiments, the disease or disorder is caused by at least muscle dysfunction (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or myocardial muscle (e.g., atrial muscle and / or ventricular muscle)). In specific embodiments, the disease or disorder is caused by at least kidney dysfunction. In a specific embodiment, the disease or disorder is caused by at least liver dysfunction and muscle dysfunction (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major)). 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 dysfunction (e.g., skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major)) and kidney dysfunction.

[0426] In specific embodiments, the disease or disorder relates to hepatocytes. In specific embodiments, the disease or disorder relates to muscle cells (e.g., 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 specific embodiments, the disease or disorder relates to renal cells. In specific embodiments, the disease or disorder relates to hepatocytes and muscle cells (e.g., 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 specific embodiments, the disease or disorder relates to hepatocytes and renal cells. In specific embodiments, the disease or disorder relates to hepatocytes, muscle cells (e.g., 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)) and renal cells.

[0427] In certain embodiments, the disease or disorder is a lysosomal storage disorder.

[0428] In a specific embodiment, the disease or disorder is Fabry disease. In a specific embodiment, the disease or disorder is functional myoclonus-renal failure syndrome. In a specific embodiment, the disease or disorder is aspartylglucosamiuria. In a specific embodiment, the disease or disorder is CLN1 disease or neuronal ceroid lipofuscinosis 1. In a specific embodiment, the disease or disorder is CLN2 disease or Jansky-Bierswazski 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 cystinosis. In a specific embodiment, the disease or disorder is Danon disease. In a specific embodiment, the disease or disorder is Faber lipogranulomatosis. In a specific embodiment, the disease or disorder is fucosidosis. In a specific embodiment, the disease or disorder is galactosialidosis. In a specific embodiment, the disease or disorder is Gaucher disease. In a specific embodiment, the disease or disorder is glycogen storage disorder type 2 (GSD II) or Pompe disease. In a specific embodiment, the disease or disorder is Glyceri syndrome 1 or Elehalde syndrome. In a specific embodiment, the disease or disorder is Glyceri syndrome 2.In a specific embodiment, the disease or disorder is Chediak-Higashi syndrome. In a specific embodiment, the disease or disorder is GM1 ganglioside deposition disorder. In a specific embodiment, the disease or disorder is GM2 ganglioside deposition disorder Tay-Sachs disease. In a specific embodiment, the disease or disorder is GM2 ganglioside deposition disorder Sandhoff disease. In a specific embodiment, the disease or disorder is GM2 ganglioside deposition disorder GM2 activator deficiency. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 1. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 2. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 3. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 4. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 5. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 6. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 7. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 8. In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 9. In a specific embodiment, the disease or disorder is Krabbe disease. In a specific embodiment, the disease or disorder is α-mannosidosis. In a specific embodiment, the disease or disorder is β-mannosidosis. In a specific embodiment, the disease or disorder is metachromatic leukodystrophy. In a specific embodiment, the disease or disorder is mucolipidosis II α / β or I-cell disease. In a specific embodiment, the disease or disorder is mucolipidosis III α / β or pseudo-Harler polydystrophy. In a specific embodiment, the disease or disorder is mucolipidosis IIIγ, pseudoharler polydystrophy. In a specific embodiment, the disease or disorder is mucolipidosis IV.In a specific embodiment, the disease or disorder is mucopolysaccharidosis type I (MPS I) or Hurler syndrome. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type II (MPS II) or Hunter 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 Maloto-Lamy syndrome. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VII (MPS VII) or Sleigh's 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 deficiencies. 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 Salla disease. In specific embodiments, the disease or disorder is Schindler disease or Kanzaki disease. In specific embodiments, the disease or disorder is sialic acid deposition disorder type I or cherry red spot myoclonus syndrome. In specific embodiments, the disease or disorder is Wolmann disease and cholesterol ester storage disorder.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 embodiment, the disease or disorder is hemophilia B.

