Adeno-associated virus vector variant

Abstract

To provide AAV variants that are able to target distinct primate brain structures.SOLUTION: Provided herein are targeting peptides, and vectors comprising a sequence that encodes the targeting peptides, where the targeting peptides deliver agents to specific substructures in the brain. Provided herein are viral vectors each comprising a modified capsid, where the modified capsid comprises at least one amino acid sequence that targets the viral vector to a distinct brain structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 939,315, filed on 22 November 2019, and U.S. Provisional Patent Application No. 63 / 084,709, filed on 29 September 2020, the entire contents of both applications are incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The aforementioned ASCII copy, created on November 19, 2020, is named CHOPP0038WO_ST25.txt and has a size of 63.8 kilobytes.

[0003] 1. Field This invention relates as a whole to the fields of medicine, virology, and neurology. More specifically, it relates to targeting peptides that target the delivery of viral vectors to different structures in the brain. [Background technology]

[0004] 2. Explanation of related technologies Various strategies have been developed to create AAV vector variants, including rational design and directional evolution. The rational design approach uses knowledge of the AAV capsid to introduce targeted changes to the capsid, such as tyrosine mutations on the capsid surface, thereby altering transduction efficiency or specificity. The directional evolution approach does not require knowledge of the capsid structure and is performed through random mutagenesis, capsid shuffling, or random peptide insertion. These strategies generally utilize in vitro systems or mice, which are ideal for cell-based or mouse studies, but do not necessarily translate to clinical applications. Indeed, AAV variants do not specifically or efficiently target different brain structures. Therefore, there is a need for AAV variants that can target different primate brain structures. [Overview of the Initiative]

[0005] overview The following viral vectors are provided herein, each containing a modified capsid, wherein the modified capsid contains at least one amino acid sequence that causes the viral vector to target different brain structures.

[0006] In one embodiment, a modified adeno-associated virus (AAV) capsid protein is provided, comprising a targeting peptide, wherein the targeting peptide causes a viral vector containing the modified AAV capsid protein to target different organs or different brain structures, and the modified AAV capsid protein is 3 to 10 amino acid lengths long. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0007] In some embodiments, the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 138), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some embodiments, the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acids in length. In some embodiments, the linker sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some embodiments, the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 141.

[0008] In some embodiments, the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 139), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some embodiments, the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acids in length. In some embodiments, the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. In some embodiments, the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 142.

[0009] In some aspects, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO: 140), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acids in length. In some aspects, the linker sequences are AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 143.

[0010] In some aspects, the targeting peptide comprises a sequence that is at most 10 amino acids in length and has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-137 and 144 in the sequence. In some aspects, the targeting peptide is 7 amino acids in length.

[0011] In some aspects, the different brain structures are the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, putamen, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. In certain aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from those listed in Table 1 to target the corresponding brain structure. In certain aspects, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from those listed in Table 2 to target the corresponding brain structure. In certain aspects, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from those listed in Table 3 to target the corresponding brain structure.

[0012] In some contexts, the different organs are the brain, kidney, heart, liver, gonads, spleen, or liver. In certain contexts, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is selected from those listed in Table 4 to target the corresponding organ. In certain contexts, the modified AAV capsid protein is the modified AAV2 capsid protein, and the targeting peptide is selected from those listed in Table 5 to target the corresponding organ. In certain contexts, the modified AAV capsid protein is the modified AAV9 capsid protein, and the targeting peptide is selected from those listed in Table 6 to target the corresponding organ.

[0013] In one embodiment, nucleic acids comprising sequences encoding any one of the modified capsid proteins of this embodiment are provided herein.

[0014] In one embodiment, recombinant adeno-associated virus (rAAV) comprising any one of the modified capsid proteins of this embodiment is provided herein. In several aspects, combinations of rAAVs are provided. For example, combinations of rAAV having a modified AAV1 capsid protein and a targeting peptide with SEQ ID NO: 21 are provided, rAAV having a modified AAV2 capsid protein and a targeting peptide with SEQ ID NO: 53, rAAV having a modified AAV2 capsid protein and a targeting peptide with SEQ ID NO: 80, and rAAV having a modified AAV9 capsid protein and a targeting peptide with SEQ ID NO: 113 are provided.

[0015] In one embodiment, a viral vector comprising a nucleic acid encoding one of the modified capsid proteins of this embodiment is provided herein. In some aspects, the viral vector further comprises a nucleic acid sequence encoding a nucleic acid of interest. In some aspects, the nucleic acid of interest is a therapeutic substance. In some aspects, the therapeutic substance is an enzyme or an RNAi molecule.

[0016] In one embodiment, cells containing any one of the viral vectors of this embodiment are provided herein. In some aspects, the cells are mammalian cells, such as human cells. In some aspects, the cells are in vitro or in vivo.

[0017] In one embodiment, a pharmaceutical composition comprising the viral vector of this embodiment and a pharmaceutically acceptable carrier is provided herein.

[0018] In one embodiment, a method for delivering an active agent to a different brain structure of a target is provided herein, comprising the step of administering the virus of this embodiment to the target. In several aspects, the different brain structures are the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. In certain aspects, an rAAV having a modified AAV1 capsid protein is used, and a targeting peptide is selected from those listed in Table 1 to target the corresponding brain structure. In certain aspects, an rAAV having a modified AAV2 capsid protein is used, and a targeting peptide is selected from those listed in Table 2 to target the corresponding brain structure. In certain aspects, an rAAV having a modified AAV9 capsid protein is used, and a targeting peptide is selected from those listed in Table 3 to target the corresponding brain structure. In various aspects, any combination of rAAVs is used. For example, combinations of rAAV with a modified AAV1 capsid protein and a targeting peptide with SEQ ID NO: 21, rAAV with a modified AAV2 capsid protein and a targeting peptide with SEQ ID NO: 53, rAAV with a modified AAV2 capsid protein and a targeting peptide with SEQ ID NO: 80, and rAAV with a modified AAV9 capsid protein and a targeting peptide with SEQ ID NO: 113 can be used.

[0019] In one embodiment, a method for delivering an active agent to a different organ of target is provided herein, comprising the step of administering the virus of this embodiment to the target. In several aspects, the organ is the brain, kidney, heart, liver, gonads, spleen, or liver. In a particular aspect, an rAAV having a modified AAV1 capsid protein is used, and a targeting peptide is selected from those listed in Table 4 to target the corresponding organ. In a particular aspect, an rAAV having a modified AAV2 capsid protein is used, and a targeting peptide is selected from those listed in Table 5 to target the corresponding organ. In a particular aspect, an rAAV having a modified AAV9 capsid protein is used, and a targeting peptide is selected from those listed in Table 6 to target the corresponding organ. In various aspects, any combination of rAAVs is used.

[0020] In some contexts, the active agent is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. In some contexts, the administration is directed to the central nervous system. In some contexts, the administration is directed to the cisterna magna, intraventricular space, ependymia, ventricles, subarachnoid space, and / or intrathecal space. In some contexts, the ventricles are the rostral lateral ventricle and / or caudal lateral ventricle and / or right lateral ventricle and / or left lateral ventricle and / or right rostral lateral ventricle and / or left rostral lateral ventricle and / or right caudal lateral ventricle and / or left caudal lateral ventricle.

[0021] In some scenarios, multiple viral particles are administered. In some scenarios, the virus contains approximately 1 × 10⁶ viral genomes per kilogram of vector genome. 6 ~Approx. 1×10 18 It is administered in doses of (vg / kg). In some cases, the virus is approximately 1 × 10⁶ vg per kg of patient. 7 ~1 × 10 17 , about 1×10 8 ~1 × 10 16, about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , about 1×10 12 ~×10 13 , or about 1×10 13 ~1×10 14 are administered. In some situations, the subject is human.

[0022] a method for treating a disease in a mammal, comprising administering the virus of this aspect to the mammal, is provided herein. In some situations, the disease is a neurodegenerative disease. In some situations, the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease. In some situations, the mammal is human.

[0023] As used herein, "essentially free of" with respect to a particular component means that none of the particular components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts. The total amount of the particular component resulting from unintentional composition contamination is, therefore, significantly lower than 0.05%, preferably lower than 0.01%. Most preferred are compositions in which the amount of such particular components cannot be detected by standard analytical methods.

[0024] As used herein, “a” or “an” may mean one or more. As used herein in the claims, “a” or “an” may mean one or more when used with the word “comprising.”

[0025] The use of the term “or” in the claims is used to mean “and / or” unless expressly indicated to mean only the options or mutually exclusive options, but this disclosure supports the definitions of only the options and “and / or.” Where used herein, “another” may mean at least a second or more.

[0026] Throughout this application, the term "about" is used to indicate that a value includes inherent error variations of the apparatus or method used to determine that value, variations that exist between the subjects of study, or values ​​that are within 10% of the stated value.

