Mutant AAV9 capsid protein and its use

Abstract

This specification provides a mutant adeno-associated virus serotype 9 (AAV9) capsid protein and recombinant AAV9 particles containing the mutant AAV9 capsid protein. Further provided are a pharmaceutical composition comprising recombinant AAV9 particles, a polynucleotide encoding the mutant AAV9 capsid protein, a vector and host cells containing the polynucleotide, a population of host cells transduced by the recombinant AAV9 particles, and various methods for using the recombinant AAV9 particles.

Description

[Technical Field] 【0001】 Cross-reference of related applications 【0002】 This application claims the benefit and priority of PCT application No. PCT / CN2023 / 085648, filed on 31 March 2023. The contents of these applications are incorporated herein by reference for all purposes. 【0003】 Technical field A mutant adeno-associated virus serotype 9 (AAV9) capsid protein and recombinant AAV9 particles containing the mutant AAV9 capsid protein are provided. Further provided are recombinant AAV9 particles, polynucleotides encoding the mutant AAV9 capsid protein, vectors and host cells containing the polynucleotide, pharmaceutical compositions comprising host cell populations transduced by recombinant AAV9 particles, and various methods for using recombinant AAV9 particles. [Background technology] 【0004】 Adeno-associated virus (AAV) is a replication-deficient parvovirus with a non-enveloped capsid and a linear single-stranded DNA genome containing two 145-nucleotide terminal inverted repeat sequences (ITRs) at its ends. Gene therapy with AAV has the potential to be a promising treatment for many diseases. However, in many cases, low transduction efficiency in some tissues is a technical obstacle to the clinical use of AAV gene therapy. 【0005】 To this day, there remains a need to develop AAV-mediated gene delivery systems with enhanced transduction into tissues. [Overview of the project] 【0006】 In one embodiment, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166 or SEQ ID A mutant adeno-associated virus serotype 9 (AAV9) capsid protein containing the amino acid sequence NO:168 is provided. 【0007】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:37. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:6. 【0008】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:39. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:7. 【0009】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:41. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:8. 【0010】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:43. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:9. 【0011】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:45. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:10. 【0012】 In certain embodiments, the mutant AAV9 capsid protein includes the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:49. In specific embodiments, the mutant AAV9 capsid protein includes the amino acid sequence of SEQ ID NO:11. 【0013】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO: 51. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO: 12. 【0014】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:53. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:13. 【0015】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:55. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:14. 【0016】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:57. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:15. 【0017】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:59 and comprises the amino acid sequence of SEQ ID NO:61. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:16. 【0018】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:63. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:17. 【0019】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:65. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO:18. 【0020】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 67. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 19. 【0021】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 69. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 20. 【0022】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 71. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 21. 【0023】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 73. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 22. 【0024】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 75. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 23. 【0025】 In certain embodiments, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 77. In a specific embodiment, the variant AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 24. 【0026】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:79. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:25. 【0027】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO: 80. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO: 26. 【0028】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:81. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:27. 【0029】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:82. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:28. 【0030】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:83. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:29. 【0031】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:84. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:30. 【0032】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:85. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:31. 【0033】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:86. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:32. 【0034】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:87. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:33. 【0035】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:116. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:115. 【0036】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:118. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:117. 【0037】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:120. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:119. 【0038】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:122. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:121. 【0039】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:124. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:123. 【0040】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:126. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:125. 【0041】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:128. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:127. 【0042】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:130. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:129. 【0043】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:132. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:131. 【0044】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:134. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:133. 【0045】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:136. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:135. 【0046】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:138. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:137. 【0047】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:140. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:139. 【0048】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:142. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:141. 【0049】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:144. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:143. 【0050】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:146. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:145. 【0051】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:148. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:147. 【0052】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:150. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:149. 【0053】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:152. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:151. 【0054】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:154. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:153. 【0055】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:156. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:155. 【0056】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:158. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:157. 【0057】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:160. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:159. 【0058】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:162. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:161. 【0059】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:164. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:163. 【0060】 In a particular embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:166. In a specific embodiment, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:165. 【0061】 In certain embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:168. In specific embodiments, the mutant AAV9 capsid protein contains the amino acid sequence of SEQ ID NO:167. 【0062】 In various embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced hepatic tropism compared to wild-type AAV9 capsid proteins containing the amino acid sequence SEQ ID NO:2. 【0063】 In various embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced tropism to the brain compared to wild-type AAV9 capsid proteins containing the amino acid sequence SEQ ID NO:2. 【0064】 In various embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced muscle tropism compared to the wild-type AAV9 capsid protein containing the amino acid sequence SEQ ID NO:2. In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to skeletal muscle and / or cardiac muscle compared to the wild-type AAV9 capsid protein. In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major, atrial muscle and / or ventricular muscle compared to the wild-type AAV9 capsid protein. 【0065】 In another embodiment, recombinant AAV9 particles containing the mutant AAV9 capsid protein described herein are provided. 【0066】 In another embodiment, a pharmaceutical composition is provided comprising recombinant AAV9 particles described herein and a pharmaceutically acceptable carrier. 【0067】 In another embodiment, polynucleotides encoding the variant AAV9 capsid protein described herein are provided. 【0068】 In another embodiment, a vector comprising the polynucleotide described herein is provided. 【0069】 In another embodiment, a host cell comprising a polynucleotide or a vector as described herein is provided. 【0070】 In another embodiment, a population of host cells stably transduced by recombinant AAV9 particles described herein is provided. 【0071】 In another embodiment, a pharmaceutical composition is provided comprising a host cell population described herein and a pharmaceutically acceptable carrier. 【0072】 In another embodiment, a method for delivering a biomolecule to one or more ex vivo or in vitro target cells is provided, comprising transducing one or more target cells using recombinant AAV9 particles described herein. In certain embodiments, the one or more target cells are one or more muscle cells, one or more hepatocytes, and / or one or more brain cells. 【0073】 In another embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering to a subject a recombinant AAV9 particle described herein, or a pharmaceutical composition described herein comprising recombinant AAV9 particles and a pharmaceutically acceptable carrier. In a particular embodiment, the one or more target cells are one or more muscle cells, one or more hepatocytes, and / or one or more brain cells. In a particular embodiment, the subject is a human. 【0074】 In another embodiment, a method is provided for treating a disease or disorder in a subject of interest, comprising administering to the subject recombinant AAV9 particles or pharmaceutical composition described herein. In certain embodiments, the subject is a human being. 【0075】 In another embodiment, a method for producing recombinant AAV9 particles is provided, comprising culturing the host cells described herein. 【0076】 Descriptive embodiment 【0077】 This disclosure includes the following non-limiting embodiments. 【0078】 1.SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166 or SEQ ID A variant adeno-associated virus serotype 9 (AAV9) capsid protein containing amino acid sequence NO:168. 【0079】 2. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:37. 【0080】 3. The mutant AAV9 capsid protein according to Embodiment 2, comprising the amino acid sequence of SEQ ID NO:6. 【0081】 4. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:39. 【0082】 5. The mutant AAV9 capsid protein according to Embodiment 4, comprising the amino acid sequence of SEQ ID NO:7. 【0083】 6. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:41. 【0084】 7. The mutant AAV9 capsid protein according to Embodiment 6, comprising the amino acid sequence of SEQ ID NO:8. 【0085】 8. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:43. 【0086】 9. The mutant AAV9 capsid protein according to Embodiment 8, comprising the amino acid sequence of SEQ ID NO:9. 【0087】 10. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:45. 【0088】 11. The mutant AAV9 capsid protein according to Embodiment 10, comprising the amino acid sequence of SEQ ID NO:10. 【0089】 12. A mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:47 and the amino acid sequence of SEQ ID NO:49. 【0090】 13. The mutant AAV9 capsid protein according to Embodiment 12, comprising the amino acid sequence of SEQ ID NO:11. 【0091】 14. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:51. 【0092】 15. The mutant AAV9 capsid protein according to Embodiment 14, comprising the amino acid sequence of SEQ ID NO:12. 【0093】 16. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:53. 【0094】 17. The mutant AAV9 capsid protein according to Embodiment 16, comprising the amino acid sequence of SEQ ID NO:13. 【0095】 18. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:55. 【0096】 19. The mutant AAV9 capsid protein according to Embodiment 18, comprising the amino acid sequence of SEQ ID NO:14. 【0097】 20. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:57. 【0098】 21. The mutant AAV9 capsid protein according to Embodiment 20, comprising the amino acid sequence of SEQ ID NO:15. 【0099】 22. A mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO: 59 and the amino acid sequence of SEQ ID NO: 61. 【0100】 23. The mutant AAV9 capsid protein according to Embodiment 22, comprising the amino acid sequence of SEQ ID NO:16. 【0101】 24. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:63. 【0102】 25. The mutant AAV9 capsid protein according to Embodiment 24, comprising the amino acid sequence of SEQ ID NO:17. 【0103】 26. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:65. 【0104】 27. The mutant AAV9 capsid protein according to Embodiment 26, comprising the amino acid sequence of SEQ ID NO:18. 【0105】 28. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:67. 【0106】 29. The mutant AAV9 capsid protein according to Embodiment 28, comprising the amino acid sequence of SEQ ID NO:19. 【0107】 30. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:69. 【0108】 31. A mutant AAV9 capsid protein according to Embodiment 30, comprising the amino acid sequence of SEQ ID NO:20. 【0109】 32. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:71. 【0110】 33. The mutant AAV9 capsid protein according to Embodiment 32, comprising the amino acid sequence of SEQ ID NO:21. 【0111】 34. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:73. 【0112】 35. The mutant AAV9 capsid protein according to Embodiment 34, comprising the amino acid sequence of SEQ ID NO:22. 【0113】 36. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:75. 【0114】 37. The mutant AAV9 capsid protein according to Embodiment 36, comprising the amino acid sequence of SEQ ID NO:23. 【0115】 38. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:77. 【0116】 39. The mutant AAV9 capsid protein according to Embodiment 38, comprising the amino acid sequence of SEQ ID NO:24. 【0117】 40. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:79. 【0118】 41. The mutant AAV9 capsid protein according to Embodiment 40, comprising the amino acid sequence of SEQ ID NO:25. 【0119】 42. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:80. 【0120】 43. The mutant AAV9 capsid protein according to Embodiment 42, comprising the amino acid sequence of SEQ ID NO:26. 【0121】 44. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:81. 【0122】 45. The mutant AAV9 capsid protein according to Embodiment 44, comprising the amino acid sequence of SEQ ID NO:27. 【0123】 46. ​​The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:82. 【0124】 47. The mutant AAV9 capsid protein according to Embodiment 46, comprising the amino acid sequence of SEQ ID NO:28. 【0125】 48. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:83. 【0126】 49. The mutant AAV9 capsid protein according to Embodiment 48, comprising the amino acid sequence of SEQ ID NO:29. 【0127】 50. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:84. 【0128】 51. The mutant AAV9 capsid protein according to Embodiment 50, comprising the amino acid sequence of SEQ ID NO:30. 