[0429] In a specific embodiment, the disease or disorder is Fabry disease, and the biomolecule is a protein that is 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 that is scavenger receptor B class member 2 (preferably human SCARB2). In a specific embodiment, the disease or disorder is aspartylglucosaminuria, and the biomolecule is a protein that is aspartylglucosaminidase (AGA) (preferably human AGA). In a specific embodiment, the disease or disorder is CLN1 disease or neuronal ceroid lipofuscinosis 1, and the biomolecule is a protein that is palmitoylthioesterase 1 (PPT1) (preferably human PPT1). In a specific embodiment, the disease or disorder is CLN2 disease or Jansky-Bielschowsky disease, and the biomolecule is the protein tripeptidyl peptidase 1 (TPP1) (preferably human TPP1). In a specific embodiment, the disease or disorder is CLN3 disease, and the biomolecule is the protein CLN3 lysosome / endosomal transmembrane protein, battenin (CLN3) (preferably human CLN3). In a specific embodiment, the disease or disorder is CLN4 disease, and the biomolecule is the protein DNAJ heat shock protein family (Hsp40) member C5 (DNAJC5) (preferably human DNAJC5). In a specific embodiment, the disease or disorder is CLN5 disease, and the biomolecule is the protein CLN5 intracellular transport protein (CLN5) (preferably human CLN5).In a specific embodiment, the disease or disorder is a CLN6 disease, and the biomolecule is a protein that is 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 that is major facilitator superfamily domain-containing protein 8 (MFSD8) (preferably human MFSD8). In a specific embodiment, the disease or disorder is a CLN8 disease, and the biomolecule is a protein that is 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 that is cathepsin D (CTSD) (preferably human CTSD). In a specific embodiment, the disease or disorder is a CLN11 disease, and the biomolecule is a protein that is granulin precursor (GRN) (preferably human GRN). In a specific embodiment, the disease or disorder is CLN12 disease, and the biomolecule is the protein ATPase cation transporting 13A2 (ATP13A2) (preferably human ATP13A2). In a specific embodiment, the disease or disorder is CLN13 disease, and the biomolecule is the protein cathepsin F (CTSF) (preferably human CTSF). In a specific embodiment, the disease or disorder is CLN14 disease, and the biomolecule is the protein potassium channel tetramerization domain protein 7 (KCTD7) (preferably human KCTD7). In a specific embodiment, the disease or disorder is cystinosis, and the biomolecule is the protein cystinosin lysosomal cystine transporter (CTNS) (preferably human CTNS). In a specific embodiment, the disease or disorder is Danon disease, and the biomolecule is a protein that is lysosome-associated membrane protein 2 (LAMP2) (preferably human LAMP2).In a specific embodiment, the disease or disorder is Faber lipogranulomatosis, and the biomolecule is the protein N-acylsphingosinamide hydrolase 1 (ASAH1) (preferably human ASAH1). In a specific embodiment, the disease or disorder is fucosidosis, and the biomolecule is the protein α-L-fucosidase 1 (FUCA1) (preferably human FUCA1). In a specific embodiment, the disease or disorder is galactosialidosis, and the biomolecule is the protein cathepsin A (CTSA) (preferably human CTSA). In a specific embodiment, the disease or disorder is Gaucher disease, and the biomolecule is the protein β-glucocerebrosidase (GBA) (preferably human GBA). In a specific embodiment, the disease or disorder is glycogen storage disorder type 2 (GSD II) or Pompe disease, and the biomolecule is a protein that 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 that is myosin VA (MYO5A) (preferably human MYO5A). In a specific embodiment, the disease or disorder is Griscelli syndrome 2, and the biomolecule is a protein that is RAB27A (RAB27A), a member of the RAS oncogene family (preferably human RAB27A). In a specific embodiment, the disease or disorder is Chediak-Higashi syndrome, and the biomolecule is a protein that is lysosome 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 Stey-Sachs disease, and the biomolecule is a protein that is hexosaminidase 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 that is hexosaminidase subunit β (HEXB) (preferably human HEXB). In a specific embodiment, the disease or disorder is GM2 gangliosidosis GM2 activator deficiency, and the biomolecule is a protein that is ganglioside GM2 activator (GM2A) (preferably human GM2A). In a specific embodiment, the disease or disorder is Hermansky-Padlak syndrome type 1, and the biomolecule is a protein that is HPS1 lysosome organelle complex biosynthesis subunit 3 1 (HPS1) (preferably human HPS1). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 2, and the biomolecule is a protein that is adapter-associated protein complex 3 subunit β1 (AP3B1 or HPS2) (preferably human HPS2). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 3, and the biomolecule is a protein that is HPS3 lysosome organelle complex biosynthesis 2 subunit 1 (HPS3) (preferably human HPS3). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 4, and the biomolecule is a protein that is HPS4 lysosome organelle complex biosynthesis 3 subunit 2 (HPS4) (preferably human HPS4). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 5, and the biomolecule is a protein that is HPS5 lysosome organelle complex biosynthesis 2 subunit 2 (HPS5) (preferably human HPS5).In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 6, and the biomolecule is a protein that is HPS6 lysosome organelle complex biosynthesis subunit 2 3 (HPS6) (preferably human HPS6). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 7, and the biomolecule is dystroblevin-binding protein 1 (DTNBP1 or HPS7) (preferably human HPS7). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 8, and the biomolecule is a protein that is lysosome organelle complex biosynthesis subunit 1 3 (BLOC1S3 or HPS8) (preferably human HPS8). In a specific embodiment, the disease or disorder is Hermanski-Padlak syndrome type 9, and the biomolecule is a protein that is lysosome organelle complex biosynthesis subunit 1 6 (BLOC1S6 or HPS9) (preferably human HPS9). In a specific embodiment, the disease or disorder is Krabbe disease, and the biomolecule is a protein that is galactosylceramidase (GALC) (preferably human GALC). In a specific embodiment, the disease or disorder is α-mannosidosis, and the biomolecule is a protein that is mannosidase α class 2B member 1 (MAN2B1) (preferably human MAN2B1). In a specific embodiment, the disease or disorder is β-mannosidosis, and the biomolecule is a protein that is mannosidase β (MANBA) (preferably human MANBA). In a specific embodiment, the disease or disorder is metachromatic leukodystrophy, and the biomolecule is a protein that is allylsulfatase A (ARSA) (preferably human ARSA). In a specific embodiment, the disease or disorder is mucolipidosis II α / β or I-cell disease, and the biomolecule is a protein that is N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably human GNPTAB).In a specific embodiment, the disease or disorder is mucolipidosis III α / β or pseudo-Harler polydystrophy, and the biomolecule is a protein that is N-acetylglucosamine-1-phosphotransferase subunits α and β (GNPTAB) (preferably human GNPTAB). In a specific embodiment, the disease or disorder is mucolipidosis III γ, pseudo-Harler polydystrophy, and the biomolecule is a protein that is N-acetylglucosamine-1-phosphotransferase subunit γ (GNPTG) (preferably human GNPTG). In a specific embodiment, the disease or disorder is mucolipidosis IV, and the biomolecule is a protein that is mucolipin-TRP cation channel 1 (MCOLN1) (preferably human MCOLN1). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type I (MPS I) or Hurler syndrome, and the biomolecule is α-L-idulonidase (IDUA) (preferably hyphenated). The protein is IDUA. In a specific embodiment, the disease or disorder is mucopolysaccharidosis type II (MPS II) or Hunter syndrome, and the biomolecule is the protein iduronate 2-sulfatase (IDS) (preferably human IDS). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IIIA (MPS IIIA) or Sanfilippo syndrome A, and the biomolecule is the protein N-sulfoglucosamine sulfohydrolase (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 the protein N-acetyl-α-aminoglucosidase (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 that is heparan-α-glucosamide 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 that is 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 that 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 that 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 that is allylsulfatase B (ARSB) (preferably human ARSB).In a specific embodiment, the disease or disorder is mucopolysaccharidosis type VII (MPS VII) or Sleigh's disease, and the biomolecule is a protein that is β-glucuronidase (GUSB) (preferably human GUSB). In a specific embodiment, the disease or disorder is mucopolysaccharidosis type IX (MPS IX), and the biomolecule is a protein that is hyaluronidase 1 (HYAL1) (preferably human HYAL1). In a specific embodiment, the disease or disorder is a plurality of sulfatase deficiencies, and the biomolecule is a protein that is sulfatase modifier 1 (SUMF1) (preferably human SUMF1). In a specific embodiment, the disease or disorder is Niemann-Pick disease types A and B, and the biomolecule is a protein that is 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 that is NPC intracellular cholesterol transporter 1 (NPC1) (preferably human NPC1). In a specific embodiment, the disease or disorder is Niemann-Pick disease type C2, and the biomolecule is a protein that is NPC intracellular cholesterol transporter 2 (NPC2) (preferably human NPC2). In a specific embodiment, the disease or disorder is Salah disease, and the biomolecule is a protein that is solute carrier family 17 member 5 (SLC17A5) (preferably human SLC17A5). In a specific embodiment, the disease or disorder is Schindler's disease or Kanzaki disease, and the biomolecule is a protein that is α-N-acetylgalactosaminidase (NAGA) (preferably human NAGA). In a specific embodiment, the disease or disorder is sialic acid deposition disorder type I or cherry red spot myoclonus syndrome, and the biomolecule is a protein that is neuraminidase 1 (NEU1) (preferably human NEU1). In a specific embodiment, the disease or disorder is Wolmann disease and cholesterol ester storage disorder, and the biomolecule is a protein that is lipase A, lysosomal acid (LIPA) (preferably human LIPA).In a specific embodiment, the disease or disorder is spinal muscular atrophy, and the biomolecule is a protein that is 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 that is microdystrophin (preferably human microdystrophin). In a specific embodiment, the disease or disorder is phenylketonuria, and the biomolecule is a protein that is phenylalanine hydroxylase (PAH) (preferably human PAH). In a specific embodiment, the disease or disorder is hemophilia A, and the biomolecule is a protein that is coagulation factor VIII (FVIII) (preferably human FVIII). In a specific embodiment, the disease or disorder is hemophilia B, and the biomolecule is a protein that is coagulation factor IX (FIX) (preferably human FIX). In a preferred embodiment, the biomolecule is a functional protein (e.g., wild-type protein).