[0027] [Invention 1001] A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide causes the viral vector comprising the modified AAV capsid protein to target different organs or different brain structures, and the modified AAV capsid protein is 3 to 10 amino acids long. [Invention 1002] The modified AAV capsid protein of Invention 1001, wherein the modified AAV capsid protein is a modified AAV9 capsid protein having a sequence at least 95% identical to SEQ ID NO: 143, the targeting peptide is SEQ ID NO: 110, and the different brain structures are the brainstem, caudate nucleus, cerebellum, cochlea (ear), cortex, cerebral cortex, deep cerebellar nuclei, ependyma, globus pallidus, hippocampus, meninges, motor cortex, optic nerve, prefrontal cortex, putamen, spinal cord, substantia nigra, subthalamic nucleus, temporal cortex, thalamus, or visual cortex. [Invention 1003] A modified AAV capsid protein according to Invention 1001, which is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein. [Invention 1004] The modified AAV capsid protein of the present invention 1001, wherein the modified AAV capsid protein is derived from the AAV1 capsid protein (see SEQ ID NO: 138), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. [Invention 1005] A modified AAV capsid protein according to the present invention 1004, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long. [Invention 1006] A modified AAV capsid protein according to Invention 1005, wherein the linker sequence is the N-terminal SSA of the targeting peptide and the C-terminal AS of the targeting peptide. [Invention 1007] The modified AAV capsid protein of the present invention 1006, wherein the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 141. [Invention 1008] The modified AAV capsid protein of the present invention 1001, wherein the modified AAV capsid protein is derived from the AAV2 capsid protein (see SEQ ID NO: 139), and the targeting peptide is inserted after residue position 587 of the AAV2 capsid protein. [Invention 1009] A modified AAV capsid protein according to Invention 1008, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long. [Invention 1010] A modified AAV capsid protein according to Invention 1009, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide. [Invention 1011] The modified AAV2 capsid protein of the present invention 1010, wherein the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 142. [Invention 1012] The modified AAV capsid protein of the present invention 1001, wherein the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO: 140), and the targeting peptide is inserted after residue position 588 of the AAV9 capsid protein. [Invention 1013] A modified AAV capsid protein according to the present invention 1012, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long. [Invention 1014] A modified AAV capsid protein according to the present invention 1013, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide. [Invention 1015] The modified AAV capsid protein of the present invention 1014, wherein the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 143. [Invention 1016] A modified AAV capsid protein according to the present invention 1001, wherein the target peptide comprises a sequence up to 10 amino acids in length, and the sequence contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 137 or 144. [Invention 1017] A modified AAV capsid protein according to Invention 1016, wherein the targeting peptide has a length of 7 amino acids. [Invention 1018] A modified AAV capsid protein according to any of the invention 1001 to 1017, wherein the aforementioned different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. [Invention 1019] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the brainstem, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1 to 9. [Invention 1020] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1, 3, 5, 7, 10-16, 25, 26, 32, and 144. [Invention 1021] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1, 3, 4, 9, and 17-21. [Invention 1022] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1, 3, 5, 12, and 21-26. [Invention 1023] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the ependymia, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 2-4, 7, 9, 21, 22, 27, and 28. [Invention 1024] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 3, 5, 12, 14, 16, 21, 22, and 29-31. [Invention 1025] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1-4, 7, and 32-34. [Invention 1026] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the meninges, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 3, 5, 7, 9, 12, 21, and 35-37. [Invention 1027] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 2, 3, 7, 14-16, 21, 31, and 38. [Invention 1028] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the putamen, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 3, 4, 12, 13, 21, 30, and 39-42. [Invention 1029] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 2-4, 7, 9, 21, 32, 33, and 43. [Invention 1030] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 2, 3, 9, 44, and 45. [Invention 1031] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 2-4, 12, 16, 30, 46, and 47. [Invention 1032] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the thalamus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NO: 1, 2, 8, 12, 21, 28, and 48-51. [Invention 1033] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the brainstem, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 52-60. [Invention 1034] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 59 and 61-69. [Invention 1035] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 56, 58, 60, and 70-75. [Invention 1036] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 53, 58, 60, 62, 63, 66, and 76-79. [Invention 1037] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the ependymia, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 53, 60, 62, 63, 66, 74-77, and 80. [Invention 1038] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 60, 75, and 81-87. [Invention 1039] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 53, 55, 58, 60, 63, 76, 79, 88, and 89. [Invention 1040] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the meninges, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 58, 60, 66, 73, 76, 80, and 90-93. [Invention 1041] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 53, 54, 57, 58, 60, 75, 79, 87, 88, and 94. [Invention 1042] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the putamen, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 55, 59, 60, 61, and 95-100. [Invention 1043] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 53, 58-61, 63, 77, 88, 95, and 101. [Invention 1044] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 52, 53, 57, 58, 75, 76, 87, 102, and 103. [Invention 1045] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 57, 58, 60, 75, 79, 87, 88, 102, 104, and 105. [Invention 1046] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the thalamus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from SEQ ID NO: 52, 55, 56, 74, 85, 88, and 106-109. [Invention 1047] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the brainstem, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110~117. [Invention 1048] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 113, 115, 116, and 118-121. [Invention 1049] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 119, and 122-125. [Invention 1050] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 114, 116, and 125-127. [Invention 1051] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the ependymia, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 118-120, and 128. [Invention 1052] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110-112, 114, 119, 120, and 129. [Invention 1053] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 116, 123, 125, 129, and 130. [Invention 1054] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is meninges, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 114, 118, 119, 122, and 131. [Invention 1055] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 114, 115, 117, 129, and 132. [Invention 1056] A modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the putamen, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 112, 113, 116, 123, 127, 133, and 134. [Invention 1057] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 113, 119, 120, 122, 123, 128, and 134. [Invention 1058] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110-114, 117, and 129. [Invention 1059] A modified AAV capsid protein according to Invention 1018, wherein the different brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 119, 120, 122, 132, and 135. [Invention 1060] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the thalamus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from SEQ ID NO: 110, 112-114, 125, 133, 136, and 137. [Invention 1061] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 1, and the different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, hippocampus, or thalamus. [Invention 1062] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 2, and the different brain structure is the brainstem, ependyma, hippocampus, optic nerve, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. [Invention 1063] The modified AAV capsid protein of Invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 3, and the different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, or subthalamic nucleus. [Invention 1064] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 4, and the different brain structure is the brainstem, cerebellar cortex, ependymia, hippocampus, putamen, spinal cord, or subthalamic nucleus. [Invention 1065] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 5, and the different brain structure is the brainstem, cerebral cortex, globus pallidus, or meninges. [Invention 1066] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 6, and the different brain structure is the brainstem. [Invention 1067] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 7, and the different brain structure is the brainstem, caudate nucleus, ependymia, hippocampus, meninges, optic nerve, or spinal cord. [Invention 1068] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 8, and the different brain structure is the brainstem or the thalamus. [Invention 1069] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 9, and the different brain structure is the brainstem, cerebellar cortex, ependyma, meninges, spinal cord, or substantia nigra. [Invention 1070] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 10 or 11, and the different brain structure is the caudate nucleus. [Invention 1071] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 12, and the different brain structure is the caudate nucleus, cerebral cortex, globus pallidus, meninges, putamen, subthalamic nucleus, or thalamus. [Invention 1072] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 13, and the different brain structure is the caudate nucleus or the putamen. [Invention 1073] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 14, and the different brain structure is the caudate nucleus, globus pallidus, or optic nerve. [Invention 1074] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 15, and the different brain structure is the caudate nucleus or the optic nerve. [Invention 1075] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 16, and the different brain structure is the caudate nucleus, globus pallidus, optic nerve, or subthalamic nucleus. [Invention 1076] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is one of SEQ ID NO: 17 to 20, and the different brain structure is the cerebellar cortex. [Invention 1077] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 21, and the different brain structure is the cerebellar cortex, cerebral cortex, ependyma, globus pallidus, meninges, optic nerve, putamen, spinal cord, or thalamus. [Invention 1078] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 22, and the different brain structure is the cerebral cortex, ependymal region, or globus pallidus. [Invention 1079] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is one of SEQ ID NO: 23 to 26, and the different brain structure is the cerebral cortex. [Invention 1080] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 27, and the different brain