【0129】 52. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:85. 【0130】 53. The mutant AAV9 capsid protein according to Embodiment 52, comprising the amino acid sequence of SEQ ID NO:31. 【0131】 54. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:86. 【0132】 55. The mutant AAV9 capsid protein according to Embodiment 54, comprising the amino acid sequence of SEQ ID NO:32. 【0133】 56. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:87. 【0134】 57. The mutant AAV9 capsid protein according to Embodiment 56, comprising the amino acid sequence of SEQ ID NO:33. 【0135】 58. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:116. 【0136】 59. The mutant AAV9 capsid protein according to Embodiment 58, comprising the amino acid sequence of SEQ ID NO:115. 【0137】 60. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:118. 【0138】 61. The mutant AAV9 capsid protein according to Embodiment 60, comprising the amino acid sequence of SEQ ID NO:117. 【0139】 62. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:120. 【0140】 63. The mutant AAV9 capsid protein according to Embodiment 62, comprising the amino acid sequence of SEQ ID NO:119. 【0141】 64. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:122. 【0142】 65. A mutant AAV9 capsid protein according to Embodiment 64, comprising the amino acid sequence of SEQ ID NO:121. 【0143】 66. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:124. 【0144】 67. The mutant AAV9 capsid protein according to Embodiment 66, comprising the amino acid sequence of SEQ ID NO:123. 【0145】 68. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:126. 【0146】 69. A mutant AAV9 capsid protein according to Embodiment 68, comprising the amino acid sequence of SEQ ID NO:125. 【0147】 70. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:128. 【0148】 71. A variant AAV9 capsid protein according to Embodiment 70, comprising the amino acid sequence of SEQ ID NO:127. 【0149】 72. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:130. 【0150】 73. A mutant AAV9 capsid protein according to Embodiment 72, comprising the amino acid sequence of SEQ ID NO:129. 【0151】 74. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:132. 【0152】 75. A variant AAV9 capsid protein according to Embodiment 74, comprising the amino acid sequence of SEQ ID NO:131. 【0153】 76. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:134. 【0154】 77. A variant AAV9 capsid protein according to Embodiment 76, comprising the amino acid sequence of SEQ ID NO:133. 【0155】 78. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:136. 【0156】 79. A mutant AAV9 capsid protein according to Embodiment 78, comprising the amino acid sequence of SEQ ID NO:135. 【0157】 80. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:138. 【0158】 81. The mutant AAV9 capsid protein according to Embodiment 80, comprising the amino acid sequence of SEQ ID NO:137. 【0159】 82. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:140. 【0160】 83. The mutant AAV9 capsid protein according to Embodiment 82, comprising the amino acid sequence of SEQ ID NO:139. 【0161】 84. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:142. 【0162】 85. The mutant AAV9 capsid protein according to Embodiment 84, comprising the amino acid sequence of SEQ ID NO:141. 【0163】 86. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:144. 【0164】 87. The mutant AAV9 capsid protein according to Embodiment 86, comprising the amino acid sequence of SEQ ID NO:143. 【0165】 88. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:146. 【0166】 89. The mutant AAV9 capsid protein according to Embodiment 88, comprising the amino acid sequence of SEQ ID NO:145. 【0167】 90. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:148. 【0168】 91. The mutant AAV9 capsid protein according to Embodiment 90, comprising the amino acid sequence of SEQ ID NO:147. 【0169】 92. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:150. 【0170】 93. The mutant AAV9 capsid protein according to Embodiment 92, comprising the amino acid sequence of SEQ ID NO:149. 【0171】 94. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:152. 【0172】 95. The mutant AAV9 capsid protein according to Embodiment 94, comprising the amino acid sequence of SEQ ID NO:151. 【0173】 96. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:154. 【0174】 97. A mutant AAV9 capsid protein according to Embodiment 96, comprising the amino acid sequence of SEQ ID NO:153. 【0175】 98. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:156. 【0176】 99. A mutant AAV9 capsid protein according to Embodiment 98, comprising the amino acid sequence of SEQ ID NO:155. 【0177】 100. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:158. 【0178】 101. The mutant AAV9 capsid protein according to Embodiment 100, comprising the amino acid sequence of SEQ ID NO:157. 【0179】 102. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:160. 【0180】 103. The mutant AAV9 capsid protein according to Embodiment 102, comprising the amino acid sequence of SEQ ID NO:159. 【0181】 104. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:162. 【0182】 105. The mutant AAV9 capsid protein according to Embodiment 104, comprising the amino acid sequence of SEQ ID NO:161. 【0183】 106. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:164. 【0184】 107. The mutant AAV9 capsid protein according to Embodiment 106, comprising the amino acid sequence of SEQ ID NO:163. 【0185】 108. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:166. 【0186】 109. The mutant AAV9 capsid protein according to Embodiment 108, comprising the amino acid sequence of SEQ ID NO:165. 【0187】 110. The mutant AAV9 capsid protein according to Embodiment 1, comprising the amino acid sequence of SEQ ID NO:168. 【0188】 111. The mutant AAV9 capsid protein according to Embodiment 110, comprising the amino acid sequence of SEQ ID NO:167. 【0189】 112. A mutant AAV9 capsid protein according to any one of embodiments 8-9, 14-15, and 24-35, relating to enhanced hepatic tropism compared to wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2. 【0190】 113. A mutant AAV9 capsid protein according to any one of embodiments 24-25, 30-31, 34-37, 42-43 and 46-47, relating to enhanced tropism to the brain compared to wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2. 【0191】 114. A mutant AAV9 capsid protein according to any one of Embodiments 1 to 113, relating to enhanced muscle tropism compared to wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2. 【0192】 115. A mutant AAV9 capsid protein according to Embodiment 114, associated with enhanced tropism to skeletal muscle and / or cardiac muscle compared to wild-type AAV9 capsid protein. 【0193】 116. The mutant AAV9 capsid protein according to Embodiment 114, which is associated with enhanced tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major, atrial muscle and / or ventricular muscle compared to wild-type AAV9 capsid protein. 【0194】 117. Recombinant AAV9 particles containing the mutant AAV9 capsid protein described in any one of Embodiments 1 to 116. 【0195】 118. A pharmaceutical composition comprising recombinant AAV9 particles as described in Embodiment 117 and a pharmaceutically acceptable carrier. 【0196】 119. A polynucleotide encoding the mutant AAV9 capsid protein described in any one of Embodiments 1 to 116. 【0197】 120. A vector comprising the polynucleotide described in Embodiment 119. 【0198】 121. A host cell comprising the polynucleotide described in Embodiment 119 or the vector described in Embodiment 120. 【0199】 122. A population of host cells stably transduced by recombinant AAV9 particles as described in Embodiment 117. 【0200】 123. A pharmaceutical composition comprising a host cell population as described in Embodiment 122 and a pharmaceutically acceptable carrier. 【0201】 124. A method for delivering a biomolecule to one or more ex vivo or in vitro target cells, comprising transducing one or more target cells using recombinant AAV9 particles described in Embodiment 117. 【0202】 125. A method for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering the recombinant AAV9 particles described in Embodiment 117 or the pharmaceutical composition described in Embodiment 118 to the subject. 【0203】 126. The method according to Embodiment 124 or 125, wherein the one or more target cells are one or more muscle cells, one or more hepatocytes and / or one or more brain cells. 【0204】 127. A method for treating a disease or disorder in a subject in need, comprising administering to a subject recombinant AAV9 particles as described in Embodiment 117, or a pharmaceutical composition as described in Embodiment 118 or 123. 【0205】 128. The method according to any one of embodiments 125 to 127, wherein the subject is a human. 【0206】 129. A method for producing recombinant AAV9 particles, comprising culturing the host cells described in Embodiment 121. [Brief explanation of the drawing] 【0207】 [Figure 1A] AAV library design. Figure 1A: Library #1. Figure 1B: Library #2. Figure 1C: Library #3. Figure 1D: Library #4. ITR: Terminal inversion repeat; p41: AAV5 p41 promoter; X: Any amino acid; pA: PolyA signal. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. 【0208】 [Figure 2] AAV library screening workflow. 【0209】 [Figure 3] Comparison of in vivo transduction by imaging. Eight-week-old C57BL / 6J mice were intravenously injected with AAV containing the CBA-GFP transgene (1e12vg / mouse). CBA: chicken β-actin promoter; GFP: green fluorescent protein. GFP expression was detected using ex vivo imaging two weeks (2wpi) after injection. MyoAAV 1A (1A) and wild-type AAV9 were used as controls. Tri: triceps; Quad: quadriceps; TA: tibialis anterior; GAS: gastrocnemius; H: heart; L: liver; Sp: spleen; Lung: lung; Ki: kidney. 【0210】 [Figure 4] Comparison of in vivo transduction by RT-PCR. Eight-week-old C57BL / 6J mice were intravenously injected with AAV containing the CBA-GFP transgene (1e12vg / mouse). CBA: chicken β-actin promoter; GFP: green fluorescent protein. Two weeks after injection (2wpi), total RNA was isolated from tissue and RT-qPCR was performed to quantify GFP expression. MyoAAV 1A (1A) and wild-type AAV9 were used as controls. The GFP expression levels (multiple changes relative to wild-type AAV9) of each mutant in the test tissue were as follows: AVT901 (Tri:6.4;Q:5.7;TA:4.4;GAS:5.3;H:1.7;Liver:1.2;S:0.6;Lung:0.7;K:1.4); AVT903 (Tri:2.5;Q:1.8;TA:1.6;GAS:2.2;H:0.9;Liver:0.9;S:0.4;Lung:0.7;K:0.9); AVT905 (Tri:11.6;Q:11.5;TA:3.4;GAS:6.1;H:2.2;Liver:2.7;S:3 .1; Lung: 5.3; K: 1.0); AVT906 (Tri: 0.6; Q: 0.9; TA: 0.3; GAS: 0.6; H: 0.8; Liver: 1.1; S: 0.6; Lung: 0.7; K: 0.8); AVT907 (Tri: 36.9; Q: 28.2; TA: 16.5; GAS: 35.2; H: 9.7; Liver: 0.3; S: 0.3; Lung: 1.1; K: 0.3); Myo1A (Tri: 22.3; Q: 16.8; TA: 16.3; GAS: 27.7; H: 7.5; Liver: 0.6; S: ​​0.7; Lung: 1.2; K: 1.1). Myo1A: MyoAAV 1A. For each variant, the values ​​for the triceps (Tri), quadriceps (Q), tibialis anterior (TA), gastrocnemius (GAS), heart (H), liver, spleen (S), lungs, and kidneys (K) are displayed from left to right. 【0211】 [Figure 5]Copy analysis of vector genomes. Eight-week-old C57BL / 6J mice were intravenously injected with AAV containing a CBA-GFP transgene (1e12vg / mouse). CBA: chicken β-actin promoter; GFP: green fluorescent protein. Two weeks after injection (2wpi), genomic DNA was isolated from tissue, and ddPCR was performed for copy analysis of the AAV vector genome. MyoAAV 1A (1A) and wild-type AAV9 were used as controls. The multiples of each AAV9 mutant's genome copy relative to wild-type AAV9 in the test tissue were as follows: AVT901 (Tri:2.6;Q:2.6;TA:1.4;GAS:1.6;H:1.3;S:3.8;Lung:1.1;K:0.9;L:0.6); AVT903 (Tri:2.2;Q:2.8;TA:1.1;GAS:0.8;H:1.9;S:2.6;Lung:0.7;K:0.9;L:0.3); AVT905 (Tri:3.9;Q:10.9;TA:3.0;GAS:6.8;H:5.0;S:1.3;Lung :12.2;K:3.0;L:3.7);AVT906(Tri:4.9;Q:1.8;TA:No data;GAS:0.4;H:1.3;S:1.7;Lung:1.0;K:0.8;L:0.8);AVT907(Tri:7.1;Q:5.4;TA:1 .9;GAS:5.9;H:10.0;S:2.2;Lung:2.0;K:1.0;L:0.2);Myo1A(Tri:7.7;Q:7.8;TA:2.8;GAS:5.1;H:6.0;S:2.3;Lung:2.3;K:2.0;L:0.5). For each variant, the values ​​for the triceps (Tri), quadriceps (Q), tibialis anterior (TA), gastrocnemius (GAS), heart (H), spleen (S), lungs, kidneys (K), and liver (L) are displayed from left to right. Myo1A: MyoAAV 1A. 【0212】 [Figure 6A]Comparison of in vitro transduction by imaging. Figure 6A: C2C12 myoblasts were seeded in a 96-well plate and incubated with a specified AAV vector containing the CB-Fluc-2A-GFP transgene (1 e6 vg / cell). Native GFP imaging was performed 24 hours after transduction under the same conditions. Figure 6B: HepG2 cells were seeded in a 96-well plate and incubated with a specified AAV vector containing the CB-Fluc-2A-GFP transgene (1 e6 vg / cell). Native GFP imaging was performed 24 hours after transduction under the same conditions. CB: Chicken β-actin promoter; Fluc: Firefly luciferase; GFP: Green fluorescent protein. MyoAAV 1A (1A), MyoAAV 2A (2A), MyoAAV 4A (4A), and wild-type AAV9 were used as controls. [Figure 6B] Same as above. 【0213】 [Figure 7A] Comparison of in vitro transduction by luciferase assay. Figure 7A: C2C12 myoblasts were seeded in a 96-well plate and incubated with a specified AAV vector containing the CB-Fluc-2A-GFP transgene (1 e6 vg / cell). A luciferase assay was performed 48 hours after transduction for quantification. Figure 7B: HepG2 cells were seeded in a 96-well plate and incubated with a specified AAV vector containing the CB-Fluc-2A-GFP transgene (1 e6 vg / cell). A luciferase assay was performed 48 hours after transduction for quantification. CB: Chicken β-actin promoter; Fluc: Firefly luciferase; GFP: Green fluorescent protein. MyoAAV 1A (1A), MyoAAV 2A (2A), MyoAAV 4A (4A), and wild-type AAV9 (AV9) were used as controls. [Figure 7B] Same as above. 【0214】 [Figure 8]Comparison of transduction efficiency using an in vivo imaging system (IVIS). 8-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). Luciferase expression was detected using IVIS 3 weeks after injection (3wpi). MyoAAV 1A, MyoAAV 2A, MyoAAV 4A, and wild-type AAV9 were used as controls. 【0215】 [Figure 9A]Comparison of in vivo transduction efficiency. Tri: Triceps; Q: Quadriceps; TA: Tibialis anterior; GAS: Gastrocnemius; H: Heart; L: Liver. Figure 9A: Comparison of in vivo transduction efficiency by RT-qPCR. 8-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). Three weeks after injection (3wpi), total RNA was isolated from tissue and RT-qPCR was performed to quantify transgene expression. MyoAAV 1A, MyoAAV 2A, MyoAAV 4A, and wild-type AAV9 were used as controls.The transgene expression levels (normalized to GAPDH) of each mutant in the test tissue were as follows: AVT901 (T:0.1;Q:0.3;TA:0.1;GAS:0.1;H:0.1;L:0.1); AVT902 (T:0.05;Q:0.1;TA:0.037;GAS:0.1;H:0.3;L:0.2); AVT907 (T:0.3;Q:0.5;TA:0.2;GAS:0.6;H:1.2) ;L:0.1);AVT908(T:0.3;Q:0.9;TA:0.2;GAS:1.1;H:0.2;L:0.8);AVT909(T:0.1;Q:0.2;TA:0.1;GAS:0.2; H:0.1;L:0.1);AVT910(T:0.2;Q:0.3;TA:0.024;GAS:0.2;H:0.1;L:0.2);AVT913(T:1.3;Q:1.6;TA:0.7;G AS:2.3;H:2.5;L:0.1);AVT914(T:1.0;Q:0.7;TA:0.4;GAS:0.6;H:0.8;L:0.2);AVT915(T:0.5;Q:0.8;TA :0.2;GAS:1.2;H:0.5;L:0.1);AVT916(T:0.7;Q:0.7;TA:0.3;GAS:0.8;H:0.7;L:0.1);AVT917(T:0.5;Q:0 .6;TA:0.3;GAS:0.9;H:0.7;L:0.1);AVT918(T:0.6;Q:0.5;TA:0.3;GAS:0.6;H:0.4;L:0.1);AVT919(T:1. 5;Q:2.2;TA:1.1;GAS:2.3;H:1.5;L:0.2);AVT920(T:0.9;Q:1.0;TA:0.5;GAS:0.9;H:0.8;L:0.1);MyoAAV 1A(T:1.0;Q:1.1;TA:0.5;GAS:1.4;H:1.0;L:0.1);MyoAAV 2A(T:0.4;Q:0.7;TA:0.2;GAS:0.8;H:0.9;L:0.1);MyoAAV 4A(T:0.4;Q:0.6;TA:0.2;GAS:0.8;H:0.6;L:0.1);AAV9(T:0.021;Q:0.049;TA:0.0095;GAS:0.027;H:0.1;L:0.3). For each variant, the values ​​for the triceps (T), quadriceps (Q), tibialis anterior (TA), gastrocnemius (GAS), heart (H), and liver (L) are shown from left to right.Figure 9B: Comparison of in vivo transduction efficiency (multiplicative change relative to wild-type AAV9). Eight-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). Three weeks after injection (3wpi), total RNA was isolated from tissue and RT-qPCR was performed to quantify transgene expression.The pages of the story are the same. AVT90 AAV9 and AVT90 1(T:5.7;Q:5.2;TA:8.8;GAS:5.2;H:1.4;L:0.4);AVT90 2(T:2.4;Q:2.1;TA:3.9;GAS:2.5;H:3.2;L:0.5);AVT907(T:14.5;Q:10.8;TA:17.6;GAS:21.1;H:12.7;L:0.2); AVT908(T:14.9;Q:19.3;TA:17.7;GAS:39.9;H:2.3;L:2.7);AVT909(T:6.3;Q:3.2;TA:8.0;GAS:6.4;H:1.6;L: 0.4);AVT910(T:7.9;Q:6.2;TA:2.5;GAS:7.6;H:1.0;L:0.7);AVT913(T:61.8;Q:32.6;TA:75.2;GAS:83.7;H:26 .7;L:0.3);AVT914(T:48.6;Q:14.9;TA:45.0;GAS:21.4;H:8.5;L:0.6);AVT915(T:23.4;Q:16.5;TA:26.2;GAS :44.5;H:5.6;L:0.3);AVT916(T:33.8;Q:14.4;TA:28.5;GAS:29.9;H:7.1;L:0.3);AVT917(T:23.3;Q:11.9;TA: 36.3;GAS:31.1;H:6.9;L:0.2);AVT918(T:27.4;Q:11.2;TA:26.4;GAS:23.7;H:3.8;L:0.4);AVT919(T:71.6;Q: 44.8;TA:113.8;GAS:85.0;H:15.3;L:0.6);AVT920(T:42.5;Q:20.3;TA:54.7;GAS:32.4;H:8.4;L:0.2);MyoAAV 1A(T:47.4;Q:22.1;TA:51.9;GAS:49.7;H:10.0;L:0.4);MyoAAV 2A(T:20.6;Q:15.3;TA:20.3;GAS:28.9;H:9.2;L:0.2);MyoAAV; 4A(T:19.5;Q:12.9;TA:23.0;GAS:29.3;H:6.7;L:0.4);AAV9(T:1.0;Q:1.0;TA:1.0;GAS:1.0;H:1.0;L:1.0)For each mutant, the values ​​for the triceps (T), quadriceps (Q), tibialis anterior (TA), gastrocnemius (GAS), heart (H), and liver (L) are shown from left to right. Figure 9C: In vivo transduction efficiency of the top three mutants shown in Figure 9B. Figures 9D-9I: Multiples of transgene expression levels for each mutant relative to wild-type AAV9 in the triceps (Figure 9D), quadriceps (Figure 9E), tibialis anterior (Figure 9F), gastrocnemius (Figure 9G), heart (Figure 9H), and liver (Figure 9I). 