[0430] In certain embodiments, the method is used to treat hereditary hemochromatosis (HH), major iron overload conditions, Wilson's disease, genetic copper overload conditions, or α1-antitrypsin deficiency. In certain embodiments, the biomolecule is human α1-antitrypsin (registration number: P01009.3), HFE protein (registration number NP_000401.1 or Q30201), or hepatic protein ATP7B (registration number P35670.4), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95 or greater.

[0431] In certain embodiments, the method is used to treat hypercholesterolemia. In certain embodiments, the biomolecule is human phenylalanine hydroxylase (registration number: P00439.1), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0432] In certain embodiments, the method is used for the treatment of hypertyrosinemia type 1. In certain embodiments, the biomolecule is fumarylacetoacetate hydrolase (registration number: P16930.2), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0433] In certain embodiments, the method is used to treat hypertyrosinemia type 2. In certain embodiments, the biomolecule is human tyrosine aminotransferase (registration number: P17735.1) or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95 or greater.

[0434] In certain embodiments, the method is used for the treatment of homocystinuria and hyperhomocysteinemia. In certain embodiments, the biomolecule is human methylenetetrahydrofolate reductase (registration number: P42898.3), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0435] In certain embodiments, the method is used for the treatment of hyperlipidemia and hypercholesterolemia. In certain embodiments, the biomolecule is human medium-chain acyl-CoA dehydrogenase (registration number: P11310.1), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0436] In certain embodiments, the method is used for the treatment of galactosemia. In certain embodiments, the biomolecule is human galactose-1-phosphate uridyltransferase (registration number: P07902.3), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0437] In certain embodiments, the method is used to treat Lesch-Nyhan syndrome. In certain embodiments, the biomolecule is human hypoxanthine phosphoribosyltransferase (registration number: P00492.2), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0438] In certain embodiments, the method is used to treat Gaucher disease. In certain embodiments, the biomolecule is human cerebrosidase (registration number: P07602.2, registration number: P04062.3) or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95 or greater.

[0439] In certain embodiments, the method is used to treat Tay-Sachs disease. In certain embodiments, the biomolecule is human β-hexosaminidase A (registration number: P06865.2), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0440] In certain embodiments, the method is used to treat Fabry disease. In certain embodiments, the biomolecule is human α-galactosidase (registration number: P06280.1) or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95 or greater.

[0441] In certain embodiments, the method is used to treat Hunter syndrome. In certain embodiments, the biomolecule is human iduronate sulfatase (registration number: P22304.1), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0442] In certain embodiments, the method is used for the treatment of glycogen storage disorder type Ia. In certain embodiments, the biomolecule is human glucose-6-phosphatase (registration number: P35575.2), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0443] In certain embodiments, the method is used for the treatment of ammonia metabolism. In certain embodiments, the biomolecule is human ornithine transcarbamylase (registration number: P00480.3), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0444] In certain embodiments, the method is used for the treatment of phenylketonuria. In certain embodiments, the biomolecule is the human low-density lipoprotein receptor (registration number: P01130.1), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95, or greater than 95.

[0445] In certain embodiments, the method is used for the treatment of propionic acidemia. In certain embodiments, the biomolecule is human propionyl-CoA carboxylase, PCCA and / or PCCB (registration numbers: P05166.3β, NP_000273.2α, NP_001121164.1α), or a variant having sequence identity or similarity of 50, 60, 70, 80, 90, 95 or greater.

[0446] measurement mRNA level analysis Changes in gene levels or expression can be measured using various methods known in this field.

[0447] For example, various methods for detecting or quantifying mRNA levels are known in this field. Exemplary methods include, but are not limited to, northern blotting, ribonuclease-protected assays, and PCR-based methods. The mRNA sequence of a gene can be used to prepare a probe complementary to at least a portion of the mRNA sequence. The mRNA in the sample can then be detected with the probe using any suitable measurement method, such as PCR-based methods, northern blotting, or dipstick measurements.

[0448] The measurement method can vary depending on the type of mRNA information desired. Exemplary methods include, but are not limited to, Northern 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.

[0449] The presence of mRNA in a sample can be determined using any suitable measurement platform. For example, the measurement may take the form of a measurement rod, membrane, tip, disk, test strip, filter, microbeads, slide, multiwell plate, or optical fiber. The measurement system may have a solid support to which nucleic acid corresponding to mRNA is attached. The solid support may include, for example, plastic, silicon, metal, resin, glass, membrane, particles, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or glass slide. The measurement components can be prepared and packaged as a kit for detecting mRNA.

[0450] If necessary, labeled mRNAs may be prepared by labeling nucleic acids. Typically, samples can be labeled using methods known in this field (e.g., the use of DNA ligases or terminal transferases, or labeling of 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. Examples of fluorescent dyes include 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)), 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) This includes, but is not limited to, R6G6 or G6), cyanine dyes (e.g., Cy3, Cy5 and Cy7), Alexa dyes (e.g., Alexa-fluor-555), coumarin, diethylaminocoumarin, umbelliferone, benzimide dyes (e.g., Hoechst 33258), phenanthridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl (Ctriazinyl), eosin dyes, tetramethylrhodamine, lisamin, naphthofluorescein, and others.

[0451] A typical mRNA assay may include the steps of: (1) obtaining a surface-bound subject probe; (2) hybridizing mRNA molecules to the surface-bound probe under conditions sufficient to provide specific binding; (3) washing after hybridization to remove nucleic acids that are not specifically bound to the surface-bound probe; and (4) detecting the hybridized mRNA. The reagents used in each of these steps and their usage conditions may vary depending on the specific application.

[0452] Hybridization can be carried out under appropriate hybridization conditions, where stringency may vary as desired. Typical conditions are sufficient to generate a probe / target complex on a solid surface between complementary binding members, i.e., between the target probe bound to the surface in the sample and complementary mRNA. In certain embodiments, stringent hybridization conditions may be employed.

[0453] Hybridization is typically performed under stringent hybridization conditions. Standard hybridization techniques (e.g., under conditions sufficient to result in specific binding of target mRNA in the sample to the probe) are described in Kallioniemi et al., Science 1992, 258:818-821 and International Publication No. 93 / 18186. Several guides to general techniques are available, e.g., 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 stringency of washing conditions, depends on the experimental design, including the source of the sample, the type of capture agent (identity), and the expected degree of complementarity, and can be determined by those skilled in the art based on ordinary experiments.