structure is the ependymia. [Invention 1081] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 28, and the different brain structure is the ependymome or the thalamus. [Invention 1082] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 29, and the different brain structure is the globus pallidus. [Invention 1083] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 30, and the different brain structure is the globus pallidus, putamen, or subthalamic nucleus. [Invention 1084] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 31, and the different brain structure is the globus pallidus or the optic nerve. [Invention 1085] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 32 or 33, and the different brain structure is the hippocampus or the spinal cord. [Invention 1086] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 34, and the different brain structure is the hippocampus. [Invention 1087] The modified AAV capsid protein according to Invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is one of SEQ ID NO: 35 to 37, and the different brain structure is meninges. [Invention 1088] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 38, and the different brain structure is the optic nerve. [Invention 1089] The modified AAV capsid protein according to Invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is one of SEQ ID NO: 39 to 42, and the different brain structure is the putamen. [Invention 1090] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 43, and the different brain structure is the spinal cord. [Invention 1091] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 44 or 45, and the different brain structure is the substantia nigra. [Invention 1092] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 46 or 47, and the different brain structure is the subthalamic nucleus. [Invention 1093] The modified AAV capsid protein of the present invention 1001 or 1007, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is one of SEQ ID NO: 48 to 51, and the different brain structure is the thalamus. [Invention 1094] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 52, and the different brain structure is the brainstem, substantia nigra, or thalamus. [Invention 1095] A modified AAV capsid protein according to Invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 53, and the different brain structure is the brainstem, cerebral cortex, ependyma, hippocampus, meninges, optic nerve, spinal cord, or substantia nigra. [Invention 1096] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 54, and the different brain structure is the brainstem or the optic nerve. [Invention 1097] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 55, and the different brain structure is the brainstem, hippocampus, putamen, or thalamus. [Invention 1098] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 56, and the different brain structure is the brainstem, cerebellar cortex, or thalamus. [Invention 1099] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 57, and the different brain structure is the brainstem, optic nerve, substantia nigra, or subthalamic nucleus. [Invention 1100] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 58, and the different brain structure is the brainstem, cerebellar cortex, cerebral cortex, hippocampus, meninges, optic nerve, spinal cord, substantia nigra, or subthalamic nucleus. [Invention 1101] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 59, and the different brain structure is the brainstem, caudate nucleus, putamen, or spinal cord. [Invention 1102] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 60, and the different brain structure is the brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, or subthalamic nucleus. [Invention 1103] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 61, and the different brain structure is the caudate nucleus, putamen, or spinal cord. [Invention 1104] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 62, and the different brain structure is the caudate nucleus, cerebral cortex, or ependymia. [Invention 1105] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 63, and the different brain structure is the caudate nucleus, cerebral cortex, ependymia, hippocampus, or spinal cord. [Invention 1106] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 64, 65, and 67-69, and the different brain structure is the caudate nucleus. [Invention 1107] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 66, and the different brain structure is the caudate nucleus, cerebral cortex, ependyma, or meninges. [Invention 1108] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 70 to 72, and the different brain structure is the cerebellar cortex. [Invention 1109] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 73, and the different brain structure is the cerebellar cortex or meninges. [Invention 1110] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 74, and the different brain structure is the cerebellar cortex, ependyma, or thalamus. [Invention 1111] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 75, and the different brain structure is the cerebellar cortex, ependyma, globus pallidus, optic nerve, substantia nigra, or subthalamic nucleus. [Invention 1112] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 76, and the different brain structure is the cerebral cortex, ependyma, hippocampus, meninges, or substantia nigra. [Invention 1113] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 77, and the different brain structure is the cerebral cortex, ependyma, or spinal cord. [Invention 1114] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 78, and the different brain structure is the cerebral cortex. [Invention 1115] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 79, and the different brain structure is the cerebral cortex, hippocampus, optic nerve, or subthalamic nucleus. [Invention 1116] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 80, and the different brain structure is the ependymia, hippocampus, or meninges. [Invention 1117] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 81-84 and 86, and the different brain structure is the globus pallidus. [Invention 1118] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 85, and the different brain structure is the globus pallidus or the thalamus. [Invention 1119] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 87, and the different brain structure is the globus pallidus, optic nerve, substantia nigra, or subthalamic nucleus. [Invention 1120] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 88, and the different brain structure is the hippocampus, optic nerve, spinal cord, subthalamic nucleus, or thalamus. [Invention 1121] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 89, and the different brain structure is the hippocampus. [Invention 1122] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 90 to 93, and the different brain structure is meninges. [Invention 1123] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 94, and the different brain structure is the optic nerve. [Invention 1124] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 95, and the different brain structure is the putamen or the spinal cord. [Invention 1125] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 96 to 100, and the different brain structure is the putamen. [Invention 1126] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 101, and the different brain structure is the spinal cord. [Invention 1127] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 102, and the different brain structure is the substantia nigra or the subthalamic nucleus. [Invention 1128] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 103, and the different brain structure is the substantia nigra. [Invention 1129] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 104 or 105, and the different brain structure is the subthalamic nucleus. [Invention 1130] The modified AAV capsid protein of the present invention 1001 or 1011, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is one of SEQ ID NO: 106 to 109, and the different brain structure is the thalamus. [Invention 1131] A modified AAV capsid protein according to Invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 110, and the different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. [Invention 1132] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 111, and the different brain structure is the brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, substantia nigra, or subthalamic nucleus. [Invention 1133] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 112, and the different brain structure is the brainstem, globus pallidus, putamen, substantia nigra, or thalamus. [Invention 1134] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 113, and the different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, hippocampus, meninges, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. [Invention 1135] A modified AAV capsid protein according to Invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 114, and the different brain structure is the brainstem, cerebral cortex, globus pallidus, meninges, optic nerve, substantia nigra, or thalamus. [Invention 1136] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 115, and the different brain structure is the brainstem or the caudate nucleus. [Invention 1137] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 116, and the different brain structure is the brainstem, caudate nucleus, cerebral cortex, hippocampus, optic nerve, or putamen. [Invention 1138] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 117, and the different brain structure is the brainstem, optic nerve, or substantia nigra. [Invention 1139] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 118, and the different brain structure is the caudate nucleus, ependyma, or meninges. [Invention 1140] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 119, and the different brain structure is the caudate nucleus, cerebellar cortex, ependyma, globus pallidus, meninges, spinal cord, or subthalamic nucleus. [Invention 1141] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 120, and the different brain structure is the caudate nucleus, ependymal globus, meninges, spinal cord, or subthalamic nucleus. [Invention 1142] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 121, and the different brain structure is the caudate nucleus. [Invention 1143] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 122, and the different brain structure is the cerebellar cortex, meninges, spinal cord, or subthalamic nucleus. [Invention 1144] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 123, and the different brain structure is the cerebellar cortex, hippocampus, putamen, or spinal cord. [Invention 1145] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 124, and the different brain structure is the cerebellar cortex. [Invention 1146] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 125, and the different brain structure is the cerebellar cortex, cerebral cortex, hippocampus, or thalamus. [Invention 1147] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 126, and the different brain structure is the cerebral cortex. [Invention 1148] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 127, and the different brain structure is the cerebral cortex or the putamen. [Invention 1149] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 128, and the different brain structure is the ependymia or spinal cord. [Invention 1150] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 129, and the different brain structure is the globus pallidus, hippocampus, optic nerve, or substantia nigra. [Invention 1151] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 130, and the different brain structure is the hippocampus. [Invention 1152] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 131, and the different brain structure is meninges. [Invention 1153] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 132, and the different brain structure is the optic nerve or the subthalamic nucleus. [Invention 1154] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 133, and the different brain structure is the putamen or the thalamus. [Invention 1155] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 134, and the different brain structure is the putamen or the spinal cord. [Invention 