8-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). Three weeks after injection (3wpi), total RNA was isolated from tissue and RT-qPCR was performed to quantify transgene expression. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA, Tukey multiple comparison test. Mutant AVT919 showed superior skeletal muscle transduction. Mutant AVT913 showed superior cardiac transduction. Mutant AVT908 showed improved transduction in both liver and muscle compared to wild-type AAV9. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 9H] Same as above. [Figure 9I] Same as above. 【0216】 [Figure 10]AAV vector genomic copies in mouse tissue. 8-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). Three weeks after injection (3wpi), genomic DNA was isolated from tissue and performed ddPCR. Mouse TFRC and luciferase primers / probes were used. Quads: quadriceps; TA: tibialis anterior; Gas: gastrocnemius. MyoAAV 1A, MyoAAV 2A, MyoAAV 4A, and wild-type AAV9 were used as controls. The multiples of each mutant's AAV vector genome copy relative to wild-type AAV9 in the test tissue were as follows: triceps (AVT908:6.8;AVT913:7.6;AVT919:22.1;MyoAAV2A:5.1;AAV9:1.0); quadriceps (AVT908:4.8;AVT913:2.3;AVT919:10.7;MyoAAV2A:2.6;AAV9:1.0); tibialis anterior (AVT908:6.6;AVT913:8.0;AVT919:22.4;My oAAV2A:4.4;AAV9:1.0);Gastrocnemius muscle (AVT908:9.5;AVT913:9.4;AVT919:13.7;MyoAAV2A:5.7;AAV9:1.0);Heart (AVT908:3.1;AVT913:11.4;AVT919:13.6;MyoAAV2A:7.1;AAV9:1.0);Liver (AVT908:4.7;AVT913:0.1;AVT919:1.0;MyoAAV2A:0.2;AAV9:1.0). 【0217】 [Figure 11] AAV-mediated GFP expression in mice. 8-week-old BALB / c mice were intravenously injected with AAV containing the CB-Fluc-2A-GFP transgene (4e11vg / mouse). AAV9 and MyoAAV 2A were used as controls. Three weeks after injection, gastrocnemius (Gas), quadriceps (Quads), and heart tissues were collected, and native GFP imaging was performed under the same conditions. 【0218】 [Figure 12]AAV-mediated transgene expression in NHP muscle. Adult male cynomolgus monkeys were intravenously injected with pooled AAVs (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Each capsid contained a cyno-FXN coding sequence with different tags (Flag, AU1, V5, HA) under the control of a chicken β-actin (CB) promoter and CMV enhancer. Three weeks post-injection, major muscle tissue was collected and RT-qPCR was performed to compare transgene mRNA levels. The macaque GAPDH housekeeping gene was used as a reference. Data are presented as mean values ​​with SD. 【0219】 [Figure 13] AAV-mediated transgene expression in the liver of NHP. Four pooled AAV capsids containing cyno-FXN coding sequences with different tags (Flag, AU1, V5, HA) were intravenously injected into adult cynomolgus monkeys under the control of a chicken β-actin (CB) promoter / CMV enhancer. Three weeks post-injection, RT-qPCR was performed on different liver lobes to compare transgene mRNA levels. Tag-specific primers / probes were used for transgene transcripts. The macaque GAPDH housekeeping gene was used as a reference. Data are presented as mean values ​​with SD. 【0220】 [Figure 14] Low expression of AAV-mediated transgenes in the CNS of NHP. Transgene-specific RT-qPCR was performed for administration of adult cynomolgus monkey brain and spinal cord samples with four pooled AAV capsids, each containing a cyno-FXN coding sequence with different tags (Flag, AU1, V5, HA) under the control of a chicken β-actin (CB) promoter / CMV enhancer. The macaque GAPDH housekeeping gene was used as the standard for normalization. Data are presented as mean values ​​with standard deviation. 【0221】 [Figure 15] Ploidy levels at the mRNA level of transgenes expressed by AAV variants relative to AAV9. Adult male cynomolgus monkeys were intravenously injected with four pooled AAVs (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks post-injection, major skeletal muscle tissue was collected and RT-qPCR was performed. Transgene-specific primers / probes and macaque GAPDH primers / probes were used in the duplex PCR reaction. The relative transgene expression level from AAV9 was set to 1.0. Data are presented as mean values ​​with SD. 【0222】 [Figure 16] Plicative changes in mRNA levels of AAV9-mediated transgenes in the heart, liver, and CNS of NHP (Nutrient-Heavy Hypovolcanic Monkeys). Adult male cynomolgus monkeys were intravenously injected with four pooled AAVs (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks post-injection, the heart, liver, brain, and spinal cord were collected and RT-qPCR was performed. Transgene-specific primers / probes and macaque GAPDH primers / probes were used in the duplex PCR reaction. The relative transgene expression level from AAV9 was set to 1.0. Data are presented as mean values ​​with SD (Stable Definition). 【0223】 [Figure 17] Vector genome copies in NHP skeletal muscle after intravenous administration of AAV variants. Adult male cynomolgus monkeys were intravenously injected with four pooled AAV variants (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks after injection, major skeletal muscle tissue was collected and genomic DNA was extracted. Transgene-specific primers / probes and RNaseP primers / probes were used in ddPCR reactions. Data are presented as mean values ​​with SD. 【0224】 [Figure 18]Vector genome copies in NHP muscle after intravenous administration of AAV variants. Adult male cynomolgus monkeys were intravenously injected with four pooled AAV variants (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks after injection, major muscle tissue was collected and genomic DNA was extracted. Transgene-specific primers / probes and RNaseP primers / probes were used in ddPCR reactions. Data are presented as mean values ​​with SD. 【0225】 [Figure 19] Vector genome copies in non-muscle tissue of NHP after intravenous administration of AAV variants. Adult male cynomolgus monkeys were intravenously injected with four pooled AAV variants (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks after injection, major muscle tissue was collected and genomic DNA was extracted. Transgene-specific primers / probes and RNaseP primers / probes were used in ddPCR reactions. Data are presented as mean values ​​with SD. 【0226】 [Figure 20] Vector genome copies in the liver, pancreas, and CNS of NHP after intravenous administration of AAV variants. Adult male cynomolgus monkeys were intravenously injected with four pooled AAV variants (AVT917, AVT919, AAV9, and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Three weeks after injection, major muscle tissue was collected and genomic DNA was extracted. Transgene-specific primers / probes and RNaseP primers / probes were used in ddPCR reactions. Data are presented as mean values ​​with SD. 【0227】 [Figure 21]mRNA expression of AAV9 and mutant AVT919-mediated transgenes in NHP. Adult female cynomolgus monkeys were intravenously injected with AAV9 or AVT919 containing an HA-tagged cynoFXN coding sequence at a dose of 2E13vg / kg under the control of a chicken β-actin (CB) promoter / CMV enhancer. Four weeks post-injection, major tissues were collected and performed RT-qPCR. Transgene-specific primers / probes and macaque GAPDH primers / probes were used in the duplex PCR reaction. Data are presented as mean values ​​with standard deviation. 【0228】 [Figure 22] Particle changes in mRNA levels of AAV9-mediated transgenes in NHP. Adult female cynomolgus monkeys were intravenously injected with AAV9 or AVT919 containing an HA-tagged cynoFXN coding sequence at a dose of 2E13vg / kg under the control of a chicken β-actin (CB) promoter / CMV enhancer. Four weeks post-injection, major tissues were collected and RT-qPCR was performed. Transgene-specific primers / probes and macaque GAPDH primers / probes were used in the duplex PCR reaction. The relative transgene expression level from AAV9 was set to 1.0. Data are presented as mean values ​​with SD. 【0229】 [Figure 23] Immunostaining of AAV9 and mutant AVT919-mediated transgene expression in NHP. Adult female cynomolgus monkeys were intravenously injected with AAV9 or AVT919 containing an HA-tagged cynoFXN coding sequence at a dose of 2E13vg / kg under the control of a chicken β-actin (CB) promoter / CMV enhancer. Major tissues were collected four weeks post-injection and performed RT-qPCR. Rabbit anti-HA monoclonal antibody was used as the primary antibody. Anti-rabbit HRP conjugate antibody and DAB substrate were used for detection. [Modes for carrying out the invention] 【0230】 This disclosure provides mutant adeno-associated virus serotype 9 (AAV9) capsid proteins and recombinant AAV9 particles containing mutant AAV9 capsid proteins. This disclosure discloses various mutant AAV9 capsid proteins and recombinant AAV9 particles containing these mutant AAV9 capsid proteins that have enhanced tropism to certain tissues compared to their wild-type counterparts. For example, some mutant AAV9 capsid proteins and recombinant AAV9 particles described herein have enhanced tropism to muscle compared to their wild-type counterparts. Some mutant AAV9 capsid proteins and recombinant AAV9 particles described herein have enhanced tropism to liver compared to their wild-type counterparts. Some mutant AAV9 capsid proteins and recombinant AAV9 particles described herein have enhanced tropism to brain compared to their wild-type counterparts. 【0231】 This disclosure further provides recombinant AAV9 particles, polynucleotides encoding mutant AAV9 capsid proteins, vectors and host cells containing polynucleotides, pharmaceutical compositions comprising cell populations transduced by recombinant AAV9 particles, and various methods for using recombinant AAV9 particles. 【0232】 definition 【0233】 As used in this disclosure, the singular forms “1,” “one,” and “the” 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. 【0234】 The terms "or" and "and" may be used interchangeably and may be understood to mean "and / or" unless the context explicitly specifies otherwise. 【0235】 As used in this disclosure, unless otherwise specified, the terms “about” and “approximately” should be interpreted as allowing for normal variation that a person skilled in the art can determine, e.g., variation of 20%, 10%, or 5% of the aforementioned value. In specific embodiments, the terms “about” and “approximately” include the exact value. All numerical values ​​provided herein are modified by the term “about” unless the context specifically indicates otherwise. 【0236】 Where the term “including” is used to describe any part of each aspect and embodiment in this disclosure, it is understood that other similar aspects described with the term “consisting of ○○” and / or “essentially consisting of ○○” are also provided. 【0237】 As used herein, the term “therapeutic dose” means an amount of biomolecules sufficient to reduce and / or improve the severity and / or duration of a given disease or disorder and / or symptoms associated therewith. The term further includes amounts necessary to reduce, delay or improve the progression or worsening of a given disease or disorder, to reduce, delay or improve the recurrence, progression or onset of a given disease or disorder, and / or to improve or enhance the preventive or therapeutic effect of another therapy. 【0238】 As used herein, the terms “subject” and “patient” may be used interchangeably. As used herein, a subject is a mammal, e.g., a non-primate (e.g., a cow, a pig, a horse, a cat, a dog, a goat, a rabbit, a rat, a mouse, etc.) or a primate (e.g., a monkey and a human), e.g., a human. In one embodiment, the subject is a mammal diagnosed with a disease or disorder, e.g., a human. In another embodiment, the subject is a mammal at risk of progression of the disease or disorder, e.g., a human. In yet another embodiment, the subject is a non-human primate. 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. 【0239】 Novel AAV9 capsid protein 【0240】 In one embodiment, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166 or SEQ ID A mutant adeno-associated virus serotype 9 (AAV9) capsid protein containing the amino acid sequence NO:168 is provided. 【0241】 A mutant AAV9 capsid protein is an AAV9 capsid protein whose amino acid sequence differs from that of the wild-type AAV9 VP1 capsid protein (shown in SEQ ID NO: 2), and also differs from the amino acid sequences of the VP2 and VP3 counterparts of AAV9 VP1 shown in SEQ ID NO: 2 (the amino acid sequences of the AAV9 VP2 and VP3 counterparts are shown in SEQ ID NO: 178 and 179, respectively). In a preferred embodiment, the mutant AAV9 capsid protein described herein is an AAV9 capsid protein that does not exist in nature. 【0242】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:37. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:6. 【0243】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:39. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:7. 【0244】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:41. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:8. 【0245】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 43. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 9. 【0246】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 45. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 10. 【0247】 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: 49. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 47 and also includes the amino acid sequence of SEQ ID NO: 49. In specific embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 11. 【0248】 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: 12. 【0249】 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: 13. 【0250】 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: 14. 【0251】 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: 15. 【0252】 In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 59. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 59 and also includes the amino acid sequence of SEQ ID NO: 61. In specific embodiments, the mutant AAV9 capsid protein provided herein includes the amino acid sequence of SEQ ID NO: 16. 【0253】 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: 17. 【0254】 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: 18. 【0255】 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: 19. 【0256】 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: 20. 【0257】 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:21. 【0258】 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:22. 【0259】 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:23. 【0260】 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:24. 【0261】 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:25. 【0262】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:80. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:26. 【0263】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:81. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:27. 【0264】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:82. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:28. 【0265】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:83. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:29. 【0266】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:84. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:30. 【0267】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:85. In a specific embodiment, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO:31. 【0268】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:86. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:32. 【0269】 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:33. 【0270】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:116. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:115. 【0271】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:118. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:117. 【0272】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 120. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 119. 【0273】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:122. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:121. 【0274】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:124. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:123. 【0275】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:126. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:125. 【0276】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:128. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:127. 【0277】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 130. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 129. 【0278】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:132. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:131. 【0279】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:134. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:133. 【0280】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:136. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:135. 【0281】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:138. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:137. 【0282】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 140. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 139. 【0283】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:142. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:141. 【0284】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:144. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:143. 【0285】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:146. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:145. 【0286】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 148. In specific embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 147. 【0287】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 150. In specific embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 149. 【0288】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 152. In specific embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 151. 【0289】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 154. In specific embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 153. 【0290】 In certain embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 156. In specific embodiments, the mutant AAV9 capsid protein provided herein comprises the amino acid sequence of SEQ ID NO: 155. 【0291】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:158. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:157. 【0292】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 160. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO: 159. 【0293】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:162. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:161. 【0294】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:164. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:163. 【0295】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:166. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:165. 【0296】 In certain embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:168. In specific embodiments, the mutant AAV9 capsid protein provided herein contains the amino acid sequence of SEQ ID NO:167. 【0297】 The AAV capsid typically consists of three viral proteins, VP1, VP2, and VP3, which are alternative splice variants. Typically, the three proteins are encoded by the same nucleotide sequence; the VP1 amino acid sequence contains the entire amino acid sequence of VP2, and the VP2 amino acid sequence contains the entire amino acid sequence of VP3. 【0298】 Generally, AAV capsid proteins contain multiple variable regions (VRs) and constant regions located between them. The "GH loop" is a loop sequence in which β-strands G and β-strand H are flanked within the internal β-barrel of the AAV capsid protein, and it includes variable regions VR IV to VR VIII. 【0299】 Those skilled in the art can easily and readily determine the sequences of the VP2 and VP3 capsid proteins and the positions of various domains (e.g., the variable region and the GH loop) from a given VP1 sequence by employing appropriate methods known in the art, such as comparing or aligning the VP1 sequence with an annotated VP1 sequence (preferably an annotated VP1 sequence from a closely related AAV species) using an appropriate algorithm. Preferably, such annotated VP1 sequence is an annotated wild-type AAV VP1 sequence (e.g., an annotated wild-type AAV9 VP1 sequence such as the amino acid sequence shown in SEQ ID NO:2). 【0300】 In various embodiments, the mutant AAV9 capsid protein described herein is the mutant AAV9 VP1 capsid protein. Further mutant AAV9 capsid proteins are provided that are the VP2 and VP3 counterparts of the VP1 capsid protein described herein. Fragments of such mutant AAV9 capsid proteins (e.g., fragments of the VP1, VP2, or VP3 capsid proteins) are further provided, 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 biological activity and associated tissue-specific properties of the corresponding capsid proteins. 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. 【0301】 In various embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced tropism to one or more tissues compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). The association of mutant AAV9 capsid proteins with enhanced tropism to tissues should be interpreted as meaning that recombinant AAV9 particles containing the mutant AAV9 capsid protein exhibit enhanced tropism to those tissues. 【0302】 In certain embodiments, the mutant AAV9 capsid protein described herein is associated with 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 and / or ventricular muscle)) compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the myocardium (e.g., atrial and / or ventricular muscle) compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major, atrial and / or ventricular muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major muscles compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the biceps compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2).In specific embodiments, the mutant AAV9 capsid protein described herein is associated with improved tropism to the triceps muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with improved tropism to the quadriceps muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with improved tropism to the tibialis anterior muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the gastrocnemius muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the rectus abdominis muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the diaphragm compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the pectoralis major muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the atrial muscle compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2).In specific embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced ventricular myotropism compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0303】 In certain embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced tropism to the liver compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0304】 In certain embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the brain (e.g., middle front brain, middle rear brain, front brain, and / or rear brain) compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the middle front brain compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced tropism to the middle rear brain compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced forebrain tropism compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the mutant AAV9 capsid protein described herein is associated with enhanced hindbrain tropism compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0305】 In certain embodiments, the variant AAV9 capsid proteins described herein are associated with enhanced tropism to both 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)) and liver compared to wild-type AAV9 capsid protein (preferably wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0306】 In certain embodiments, the variant AAV9 capsid proteins described herein are associated with enhanced tropism to both muscles (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)) and the brain (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain) compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0307】 In certain embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced tropism to both the liver and the brain (e.g., the mesoforebrain, mesopostratum, forebrain, and / or hindbrain) compared to the wild-type AAV9 capsid protein (preferably the wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0308】 In certain embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced tropism to the kidney compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0309】 In certain embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced pulmonary tropism compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0310】 In certain embodiments, the mutant AAV9 capsid proteins described herein are associated with enhanced spleen tropism compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). 【0311】 In various embodiments, the variant AAV9 capsid proteins described herein are associated with enhanced tropism to one or more tissues of the MyoAAV 1A capsid protein (preferably MyoAAV 1A capsid protein containing the amino acid sequence of SEQ ID NO:3), MyoAAV 2A capsid protein (preferably MyoAAV 2A capsid protein containing the amino acid sequence of SEQ ID NO:4), MyoAAV 3A capsid protein (preferably MyoAAV 3A capsid protein containing the amino acid sequence of SEQ ID NO:111), and / or MyoAAV 4A capsid protein (preferably MyoAAV 4A capsid protein containing the amino acid sequence of SEQ ID NO:5). In particular embodiments, the one or more tissues are or include muscle (e.g., skeletal muscle and / or cardiac muscle). In a specific embodiment, the one or more tissues are or include skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major). In a specific embodiment, the one or more tissues are or include cardiac muscle (e.g., atrial muscle and / or ventricular muscle). In a particular embodiment, the one or more tissues are or include the liver. In a particular embodiment, the one or more tissues are or include the brain (e.g., mesoforebrain, mesoposterior brain, forebrain and / or hindbrain). In a particular embodiment, the one or more tissues are 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)) and the liver. In a particular embodiment, the one or more tissues are or include 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 muscle and / or ventricular muscle)) and the brain (e.g., mesoforebrain, mesoposterior brain, forebrain and / or hindbrain). In a particular embodiment, the one or more tissues are or include the liver and the brain (e.g., mesoforebrain, mesoposterior brain, forebrain and / or hindbrain).In certain embodiments, the one or more tissues are or include a kidney. In certain embodiments, the one or more tissues are or include a lung. In certain embodiments, the one or more tissues are or include a spleen. 【0312】 In various embodiments, the mutant AAV9 capsid proteins described herein are associated with reduced tropism to one or more tissues compared to wild-type AAV9 capsid proteins (preferably wild-type AAV9 capsid proteins containing the amino acid sequence of SEQ ID NO:2). The association of mutant AAV9 capsid proteins with reduced tropism to tissues should be interpreted as meaning that recombinant AAV9 particles containing the mutant AAV9 capsid protein have reduced tropism to said tissues. In particular embodiments, the one or more tissues are or include 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)). In particular embodiments, the one or more tissues are or include the liver. In certain embodiments, the one or more tissues are or include the brain (e.g., the mesoforebrain, mesoposterior brain, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include the kidney. In certain embodiments, the one or more tissues are or include the lung. In certain embodiments, the one or more tissues are or include the spleen. 【0313】 In various embodiments, the variant AAV9 capsid proteins described herein are associated with reduced tropism in one or more of the MyoAAV 1A capsid proteins (preferably MyoAAV 1A capsid proteins containing the amino acid sequence of SEQ ID NO:3), MyoAAV 2A capsid proteins (preferably MyoAAV 2A capsid proteins containing the amino acid sequence of SEQ ID NO:4), MyoAAV 3A capsid proteins (preferably MyoAAV 3A capsid proteins containing the amino acid sequence of SEQ ID NO:111), and / or MyoAAV 4A capsid proteins (preferably MyoAAV 4A capsid proteins containing the amino acid sequence of SEQ ID NO:5). In certain embodiments, the one or more tissues are or include 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)). In certain embodiments, the one or more tissues are or include the liver. In certain embodiments, the one or more tissues are or include the brain (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include the kidney. In certain embodiments, the one or more tissues are or include the lung. In certain embodiments, the one or more tissues are or include the spleen. 【0314】 In various embodiments, the variant AAV9 capsid protein described herein is associated with enhanced tropism to one or more tissues as described above, and with reduced tropism to one or more other tissues as described above. 【0315】 In various embodiments, the tropism to the tissues described herein is measured after systemic administration of the AAV9 particles corresponding to the subject. In specific embodiments, the tropism to the tissues described herein is measured after intravenous administration of the AAV9 particles corresponding to the subject. 【0316】 In certain embodiments, the mutant AAV9 capsid protein described herein is isolated. In certain embodiments, the mutant AAV9 capsid protein described herein is purified. 【0317】 Recombinant AAV9 particles 【0318】 In another embodiment, recombinant AAV9 particles are provided that contain the mutant AAV9 capsid protein described herein (e.g., the mutant AAV9 capsid protein described in Section 5.2). Further recombinant AAV9 particles are provided that contain a fragment of the mutant AAV9 capsid protein described herein (e.g., the fragment described in Section 5.2). 【0319】 An "AAV particle" refers to an AAV virus consisting of at least one AAV capsid protein and a packaged AAV genome. Therefore, an AAV9 particle is an AAV serotype 9 virus. 【0320】 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 cis-acting origins for DNA replication and as packaging signals for the viral genome. 【0321】 In various embodiments, the recombinant AAV9 particles described herein comprise the mutant AAV9 capsid protein described herein and a recombinant AAV genome containing a heterologous nucleotide sequence flanked with an AAV ITR (e.g., AAV9 ITR or AAV2 ITR), wherein the heterologous nucleotide sequence is heterologous to the AAV ITR. In a preferred embodiment, the recombinant AAV genome does not contain a functional AAV cap gene. In a preferred embodiment, the recombinant AAV genome does not contain a functional AAV rep gene. In a preferred embodiment, the recombinant AAV genome does not contain a functional AAV cap gene and does not contain a functional AAV rep gene. 【0322】 In certain embodiments, the heterologous nucleotide sequence includes a nucleotide sequence encoding a biomolecule. The biomolecule may, but is not limited to, a polypeptide, a protein, a nucleic acid (e.g., DNA or RNA), or an oligonucleotide (e.g., siRNA, shRNA, miRNA, or aptamer). 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 molecule such as a therapeutic protein. The therapeutic molecule can be used to correct or improve a gene deficiency associated with a 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, and interleukins. In a specific embodiment, the biomolecule is expressed in muscle cells (e.g., expressed at a higher level in muscle cells than in other cells). In a specific embodiment, the biomolecule is , expressed in hepatocytes (for example, expressed at a higher level in hepatocytes than in other cells). In a specific embodiment, the biomolecule is expressed in brain cells (for example, expressed at a higher level in brain cells than in other cells). In a specific embodiment, the biomolecule is expressed in both muscle cells and hepatocytes (for example, expressed at a higher level in both muscle cells and hepatocytes than in other cells). In a specific embodiment, the biomolecule is expressed in both muscle cells and brain cells (for example, expressed at a higher level in both muscle cells and brain cells than in other cells). In a specific embodiment, the biomolecule is expressed in both hepatocytes and brain cells (for example, expressed at a higher level in both hepatocytes and brain cells than in other cells).In specific embodiments, the biomolecule is expressed in kidney cells (for example, at a higher level in kidney cells than in other cells). In specific embodiments, the biomolecule is expressed in lung cells (for example, at a higher level in lung cells than in other cells). In specific embodiments, the biomolecule is expressed in spleen cells (for example, at a higher level in spleen cells than in other cells). 