[0454] Those skilled in the art will readily recognize that similar stringency conditions can be provided by utilizing alternative but equivalent hybridization and washing conditions.

[0455] After the mRNA hybridization procedure, surface-bound polynucleotides are generally washed to remove unbound nucleic acids. Washing can be performed using a stringent washing protocol, as described above. The hybridization of target mRNA to the probe is then detected using standard techniques.

[0456] Other methods, such as PCR-based methods, can also be used to detect gene expression. An example of a PCR method can be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of an RT-PCR method can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety. A method for fluorescent in situ PCR is described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

[0457] In some embodiments, quantitative reverse transcription PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). Quantitative results obtained by qRT-PCR generally provide more information than qualitative data. Therefore, in some embodiments, qRT-PCR-based assays may be useful for measuring mRNA levels during cell-based assays. The qRT-PCR method is also useful for monitoring patient treatment. An example of a qRT-PCR-based method can be found, for example, in U.S. Patent No. 7,101,663, which is incorporated herein by reference in its entirety.

[0458] In contrast to conventional reverse transcriptase-PCR and agarose gel analysis, qRT-PCR provides quantitative results. A further advantage of qRT-PCR is its relatively easy and convenient use. Instruments for qRT-PCR, such as the Applied Biosystems 7500, are commercially available, as are reagents such as TaqMan® Sequence Detection Chemistry. For example, TaqMan® gene expression assays can be used according to the manufacturer's instructions. These kits are pre-formulated gene expression assays for rapid and reliable detection and quantification of human, mouse, and rat mRNA transcripts. To determine the number of cycles at which the fluorescence signal associated with a particular amplicon accumulation crosses a threshold (called CT), data can be analyzed using, for example, the 7500 Real-Time PCR System Sequence Detection Software or the Comparative CT Relative Quantification Method. Using this method, the output is expressed as a multiplier 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 low enough to be above the baseline but within the exponential growth region of the amplification curve.

[0459] In other embodiments, the target RNA can be detected or quantified by next-generation sequencing (NGS).

[0460] Protein-level analysis Changes in protein expression levels can be assessed by measuring the level of the protein of interest. Protein levels can be evaluated or quantified by various methods known in the art, including immunoprecipitation, Western blot analysis (immunoblot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS), LC-MS (liquid chromatography-mass spectrometry), and other methods. Antibodies against targets can be identified and obtained from various sources, such as the MSRS antibody catalog (Aerie Corporation, Birmingham, Mich.), or prepared by conventional monoclonal or polyclonal antibody production methods known in the art. Antibodies for detecting proteins of interest in mouse, rat, monkey, and human are commercially available. In the case of MassSpec, protein levels can be measured by labeled or unlabeled methods.

[0461] In-vivo analysis In vivo measurements can be used to evaluate the therapeutic effects of recombinant viral particles (including recombinant AAV particles, nonviral gene delivery systems, or pharmaceutical compositions as disclosed herein).

[0462] In some embodiments, motor function is measured by the animal's righting and open field performance. In certain embodiments, respiration is measured by whole-body plethysmography, invasive resistance, and compliance measurement within the animal's body.

[0463] In some embodiments, overall survival (OS) and disease-free survival (DFS) are measured by observing the body weight and health status of living animals twice a day.

[0464] The tests may be carried out in normal animals or experimental disease models. For administration to animals, oligonucleotides can be prepared in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral administration routes such as intraperitoneal, intravenous, and subcutaneous. The calculation of doses and administration frequencies of recombinant viral particles of this disclosure (including recombinant AAV particles, nonviral gene delivery systems, or pharmaceutical compositions) is within the scope of the skills of those skilled in the art and depends on factors such as the route of administration and animal body weight. After treatment with recombinant viral particles of this disclosure (including recombinant AAV particles, nonviral gene delivery systems, or pharmaceutical compositions) for a certain 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 proteins of interest can be measured, for example, using NGS.

[0465] Kits and Products Furthermore, kits, unit doses, and manufactured products comprising any of the compositions described herein are provided. In some embodiments, kits comprising any of the pharmaceutical compositions described herein are provided, preferably with instructions for use thereof.

[0466] The kit of this application is used in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, and packaging (e.g., sealed polyester film (Mylar) or plastic bags). The kit may be provided with additional components such as buffer and interpretation information, as needed. Accordingly, this application further provides manufactured products including vials (e.g., sealed vials), bottles, cans, flexible packaging, etc.

[0467] The manufactured product may include a container and labels or package inserts on or associated with the container. Suitable containers include, for example, bottles, vials, injectors, etc. Containers can be formed from various materials such as glass or plastic. Typically, the container contains a composition for the effective treatment of a disease or disorder described herein and may have a sterile inlet (for example, the container may be a vial with a stopper that can be punctured by an intravenous solution bag or a subcutaneous needle). Labels or package inserts indicate that the composition is for the treatment of a specific disease in an individual. Labels or package inserts further include instructions for administering the composition to an individual. Labels may indicate a guide for redissolution and / or use. The container containing the pharmaceutical composition may be a multipurpose vial that allows for repeated administration (e.g., 2 to 6 doses) of the reconstituted formulation. Package inserts generally refer to instructions included in the commercial packaging of a therapeutic product and include information on indications, uses, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic product. The manufactured product may further include a second container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. From a commercial and user perspective, it may further include other desired materials, including other buffers, diluents, filters, needles, and syringes.

[0468] The kit or product may include multiple unit doses of pharmaceutical compositions and instructions for use, and may be packaged in quantities sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy). array [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-22 [Table 1-23] [Table 1-24] [Table 1-25] [Table 1-26] [Table 1-27] [Table 1-28] [Table 1-29]

[0469] Examples Several embodiments provided herein, described by the following non-limiting examples, illustrate the design and testing of various human α-galactosidase A coding sequences, promoters, expression cassettes, and recombinant glandular virus (AAV) particles, demonstrating that they are particularly suitable for gene therapy, and especially for lysosomal storage disorders, including Fabry disease.

[0470] Example 1: Calibration of substrate levels in a mouse model of Fabry disease by intravenous administration of AAV-GLA. 1. Codon optimization of human α-galactosidase A (GLA). Two codon-optimized versions of the wild-type (WT) human GLA coding sequence (hGLAco1 and hGLAco2, each with nucleotide sequences corresponding to SEQ ID NO:1 and SEQ ID NO:2) were synthesized and cloned into an expression cassette backbone along with a chicken-β-actin (CB) promoter and a bovine growth hormone (bGH) poly A sequence. HepG2 cells were inoculated into 48-well plates (1e5 cells / well). Approximately 20 hours after inoculation, 500 ng 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 minutes. GLA samples were diluted 100-fold or 1000-fold, and undiluted GFP samples were used in ELISA with the human α-galactosidase A ELISA pair kit (Cat. No. SEK12078) (Figure 1A). Samples were diluted 10-fold, and α-galactosidase A activity was measured using the kit (ab239716) (Figure 1B). 10 microliters of PNGase F-treated or untreated samples were used for Western blot analysis with an anti-α-galactosidase polyclonal antibody (1:1000, PA5-27349, Thermo Fisher) (Figure 1C). The PNGase F-treated pattern also demonstrated proper glycosylation of α-galactosidase A expressed from HepG2 cells. hGLAco1 showed twice as high protein expression as wild-type GLA. Therefore, hGLAco1 (SEQ ID NO: 1) was selected for further investigation.