1156] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 135, and the different brain structure is the subthalamic nucleus. [Invention 1157] The modified AAV capsid protein of the present invention 1001 or 1015, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 136 or 137, and the different brain structure is the thalamus. [Invention 1158] A nucleic acid comprising a sequence encoding a modified capsid protein according to any of inventions 1001 to 1157. [Invention 1159] Recombinant adeno-associated virus (rAAV) virus comprising a modified capsid protein according to any of invention 1001 to 1157. [Invention 1160] A viral vector comprising a nucleic acid encoding a modified capsid protein according to any of invention 1001 to 1157. [Invention 1161] A viral vector according to the present invention 1160, further comprising a nucleic acid sequence encoding a nucleic acid of interest. [Invention 1162] A viral vector according to the present invention 1161, wherein the nucleic acid of interest is a therapeutic substance. [Invention 1163] The viral vector of the present invention 1162, wherein the therapeutic substance is an enzyme or an RNAi molecule. [Invention 1164] A cell containing any of the viral vectors described in invention 1160 to 1163. [Invention 1165] A mammalian cell, the cell of the present invention 1164. [Invention 1166] Human cells, according to the present invention 1164. [Invention 1167] Cells of the present invention 1164, obtained in vitro. [Invention 1168] Cells of the present invention 1164, obtained in vivo. [Invention 1169] A pharmaceutical composition comprising a viral vector and a pharmaceutically acceptable carrier according to the present invention 1159. [Invention 1170] A method for delivering an active substance to different brain structures of a target, comprising the step of administering the virus of the present invention 1159 to the target. [Invention 1171] A method for delivering an active substance to a target brainstem, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1 to 9, an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 52 to 60, or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110 to 117. [Invention 1172] A method for delivering an active substance to a target tail nucleus, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 7, and 10-16; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 59 and 61-69; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 113, 115, 116, and 118-121. [Invention 1173] A method for delivering an active substance to a target cerebellar cortex, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 4, 9, and 17-21; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 56, 58, 60, and 70-75; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 119, and 122-125. [Invention 1174] A method for delivering an active substance to a target cerebral cortex, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 5, 12, and 21-26; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 58, 60, 62, 63, 66, and 76-79; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 114, 116, and 125-127. [Invention 1175] A method for delivering an active substance to the genitalia of a target, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2-4, 7, 9, 21, 22, 27, and 28; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 60, 62, 63, 66, 74-77, and 80; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 118-120, and 128. [Invention 1176] A method for delivering an active substance to a target globus pallidus, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 3, 5, 12, 14, 16, 21, 22, and 29-31; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 60, 75, and 81-87; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110-112, 114, 119, 120, and 129. [Invention 1177] A method for delivering an active substance to a target hippocampus, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1-4, 7, and 32-34, and 28; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 55, 58, 60, 63, 76, 79, 80, 88, and 89; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 116, 123, 125, 129, and 130. [Invention 1178] A method for delivering an active substance to a target meninges, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 3, 5, 7, 9, 12, 21, and 35-37, and 28; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 58, 60, 66, 73, 76, 80, and 90-93; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 114, 118, 119, 122, and 131. [Invention 1179] A method for delivering an active substance to a target optic nerve, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2, 3, 7, 14-16, 21, 31, and 38; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 54, 57, 58, 60, 75, 79, 87, 88, and 94; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 114, 115, 117, 129, and 132. [Invention 1180] A method for delivering an active substance to a target capsule, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 3, 4, 12, 13, 21, 30, and 39-42; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 55, 59, 60, 61, and 95-100; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 112, 113, 116, 123, 127, 133, and 134. [Invention 1181] A method for delivering an active substance to a target spinal cord, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2-4, 7, 9, 21, 32, 33, and 43; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 53, 58-61, 63, 77, 88, 95, and 101; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 113, 119, 120, 122, 123, 128, and 134. [Invention 1182] A method for delivering an active substance to a target substantia nigra, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2, 3, 9, 44, and 45, and 28; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 52, 53, 57, 58, 75, 76, 87, 102, and 103; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110-114, 117, and 129. [Invention 1183] A method for delivering an active substance to a target subthalamic nucleus, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2-4, 12, 16, 30, 46, and 47; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 57, 58, 60, 75, 79, 87, 88, 102, 104, and 105; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 119, 120, 122, 132, and 135. [Invention 1184] A method for delivering an active substance to a target thalamus, comprising the step of administering an AAV1 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 2, 8, 12, 21, 28, and 48-51; an AAV2 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 52, 55, 56, 74, 85, 88, and 106-109; or an AAV9 virus containing a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 112-114, 125, 133, 136, and 137. [Invention 1185] The method according to any one of the present invention 1170 to 1184, wherein the active substance is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. [Invention 1186] Any method according to invention 1170 to 1184, wherein the administration is directed to the central nervous system. [Invention 1187] The method of the present invention 1186, wherein the administration is directed to the cisterna magna, intraventricular space, ependymia, ventricles, subarachnoid space, cochlea, and / or intramedullary space. [Invention 1188] The method of the present invention 1187, wherein the ventricles are a rostral lateral ventricle and / or caudal lateral ventricle and / or right lateral ventricle and / or left lateral ventricle and / or right rostral lateral ventricle and / or left rostral lateral ventricle and / or right caudal lateral ventricle and / or left caudal lateral ventricle. [Invention 1189] A method according to any of the present invention 1170 to 1188, wherein multiple virus particles are administered. [Invention 1190] The aforementioned virus contains approximately 1 × 10⁶ vector genomes per kilogram. 6 ~Approx. 1×10 18 The method of the present invention 1189, administered in doses of (vg / kg). [Invention 1191] The aforementioned virus is present in approximately 1 x 10⁶ grams per kg of patient. 7 ~1 × 10 17 , about 1×10 8 ~1 × 10 16 , about 1×10 9 ~1 × 10 15 , about 1×10 10 ~1 × 10 14 , about 1×10 10 ~1 × 10 13 , about 1×10 10 ~1 × 10 13 , about 1×10 10 ~1 × 10 11 , about 1×10 11 ~1 × 10 12 , about 1×10 12 ~×10 13 , or approximately 1 x 10 13 ~1 × 10 14 The method of the present invention 1189, administered in individual doses. [Invention 1192] Any method of the present invention 1170 to 1191, wherein the subject is a human. [Invention 1193] A method for treating a disease in a mammal, comprising the step of administering the virus of the present invention 1159 to the mammal. [Invention 1194] The method of the present invention 1193, wherein the disease is a neurodegenerative disease. [Invention 1195] The method of the present invention 1194, wherein the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease. [Invention 1196] The method of the present invention 1193, wherein the mammal is a human. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given only as examples, for various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0028] The following drawings form part of this specification and are provided to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Figure 1] Schematic diagram of the AAV peptide display library. [Figure 2] A schematic diagram of an in vivo screening strategy. [Figure 3] Graph display of input library diversity. Diversity of input viral libraries measured from aliquots of AAV1, AAV2, and AAV9 viral vectors before Round 1 ICV injection. [Figure 4-1] A graph showing round-over-round barcode enrichment. The total number of unique barcodes recovered after round 1 and round 2 enrichment in rhesus macaque for each collected tissue. Round 2 values ​​for DNA and RNA are shown. [Figure 4-2] Refer to the explanation in Figure 4-1. [Figure 4-3] Refer to the explanation in Figure 4-1. [Figure 5-1] Illustration of round-over-round enrichment of barcodes in the cerebellar cortex of AAV1 serotypes. [Figure 5-2] Illustration of round-over-round enrichment of barcodes in the cerebellar cortex under AAV2 serotype. [Figure 5-3] Illustration of round-over-round enrichment of barcodes in AAV9 serotypes of the cerebellar cortex. [Figure 6-1] An illustration of AAV9 1999 enrichment. This is a heatmap depicting barcode enrichment from AAV9, with cells color-coded according to the percentage of barcodes detected in the displayed tissue. Barcodes recovered from DNA are shown on the left, and barcodes recovered from RNA are shown on the right. [Figure 6-2] Refer to the explanation in Figure 6-1. [Figure 7A] Heatmap rendering of opool barcode concentration from AAV1. [Figure 7B] Heatmap rendering of pool barcode enrichment from AAV2. [Figure 7C] Heatmap rendering of pool barcode enrichment from AAV9. [Figure 8] In vivo validation of AAV9 1999 in rhesus monkeys. The eGFP expression construct was packaged into AAV9 1999 driven by a CAG promoter. AAV9 1999 1.5E13 vg was delivered to 5-year-old female rhesus monkeys by ICV injection. Representative images of the H&E-stained cerebellum illustrating the transduction pattern of AAV9 1999 are shown. [Figure 9] Figures 9A-D. In vivo mouse validation of AAV9 1999. The eGFP expression construct was packaged into AAV9 1999 driven by a CAG promoter. AAV9 1999 containing the eGFP construct and the AAV9 capsid were delivered to C57BL / 6 p0 mouse pups by ICV injection at 1E10 vg. Representative images of the eGFP fluorescence signal are whole brain (Figure 9A), whole brain sagittal section (Figure 9B), S1 cortical section (Figure 9C, left), hippocampal section (Figure 9C, center), cerebellar sagittal section (Figure 9C, right), and lumbar coronal section (Figure 9D). [Figure 10A] Fluorescence image of the lateral ventricle of a rhesus monkey in vivo with AAV mixture. [Figure 10B] Fluorescence image of the fourth ventricle of a rhesus monkey in vivo with AAV mixture. [Figure 10C] Fluorescence images of in vivo rhesus monkey meninges in AAV mixture. [Figure 11A]Fluorescence images of cochlear turns after administration of AAV9 1999 capsid containing the eGFP construct to mice. [Figure 11B] Fluorescence images of inner hair cells in mice after administration of AAV9 1999 capsid containing the eGFP construct to the cochlea. [Figure 11C] Fluorescence images of the organ of Corti and distal cochlear axis after administration of AAV9 1999 capsid containing the eGFP construct to the cochlea of ​​mice. [Modes for carrying out the invention]