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 hepatocytes (for example, necessary or important for the normal function of hepatocytes). In specific embodiments, the biomolecule acts in brain cells (for example, necessary or important for the normal function of brain cells). In specific embodiments, the biomolecule acts in both muscle cells and hepatocytes (for example, necessary or important for the normal function of both muscle cells and hepatocytes). In specific embodiments, the biomolecule acts in both muscle cells and brain cells (for example, necessary or important for the normal function of both muscle cells and brain cells). In specific embodiments, the biomolecule acts in both hepatocytes and brain cells (for example, necessary or important for the normal function of both hepatocytes and brain cells). In specific embodiments, the biomolecule acts in renal cells (for example, necessary or important for the normal function of renal cells). In specific embodiments, the biomolecule acts in lung cells (for example, necessary or important for the normal function of lung cells). In specific embodiments, the biomolecule acts in spleen cells (for example, necessary or important for the normal function of spleen cells). In specific embodiments, the muscle cells described herein are skeletal muscle cells (for example, biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragmatic and / or pectoralis major muscle cells) and / or cardiomyocytes (for example, atrial and / or ventricular muscle cells). In specific embodiments, the brain cells described herein are mesoforebrain, mesoposterior, forebrain and / or posterior brain cells.In a specific embodiment, the biomolecule is functional motor neuron (SMN) protein (e.g., wild-type SMN protein). In a specific embodiment, the biomolecule is functional microdystrophin (e.g., wild-type microdystrophin). In a specific embodiment, the biomolecule is functional α-galactosidase (e.g., wild-type α-galactosidase). In a specific embodiment, the biomolecule is functional phenylalanine hydroxylase (PAH) (e.g., wild-type PAH). In a specific embodiment, the biomolecule is functional coagulation factor VIII (FVIII) (e.g., wild-type FVIII). In a specific embodiment, the biomolecule is functional coagulation factor IX (FIX) (e.g., wild-type FIX). In a specific embodiment, the biomolecule is functional β-glucocerebrosidase (GBA) (e.g., wild-type GBA). In a specific embodiment, the biomolecule is NPC intracellular cholesterol transporter 1 (NPC1) (e.g., wild-type NPC1). In a specific embodiment, the biomolecule is NPC intracellular cholesterol transporter 2 (NPC2) (e.g., wild-type NPC2). In a specific embodiment, the biomolecule is acid α-glucosidase (GAA) (e.g., wild-type GAA). 【0323】 In certain embodiments, the heterologous nucleotide sequence further comprises one or more nucleotide sequences encoding one or more regulatory elements, the one or more nucleotide sequences encoding one or more regulatory elements being operably ligated to a nucleotide sequence encoding a biomolecule. In this specification, “operably ligated” means ligating a nucleotide sequence in a cell transduced by the AAV virus such that the biomolecule is transcribed, translated, and / or expressed, the cell comprising a recombinant AAV genome containing the heterologous nucleotide sequence. 【0324】 The regulatory element includes an expression regulatory element that is continuous with a nucleotide sequence encoding a biomolecule of interest, and an expression regulatory element that acts trans or at a distance to control the expression of the biomolecule of interest. 【0325】 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 covalent sequences); sequences that enhance protein stability; secretory signals; and nuclear localization sequences. 【0326】 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 encoding a nucleotide sequence of a biomolecule. In a particular embodiment, the promoter is a tissue-specific promoter. In a preferred embodiment, the tissue-specific promoter is a muscle-specific promoter. In a specific embodiment, the tissue-specific promoter is a skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major)-specific promoter. In a specific embodiment, the tissue-specific promoter is a cardiac muscle (e.g., atrial muscle and / or ventricular muscle)-specific promoter. In a specific embodiment, the tissue-specific promoter is a liver-specific promoter. In a specific embodiment, the tissue-specific promoter is a brain (e.g., mesoforebrain, mesopostracheal, forebrain, and / or hindbrain)-specific promoter. In a specific embodiment, the tissue-specific promoter is a kidney-specific promoter. In specific embodiments, the tissue-specific promoter is a lung-specific promoter. In specific embodiments, the tissue-specific promoter is a spleen-specific promoter. Non-limiting exemplary muscle-specific promoters include promoters from genes encoding skeletal β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, and synthetic muscle promoters that are more active than naturally occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). 【0327】 In various embodiments, the heterogeneous nucleotide sequence includes an expression cassette comprising a nucleotide sequence encoding a biomolecule described herein and one or more nucleotide sequences encoding one or more regulatory elements described herein that are operably linked thereto (e.g., a nucleotide sequence encoding a promoter and a nucleotide sequence encoding a polyA signal). 【0328】 In certain embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to one or more tissues compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0329】 In certain embodiments, the recombinant AAV9 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 and / or ventricular muscle)) compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein have improved tropism to the myocardium (e.g., atrial and / or ventricular muscle) compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein have improved tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major, atrial and / or ventricular muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein have improved tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, and / or pectoralis major muscles compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2).In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the triceps muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the quadriceps muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the tibialis anterior muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the gastrocnemius muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the rectus abdominis muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the diaphragm compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the pectoralis major muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced tropism to the atrial muscle compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2).In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced ventricular myocardial tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0330】 In certain embodiments, the recombinant AAV9 particles described herein exhibit enhanced hepatic tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0331】 In certain embodiments, the recombinant AAV9 particles described herein have enhanced tropism to the brain (e.g., mesoforebrain, mesopostracheal, forebrain and / or hindbrain) compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein have enhanced tropism to the mesoforebrain compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein have enhanced tropism to the mesopostracheal compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced forebrain tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In specific embodiments, the recombinant AAV9 particles described herein exhibit enhanced hindbrain tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0332】 In certain embodiments, the recombinant AAV9 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 and / or ventricular muscle)) and liver compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0333】 In certain embodiments, the recombinant AAV9 particles described herein have enhanced tropism to both muscles (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)) and brain (e.g., mesoforebrain, mesoposterior, forebrain, and / or hindbrain) compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0334】 In certain embodiments, the recombinant AAV9 particles described herein have enhanced tropism to both the liver and the brain (e.g., mesoforebrain, mesopostratum, forebrain and / or hindbrain) compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0335】 In certain embodiments, the recombinant AAV9 particles described herein exhibit improved renal tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0336】 In certain embodiments, the recombinant AAV9 particles described herein exhibit enhanced pulmonary tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0337】 In certain embodiments, the recombinant AAV9 particles described herein exhibit enhanced spleen tropism compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). 【0338】 In various embodiments, the recombinant AAV9 particles described herein have enhanced tropism to one or more tissues with respect to MyoAAV 1A (preferably MyoAAV 1A having a capsid protein containing the amino acid sequence of SEQ ID NO:3), MyoAAV 2A (preferably MyoAAV 2A having a capsid protein containing the amino acid sequence of SEQ ID NO:4), MyoAAV 3A (preferably MyoAAV 3A having a capsid protein containing the amino acid sequence of SEQ ID NO:111), and / or MyoAAV 4A (preferably MyoAAV 4A having a capsid protein containing the amino acid sequence of SEQ ID NO:5). In particular embodiments, the one or more tissues are or include muscle (e.g., skeletal muscle and / or cardiac muscle). In a specific embodiment, the one or more tissues are or include skeletal muscle (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major). In a specific embodiment, the one or more tissues are or include cardiac muscle (e.g., atrial muscle and / or ventricular muscle). In a particular embodiment, the one or more tissues are or include the liver. In a particular embodiment, the one or more tissues are or include the brain (e.g., mesoforebrain, mesoposterior brain, forebrain and / or hindbrain). In a particular embodiment, the one or more tissues are 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)) and the liver. In certain embodiments, the one or more tissues are or include 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 muscle and / or ventricular muscle)) and brains (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include livers and brains (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include kidneys.In certain embodiments, the one or more tissues are the lungs or include them. In certain embodiments, the one or more tissues are the spleen or include them. 【0339】 In various embodiments, the recombinant AAV9 particles described herein have reduced tropism to one or more tissues compared to wild-type AAV9 particles (preferably wild-type AAV9 particles having a capsid protein containing the amino acid sequence of SEQ ID NO:2). In certain embodiments, the one or more tissues are or include 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)). In certain embodiments, the one or more tissues are or include the liver. In certain embodiments, the one or more tissues are or include the brain (e.g., mesoforebrain, mesopostracheal, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include the kidney. In certain embodiments, the one or more tissues are or include the lung. In certain embodiments, the one or more tissues are the spleen or include it. 【0340】 In various embodiments, the recombinant AAV9 particles described herein exhibit reduced tropism to one or more tissues with respect to MyoAAV 1A (preferably MyoAAV 1A having a capsid protein containing the amino acid sequence of SEQ ID NO:3), MyoAAV 2A (preferably MyoAAV 2A having a capsid protein containing the amino acid sequence of SEQ ID NO:4), MyoAAV 3A (preferably MyoAAV 3A having a capsid protein containing the amino acid sequence of SEQ ID NO:111), and / or MyoAAV 4A (preferably MyoAAV 4A having a capsid protein containing the amino acid sequence of SEQ ID NO:5). In certain embodiments, the one or more tissues are or include 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)). In certain embodiments, the one or more tissues are or include the liver. In certain embodiments, the one or more tissues are or include the brain (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain). In certain embodiments, the one or more tissues are or include the kidney. In certain embodiments, the one or more tissues are or include the lung. In certain embodiments, the one or more tissues are or include the spleen. 【0341】 In various embodiments, the recombinant AAV9 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. 【0342】 In various embodiments, recombinant AAV9 particles are administered systemically to a subject, and then tropism to the tissues described herein is measured. In specific embodiments, recombinant AAV9 particles are administered intravenously to a subject, and then tropism to the tissues described herein is measured. 【0343】 In preferred embodiments, the recombinant AAV9 particles described herein are AAV9 particles that do not exist in nature. In specific embodiments, the recombinant AAV9 particles described herein are isolated. In specific embodiments, the recombinant AAV9 particles described herein are purified. 【0344】 Polynucleotides, vectors, and cells 【0345】 In another embodiment, polynucleotides encoding the mutant AAV9 capsid protein described herein (e.g., the mutant AAV9 capsid protein described in Section 5.2) are provided. Further, polynucleotides encoding fragments of the mutant AAV9 capsid protein described herein (e.g., the fragments described in Section 5.2) are provided. 【0346】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:38. 【0347】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:40. 【0348】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:42. 【0349】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:44. 【0350】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:46. 【0351】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 48. In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 50. In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 48 and also includes the nucleotide sequence of SEQ ID NO: 50. 【0352】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:52. 【0353】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:54. 【0354】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:56. 【0355】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:58. 【0356】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 60. In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 62. In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein includes the nucleotide sequence of SEQ ID NO: 60 and also includes the nucleotide sequence of SEQ ID NO: 62. 【0357】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:64. 【0358】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:66. 【0359】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:68. 【0360】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:70. 【0361】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:72. 【0362】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:74. 【0363】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:76. 【0364】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:78. 【0365】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:100. 【0366】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence SEQ ID NO:101. 【0367】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:102. 【0368】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:103. 【0369】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:104. 【0370】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:105. 【0371】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence of SEQ ID NO:106. 【0372】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence SEQ ID NO:107. 【0373】 In certain embodiments, the polynucleotide encoding the mutant AAV9 capsid protein or fragment thereof described herein comprises the nucleotide sequence SEQ ID NO:108. 【0374】 In a particular embodiment, the polynucleotide described herein is a DNA polynucleotide. In a specific embodiment, the polynucleotide described herein is single-stranded DNA. In a specific embodiment, the polynucleotide described herein is double-stranded DNA. In a specific embodiment, the polynucleotide described herein is cDNA. In a particular embodiment, the polynucleotide described herein is an RNA polynucleotide. In a specific embodiment, the polynucleotide described herein is single-stranded RNA. In a specific embodiment, the polynucleotide described herein is double-stranded RNA. In a specific embodiment, the polynucleotide described herein is mRNA. 【0375】 In certain embodiments, the polynucleotides described herein are optimized by alternative or preferred codon usage for a particular type of host cell or delivery target cell. Codon optimization can be performed using any suitable method known in the art (e.g., suitable software known in the art). 【0376】 In another embodiment, a vector comprising a polynucleotide as described herein (e.g., plasmid, bacmid, cosmid, construct, etc.) encoding the variant AAV9 capsid protein or a fragment thereof as described herein is provided. 【0377】 In another embodiment, a host cell comprising a polynucleotide or vector as described herein is provided. In a particular embodiment, the host cell further comprises a recombinant AAV genome comprising a nucleotide sequence encoding a biomolecule as described in Section 5.3, or a vector encoding such a recombinant AAV genome (e.g., plasmid, bacmid, cosmid, construct, etc.). In a particular embodiment, the host cell further comprises a nucleotide sequence encoding an AAV rep gene, which is the same or a different polynucleotide or vector encoding a variant AAV9 capsid protein as described herein (e.g., plasmid, bacmid, cosmid, construct, etc.). In a particular embodiment, 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). 【0378】 In another embodiment, a host cell containing recombinant AAV9 particles as described herein is provided. 【0379】 In another embodiment, a host cell that produces recombinant AAV9 particles as described herein is provided. 【0380】 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. 