[0471] 2. Hepatocyte-cardiomyocyte (HC) dual-specific promoter in in vitro studies. Five promoters (HC1-HC5, each containing nucleotide sequences corresponding to SEQ ID NO:4-8) were cloned upstream of the reniral luciferase (RLuc) reporter gene, including an immobilized 59-bp hepatocyte promoter (SEQ ID NO:3) linked to various cardiomyocyte-specific transcription factor binding sites (C1-C5). Firefly luciferase (FLuc), another reported gene driven by the HSV-TK promoter, resides on the same construct and is used as an internal control. Rat cardiomyocyte lineage H9c2 (ATCC) and HepG2 cells were inoculated into 96-well plates. One day after inoculation, plasmids containing promoters HC1-HC5 were transfected using Lipofectamine 3000. Two days after transfection, luminescence was quantified using a dual luciferase assay kit (Cat. No. E1500, Promega) and a SpectraMax i3x Multi-Mode Microplate Reader with a SpectraMax Injector Cartridge, according to the manufacturer's guidelines. The RLuc / FLuc ratio was used to measure HC promoter strength (Figure 2). RLuc, driven by the SV40 promoter, and FLuc, 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). This was selected for in vivo studies.

[0472] 3. Severed mouse muscle creatine kinase (MCK) promoter in in vitro studies. Three MCK promoters (MCK-D1 to MCK-D3, each possessing nucleotide sequences from SEQ ID NO: 11 to 13) with 50-bp, 55-bp, and 105-bp cleavage forms derived from the CK8e promoter (SEQ ID NO: 14) were cloned upstream of the reniral luciferase (RLuc) reporter gene. C2C12 myoblasts were inoculated into 96-well plates. One day after inoculation, plasmids containing the MCK promoters were transfected using Lipofectamine 3000. Two days after transfection, luminescence was quantified using a dual luciferase assay kit (Cat. No. E1500, Promega) and a SpectraMax i3x Multi-Mode Microplate Reader with a SpectraMax Injector Cartridge. The RLuc / FLuc ratio was used to measure promoter strength (Figure 3A). RLuc plasmids driven by the CK8e promoter and FLuc plasmids driven by the HSV-TK promoter were used as controls. Compared to the CK8e promoter, cleavage at 50-bp, 55-bp, and 105-bp did not cause a decrease in activity (Figure 3A). The MCK-D3 promoter (SEQ ID NO. 13) was selected and used for further in vivo studies. A synthetic promoter HC7 (SEQ ID NO: 10) was constructed by combining the 59-bp hepatocyte promoter and the MCK-D3 promoter. The activity of promoter HC7 was evaluated using an in vivo imaging system (IVIS). Six-week-old BALB / c male mice were intravenously (tail vein) injected with AAV containing a firefly luciferase transgene driven by the HC7 promoter (1E11vg / mouse). Strong luminescence signals were observed in the liver and skeletal muscle one week (Figure 3B) and three weeks (Figure 3C) after injection.

[0473] 4. Expression and activity of α-galactosidase A mediated by a human GLA expression cassette in HepG2. Four constructs (Table 2) with different promoters or GLA variants were cloned into the autocomplementary adeno-associated virus (scAAV) vector backbone. HepG2 cells were inoculated into 48-well plates (1e5 cells / well). One day after inoculation, cells were transfected with 500 ng plasmids containing either the hGLA cassette (Table 2) or GFP (negative control, Neg.ctrl) using Lipofectamine 3000. Promoter HC6 (SEQ ID NO: 9) was constructed by combining a 59-bp hepatocyte promoter with a cleaved 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. GLA samples were diluted 100-fold or 1000-fold, and undiluted samples from the GFP group were used in ELISA using a human α-galactosidase A ELISA pair kit (Cat. No. SEK12078) (Figure 4A). The samples were diluted 10-fold, and α-galactosidase A activity was measured using the kit (ab239716) (Figure 4B). thGLAco1: a cleaved form of human α-galactosidase A with two amino acids deleted from the C-terminus (nucleotide sequence shown in SEQ ID NO: 20). All constructs showed high expression and activity of α-galactosidase A protein in HepG2.

[0474] [Table 2]

[0475] 5. Expression and activity of α-galactosidase A mediated by a human GLA expression cassette in H9c2 cells. Four constructs (cassettes 1-4) were transfected into H9c2 cells using Lipofectamine 3000. A plasmid expressing GFP was used as a negative control. Three days after transfection, the supernatant was collected and used for human α-galactosidase A ELISA (Figure 5A) and activity analysis (Figure 5B). Construct cassette 3 showed 3.1-fold and 3.8-fold expression compared to cassettes 1 and 2, respectively (Figure 5A). Deletion of the last two amino acids resulted in a 1.4-fold decrease in α-galactosidase A activity in vitro (Figure 5B).

[0476] 6. In a mouse model of Fabry disease, the activity and effects mediated by cassette 1 were characterized. Expression cassette 1 was packaged into scAAV9. scAAV9 was produced by transfection of a triple plasmid and purified by iodoxanol gradient ultracentrifugation. The titer of the vector genome was measured by ddPCR and purity was confirmed by SDS-PAGE. The scAAV9 vector was filtered through 0.22 μm 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 with scAAV9 containing the expression cassette 1 at four doses (2E11 vg / kg, 2E12 vg / kg, 6E12 vg / kg, 2E13 vg / kg) via the tail vein. Two groups of GLA-KO and wild-type mice were intravenously injected with formulation buffer as controls. Serum was collected every two weeks using K2-EDTA tubes and used 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, GLA-KO mice treated with scAAV9 cassette 1 showed 178-fold, 2360-fold, 7285-fold, and 18485-fold higher human α-Gal A activity in serum at doses of 2E11vg / kg, 2E12vg / kg, 6E12vg / kg, and 2E13vg / kg, respectively, compared to wild-type mice treated with buffer (Figure 6B). At the endpoint, mice were killed and perfused with cold PBS. Total RNA was isolated from the liver and heart. Total RNA treated with DNA polymerase 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 reference for normalization. hGLA transgene mRNA levels were clearly dose-dependent in the liver (Figure 6C). Higher hGLA transgene mRNA levels were observed in the heart in the high-dose groups (2E12vg / kg, 6E12vg / kg, 2E13vg / kg) compared to the low-dose group (2E11vg / kg) (Figure 6D). Genomic DNA was isolated from the liver and heart using the DNeasy Blood & Tissue Kit (Qiagen).AAV vector genome copies in these tissues were measured by droplet digital PCR (ddPCR) using hGLAco1 and mouse TFRC primers / probes (Figure 5). Vector genome copies showed dose-dependent effects in both liver (Figure 6E) and heart (Figure 6F). Approximately 15 mg of liver samples were lysed in RIPA buffer. The supernatant after centrifugation was either treated with PNGase F or left untreated. Western blotting was performed using 2 μg protein / sample loaded with an anti-α-galactosidase polyclonal antibody (1:2000, PA5-27349, Thermo Fisher). After treatment with scAAV9-cassette 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 GLA-KO and wild-type mice treated with buffer. Degradation samples from the liver, heart, and kidney were used for α-Gal A activity analysis (Figure 6H). Compared to wild-type mice treated with buffer, GLA-KO mice treated with the lowest dose (2E11vg / kg) of scAAV9 cassette 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 2E12vg / kg of scAAV9 cassette 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 6E12vg / kg of scAAV9 cassette 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 2E13vg / kg scAAV9 cassette 1 showed 5005-fold, 1215-fold, and 87-fold increases in α-Gal A activity in the liver, heart, and kidney, respectively.