[0029] Detailed explanation Viral vectors are provided herein, each containing a modified capsid, wherein the modified capsid contains at least one amino acid sequence that causes the viral vector to target a different brain structure. In certain embodiments, the brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. Targeting peptides for each brain structure are provided in Tables 1-3.

[0030] In a particular embodiment, the viral vector is an adeno-associated viral vector (AAV). In a particular embodiment, the AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is provided at SEQ ID NO: 138. An exemplary wild-type reference AAV2 capsid protein sequence is provided at SEQ ID NO: 139. An exemplary wild-type reference AAV9 capsid protein sequence is provided at SEQ ID NO: 140. In a particular aspect, the targeting peptide is inserted at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 141, which shows a targeting peptide insertion after position 590 as SSAX7AS, where the leading SSA and trailing AS are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO: 142, which shows a targeting peptide insertion after position 587 as AAAX7AA, where the leading AAA and trailing AA are linker sequences and X7 represents the targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO: 143, which shows a targeting peptide insertion after position 588 as AAAX7AS, where the leading AAA and trailing AS are linker sequences and X7 represents the targeting peptide.

[0031] (Table 1) AAV1 targeting peptides for each brain structure TIFF0007888221000001.tif52154TIFF0007888221000002.tif227154TIFF0007 888221000003.tif227154TIFF0007888221000004.tif227154TIFF00078882210 00005.tif227154TIFF0007888221000006.tif227154TIFF0007888221000007.t if227154TIFF0007888221000008.tif227154TIFF0007888221000009.tif129154

[0032] (Table 2) AAV2 targeting peptides for each brain structure TIFF0007888221000010.tif83154TIFF0007888221000011.tif222154TIFF0007 888221000012.tif232154TIFF0007888221000013.tif227154TIFF00078882210 00014.tif227154TIFF0007888221000015.tif227154TIFF0007888221000016.t if227154TIFF0007888221000017.tif227154TIFF0007888221000018.tif170154

[0033] (Table 3) AAV9 targeting peptides for each brain structure TIFF0007888221000019.tif42154TIFF0007888221000020.tif227154TIFF0007 888221000021.tif227154TIFF0007888221000022.tif227154TIFF00078882210 00023.tif227154TIFF0007888221000024.tif227154TIFF0007888221000025.t if227154TIFF0007888221000026.tif227154TIFF0007888221000027.tif57154

[0034] (Table 4) AAV1 targeting peptides for various organs TIFF0007888221000028.tif42154

[0035] (Table 5) AAV2 targeting peptides for various organs TIFF0007888221000029.tif217154

[0036] (Table 6) AAV9 targeting peptides for various organs TIFF0007888221000030.tif201154

[0037] I. Adeno-associated virus (AAV) vectors Adeno-associated viruses (AAVs) are small, non-pathogenic viruses belonging to the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, including more than 12 species from humans and primates. AAVs differ from other members of this family in that they rely on helper viruses for replication.

[0038] The AAV genome exists extrachromosomally without being integrated into the host cell genome, has a broad host range, can transduce into both dividing and non-dividing cells in vitro and in vivo, and can maintain high levels of expression of transduced genes. AAV virus particles are thermally stable, resistant to changes in solvents, detergents, pH, and temperature, and can be concentrated by column purification and / or CsCl gradient or other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb genome of AAV consists of one segment of single-stranded DNA with polarity of either positive or negative. Both ends of the genome are short reverse end repeats (ITRs) that can fold into hairpin structures and function as origins for viral DNA replication.

[0039] The AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. AAV particles often contain the AAV genome packaged with the AAV capsid protein. When a recombinant plasmid is used to construct or manufacture a recombinant vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," and while it is crucial for plasmid cloning and amplification—processes necessary for plasmid replication and production—it is not packaged or encapsulated in the viral particle itself. Therefore, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by the AAV capsid protein.

[0040] AAV virions (particles) are unenveloped icosahedral particles with a diameter of approximately 25 nm, containing the AAV capsid. AAV particles exhibit icosahedral symmetry, composed of three related capsid proteins, VP1, VP2, and VP3, which interact with each other to form the capsid. Most native AAV genomes often contain two read frames (ORFs), sometimes referred to as left-hand and right-hand ORFs. The right-hand ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a 1:1:10 ratio, though this ratio can vary, and all originate from the right-hand ORF. The VP1, VP2, and VP3 capsid proteins are distinct from each other due to alternative splicing and the use of unusual start codons. Deletion analysis has shown that the removal or modification of VP1, translated from messages undergoing alternative splicing, results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in impaired production of single-stranded progeny DNA or infectious particles. In a particular embodiment, the genome of an AAV particle encodes one, two, or all three of the VP1, VP2, and VP3 polypeptides.

[0041] Left-hand ORFs often encode non-structural Rep proteins Rep40, Rep52, Rep68, and Rep78, which are involved in the regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins are associated with the preferential inclusion of the AAV genome into a region of arm q of human chromosome 19. Rep68 / 78 has been shown to possess NTP-binding activity in addition to DNA helicase activity and RNA helicase activity. Some Rep proteins have nuclear localization signals, along with several potential phosphorylation sites. In certain embodiments, the genome of an AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of an AAV (e.g., rAAV) does not encode any Rep proteins. In certain embodiments, one or more of the Rep proteins are not included in AAV particles containing nucleic acids encoding polypeptides, as they can be delivered trans.

[0042] The ends of the AAV genome contain short reverse end repeats (ITRs) with the potential to fold into a T-shaped hairpin structure that serves as a starting point for viral DNA replication. Therefore, the AAV genome contains one or more (e.g., a pair) ITR sequences adjacent to the single-stranded viral DNA genome. ITR sequences are often approximately 145 base pairs long. Within the ITR region, two elements considered central to ITR function—the GAGC repeat motif and terminal resolution sites (trs)—have been described. The repeat motif has been shown to bind to Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for site- and strand-specific cleavage at the trs. These two elements, in addition to their role in replication, also appear to be central to viral inclusion. The chromosome 19 integration locus contains a Rep-binding site adjacent to trs. These elements have been shown to be functional and necessary for locus-specific inclusion.

[0043] The term "recombinant" as a modifier for vectors, such as recombinant viral vectors, for example, recombinant lentivirus or recombinant parvovirus (e.g., AAV) vectors, and as a modifier for sequences, such as recombinant nucleic acid sequences or recombinant polypeptides, means that the composition has been manipulated (i.e., engineered) in a way that does not commonly occur in nature. Specific examples of recombinant vectors, such as AAV vectors, retroviral vectors, or lentiviral vectors, are those in which nucleic acid sequences not normally present in the wild-type viral genome have been inserted into the viral genome. An example of a recombinant nucleic acid sequence is one in which a nucleic acid (e.g., a gene) encodes inhibitory RNA cloned into the vector, with or without the 5', 3', and / or intron regions that the gene normally accompanies in the viral genome. The term "recombinant" is not always used herein in relation to vectors such as viral vectors and sequences such as polynucleotides, but the "recombinant" type, including nucleic acid sequences, polynucleotides, and transgenes, is clearly encompassed, even with such omissions.

[0044] Recombinant viral "vectors" are derived from the wild-type genome of a virus by using molecular methods to remove portions of the wild-type genome from the virus and replace them with non-native nucleic acids, such as nucleic acid sequences. Typically, for example in the case of AAV, one or both of the reverse terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. Recombinant viral vectors (e.g., rAAV) are distinguished from the viral genome (e.g., AAV) in that portions of the viral genome are replaced with non-native sequences, such as nucleic acids encoding transactivators, inhibitory RNAs, or therapeutic proteins, compared to the viral genome nucleic acid. Therefore, the incorporation of such non-native nucleic acid sequences defines the viral vector as a "recombinant" vector, and in the case of AAV, it can be called an "rAAV vector."

[0045] In certain embodiments, an AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs adjacent to a nucleic acid sequence encoding a polypeptide having at least some function or activity (i.e., located at the 5' and 3' ends of the nucleic acid sequence, respectively).

[0046] AAV vectors (e.g., rAAV vectors) can be packaged, and in this specification, these are referred to as “AAV particles” for ex vivo, in vitro, or in vivo cell infection (transduction). When recombinant AAV vectors are encapsulated or packaged within AAV particles, these particles may also be referred to as “rAAV particles.” In certain embodiments, an AAV particle is an rAAV particle. An rAAV particle often contains an rAAV vector or a portion thereof. An rAAV particle can be one or more rAAV particles (e.g., multiple AAV particles). An rAAV particle typically contains a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. Note that references to rAAV vectors can also be used to refer to rAAV particles.

[0047] Any suitable AAV particles (e.g., rAAV particles) may be used in the methods or uses described herein. The rAAV particles and / or the genome contained therein may be derived from any suitable serotype or strain of AAV. The rAAV particles and / or the genome contained therein may be derived from two or more serotypes or strains of AAV. Thus, rAAV may contain proteins and / or nucleic acids or portions thereof of any serotype or strain of AAV, and its AAV particles are suitable for infection and / or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0048] In certain embodiments, the plurality of rAAV particles include particles of the same strain or serotype (or subgroup or variant), or particles derived from the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles include a mixture of two or more different (e.g., different serotypes and / or different strains) rAAV particles.

[0049] As used herein, the term “serotype” is a characteristic used to refer to an AAV having a capsid that is serologically different from other AAV serotypes. Serological characteristics are determined based on the absence of cross-reactivity between antibodies against a given AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of an AAV serotype). Even if an AAV variant containing a capsid variant is not serologically different from a reference AAV serotype or other AAV serotypes, they differ from the reference serotype or other AAV serotypes by at least one nucleotide or amino acid residue.