【0381】 As used herein, the term “host” means a cell containing polynucleotides, vectors, AAV particles, or AAV genomes (e.g., cells derived from insects, animals (including humans and non-human animals), yeast, and bacteria, etc.). This disclosure is not limited to any particular type of host cell. In practice, any suitable cell is expected to be used as a host herein. Host cells may exist or be derived from single cells, groups, cultures (e.g., liquid cultures or cultures on solid substrates), cell lines, living organisms or parts thereof. 【0382】 Accordingly, the host cells of this disclosure may be, for example, bacterial cells, yeast cells, insect cells, or mammalian cells (e.g., human cells or non-human mammalian cells). Non-limiting exemplary insect cells that can be used as host cells include Ao38, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, and Hz2E5. Non-limiting exemplary mammalian cells that can be used as host cells include HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells. In certain embodiments, the host cells are human cells. In a specific embodiment, the human cells are autologous to the subject (e.g., a human patient) being treated with recombinant AAV9 particles produced by the human cells. In a specific embodiment, the human cells are allogeneic to the subject (e.g., a human patient) being treated with recombinant AAV9 particles produced by the human cells. 【0383】 The polynucleotides, vectors, and host cells described herein may be used to produce biomolecules encoded by the mutant AAV9 capsid protein, recombinant AAV9 particles, and / or recombinant AAV9 genome of the recombinant AAV9 particles described herein. 【0384】 In another embodiment, a population of host cells stably transduced by recombinant AAV9 particles described herein is provided. In a specific embodiment, the host cell population is an ex vivo host cell population. In a specific embodiment, the host cell population is an in vitro host cell population. In a specific embodiment, the host cell population is an in vivo host cell population. In a specific embodiment, the host cell population is a human host cell population. In a specific embodiment, the host cell population is an ex vivo human host cell population. In a specific embodiment, the host cell population is an in vitro human host cell population. 【0385】 In certain embodiments, such host cell populations may be used to generate recombinant AAV9 particle populations that are used as therapeutic agents and administered to subjects in need (e.g., human patients). 【0386】 In certain embodiments, such host cell populations may be used to generate a group of biomolecules encoded by the recombinant AAV genome described in Section 5.3, which are used as therapeutic agents and administered to the subject in need (e.g., a human patient). 【0387】 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 being 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 being treated by the population of host cells (e.g., a human patient). 【0388】 Production method 【0389】 In another embodiment, a method for producing recombinant AAV9 particles as described herein (e.g., recombinant AAV9 particles as described in Section 5.3) is provided. A method for producing polynucleotides as described herein (e.g., polynucleotides as described in Section 5.4), vectors as described herein (e.g., vectors as described in Section 5.4), host cells as described herein (e.g., host cells as described in Section 5.4), mutant AAV9 capsid proteins or fragments thereof as described herein (e.g., mutant AAV9 capsid proteins or fragments thereof as described in Section 5.2), or recombinant AAV genomes as described herein (e.g., recombinant AAV genomes as described in Section 5.3) is further described. 【0390】 The polynucleotides, vectors, host cells, mutant AAV9 capsid proteins and their fragments, recombinant AAV9 particles, and recombinant AAV genomes of the present invention 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. 【0391】 The generation of the polynucleotides, vectors, recombinant AAV genomes and / or mutant AAV9 capsid proteins or fragments thereof 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). 【0392】 The polynucleotides, vectors, recombinant AAV particles, or host cells for holding recombinant AAV genomes of this disclosure may be prepared by any suitable method, which includes, but is not limited to, transfection, electroporation, transduction, liposome delivery, membrane fusion techniques, fast DNA coated pellets, viral infection, and protoplast fusion. 【0393】 Recombinant AAV particles can be produced by host cells that allow the production and replication of AAV particles.Methods for producing recombinant AAV particles are well known in this field, 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, Alaboratory Manual, Oxford Univ. Press (1994); *Samulski et al., *J.Vir. 63:3822-8 (1989); *Kajigaya et al., *Proc. Nat'l. Acad. Sci. USA*. 88:4646-50 (1991); Ruffing et al., J.Vir.66:6922-30 (1992); Kimbauer et al., Vir.219:37-44 (1996); Zhao et al., Vir.272:382-93 (2000); US Patent Nos US5064764, US5756283, US6194191, US6204059, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6566118, US6943019, US6953690, US7022519, US This is described in 7238526, US7291498, US7491508 and US8137948; and in international patent application publication numbers WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353, WO2001023597, WO2015191508, WO2018022608, WO2019217513, WO2019222132, WO2019222136 and WO2020232044, the disclosures of which are incorporated herein by reference in their entirety. 【0394】 AAV viruses proliferate only in cells that generate helper functions for productive AAV infection. In certain embodiments, helper functions are provided by one or more helper plasmids and / or helper viruses (e.g., adenoviruses, baculoviruses, vaccinia viruses, herpesviruses, or papillomaviruses) containing helper genes. Those skilled in the art will understand that any helper virus and / or helper plasmid capable of providing sufficient helper functions for AAV may be used herein. Non-limiting examples of helper genes from adenoviruses or baculoviruses include, but are not limited to, E1A, E1B, E2A, E4, and VA. 【0395】 The AAV virus also requires the AAV cap gene product and the AAV rep gene product to produce productive AAV infection. In various embodiments, the AAV cap gene product is provided in trans. In other words, the AAV genome does not contain the AAV cap gene. In various embodiments, the AAV rep gene product is provided in trans. In other words, the AAV genome does not contain the AAV rep gene. In various embodiments, both the AAV cap gene product and the AAV rep gene product are provided in trans. In other words, the AAV genome does not contain the AAV cap gene and does not contain the AAV rep gene. In certain embodiments, the AAV cap gene is present in a vector (e.g., plasmid, bacmid, cosmid, construct, etc.), which is transfected into a host cell containing other essential components (e.g., a vector encoding a recombinant AAV genome (e.g., plasmid, bacmid, cosmid, construct, etc.)), and the recombinant AAV genome comprises a nucleotide sequence of interest encoding a biomolecule flanked by ITR, an AAV rep gene, and helper functions, thereby producing infectious AAV particles.In certain embodiments, the AAV cap gene and the AAV rep gene are present in one or two vectors (e.g., plasmids, bacmids, cosmids, constructs, etc.), which are transfected into a host cell containing other essential components (e.g., a recombinant AAV genome vector containing nucleotide sequences of interest encoding biomolecules flanked by ITRs (e.g., plasmids, bacmids, cosmids, constructs, etc.)) and helper functions, thereby producing infectious AAV particles. 【0396】 Pharmaceutical composition 【0397】 In another embodiment, a pharmaceutical composition is provided comprising recombinant AAV9 particles described herein (for example, recombinant AAV9 particles described in Section 5.3) and a pharmaceutically acceptable carrier. 【0398】 In another embodiment, a pharmaceutical composition is provided comprising a population of host cells described herein (for example, a population of host cells described herein that have been stably transduced by recombinant AAV9 particles described herein) and a pharmaceutically acceptable carrier. 【0399】 In certain embodiments, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1 × 10⁻⁶ 8 vg / ml to 1x10 20 The concentration may be within the range of vg / ml. In a specific embodiment, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1 × 10⁻⁶ 9 vg / ml to 1x10 19 The concentration may be within the range of vg / ml. In a specific embodiment, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1 × 10⁻⁶ 10 vg / ml to 1x10 18 The concentration may be within the range of vg / ml. In a specific embodiment, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1 × 10⁻⁶ 11 vg / ml to 1x10 17It may also be within the range of vg / ml. In a specific embodiment, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1×10 12 vg / ml to 1×10 16 vg / ml. In a specific embodiment, the concentration of recombinant AAV9 particles in the pharmaceutical composition described herein is 1×10 13 vg / ml to 1×10 15 vg / ml. 【0400】 In a particular embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1×10 2 cells / ml to ~1×10 12 cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1×10 3 cells / ml to 1×10 11 cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1×10 4 cells / ml to 1×10 10 cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1×10 5 cells / ml to 1×10 9 cells / ml. In a specific embodiment, the concentration of host cells in the pharmaceutical composition described herein is 1×10 6 cells / ml to 1×10 8 cells / ml. 【0401】 Generally, if a drug (e.g., an excipient or a carrier) is safe, non-toxic, and not undesirable from a biological or other perspective, the drug is pharmaceutically acceptable and acceptable for veterinary use and human pharmaceutical use. 【0402】 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. 【0403】 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. 【0404】 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 high-concentration drugs. 【0405】 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, intranasally, 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. 【0406】 Delivery and treatment methods 【0407】 In another embodiment, a method for delivering a biomolecule to one or more ex vivo target cells is provided, comprising transducing one or more target cells using recombinant AAV9 particles described herein (for example, recombinant AAV9 particles described in Section 5.3). 【0408】 In another embodiment, a method for delivering a biomolecule to one or more in vitro target cells is provided, comprising transducing one or more target cells using recombinant AAV9 particles described herein (for example, recombinant AAV9 particles described in Section 5.3). 【0409】 In another embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering recombinant AAV9 particles described herein (e.g., recombinant AAV9 particles described in Section 5.3) to the subject. 【0410】 In another embodiment, a method is provided for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering a pharmaceutical composition described herein (for example, a pharmaceutical composition described in Section 5.6) to the subject. 【0411】 The biomolecule may be, but is not limited to, a polypeptide, a protein, a nucleic acid (e.g., DNA or RNA), or an oligonucleotide (e.g., siRNA, shRNA, miRNA, or aptamer). In a specific embodiment, the biomolecule is a polypeptide or a protein. In a preferred embodiment, 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 molecule 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, and interleukins. In specific embodiments, the biomolecule is expressed in muscle cells (e.g., expressed at a higher level in muscle cells than in other cells). In specific embodiments, the biomolecule is expressed in hepatocytes (e.g., expressed at a higher level in hepatocytes than in other cells). (expressed at a higher level). In a specific embodiment, the biomolecule is expressed in brain cells (for example, expressed at a higher level in brain cells than in other cells). In a specific embodiment, the biomolecule is expressed in both muscle cells and hepatocytes (for example, expressed at a higher level in both muscle cells and hepatocytes than in other cells). In a specific embodiment, the biomolecule is expressed in both muscle cells and brain cells (for example, expressed at a higher level in both muscle cells and brain cells than in other cells). In a specific embodiment, the biomolecule is expressed in both hepatocytes and brain cells (for example, expressed at a higher level in both hepatocytes and brain cells than in other cells). In a specific embodiment, the biomolecule is expressed in kidney cells (for example, expressed at a higher level in kidney cells than in other cells).In a specific embodiment, the biomolecule is expressed in lung cells (for example, at a higher level in lung cells than in other cells). In a specific embodiment, the biomolecule is expressed in spleen cells (for example, at a higher level in spleen cells than in other cells). In a specific embodiment, the biomolecule acts in muscle cells (for example, necessary or important for the normal function of muscle cells). In a specific embodiment, the biomolecule acts in hepatocytes (for example, necessary or important for the normal function of hepatocytes). In a specific embodiment, the biomolecule acts in brain cells (for example, necessary or important for the normal function of brain cells). In a specific embodiment, the biomolecule acts in both muscle cells and hepatocytes (for example, necessary or important for the normal function of both muscle cells and hepatocytes). In a specific embodiment, the biomolecule acts in both muscle cells and brain cells (for example, necessary or important for the normal function of both muscle cells and brain cells). In a specific embodiment, the biomolecule acts in both hepatocytes and brain cells (for example, necessary or important for the normal function of both hepatocytes and brain cells). In specific embodiments, the biomolecule acts in renal cells (for example, necessary or important for the normal function of renal cells). In specific embodiments, the biomolecule acts in lung cells (for example, necessary or important for the normal function of lung cells). In specific embodiments, the biomolecule acts in spleen cells (for example, necessary or important for the normal function of spleen cells). In specific embodiments, the muscle cells described herein are skeletal muscle cells (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragmatic and / or pectoralis major muscle cells) and / or cardiomyocytes (e.g., atrial and / or ventricular muscle cells). In specific embodiments, the brain cells described herein are mesoforebrain, mesoposterior, forebrain and / or hindbrain cells. In specific embodiments, the biomolecule is functional motor neuron survival (SMN) protein (e.g., wild-type SMN protein).In a specific embodiment, the biomolecule is functional microdystrophin (e.g., wild-type microdystrophin). In a specific embodiment, the biomolecule is functional α-galactosidase (e.g., wild-type α-galactosidase). In a specific embodiment, the biomolecule is functional phenylalanine hydroxylase (PAH) (e.g., wild-type PAH). In a specific embodiment, the biomolecule is functional coagulation factor VIII (FVIII) (e.g., wild-type FVIII). In a specific embodiment, the biomolecule is functional coagulation factor IX (FIX) (e.g., wild-type FIX). In a specific embodiment, the biomolecule is functional β-glucocerebrosidase (GBA) (e.g., wild-type GBA). In a specific embodiment, the biomolecule is NPC intracellular cholesterol transporter 1 (NPC1) (e.g., wild-type NPC1). In a specific embodiment, the biomolecule is NPC intracellular cholesterol transporter 2 (NPC2) (e.g., wild-type NPC2). In a specific embodiment, the biomolecule is acid α-glucosidase (GAA) (e.g., wild-type GAA). 【0412】 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 hepatocytes. In a particular embodiment, the one or more target cells are one or more brain cells. In a particular embodiment, the one or more target cells are one or more muscle cells and one or more hepatocytes. In a particular embodiment, the one or more target cells are one or more muscle cells and one or more brain cells. In a particular embodiment, the one or more target cells are one or more hepatocytes and one or more brain cells. In a particular embodiment, the one or more target cells are one or more muscle cells, one or more hepatocytes and / or one or more brain cells. In a particular embodiment, the one or more target cells are one or more kidney cells. In a particular embodiment, the one or more target cells are one or more lung cells. In a particular embodiment, the one or more target cells are one or more spleen cells. In a particular embodiment, the one or more target cells are one or more muscle cells, one or more hepatocytes, one or more brain cells, one or more renal cells, one or more lung cells and / or one or more spleen 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 muscle and / or ventricular muscle cells). In a specific embodiment, the one or more brain cells described herein are one or more mesoforebrain, mesoposterior, forebrain and / or hindbrain cells. 