[0477] Since lyso-Gb3 is a biomarker for Fabry disease, its substrate levels were quantified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Compared to GLA-KO mice treated with a solvent control, all GLA-KO mice treated with AAV showed significantly reduced serum lyso-Gb3 levels (Figure 6I). Treatment with 2E11 vg / kg of scAAV9-cassette 1 reduced lyso-Gb3 to 2.5% of that of 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 of scAAV9-cassette 1 completely restored lyso-Gb3 levels in the liver, heart, and kidneys to normal (Figure 6J).

[0478] 7. In a mouse model of Fabry disease, the activity and efficacy mediated by AAV-hGLA were characterized. Two expression cassettes (Cassette 1 and Cassette 3) were packaged with AAV9-derived liver-muscle oriented mutant AVT908. AAV production, purification, and characterization were the same as described above. Each hGLA expression cassette was intravenously injected into four groups of adult male GLA-KO mice (JAX stock #003535) via the tail vein at two doses (6E11vg / kg, 2E12vg / kg) (n=5 / group). Serum was collected every two weeks using K2-EDTA tubes and used to measure α-galactosidase A activity (Figure 7A). Eight weeks after treatment, α-Gal A activity in the serum of Cassette 3 was 134% of that of Cassette 1. Similar hGAL transgene mRNA levels were observed in the liver for both expression cassettes. In muscle tissue with similar or lesser vector genome copies (Figure 7B), Cassette 3 showed significantly higher hGLA transgene mRNA levels in the heart and quadriceps than Cassette 1 (Figure 7C). Similarly, for both doses, Cassette 3 showed 84-fold activity in the heart compared to Cassette 1. RT-qPCR data confirmed the activity of the hepatocyte-muscle bispecific promoter HC7 in both liver and muscle. Since mice were fully perfused with PBS before tissue sampling, the α-Gal A protein detected in the heart originated from and / or was taken up by cardiomyocytes. Western blotting revealed that higher doses of Cassette 3 resulted in even higher levels of α-Gal A protein in the heart (Figure 7D). Cassette 1 at a dose of 2E12vg / kg also showed the presence of α-Gal A protein in the heart. Mouse GAPDH was used as a control. Liver, heart, and kidney tissues were degraded and used for α-Gal A activity measurement. All tissues showed high activity after AAV-hGLA treatment. Administration of 2E12vg / kg of Cassette 3 resulted in α-Gal A activity in the liver, heart, and kidneys at 274%, 632%, and 189% of that of the 6E11vg / kg administration, respectively (Figure 7E). Compared to GLA-KO mice treated with buffer, immunohistochemical staining in the liver and heart of GLA-KO mice treated with 2E12vg / kg of Cassette 3 detected robust and widely expressed human α-Gal A protein in an AAV background (Figure 7F).The presence of human α-Gal A protein expressed by AAV in the kidney was also observed (Figure 7F).

[0479] Example 2: Calibration of substrate levels in a Fabry disease mouse model 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) resulting from pathogenic mutations in the GLA gene. The accumulation of substrates within lysosomes gradually leads to cellular dysfunction and multi-organ damage. While current enzyme replacement therapies are effective in treating FD, lifelong infusions every two weeks are a significant burden for patients. To provide a sustained-acting single dose, this study aims to develop a recombinant adeno-associated virus (rAAV) vector-based gene therapy for FD. Two algorithms were used to generate codon-optimized human GLA (hGLA) cDNA with minimal CpG. A series of synthetic hepatophile promoters were evaluated in vitro. Subsequently, an optimized transgene expression cassette was selected and packaged into AAV9 (scAAV9-hGLA). Single intravenous injection of scAAV9-hGLA into 10-week-old α-Gal A knockout male mice resulted in dose-dependent α-Gal A activity in the serum, liver, heart, and kidneys. Stable serum expression of α-Gal A persisted until the end of the 8-week mouse study. Compared to wild-type mice treated with a solvent control (buffer), the lowest dose (2 × 10⁶) showed significant improvement. 11 GLA-KO mice treated with vg / kg showed α-Gal A activity of 178x (×), 58×, 3×, and 0.8× in serum, liver, heart, and kidney, respectively. 2×10 12 GLA-KO mice treated with vg / kg scAAV9-hGLA showed α-Gal A activity of 2360×, 1378×, 119×, and 10×. The highest dose was (2×10⁻⁶). 13(vg / kg) resulted in an increase in serum α-Gal A activity to 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 GLA-KO mice treated with a solvent control, all GLA-KO mice treated with AAV showed significantly reduced serum lyso-Gb3 levels. 2×10 11 Treatment with scAAV9-hGLA at vg / kg reduced lyso-Gb3 to 2.5% of that in GLA-KO control mice. Three relatively high doses (2 × 10) were used. 12 , 6×10 12 and 2 × 10 13 Treatment with vg / kg restored lyso-Gb3 levels in serum, liver, heart, and kidney to normal levels. Codon-optimized hGLA transgene mRNA levels were associated with AAV vector genomic copies in the liver and showed dose-dependent effects. Western blotting demonstrated robust expression and proper glycosylation of liver-derived human α-Gal A protein. Immunostaining confirmed the presence of α-Gal A in the liver, heart, and kidney. Histopathological examination revealed no toxicity at any dose. Finally, this study demonstrated that the effective dose can be further minimized by using a newly developed, in-house developed liver-muscle-targeted capsid AVT908 and promoter. The data support the development of low-dose AAV-GLA gene therapy for Fabry disease.