[0050] In a particular embodiment, an rAAV vector based on a first serotype genome corresponds to one or more serotypes of the capsid proteins that package the vector. For example, the serotypes of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome correspond to the serotypes of the capsids that make up the rAAV particles.

[0051] In a particular embodiment, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome derived from one or more serotypes of the AAV capsid proteins that package the vector. For example, the rAAV vector genome may contain nucleic acids (e.g., ITR) derived from AAV2, while at least one or more of the three capsid proteins are derived from different serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotypes or variants thereof.

[0052] In a particular embodiment, the rAAV particle or its vector genome associated with a reference serotype contains or has a polynucleotide, polypeptide, or a subsequence thereof that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof of the polynucleotide, polypeptide, or subsequence thereof that is identical to In a particular embodiment, the rAAV particle or its vector genome associated with a reference serotype contains or has a capsid or ITR sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to the capsid or ITR sequence of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0053] In a particular embodiment, the methods described herein include the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0054] In certain embodiments, the methods herein involve the use, administration, or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particles comprise an AAV2 capsid. In certain embodiments, the rAAV2 particles comprise one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75%, or more identical to the corresponding capsid protein of a native AAV2 particle or a wild-type AAV2 particle, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and up to 100% identical. In a particular embodiment, the rAAV2 particle contains VP1, VP2, and VP3 capsid proteins that are at least 75% or more identical to the corresponding capsid proteins of native AAV2 particles or wild-type AAV2 particles, up to 100% identical, for example, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. In a particular embodiment, the rAAV2 particle is a variant of native AAV2 particles or wild-type AAV2 particles. In some aspects, one or more capsid proteins of AAV2 variants have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of native AAV2 particles or wild-type AAV2 particles.

[0055] In a particular embodiment, the rAAV9 particle contains an AAV9 capsid. In a particular embodiment, the rAAV9 particle contains one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75%, or more identical to the corresponding capsid protein of a native AAV9 particle or a wild-type AAV9 particle, for example, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and up to 100% identical. In a particular embodiment, the rAAV9 particles contain VP1, VP2, and VP3 capsid proteins that are at least 75% or more identical to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles, up to 100% identical, for example, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc. In a particular embodiment, the rAAV9 particles are a variant of native AAV9 particles or wild-type AAV9 particles. In some aspects, one or more capsid proteins of AAV9 variants have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of native AAV9 particles or wild-type AAV9 particles.

[0056] In a particular embodiment, rAAV particles have one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, AAV to the host cell genome). It includes one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITR of a native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, up to 100% identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., as long as they retain the inclusion of DNA and / or packaging, if desired.

[0057] In a particular embodiment, rAAV2 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITRs of native or wild-type AAV2 particles, up to 100% identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., insofar as they retain one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, the inclusion of AAV DNA into the host cell genome, and / or packaging, if desired).

[0058] In a particular embodiment, rAAV9 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical to the corresponding ITRs of native or wild-type AAV2 particles, up to 100% identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., insofar as they retain one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, the inclusion of AAV DNA into the host cell genome, and / or packaging, if desired).

[0059] rAAV particles may contain ITRs having any appropriate number of "GAGC" repeats. In a particular embodiment, the ITR of an AAV2 particle contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In a particular embodiment, an rAAV2 particle contains an ITR containing 3 "GAGC" repeats. In a particular embodiment, an rAAV2 particle contains an ITR having fewer than 4 "GAGC" repeats. In a particular embodiment, an rAAV2 particle contains an ITR having more than 4 "GAGC" repeats. In a particular embodiment, the ITR of an rAAV2 particle contains a Rep-binding site where the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0060] Examples of suitable DNA lengths that can be incorporated into rAAV vectors for packaging / capsidization into rAAV particles can be about 5 kilobases (kb) or less. In certain embodiments, the DNA length may be less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0061] rAAV vectors containing nucleic acid sequences that instruct the expression of RNAi or polypeptides can be prepared using appropriate recombinant techniques known in the art (see, e.g., Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and grown using an AAV viral packaging system. Transducible AAV particles have the ability to bind to and enter mammalian cells and subsequently deliver their nucleic acid cargo (e.g., heterologous genes) to the cell nucleus. Thus, intact transducible rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often non-replicable and require additional protein mechanisms for self-replication. Therefore, rAAV particles configured to transduce mammalian cells are engineered to bind to and enter mammalian cells and deliver nucleic acids to those cells, often located between a pair of AAV ITRs in the rAAV genome.

[0062] Suitable host cells for producing transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be used as recipients of heterologous rAAV vectors or have been used as recipients of heterologous rAAV vectors. A stable human cell line, HEK293 (e.g., readily available from the American Type Culture Collection under accession number ATCC CRL1573), can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing adenovirus E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly favorable platform for producing rAAV particles. Methods for producing high-titer AAV particles capable of transducing into mammalian cells are known in the art. For example, AAV particles can be produced as described in Wright, 2008 and Wright, 2009.

[0063] In certain embodiments, AAV helper function is introduced into host cells by transfecting the host cells with an AAV helper construct either before or concurrently with the transfection of the AAV expression vector. Thus, in some cases, AAV helper constructs are used to express, at least transiently, the rep and / or cap genes of AAV, in order to complement the missing AAV function required for productive AAV transduction. AAV helper constructs often lack an AAV ITR and are unable to replicate or package themselves. These constructs can take the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, including the commonly used plasmids pAAV / Ad and pIM29+45, which encode both the rep and cap expression products. Several other vectors encoding both the rep and / or cap expression products are also known.

[0064] An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence along with essential regulatory regions necessary for its expression in a host cell. An expression vector may contain at least an origin of replication for intracellular proliferation, and optionally additional elements such as heterologous nucleic acid sequences, expression regulatory elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0065] II. Therapeutic Substances In some embodiments, nucleic acids can be introduced into mammalian cells or target tissues using viral gene transfer methods. Using such methods, nucleic acids encoding inhibitory RNA, non-coding RNA, and / or therapeutic proteins can be administered to cells in culture or in a host organism.

[0066] A. Inhibitory RNA RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA, resulting in sequence-specific inhibition of gene expression.

[0067] "Inhibitory RNA," "RNAi," "small interfering RNA," or "short interfering RNA," or "siRNA" molecule, "short hairpin RNA," or "shRNA" molecule, or "miRNA" is an RNA double helix of nucleotides that targets a nucleic acid sequence of interest. As used herein, the term "siRNA" is a general term encompassing a subset of shRNA and miRNA. "RNA double helix" refers to a structure formed by the complementary pairing of two regions of an RNA molecule. Since the nucleotide sequence of the double helix portion of siRNA is complementary to the nucleotide sequence of the target gene, siRNA "targets" that gene. In certain embodiments, siRNA targets the sequence encoding Huntington. In some embodiments, the length of the siRNA double helix is ​​less than 30 base pairs. In some embodiments, the double helix can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs long. In some embodiments, the length of the double helix is ​​19–25 base pairs. In certain embodiments, the length of the double helix is ​​19 base pairs or 21 base pairs. The RNA double helix portion of siRNA can be part of a hairpin structure. In addition to the double helix portion, the hairpin structure includes a loop portion located between the two sequences that form the double helix. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In certain embodiments, the loop is 18 nucleotides long. The hairpin structure can also include a 3' overhang and / or a 5' overhang. In some embodiments, the overhang is a 3' overhang and / or a 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides long.

[0068] shRNAs consist of a stem-loop structure designed to include a 5' facile region, an siRNA region segment, a loop region, a 3' siRNA region, and another 3' facile region. Most RNAi expression strategies have utilized short hairpin RNAs (shRNAs) driven by potent polIII promoters. While many shRNAs exhibit effective knockdown of target sequences both in vitro and in vivo, some shRNAs that effectively knock down target genes have also been found to be toxic in vivo.

[0069] miRNAs are small cellular RNAs (approximately 22 nt) processed from precursor stem-loop transcripts. Known miRNA stem-loops can be modified to contain RNAi sequences specific to the gene of interest. miRNA molecules may be preferred over shRNA molecules because they are endogenously expressed. Therefore, miRNA molecules are less likely to induce the dsRNA-responsive interferon pathway, are processed more efficiently than shRNA, and have been shown to silencing up to 80% more effectively than shRNA.

[0070] A recently discovered alternative approach is the use of artificial miRNAs (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more naturally similar to endogenous RNAi substrates and are better suited to Pol-II transcription (e.g., enabling tissue-specific expression of RNAi) and polycistronic strategies (e.g., enabling delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, incorporated herein by reference.