【0413】 In another embodiment, a method is provided for treating a disease or disorder in a subject in need, comprising administering to the subject (preferably in a therapeutically effective amount) recombinant AAV9 particles described herein (e.g., recombinant AAV9 particles described in Section 5.3). 【0414】 In another embodiment, a method is provided for treating a disease or disorder in a subject in need, comprising administering to the subject (preferably in a therapeutically effective amount) a pharmaceutical composition described herein (e.g., a pharmaceutical composition described in Section 5.6). 【0415】 Based on the nature of the disease or disorder, the subject's condition, and the physician's knowledge, the route of administration or delivery to the subject and the amount of recombinant AAV9 particles or pharmaceutical composition to be administered can be determined. Non-limiting examples of usable routes of administration or delivery include direct delivery to a target organ, orally, by inhalation, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrathecally, intrapancreatally, intraperitoneally, intratumorally, and other parenteral routes. In specific embodiments, the recombinant AAV9 particles or pharmaceutical composition described herein are administered or delivered systemically. In specific embodiments, the recombinant AAV9 particles or pharmaceutical composition described herein are administered or delivered intravenously. 【0416】 The delivery and treatment methods described herein may be used to treat a variety of diseases or disorders, including but not limited to cancers such as cancer, sarcomas, leukemia, lymphoma, germ cell tumors and blastomas, autoimmune diseases, infectious diseases and hereditary disorders. In specific embodiments, the disease or disorder is caused by dysfunction of at least the muscles (e.g., skeletal muscles (e.g., biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm and / or pectoralis major) and / or the myocardium (e.g., atrial and / or ventricular muscles)). In specific embodiments, the disease or disorder is caused by dysfunction of at least the liver. In specific embodiments, the disease or disorder is caused by dysfunction of at least the brain (e.g., mesoforebrain, mesoposterior, forebrain and / or hindbrain). 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 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 brain dysfunction (e.g., mesoforebrain, mesoposteriorbrain, forebrain and / or posteriorbrain). In an embodiment, the disease or disorder is caused by at least liver dysfunction and brain dysfunction (e.g., mesoforebrain, mesoposteriorbrain, forebrain and / or hindbrain). In a specific embodiment, the disease or disorder is caused by at least kidney dysfunction. In a specific embodiment, the disease or disorder is caused by at least lung dysfunction. In a specific embodiment, the disease or disorder is caused by at least spleen dysfunction. 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 Fabry disease. In a specific embodiment, the disease or disorder is Gaucher disease.In a specific embodiment, the disease or disorder is Niemann-Pick disease type C (NPC). In a specific embodiment, the disease or disorder is Pompe disease. 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. 【0417】 array 【0418】 [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 1-21] 【0419】 Examples 【0420】 Some embodiments provided herein are illustrated by the following non-limiting examples, which describe the identification and testing of numerous mutant AAV9 capsid proteins having improved tissue transduction. 【0421】 Example 1: Engineered AAV9 variant for enhanced tissue targeting 【0422】 introduction 【0423】 The directional evolution of AAV capsids enables the rapid selection of novel capsids with improved tissue transduction. In this study, we designed AAV9-based capsid engineering by performing library screening of peptide insertions and substitutions (in variable regions IV and VIII) in mice and cynomolgus monkeys using viral mRNA driven by a synthetic promoter recovered from specific tissues. 【0424】 AAV Library Design 【0425】 The peptide display random libraries (Lib#1~4) were designed by inserting seven amino acids (7mer) or nine-mer after Q588 (VP1 position in AAV9) in the variable region (VR) VIII, or after G453 in VR IV of AAV9 (Figure 1A~1D). Inserted DNA libraries #1~4 were generated by PCR using the following primers. 【0426】 [Table 2] 【0427】 AAV Library Screening Workflow 【0428】 A DNA fragment library was generated using an AAV9 library backbone DNA template for PCR. The purified PCR product was incorporated into a library backbone containing the synthetic promoter p41, AAV9 (Cap9)VP1, bovine growth hormone (bGH) poly A, and terminal inversion repeats (ITRs). The p41 promoter was used to drive capsid gene expression only in the presence of the Ad5 helper gene product to produce the AAV library. The synthetic promoter was intended to recover Cap9 mRNA expressed in specific tissues. The incorporated library plasmid DNA was transformed into competent cells for amplification. The amplified cap9 library plasmid was used to produce the AAV library by co-transfecting HEK293 cells with pHelper and pRep2-AAP (SEQ ID NO.1). Purified AAV virus libraries (Lib#1, Lib#2, Lib#3) were intravenously injected into 8-week-old mice at a dose of 4e12vg / mouse (C57BL / 6J, DBA / 2J-mdx, BALB / cJ). Twenty days after injection, muscle tissue (triceps, quadriceps, gastrocnemius, abdominal muscles, tibialis anterior, diaphragm, myocardium) and liver were collected for total RNA isolation. Viral mRNA was converted to cDNA using Cap9-specific reverse transcription primers and used for specific PCR amplification and the generation of a second library. 【0429】 To screen in non-human primates (NHPs), viral libraries (Lib#2, Lib#4) were intravenously injected into cynomolgus monkeys, and tissue samples were collected 21 days post-injection. Total RNA was isolated and treated with DNase I. Cap9-specific mRNA was reverse transcribed and amplified by PCR (RT-PCR) for next-generation sequencing (NGS). Enriched candidates were selected by customized enrichment score analysis. A total of 810 candidates, including variants predicted by artificial intelligence modeling, were synthesized for a second screening. Simultaneously, 27 top variants were packaged individually for in vitro and in vivo characterization. These top variants were selected by NSG enrichment score. All variants were enriched in muscle, except for AVT905, which was enriched in liver. The nucleotide sequence encoding the peptide insertion of one mutant AVT905 was codon-optimized, and the corresponding mutant was named and characterized as AVT908 (see Table 3 below; lowercase letters in the nucleotide sequence of AVT908 indicate codon optimization). Since AVT908 and AVT905 have the same amino acid sequence, AVT908 is also expected to be enriched in the liver. 【0430】 Figure 2 shows a schematic diagram of the screening workflow. Table 3 shows the top mutant candidates selected for validation. 【0431】 [Table 3-1] [Table 3-2] 【0432】 Example 2: Characterization of the expression of AAV9-derived capsid-mediated transgenes in C57BL / 6J mice. 【0433】 Five variants (AVT901, AVT903, AVT905, AVT906, AVT907) and two controls (AAV9 and MyoAAV 1A(1A)) were individually packaged as green fluorescent protein (GFP) transgenes driven by the chicken β-actin (CB) promoter. All seven AAVs were produced in HEK293 cells by EIpro(Polyplus) triple plasmid transfection. Three days after transfection, cells were harvested, lysed, treated with benzonase (Sigma, E1014-25KU), and purified by iodixanol gradient ultracentrifugation. After buffer exchange with 0.001% F68-containing PBS, the AAVs were filtered through a 0.22 μm filter. Titer was measured by ddPCR using ITR primers and probes. AAV purity was evaluated by SDS-PAGE. 【0434】 These seven types of AAVs were intravenously injected into 8-week-old C57BL / 6J mice (1e12vg / mouse). Two weeks after injection (2wpi), tissue samples were collected and used for ex vivo imaging to detect innate GFP expression (Figure 3). AVT907, AVT905, and AVT903 showed significantly stronger GFP intensity than AAV9 in skeletal muscle. Specifically, in the triceps and gastrocnemius muscles, the order was AVT907 > AVT905 > AVT903 > AAV9, in the quadriceps muscle, AVT907 ≈ MyoAAV 1A > AVT905 > AVT901 > AVT903 > AAV9 ≈ AVT906, and in the tibialis anterior muscle, AVT907 > AVT905 ≈ AVT903 > AAV9. AVT907 and AVT905 showed stronger GFP intensity than AAV9 in the heart. AVT905 showed the strongest GFP signal in the liver, followed by AAV9. AVT903 showed weaker GFP in the liver than AAV9. No visible signal was captured in the liver for AVT907. No visible signal was captured in the lungs, spleen, or kidneys for any of the variants in the tests or for AAV9. 【0435】 Then, total RNA was isolated from the tissue using Trizol and treated with DNase I. After reverse transcription (RT), transgene mRNA was quantified by real-time qPCR. GFP and mouse GAPDH primers / probes were used for RT-qPCR (Figure 4). For comparison, the normalization level of AAV9-mediated GFP expression was set to 1.0 (Figure 4). Compared to the levels of AAV9-mediated transgenes, the levels of AVT907-mediated transgenes were 17-37 times higher in skeletal muscle and 10 times higher in the heart; the levels of AVT905-mediated transgenes were 3-12 times higher in skeletal muscle, 2 times higher in the heart, 3 times higher in the liver, 3 times higher in the spleen, and 5 times higher in the lungs; and the levels of AVT901-mediated transgenes were 4-6 times higher in skeletal muscle. 【0436】 Furthermore, using the DNasy Blood & Tissue Kit (Qiagen), genomic DNA from the triceps, quadriceps, gastrocnemius, tibialis anterior, heart, spleen, kidney, and liver was isolated two weeks (2wpi) after injection. The AAV vector genome copies in these tissues were measured by droplet digital PCR (ddPCR) with GFP and mouse TFRC primers / probes (Figure 5). The level of the AAV9 vector genome was set to 1.0 (Figure 5). Compared to the AAV9 vector genome copy, the AVT907 vector genome copy was 2-7 times higher in skeletal muscle and 10 times higher in the heart; the AVT905 vector genome copy was 3-11 times higher in skeletal muscle, 5 times higher in the heart, 4 times higher in the liver, 3 times higher in the kidney, and 12 times higher in the lung; and the AVT901 vector genome copy was 3 times higher in the triceps and quadriceps. 【0437】 Example 3: In vitro expression of a novel AAV9 mutant-mediated transgene. 【0438】 Sixteen mutants (AVT901, AVT902, AVT905, AVT907, AVT908, AVT909, AVT910, AVT911, AVT913, AVT914, AVT915, AVT916, AVT917, AVT918, AVT919, AVT920) and four controls (AAV9 MyoAAV 1A, MyoAAV 2A, MyoAAV 4A) were individually packaged as transgenes expressing 2A-linked GFP and firefly luciferase (Fluc), driven by a chicken β-actin (CB) promoter. Production and titer analysis were performed in this manner. AVT905 and AVT908 have the same amino acid sequence but use different codons. C2C12 myoblasts and HepG2 cells were seeded in 96-well plates and incubated with a specified AAV vector containing the CB-Fluc-2A-GFP transgene (1 e6 vg / cell). 【0439】 Native GFP imaging was performed under the same conditions 24 hours after transduction (Figures 6A and 6B). AVT905 and AVT908 showed nearly 100% GFP positivity in both C2C12 and HepG2 cells. In C2C12 cells, no GFP signals were observed for AAV9, 1A, AVT901, AVT902, AVT909, AVT910, and AVT911. In HepG2 cells, no GFP signals were observed for AAV9, 1A, 4A, AVT902, AVT907, AVT909, AVT910, AVT913, AVT918, and AVT920. 48 hours after transduction, cells were lysed and Fluc expression was quantified using a luciferase assay system (Promega) (Figures 7A and 7B). AVT905 and AVT908 showed significantly stronger luminescence signals than the other mutants. MyoAAV 1A, MyoAAV 2A, MyoAAV in C2C12 myoblasts The expression of 4A, AVT901, AVT902, AVT905, AVT907, AVT908, AVT909, AVT910, AVT911, AVT913, AVT914, AVT915, AVT916, AVT917, AVT918, AVT919, and AVT920-mediated luciferase was 0.7 times, 4.2 times, 7.8 times, 1.9 times, 2.3 times, 21.9 times, 6.1 times, 18.5 times, 2.3 times, 2.4 times, 1.9 times, 4.4 times, 3.7 times, 12 times, 9.7 times, 3.2 times, 2.1 times, 6.1 times, and 1.5 times, respectively, compared to the expression of AAV9-mediated luciferase in C2C12 myoblasts. MyoAAV 1A, MyoAAV 2A, MyoAAV in HepG2 cells The expression of 4A, AVT901, AVT902, AVT905, AVT907, AVT908, AVT909, AVT910, AVT911, AVT913, AVT914, AVT915, AVT916, AVT917, AVT918, AVT919, and AVT920-mediated luciferase was 0.4 times, 1.7 times, 1.4 times, 1.2 times, 0.8 times, 5.0 times, 0.7 times, 4.8 times, 1.0 times, 1.5 times, 1.9 times, 0.7 times, 1.0 times, 1.2 times, 1.5 times, 1.2 times, 0.7 times, 0.1 times, and 0.6 times, respectively, compared to the expression of AAV9-mediated luciferase in HepG2 cells. 【0440】 Example 4: Expression of a novel AAV9 mutant-mediated transgene in Balb / c mice. 【0441】 Sixteen mutants (AVT901, AVT902, AVT907, AVT908, AVT909, AVT910, AVT911, AVT912, AVT913, AVT914, AVT915, AVT916, AVT917, AVT918, AVT919, AVT920) and four controls (AAV9 MyoAAV 1A, MyoAAV 2A, MyoAAV 4A) were individually packaged as transgenes expressing 2A-linked GFP and firefly luciferase (Fluc), driven by the chicken β-actin (CB) promoter. 【0442】 Twenty different AAVs were intravenously (tail vein) injected into 8-week-old Balb / c mice (4e11vg / mouse). Three weeks after injection (3wpi), luciferin was injected intraperitoneally into the mice. After 10 minutes, the mice were sent to an in vivo imaging system (IVIS) to capture the expression of firefly luciferase (Figure 8). Compared to AAV9-mediated luminescence signals in skeletal muscle, AVT911 and AVT912 showed similar levels, AVT902, AVT909, and AVT910 showed stronger luminescence signals, and AVT901, AVT908, AVT914, AVT915, AVT916, AVT917, and AVT918 showed significantly stronger luminescence signals in skeletal muscle. Furthermore, AVT907, AVT913, AVT919, and AVT920 showed stronger luminescence signals in skeletal muscle compared to the disclosed MyoAAV2A (a reference muscle-directed AAV capsid). AAV908 also showed a significantly stronger luminescence signal in the liver than AAV9. Three weeks after injection (3 wpi), tissue samples (triceps, quadriceps, gastrocnemius, tibialis anterior, heart, and liver) were collected for the isolation of total RNA and genomic DNA. mRNA levels of AAV-mediated transgenes were quantified and analyzed by RT-qPCR (Figures 9A-9I). Mouse GAPDH (VIC® marker) was used as the reference. Compared to AAV9 wild-type, the expression levels of AVT919-mediated transgenes in the triceps, quadriceps, tibialis anterior, gastrocnemius, and heart were 72-fold, 45-fold, 113-fold, 85-fold, and 15-fold, respectively (Figures 9B-9H). The expression levels of AVT913-mediated transgenes in the triceps, quadriceps, tibialis anterior, gastrocnemius, and heart were 62-fold, 33-fold, 75-fold, 84-fold, and 27-fold, respectively (Figures 9B-9H). The expression levels of AVT908-mediated transgenes in the triceps, quadriceps, tibialis anterior, gastrocnemius, and heart were 15-fold, 19-fold, 18-fold, 40-fold, and 2-fold, respectively (Figures 9B-9H). AVT908, AVT913, and AVT919 showed AAV9-mediated transgene expression levels of 270%, 30%, and 60% in the liver, respectively (Figures 9B, 9C, and 9I). AAV vector genome copies were analyzed by ddPCR (Figure 10). Mouse TFRC (VIC® marker) was used as the reference.Primers / probes (FAM markers) for fluc were used on the AAV genome. The novel capsids AVT908, AVT913, and AVT919 showed improvement at the genomic DNA level in the triceps, quadriceps, tibialis anterior, gastrocnemius, and cardiac muscle compared to AAV9, with AVT908 showing improvement in the liver at the genomic DNA level (Figure 10). Specifically, in the tibialis anterior muscle, the genomic DNA levels of AVT908, AVT913, and AVT919 were 6.8 times, 7.6 times, and 22.1 times, respectively, compared to the genomic DNA level of AAV9 (Figure 10). In the heart, the genomic DNA levels of AVT913 and AVT919 were greater than 10 times that of AAV9 (Figure 10). 【0443】 In repeated studies using mice, two mutants (AVT913 and AVT919) containing the same expression cassette (GFP and firefly luciferase linked by a 2A sequence, driven by the chicken β-actin promoter) and two control capsids (AAV9 and MyoAAV 2A) were intravenously (tail vein) injected into 8-week-old male Balb / c mice. Three weeks after injection, muscle tissue was collected and used for natural GFP imaging. In AAV9-treated mouse tissue, no visible imaging signal was observed, or the imaging signal was very weak (Figure 11). The mutant AVT917 showed the strongest GFP intensity in muscle tissue. Specifically, in the gastrocnemius and quadriceps muscles, AVT919 > AVT913 > MyoAAV 2A > AAV9, and in the heart, AVT919 > AVT913 ≈ MyoAAV 2A > AAV9 (Figure 11). 【0444】 Example 5: Validation of pooled novel capsids in NHP. 