[0480] Example 3: Robust hGLA expression in NHP mediated by an engineered AAV capsid Human GLA transgene expression cassette 3 (SEQ ID NO: 18) was packaged into 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 using a triple transfection method. After purification by affinity chromatography and filtration through 0.22 μm, the titer of the vector genome was determined by ddPCR using hGLA primers / probes. Purity was analyzed by SDS-PAGE. AAV products with endotoxin levels below 1 EU / mL were used in non-human primate (NHP) studies.

[0481] In three adult (>3 years old) male cynomolgus monkeys that did not have pre-existing neutralizing antibodies against AAV9, we compared in vivo hGLA expression mediated by different AAV capsids. Without the use of immunosuppressants, each animal was intravenously administered a single AAV expressing hGLA at a dose of 2E13vg / kg.

[0482] Eight weeks after administration, animals injected with AVT918 containing hGLA expression cassette 3 were killed. Plasma, liver, heart, kidney, and skeletal muscle were collected and processed for α-Gal A activity measurement.

[0483] Animals injected with AVT917 containing hGLA expression cassette 3 were killed 13 weeks after administration. Plasma, liver, heart, kidney, and skeletal muscle were collected, and α-Gal A activity was measured. Total genomic DNA was extracted from major tissues using the DNeasy Blood & Tissue Kit (Qiagen, 69506). AAV vector genomic copies in tissues were measured by ddPCR (Bio-Rad, QX200) using transgene hGLA-specific primers / probes (forward primer: forward: 5'-GGCAGCTTTGGCTACTATGA 3', reverse: 5'-GTCACAGTAGCAGCCATCAA 3', probe: 5'-FAM-TTGATGCCCAGACCTTTGCTGACT-BHQ1-3'). RNA polymerase P primer / probe (Thermo Fisher Scientific, 4403326) was used as a reference. To quantify transgene mRNA levels in tissue, 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. Macacu GAPDH (forward: 5'-GCACCGTGAAGGCTGAGAAC-3', reverse: 5'-GGATCTCGCTCCTGGAAGATG-3', probe: 5-HEX-CTCGTCATCAATGGAAGCCCCATCA-BHQ1-3') was used as a housekeeping gene. The relative transgene mRNA levels were 2 - Δ Ct It was calculated using the formula.

[0484] Animals injected with AAV9 containing hGLA expression cassette 3 were released after monitoring plasma α-Gal A activity for 26 weeks. From week 6 onward, plasma α-Gal A activity and human α-Gal A protein levels in these animals were stable (triangles in Figure 8). Long-term data over 26 weeks demonstrated that the HC7 promoter (SEQ ID NO: 10) has stable activity in NHP in the case of AAV.

[0485] Plasma and serum samples were collected at different time points. α-Gal A activity in plasma was measured using fluorescence-based enzyme activity assays. Specifically, 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 the substrate. Furthermore, N-acetylgalactosamine (Sigma-Aldrich, A2795-500MG), an α-galactosidase B inhibitor, was added to the samples. A black 96-well plate (Corning, 3603) was shaken at 100 rpm and incubated at 37°C for 2 hours. After incubation, 0.1 mL of 0.5 M glycine-NaOH buffer (pH 10.5) was added to each well to stop the reaction. Fluorescence intensity was measured using endpoint setting (Ex / Em = 365 / 450 nm). α-Gal A activity was calculated according to a 4MU calibration curve and expressed as nmol / h / mL plasma or nmol / h / mg protein.

[0486] All three capsids (AAV9, AVT917, and AVT918) mediated high levels of α-Gal A activity in NHP plasma after intravenous administration (Figure 8A). AVT917 mediated the highest level of α-Gal A activity in plasma, showing a 1,778-fold increase from baseline levels (Table). [Table 3]

[0487] All three capsids (AAV9, AVT917, and AVT918) mediated the expression of specific human α-Gal A protein in NHP plasma after intravenous administration (Figure 8B). AVT917 showed the highest GLA expression based on the concentration of human α-Gal A protein in plasma (Table). [Table 4]

[0488] AAV vector genome (vg) copies were measured in NHP tissue treated with AVT917 by ddPCR. Vector genome copies in the left and right lobes of the liver, heart, kidney, triceps, quadriceps, and calf were 990, 1126, 8.6, 0.7, 1.2, 1.6, and 2.0 vg / diploid genome (dpg), respectively. Vector genome copies in central nervous system (CNS) tissue were less than 1 vg / dpg (Figure 9A). The level and tissue specificity of transgene hGLA mRNA driven by the HC7 promoter (SEQ ID NO: 10) were evaluated by RT-qPCR. The HC7 promoter showed robust activity in the liver, followed by activity in 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 AVT917 capsid and HC7 promoter were monitored in plasma. Thirteen weeks after administration of AVT917 containing HC7-hGLA expression cassette 3, major tissues were collected and processed to measure α-Gal A activity. Compared to α-Gal A activity in tissues from animals not treated with AAV-hGLA (Neg Ctrl), improvements of approximately 80×, 54×, 1.6×, 197×, 68×, and 138× were observed in the heart, liver, kidney, gastrocnemius, quadriceps, and triceps muscles, respectively (Figure 9C).

[0489] Example 4: Efficacy of the capsid AVT917 containing HC7-hGLA in Fabry disease mice. Human GLA transgene expression cassette 3 (SEQ ID NO: 18) was packaged into capsid AVT917 (SEQ ID NO: 56) and produced in HEK293 cells by triple transfection. The purified AAV preparation is called AVT917-HC7-hGLA. To explore the minimum effective dose, adult (~16 weeks old) male Fabry disease mice (GIa-KO) were intravenously injected with three doses (2E11vg / kg, 6E11vg / kg, 2E12vg / kg, n=10 / group) of solvent (formulation buffer) or AVT919-HC7-hGLA (cassette 3). Plasma was collected every two weeks and used for α-Gal A activity measurement. Compared to solvent-group mice, all mice treated with AVT917-HC7-hGLA showed hyperphysiological α-Gal A activity in plasma over an 8-week study (Figure 10A). 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 activity levels in the solvent, low-dose (2E11vg / kg), medium-dose (6E11vg / kg), and high-dose (2E13vg / kg) mice 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 improvements of 1×, 1710×, 4495×, and 13314× compared to the mean plasma α-Gal A activity levels in solvent-treated Fabry disease mice.

[0490] The levels of transgene hGLA mRNA in tissues of Fabry disease mice treated with AVT917-HC7-hGLA were evaluated by RT-qPCR. First, hGLA transcripts were present in the liver and heart (Figure 11A, B). Weak hGLA mRNA levels were observed in the quadriceps and gastrocnemius skeletal muscles (Figure 11D, E). Negligible transgene expression was observed in the kidneys and spleen (Figure 11C, F). These data support the in vivo hepatocyte-myocyte bispecific activity of the HC7 promoter.