[0071] The transcription unit of "shRNA" consists of a sense sequence and an antisense sequence linked by an unpaired nucleotide loop. shRNA is exported from the nucleus by exportin-5 and, upon entering the cytoplasm, is processed by Dicer to produce functional siRNA. The stem-loop of "miRNA" consists of a sense sequence and an antisense sequence linked by an unpaired nucleotide loop. This is typically expressed as part of a larger primary transcript (pri-miRNA), which is then cleaved by the Droscha-DGCR8 complex to produce an intermediate known as pre-miRNA, which is then exported from the nucleus by exportin-5 and, upon entering the cytoplasm, is processed by Dicer to produce functional siRNA. Where used interchangeably in this specification, “artificial miRNA” or “artificial miRNA shuttle vector” refers to a primary miRNA transcript in which a double-stranded stem-loop region (at least about 9–20 nucleotides) excised by drotherapeutic and dicer processing is replaced with an siRNA sequence for a target gene, while retaining the structural elements within the stem-loop necessary for effective drotherapeutic processing. The term “artificial” derives from the fact that the adjacent sequences (approximately 35 nucleotides upstream and approximately 40 nucleotides downstream) originate from restriction enzyme sites within the multi-cloning site of the siRNA. Where used in this specification, the term “miRNA” encompasses both natural miRNA sequences and artificially constructed miRNA shuttle vectors.

[0072] siRNA can be encoded by a nucleic acid sequence, which may also include a promoter. This nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a 6T sequence.

[0073] Several factors must be considered in RNAi design, including the properties of the siRNA, the persistence of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA introduced into the organism typically contains an exonic sequence. Furthermore, since the RNAi process is homology-dependent, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, 85%, 90%, 95%, or even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with less than approximately 80% identity to the target gene are considerably less effective. Therefore, the higher the homology between the siRNA and the gene to be inhibited, the less likely it is that the expression of unrelated genes will be affected.

[0074] In addition, the size of the siRNA is also an important consideration. In some embodiments, the present invention relates to an siRNA molecule comprising at least about 19 to 25 nucleotides and capable of regulating gene expression. With respect to the present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is about 19 to about 25 nucleotides in length.

[0075] An siRNA target generally refers to a polynucleotide containing a polypeptide-coding region, or a polynucleotide containing a polynucleotide region that modulates replication, transcription, translation, or other processes important for polypeptide expression, or a polynucleotide containing both a polypeptide-coding region and a functionally linked region that modulates expression. Any gene expressed in a cell can be targeted. Preferably, the target gene is involved in or associated with the progression of cellular activity important for disease, or the progression of cellular activity of particular interest as a research subject.

[0076] B. Non-coding RNA As demonstrated by cDNA cloning projects and genome tiling arrays, over 90% of the human genome is transcribed but does not code for proteins. These transcripts are called non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNA, transfer RNA, competitive endogenous RNA (ceRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are essential for cellular function. Similarly, numerous short ncRNAs, such as microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that exhibit cell-type-specific expression and localize to specific intracellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the view that they were selected during evolution.

[0077] LncRNAs appear to have many different functions. Often, they seem to play a role in regulating protein activity or localization, or functioning as an organizational framework for intracellular structures. In other cases, lncRNAs may be processed to produce multiple small RNAs, or modulate the processing methods of other RNAs. The latest version of data generated by the public research consortium GenCode (version 27) catalogs slightly less than 16,000 lncRNAs in the human genome, generating nearly 28,000 transcripts; if other databases are included, more than 40,000 lncRNAs are known.

[0078] Interestingly, lncRNAs can influence the expression of specific target proteins at particular genomic loci, modulate the activity of protein-binding partners, induce chromatin modification complexes at their sites of action, and undergo post-transcriptional processing to generate numerous 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0079] There is growing evidence suggesting that abnormally expressed lncRNAs play crucial roles in normal physiological processes and multiple disease states. lncRNAs are misregulated in a variety of diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation has also been observed in various types of cancer, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Some lncRNAs, such as gadd74 and lncRNA-RoR5, modulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thus providing greater flexibility and robustness to cell cycle progression. Furthermore, some lncRNAs are involved in mitotic processes, such as centromere satellite RNA, which is essential for kinetochore formation and therefore crucial for chromosome segregation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, regulates M-phase exit by functioning in cis and repressing the expression of the adjacent gene Pura, a regulator of cell proliferation.

[0080] lncRNAs are generally defined as transcripts longer than 200 nucleotides (e.g., approximately 200–1200 nt, approximately 2500 nt, or more) that lack an extended reading frame (ORF). The term "non-coding RNA" (ncRNA) includes both lncRNAs and shorter transcripts less than approximately 200 nt, such as approximately 30–200 nt.

[0081] Therefore, in some embodiments, delivery of ncRNA to a specific brain structure of interest, for example, corrects abnormal RNA expression levels or modulates disease-causing lncRNA levels. Therefore, in some embodiments, the present invention provides rAAV in which the viral genome is engineered to encode therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) of about 200 nucleotides (nt) or longer. In some embodiments, the therapeutic substance is an ncRNA of about 25 nt or about 30 nt to about 200 nt in length. In some embodiments, the lncRNA is of about 200 nt to about 1,200 nt in length. In some embodiments, the lncRNA is of about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nt in length.

[0082] C. CRISPR system Gene editing is a technology that enables the modification of target genes within living cells. In recent years, the implementation of on-demand gene editing using the bacterial CRISPR immune system has revolutionized how scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be relatively easily engineered to target new sites by altering its guide RNA sequence. This discovery has made sequence-specific gene editing functionally viable.

[0083] Generally, the “CRISPR system” refers collectively to the transcripts and other elements involved in the expression or orientation of the activity of CRISPR-related ("Cas") genes, such as the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (including “direct repeats” and, in the case of endogenous CRISPR systems, partial direct repeats processed by tracrRNA), the guide sequence (also called a “spacer” in the case of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0084] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that binds sequence-specifically to DNA, and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, and may be derived from certain organisms containing an endogenous CRISPR system, such as Streptococcus pyogenes.

[0085] The CRISPR system can induce double-stranded breaks (DSBs) and subsequent fragmentation at target sites, as discussed herein. In another embodiment, a Cas9 variant considered a "nickase" is used to introduce a nick into a single strand at a target site. For example, to improve specificity, a pair of nickases can be used, each directed by a pair of different gRNAs that target the sequence such that a 5' overhang is introduced when the nicks are introduced simultaneously. In another embodiment, a catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcription repressor (e.g., KRAB) or transcription activator, to affect gene expression. Alternatively, a CRISPR system with catalytically inactive Cas9 further includes a transcription repressor or transcription activator fused to a ribosomal binding protein.

[0086] In some cases, Cas nucleases and gRNAs (including fusions of target-specific crRNA and immutable tracrRNA) are introduced into cells. Generally, the target site at the 5' end of the gRNA uses complementary base pairing to allow the Cas nuclease to target the target site, such as a gene. The target site may be selected based on its location, such as immediately 5' to a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA targets the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" refers to a sequence designed so that a guide sequence is complementary to it, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex.

[0087] The target sequence may contain any polynucleotide, such as DNA polynucleotides or RNA polynucleotides. The target sequence may be located within the cell nucleus or cytoplasm, such as within a cell organelle. Generally, a sequence or template that can be used for recombination to a target gene locus containing the target sequence is called an “editing template,” “edited polynucleotide,” or “editing sequence.” In some contexts, an exogenous template polynucleotide may be called an editing template. In some contexts, the recombination is homologous recombination.

[0088] Typically, in endogenous CRISPR systems, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and forms a complex with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs, or more, from the target sequence). Tracr sequences that may contain or consist of all or part of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides from a wild-type tracr sequence, or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides) may also form part of a CRISPR complex, for example, by hybridization to all or part of a tracr mate sequence functionally linked to a guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex, for example, having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity across the entire length of the tracr mate sequence when optimally aligned.

[0089] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into cells so that the expression of those elements of the CRISPR system instructs the formation of a CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence can be functionally linked to separate regulatory elements on separate vectors. The Cas enzyme may be a target gene regulated by a regulatory alternative splicing event disclosed herein, either as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA may be regulated by a constitutive promoter.

[0090] Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector to provide any component of the CRISPR system that is not included in the first vector, with one or more additional vectors. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also called “cloning sites”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. By using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple corresponding target sequences within a cell.

[0091] Vectors may contain regulatory elements functionally linked to enzyme-coding sequences that encode CRISPR enzymes such as Cas proteins. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. Examples include Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are well known. For example, the amino acid sequence of the *Streptococcus pyogenes* Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0092] The CRISPR enzyme can be Cas9 (e.g., derived from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumonia)). The CRISPR enzyme can instruct the cleavage of one or both strands at a location within a target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. A vector can encode a CRISPR enzyme in which the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence, compared to the corresponding wild-type enzyme. For example, the substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 derived from Streptococcus pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (which cleaves one strand). In some embodiments, the Cas9 nickase can be used in combination with guide sequences, for example, two guide sequences targeting the sense and antisense strands of a DNA target, respectively. This combination allows for nicking both strands and using them to induce either NHEJ or HDR.