【0445】 The coding sequences of 19 novel capsids and 5 publicly available controls (Myo1A, Myo2A, Myo3A, Myo4A, and AAV9) were individually cloned into a library backbone containing the p41 promoter, AAV2 ITR, synthetic promoter, and bGH polyA. Twenty-four capsids were individually produced in HEK293 cells by co-transfection with Rep2-AAP and Ad5 helper plasmids. Purification was performed by iodixanol gradient ultracentrifugation, filtered to 0.22 μm, and titer was measured by ddPCR using ITR primers / probes. Purity was analyzed by SDS-PAGE. 【0446】 Adult female cynomolgus monkeys (4 years old, ~3 kg) were intravenously injected with a pool of 24 capsids. Three weeks after injection (3 wpi), tissue samples were collected for total RNA and genomic DNA isolation. Total RNA was treated with DNase I to remove the AAV genome. Cap9-specific mRNA was reverse transcribed and amplified by PCR (RT-PCR). Genomic DNA was used for cap9-specific PCR. Pooled AAV vector DNA (for NHP injection) was amplified and synthesized using the minimum number of cycles. All purified PCR products were subjected to NGS (NovaSeq 6000 S4 Reagent Kit v1.5, 300 cycles). Enrichment scores (ES) were analyzed using a customized pipeline. 【0447】 The mRNA transgene levels of a total of 24 viral capsids were compared to wild-type AAV9 and expressed as a multiplicative change (FC) (Table 4). 【0448】 Example 6: Multiple validation of a single novel capsid in NHP. 【0449】 Adult male cynomolgus monkeys (4.25 kg) were intravenously injected with four pooled capsids: two mutants (AVT917 and AVT919) and two control capsids (AAV9 and MyoAAV 4A) at a dose of 1E13vg / kg / capsid. Each capsid contained a cyno-FXN coding sequence with different tags (Flag, AU1, V5, HA) under the control of a chicken β-actin (CB) promoter and CMV enhancer. Methylprednisolone was orally administered at an animal dose of 1 mg / kg / day for a total of eight days, starting three days before AAV injection. Three weeks after injection (3 wpi), the animals were anesthetized and sacrificed. Tissue was collected for total RNA and genomic DNA isolation. Multiple locations of the same tissue were collected for sampling. HiScript III 1 st mRNA was reverse transcribed using a cDNA synthesis kit (+g DNA wiper) (Vazyme, R412). Transgene-specific transcripts were quantified by qPCR using primers / probes for the tag. The endogenous macaque GAPDH housekeeping gene was used for normalization. The Bio-Rad CFX96 real-time PCR detection system was used for data collection and analysis. Relative transgene mRNA levels were 2 - Δ CtThe calculations were performed using the following method. To identify AAV vector genome copies in the tissue, 10–300 ng of purified genomic DNA was used in 20 μL of ddPCR reaction mixture, following the instructions for ddPCR Supermix for Probes (without dUTP, catalog number 1863025, Bio-Rad). Primers / probes for tags (FAM markers) were used for detection of the vector genome, and RNA polymerase P primers / probes (VIC markers, catalog number 4403328, Thermo Fisher Scientific) were used as a reference for macaque genome copy number. A QX200 droplet generator and reader (Bio-Rad) were used for data collection and analysis. The expression level of AAV9-mediated transgenes was set to 1.0. Improvements in capsids AVT917 and AVT919 were shown by calculating the multiplicative changes of AAV9. 【0450】 By comparing transgene expression at the mRNA level, AVT919 and the control capsid MyoAAV 4A showed significantly higher transduction than AAV9 in the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, diaphragm, atria, and ventricles (Figure 12). AVT917 also showed higher expression than AAV9 in muscle tissue (Figure 12). In other words, in NHP muscle tissue, the order was AVT919 ≈ MyoAAV 4A > AVT917 > AAV9. 【0451】 In the livers of NHP (non-hemolytic pulmonary hematopoietic) monkeys, AVT919 showed the highest expression levels in all lobes of the liver (Figure 13). In other words, after intravenous administration, the order of expression in monkey livers was AVT919 > MyoAAV 4A > AAV9 > AVT917 (Figure 13). 【0452】 No significant differences were observed between brain and spinal cord tissue for AVT919, MyoAAV4A, and AAV9 (Figure 14). AVT917 showed relatively low transgene expression in the brain and spinal cord after intravenous administration (Figure 14). 【0453】 The mutant AVT919 (square in Figure 15) showed 33.6-fold, 35.8-fold, 30-fold, 34.7-fold, 33.2-fold, and 35.3-fold improvements in mRNA levels in the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, and diaphragm, respectively, compared to AAV9 (inverted triangle in Figure 15) (Figure 15). 【0454】 The mutant AVT917 (upper triangle in Figure 15) showed improvements of 6.8-fold, 8.0-fold, 7.7-fold, 9.6-fold, 6.4-fold, and 4.8-fold at the mRNA level in the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, and diaphragm, respectively, compared to AAV9 (inverted triangle in Figure 15) (Figure 15). 【0455】 The mutant AVT919 (square in Figure 16) showed improvements of 5.1-fold, 4.6-fold, 6.7-fold, 1.7-fold, and 1.0-fold in mRNA levels in the atria, ventricles, liver, brain, and spinal cord, respectively, compared to AAV9 (inverted triangle in Figure 16) (Figure 16). 【0456】 The mutant AVT917 (upper triangle in Figure 16) showed mRNA levels 1.2 times, 1.1 times, 0.5 times, 0.3 times, and 0.2 times higher than AAV9 in the atria, ventricles, liver, brain, and spinal cord, respectively (Figure 16). 【0457】 By comparing vector genome copies, AVT919 (square in Figure 17) and the control capsid MyoAAV 4A (black circle in Figure 17) showed significantly higher transduction than AAV9 in the tibialis anterior, triceps, quadriceps, and gastrocnemius muscles (Figure 17). Three weeks after intravenous administration to NHP at a dose of 1E13vg / kg, AVT919 showed vector genome copy / diploid genome ratios of 1.0, 1.0, 1.5, and 1.9 in the tibialis anterior, triceps, quadriceps, and gastrocnemius muscles, respectively (square symbol in Figure 17). 【0458】 By comparing vector genome copies, AVT919 and the control capsid MyoAAV 4A showed significantly higher transduction than AAV9 in the rectus abdominis, diaphragm, atria, and ventricles (Figure 18). Three weeks after intravenous administration to NHP at a dose of 1E13vg / kg, AVT919 showed vector genome copies / diploid genome ratios of 5.8, 9.4, 15.8, and 14.9 in the rectus abdominis, diaphragm, atria, and ventricles, respectively (square symbols in Figure 18). 【0459】 AVT917 (upper triangle in Figure 18) showed higher transduction in the rectus abdominis, diaphragm, atria, and ventricles compared to AAV9 (Figure 18). 【0460】 The mutants AVT917 and AVT919 showed similar transduction at the DNA level in the spleen, kidney, and lung compared to AAV9 (Figure 19). 【0461】 AVT917, AVT919, and the control capsid MyoAAV 4A showed higher transduction at the DNA level in the adrenal gland than AAV9 (Figure 19). Specifically, after intravenous administration to NHP at a dose of 1E13vg / kg, MyoAAV4A, AVT919, AVT917, and AAV9 showed 24.2, 6.8, 10.1, and 2.4 vector genome copies / diploid genomes, respectively, in the adrenal gland three weeks later (Figure 19). 【0462】 The mutants AVT917 and AVT919 showed similar transduction at the DNA level in the pancreas, brain, and spinal cord compared to AAV9 (Figure 20). 【0463】 AVT919 showed significantly higher vector genome copies in the liver of NHP patients compared to the control capsids MyoAAV 4A and AAV9 (Figure 20). Specifically, three weeks after intravenous administration of 1E13vg / kg to NHP patients, MyoAAV4A, AVT919, AVT917, and AAV9 showed vector genome copies / diploid genome ratios of 148.0, 226.7, 60.2, and 84.9, respectively, in the liver (Figure 20). 【0464】 Example 7: Single-pronged validation of a novel capsid in NHP. 【0465】 A 4-year-old female cynomolgus monkey (3.35 kg) was intravenously injected with the AAV9 capsid at a dose of 2E13 vg / kg. A 5-year-old female cynomolgus monkey (3.0 kg) was intravenously injected with the same dose of the mutant AVT919. Both animals were negative for pre-existing neutralizing anti-AAV9 capsid antibodies. Both AAV9 and AVT919 capsids contained the same AAV genome sequence, which was a hemagglutinin (HA)-tagged Cyno FXN transgene driven by the chicken β-actin promoter. Four weeks after intravenous injection, the animals were anesthetized and sacrificed. Tissue was collected for total RNA extraction. For sampling, multiple locations of the same tissue were collected. HiScript III 1 st mRNA was reverse transcribed using a cDNA synthesis kit (+gDNA wiper) (Vazyme, R412). Transgene-specific transcripts were quantified by RT-qPCR using primers / probes for HA tags. The endogenous macaque GAPDH housekeeping gene was used for normalization. Relative transgene mRNA levels were 2 - Δ Ct It was calculated using the formula. 【0466】 Compared to AAV9 (square in Figure 21), the mutant AVT919 showed significantly improved transgene expression at the mRNA level in skeletal muscles, including the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, diaphragm, and heart (Figure 21). 【0467】 Specifically, AVT919 showed 57-fold, 21-fold, 6-fold, 4-fold, 47-fold, 7-fold, and 2-fold improvements compared to AAV9 at the mRNA level in the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, diaphragm, and heart, respectively (Figure 22). 【0468】 To detect the protein products of AAV-mediated transgenes, tissues collected after treatment with rabbit anti-HA monoclonal primary antibody (catalog number 37245, Cell Signaling Technology) were immunostained. Peroxidase AffiniPure goat anti-rabbit IgG(H+L) secondary antibody (Jackson, catalog number 111-035-003) diluted 1:5000 and DAB substrate were used for staining (brown). 【0469】 Four weeks after intravenous administration of AAV at a dose of 2E13vg / kg via NHP, AAV9 showed weak or low transgene expression at the protein level in the tibialis anterior, triceps, quadriceps, gastrocnemius, and rectus abdominis muscles (Figures 23A-C). In contrast, the mutant AVT919 showed strong or high transgene expression in muscle tissue including the tibialis anterior, triceps, quadriceps, gastrocnemius, rectus abdominis, diaphragm, and heart (Figures 23A-D). 【0470】 The mutant AVT919 showed higher transgene expression at the protein level compared to AAV9, and also higher than in NHP liver (Figure 23E). 【0471】 [Table 4] 【0472】 All capsids (except AVT905 and AV9ML018) showed improvement in muscle compared to AAV9. Furthermore, AVT919, AVT913, AVT915, AVT914, AVT917, AVT916, and AV9ML012 showed improvement at the mRNA level in various muscle tissues compared to MyoAAV 4A (Table 5). 【0473】 [Table 5] 【0474】 AVT918, AV9ML005, AVT913, AVT919, AV9ML002, and AVT916 showed improvement in the brains of NHP patients after intravenous administration, and their mRNA levels were more than twice that of AAV9 (Table 6). 【0475】 [Table 6] 【0476】 AVT914, AVT916, AVT917, AVT918, AVT913, and AVT915 showed improvement in NHP in the liver after intravenous administration, and their mRNA levels were more than twice that of AAV9 (Table 7). 【0477】 [Table 7] 【0478】 Furthermore, 27 capsid variants (AV9ML025~051) were identified as superior to the standard MyoAAV 4A at the mRNA level in NHP (Table 8). 【0479】 [Table 8-1] [Table 8-2] 【0480】 Example 8: Engineered AAV9 variant for effective muscle transduction 【0481】 Systemic administration of adeno-associated virus (AAV) vectors expressing functional genes is considered a promising treatment for muscular dystrophy. However, due to the low transduction efficiency in muscle after intravenous delivery, very high doses of AAV are required. This study aims to engineer novel AAV capsids with enhanced transduction efficiency in muscle using AI-assisted AAV capsid evolution discovery technology. Based on the AAV9 capsid, random peptides were generated and screened in mice and non-human primates (NHPs) and inserted into an AAV capsid library. The screening process relied on the recovery of muscle-specific viral mRNA using the proprietary robust synthetic muscle-specific promoter SCC45. Two screenings were performed in three mouse strains (C57BL / 6, BALB / c, and DBA / 2j-mdx), and one screening was performed in NHPs, after which top capsid candidates were selected and validated individually. By using GFP and a firefly luciferase reporter gene, many mutants showed significant improvement (up to 113-fold) in mouse skeletal muscle compared to AAV9. One mutant, AVT913, showed 26-fold enhanced transduction at the mRNA level in the heart. 【0482】 Furthermore, the AVT919 mutant showed significant improvement over the standard muscle-oriented AAV capsid, MyoAAV2A, in the triceps and tibialis anterior muscles. While most top mutants exhibited improved muscle transduction and reduced liver orientation, the specific mutant AVT908 demonstrated enhanced efficiency in both muscle and liver. 【0483】 To confirm whether similar tissue-targeting patterns are reproduced in NHP, these mutants were packaged individually with other candidates and intravenously injected into adult cynomolgus monkeys. Characteristic results revealed cross-species translationability. These engineered muscle-targeted AAV capsids would provide an efficient delivery platform for muscle-targeted gene therapy. 【0484】 Incorporated by reference 【0485】 All references cited herein are incorporated herein by collective citation for all purposes, to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by collective citation for all purposes. 【0486】 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 provided merely as examples, and the present invention is limited only by the terms of the appended claims and the equivalents thereof.

Claims

[Claim 1] SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ A variant adeno-associated virus serotype 9 (AAV9) capsid protein containing the amino acid sequences of ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, or SEQ ID NO:

168. [Claim 2] SEQ ID NO: 6 to 33, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ A mutant AAV9 capsid protein according to claim 1, comprising the amino acid sequence of ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, or SEQ ID NO:

167. [Claim 3] A mutant AAV9 capsid protein comprising SEQ ID NO: 43, SEQ ID NO: 9, SEQ ID NO: 51, SEQ ID NO: 12, SEQ ID NO: 63, SEQ ID NO: 17, SEQ ID NO: 65, SEQ ID NO: 18, SEQ ID NO: 67, SEQ ID NO: 19, SEQ ID NO: 69, SEQ ID NO: 20, SEQ ID NO: 71, SEQ ID NO: 21, SEQ ID NO: 73, or SEQ ID NO: 22 is associated with improved tropism to the liver compared to the wild-type AAV9 capsid protein comprising the amino acid sequence of SEQ ID NO: 2, as described in claim 2. [Claim 4] The mutant AAV9 capsid protein according to claim 2, wherein SEQ ID NO: 63, SEQ ID NO: 17, SEQ ID NO: 69, SEQ ID NO: 20, SEQ ID NO: 73, SEQ ID NO: 22, SEQ ID NO: 75, SEQ ID NO: 23, SEQ ID NO: 80, or SEQ ID NO: 26 is associated with enhanced tropism to the brain compared to the wild-type AAV9 capsid protein containing the amino acid sequence of SEQ ID NO:

2. [Claim 5] The mutant AAV9 capsid protein according to claim 4, which is associated with enhanced tropism to skeletal muscle and / or cardiac muscle compared to wild-type AAV9 capsid protein. [Claim 6] The mutant AAV9 capsid protein according to claim 4, which is associated with enhanced tropism to the biceps, triceps, quadriceps, tibialis anterior, gastrocnemius, rectus abdominis, diaphragm, pectoralis major, atrial muscle and / or ventricular muscle compared to wild-type AAV9 capsid protein. [Claim 7] Recombinant AAV9 particles comprising the mutant AAV9 capsid protein described in any one of claims 1 to 6. [Claim 8] A pharmaceutical composition comprising recombinant AAV9 particles according to claim 7 and a pharmaceutically acceptable carrier. [Claim 9] A polynucleotide encoding the mutant AAV9 capsid protein according to any one of claims 1 to 6. [Claim 10] A vector comprising the polynucleotide described in claim 9. [Claim 11] A host cell comprising the polynucleotide described in claim 9 or the vector described in claim 10. [Claim 12] A group of host cells stably transduced by recombinant AAV9 particles according to claim 7. [Claim 13] A pharmaceutical composition comprising a host cell population according to claim 12 and a pharmaceutically acceptable carrier. [Claim 14] A method for delivering a biomolecule to one or more ex vivo or in vitro target cells, comprising transducing one or more target cells using recombinant AAV9 particles as described in claim 7. [Claim 15] A method for delivering a biomolecule to one or more in vivo target cells in a subject, comprising administering the recombinant AAV9 particles described in claim 7 or the pharmaceutical composition described in claim 8 to the subject. [Claim 16] The method according to claim 14 or 15, wherein the one or more target cells are one or more muscle cells, one or more hepatocytes and / or one or more brain cells. [Claim 17] A method for treating a disease or disorder in a subject in need, comprising administering to a subject the recombinant AAV9 particles described in claim 7, or the pharmaceutical composition described in claim 8 or 13. [Claim 18] The method according to any one of claims 15 to 17, wherein the subject is a human. [Claim 19] A method for producing recombinant AAV9 particles, comprising culturing the host cells described in claim 11.