[0491] After a single intravenous administration of AVT917-HC7-hGLA to Fabry disease mice, key tissues were collected 8 weeks later and processed using liquid chromatography and tandem mass spectrometry (LC-MS-MS) for measurement of the substrate Lyso-Gb3. All Fabry disease mice treated with AVT917-HC7-hGLA showed a significant reduction in substrate in plasma, liver, heart, and kidney compared to Fabry disease mice treated with the solvent (Figure 12). Fabry disease mice treated with a low dose (2E11vg / kg) of AVT917-HC7-hGLA retained only 2.13%, 4.16%, 0.89%, and 8.73% Lyso-Gb3 in plasma, liver, heart, and kidney compared to Fabry disease mice treated with the solvent (Figure 12). Lyso-Gb3 levels in the tissues of Fabry disease mice treated with the solvent were set to 100%.

[0492] Example 4: Expression of hGLA transgenes mediated by AVT917 in NHP Adult male cynomolgus monkeys were intravenously administered AVT917-HC7-hGLA at doses of 2E12vg / kg (N=3) and 6E12vg / kg (N=2). Plasma was collected, and α-Gal A activity was measured by fluorescence-based 4-MU assay. Before AAV administration, the mean α-Gal A activity of 5 animals was 15.6 nmol / h / L. One week after administration, α-Gal A activity increased by 234× and 711× from baseline levels at 2E12vg / kg and 6E12vg / kg, respectively. After week 6, α-Gal A activity in the plasma of the three animals treated with 2E12vg / kg remained stable (Figure 13A). The observation period for one animal was long, at 26 weeks. α-Gal A activity in the plasma of two animals treated with 6E12vg / kg was stable between weeks 2 and 6 (Figure 13A). Enzyme activity increased slightly after week 6 (Figure 13A). These data support the ability of low-dose AVT917-HC7-hGLA to provide hyperphysiological and stable α-Gal A activity in NHP after a single intravenous administration.

[0493] Human α-Gal A protein expressed from NHP treated with AVT917-HC7-hGLA was measured by ELISA. Prior to administration, no human α-Gal A protein was detected in the plasma. One week after treatment with AVT917-HC7-hGLA, three animals receiving a dose of 2E12vg / kg showed plasma levels of 53 ng / mL, 105 ng / mL, and 224 ng / mL of human α-Gal A. From week 2 onward, human α-Gal protein levels remained stable in these three animals (mean levels: 27-36 ng / mL) (Figure 13B). At week 26, one animal showed a plasma level of 63 ng / mL of human α-Gal A protein. These data demonstrate that HC7 can provide stable promoter activity in NHP. In week 1, two animals treated with 6E12vg / kg of AVT917-HC7-hGLA showed human α-Gal A levels of 192 ng / mL and 433 ng / mL in their plasma. The mean human α-Gal A protein levels at weeks 2 and 13 were 123 ng / mL and 111 ng / mL, respectively (Figure 13B).

[0494] Example 5: Comparison of hGLA transgene expression mediated by AVT917 and AVT919 in NHP Adult male cynomolgus monkeys were intravenously administered AVT917 or AVT919 containing cassette 3 (SEQ ID NO: 18) at a dose of 2E12 vg / kg (N=1 / capsid). Plasma was collected and used for ELISA to measure human α-Gal A protein concentration. Before AAV administration, the human α-Gal A level in plasma was 0 ng / mL. After 1 week of administration, AVT917 and AVT919 generated 224 ng / mL and 147 ng / mL of human α-Gal A in plasma, respectively (Figure 14). The plasma human α-Gal A level mediated by AVT917 was higher than that mediated by AVT919 (Figure 14).

[0495] Embedding by reference All references cited herein are incorporated herein by whole citation and used for all purposes, to the extent indicated specifically and individually with respect to the individual publications or patents or patent applications, and are incorporated herein by whole citation for all purposes.

[0496] It will be apparent to those skilled in the art that many modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The specific embodiments described herein are presented as examples only, and the present invention is limited only by the conditions of the appended claims and the entire scope of the equivalents thereof.

[0497] This disclosure is made by describing many embodiments using generally positive language. Specifically, this disclosure includes embodiments that completely or partially exclude certain subject matter, such as substances or materials, steps and conditions of a method, operating procedures, processes, measurements or analyses. Therefore, even if this disclosure is not expressed based on content not included in this disclosure, this disclosure still discloses aspects that are not expressly included in this disclosure.

Claims

1. A polynucleotide containing a nucleotide sequence encoding human α-galactosidase A, The nucleotide sequence encoding human α-galactosidase A is a polynucleotide containing SEQ ID NO:

1.

2. An expression cassette comprising a polynucleotide containing a nucleotide sequence encoding human α-galactosidase A as described in claim 1, and a polynucleotide containing a nucleotide sequence encoding a promoter, An expression cassette in which the nucleotide sequence encoding the promoter is operably 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 includes 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-myocyte dual-specific promoter.

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

7. The expression cassette according to claim 6, wherein the nucleotide sequence encoding the expression cassette includes SEQ ID NO: 16 or SEQ ID NO:

18.

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

10.

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

10. The expression cassette according to claim 9, wherein the nucleotide sequence encoding human α-galactosidase A includes 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-myocyte dual-specific promoter.

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

14. The expression cassette according to claim 13, wherein the nucleotide sequence encoding the expression cassette includes SEQ ID NO: 16 or SEQ ID NO:

18.

15. A vector comprising a polynucleotide according to claim 1 or 8, or an expression cassette according to any one of claims 2 to 7 or 9 to 14.

16. Recombinant viral particles comprising a recombinant viral genome, comprising an expression cassette according to any one of claims 2 to 7 or 9 to 14.

17. Recombinant adeno-associated virus (AAV) particles comprising (a) an AAV capsid and (b) a recombinant AAV genome comprising an expression cassette according to any one of claims 2 to 7 or 9 to 14, wherein the AAV terminal inverted repeat sequence (ITR) is flanked.

18. Recombinant AAV particles according to claim 17, which are recombinant AAV serotype 9 (rAAV9) particles.

19. The recombinant AAV particle according to claim 17 or 18, wherein the AAV capsid comprises a mutant AAV9 capsid protein.

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

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

22. A host cell comprising a polynucleotide according to claim 1 or 8, a vector according to claim 15, a recombinant virus particle according to claim 16, or a recombinant AAV particle according to any one of claims 17 to 21.

23. A group of host cells stably transduced by recombinant virus particles according to claim 16 or recombinant AAV particles according to any one of claims 17 to 21.

24. A pharmaceutical composition comprising recombinant virus particles according to claim 16, or recombinant AAV particles according to any one of claims 17 to 21, a group of host cells according to claim 23, and a pharmaceutically acceptable carrier.

25. A method for producing recombinant virus particles or recombinant AAV particles, comprising culturing the host cells described in claim 22.

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

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

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

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