[0093] In some embodiments, the enzyme-coding sequences encoding CRISPR enzymes are codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells are those of, or may originate from, specific organisms, including, but not limited to, humans, mice, rats, rabbits, dogs, or mammals, such as non-human primates. Generally, codon optimization is the process of modifying a nucleic acid sequence by replacing at least one codon in the native sequence with a codon that is more frequently used or most frequently used in the host cell's gene, while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit specific biases for certain codons of specific amino acids. Codon bias (differences in codon usage frequency between organisms) is often thought to correlate with messenger RNA (mRNA) translation efficiency, and that this depends, above all, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of a selected tRNA in a given cell is generally a reflection of the codon most frequently used in peptide synthesis. Therefore, genes can be adapted to the optimal gene expression in a given organism based on codon optimization.

[0094] Generally, the guide sequence is any polynucleotide sequence that has sufficient complementarity to the target polynucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher, or greater than approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or higher, when optimally aligned using a suitable alignment algorithm.

[0095] The optimal alignment can be determined using any suitable algorithm for aligning sequences, and non-restrictive examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, California), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0096] A CRISPR enzyme may be part of a fusion protein containing one or more heterologous protein domains. A CRISPR enzyme fusion protein may include any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or fragments of such proteins that bind to DNA molecules or other cellular molecules, such as, but are not limited to, maltose-binding protein (MBP), S-tags, Lex A DNA-binding domain (DBD) fusions, GAL4A DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Further domains that may form part of a fusion protein containing a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0097] D. Therapeutic proteins Some embodiments relate to the expression of recombinant proteins and recombinant polypeptides. In some aspects, proteins or polypeptides may be modified to increase serum stability. Therefore, when this application refers to the function or activity of a “modified protein” or “modified polypeptide,” it will be understood by those skilled in the art that this includes proteins or polypeptides that have additional advantages compared to, for example, unmodified proteins or polypeptides. It is particularly conceivable that embodiments relating to “modified proteins” can also be carried out for “modified polypeptides,” and vice versa.

[0098] Recombinant proteins may contain amino acid deletions and / or substitutions. Therefore, proteins with deletions, proteins with substitutions, and proteins with both deletions and substitutions are modified proteins. In some embodiments, these proteins may further contain inserted or added amino acids, such as fusion proteins or proteins with linkers. A "modified deletion protein" lacks one or more residues of the native protein but may retain the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one in which amino acid residues are deleted from at least one antigenic region, i.e., from a region of the protein that has been determined to be antigenic in a particular organism, for example, in the organism to which the modified protein is administered.

[0099] Substitutional variants, or replacement variants, typically involve the exchange of one amino acid for another at one or more sites within a protein and may be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. The substitution may be a conservative substitution, i.e., a substitution in which one amino acid is replaced with one having a similar shape and charge, or it may not. Conservative substitutions are well known in the art and include, for example, the substitution of alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine.

[0100] In addition to deletions or substitutions, modified proteins can harbor residue insertions. This typically involves the addition of at least one residue in a polypeptide. This can include the insertion of a targeting peptide or targeting polypeptide, or simply a single residue. Terminal additions, known as fusion proteins, will be discussed later.

[0101] The term “biologically functional equivalent” is well understood in the art and will be further elaborated herein. Therefore, sequences in which approximately 70%–80%, 81%–90%, or even 91%–99% of the amino acids are identical or functionally equivalent to those of a control polypeptide are included, provided that the biological activity of the protein is maintained. Recombinant proteins can be, in certain aspects, biologically functional equivalents to their corresponding native proteins.

[0102] It will be understood that amino acid sequences and nucleic acid sequences may include additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, and that such sequences are still essentially as described in one of the sequences disclosed herein, provided that they satisfy the aforementioned criteria, including the maintenance of biological protein activity, if protein expression is involved. Terminal additions are particularly applicable to nucleic acid sequences that may include various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences known to exist within a gene, i.e., introns.

[0103] As used herein, protein or peptide generally refers to proteins translated from genes, comprising more than approximately 200 amino acids, up to a full-length sequence; polypeptides exceeding approximately 100 amino acids; and / or peptides ranging from approximately 3 to approximately 100 amino acids, but not limited to these. For convenience, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein.

[0104] As used herein, “amino acid residue” means any natural amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are continuous and do not have any non-amino acid sequences that interrupt the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0105] Therefore, the term “protein or peptide” encompasses an amino acid sequence that includes at least one of the 20 common amino acids found in natural proteins, or at least one modified or abnormal amino acid.

[0106] A particular aspect of the present invention relates to fusion proteins. These molecules may have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, the fusion may also use a leader sequence from another species to enable recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, which is preferably cleavable to facilitate protein purification. Non-limiting examples of affinity tags include polyhistidines, chitin-binding proteins (CBPs), maltose-binding proteins (MBPs), and glutathione-S-transferase (GST).

[0107] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of a complete fusion protein, or by attachment of DNA sequences encoding heterologous domains, followed by expression of an intact fusion protein.

[0108] The production of fusion proteins that restore the functional activity of parent proteins can be facilitated by linking genes with cross-linking DNA segments that encode peptide linkers, which are joined between tandem polypeptides. The linkers are of sufficient length to allow for the proper folding of the resulting fusion protein.

[0109] III. Method of Administration Viral vectors may, in some cases, be administered directly to the patient (in vivo), or they may be used to treat cells in vitro or ex vivo before being administered to the patient. The term “vector” refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other medium that can be manipulated by insertion or incorporation of nucleic acids. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acids into cells so that the nucleic acid sequence within the nucleic acid is transcribed by the cell and subsequently translated, if it codes for a protein.

[0110] Any suitable cells or mammals can be administered or treated by the methods or uses described herein. Typically, mammals requiring the methods described herein are those that have or are suspected of having abnormal proteins or abnormal proteins associated with a disease condition. Alternatively, a mammalian recipient may have a condition suitable for gene replacement therapy. As used herein, “gene replacement therapy” means the administration of exogenous genetic material encoding a therapeutic substance to a recipient and the subsequent in situ expression of the administered genetic material. Thus, the phrase “condition suitable for gene replacement therapy” encompasses conditions such as hereditary diseases (i.e., disease conditions resulting from the deletion of one or more genes), acquired conditions (i.e., pathological conditions not resulting from congenital defects), cancer, and prophylactic processes (i.e., prevention of disease or undesirable medical conditions). Thus, as used herein, the term “therapeutic substance” means any active substance or material that has a beneficial effect on a mammalian recipient. Thus, “therapeutic substance” encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0111] Non-limiting examples of mammals include humans, non-human primates (apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, a mammal is a human. In certain embodiments, a mammal is a non-rodent mammal (e.g., humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, a non-rodent mammal is a human. A mammal can be of any age or developmental stage (e.g., adult, teenager, child, infant, or mammal in utero). A mammal can be male or female. In certain embodiments, a mammal can be an animal disease model, such as an animal model having or expressing an abnormal protein or abnormal protein associated with a disease condition, or an animal model in which protein expression is insufficient and causes disease.

[0112] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years or older) and children (under 18 years). Adults include the elderly. Typical adults are 50 years or older. Children's ages range from 1–2 years or 2–4 years, 4–6 years, 6–18 years, 8–10 years, 10–12 years, 12–15 years, and 15–18 years. Children also include infants. Infants are typically in the range of 1–12 months of age.

[0113] In certain embodiments, the method includes the step of administering a plurality of viral particles to a mammal, as described herein, to reduce, mitigate, prevent, inhibit, or delay the severity, frequency, progression, or onset time of one or more symptoms of a disease condition, such as a neurodegenerative disease. In certain embodiments, the method includes the step of administering a plurality of viral particles to a mammal to treat adverse symptoms of a disease condition, such as a neurodegenerative disease. In certain embodiments, the method includes the step of administering a plurality of viral particles to a mammal to stabilize, delay, or prevent the worsening, progression, or reversal of a disease condition, such as a neurodegenerative disease, and adverse symptoms.

[0114] In a particular embodiment, the method, as described herein, includes the step of administering a plurality of viral particles to the central nervous system or a portion thereof, thereby reducing, mitigating, preventing, inhibiting, or delaying by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days the severity, frequency, progression, or time of onset of one or more symptoms of a disease state such as a neurodegenerative disease.

[0115] In certain embodiments, symptoms or adverse effects include early, middle, or late symptoms, behavioral symptoms, personality or language symptoms, swallowing, motor, seizure, tremor or restlessness symptoms, ataxia, and / or cognitive symptoms such as memory or the ability to organize.

[0116] IV. Pharmaceutical Compositions As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a bioacceptable composition, formulation, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or in vivo contact. A “pharmaceutically acceptable” composition or a “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Such compositions, “pharmaceutically acceptable” and “physiologically acceptable” formulations and compositions can be sterile. Such pharmaceutical formulations and compositions can be used, for example, when administering viral particles to a subject.

[0117] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspensions, coatings, isotonic and absorption enhancers or absorption retarders, suitable for pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickeners. Auxiliary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) may also be incorporated into formulations and compositions.

[0118] Pharmaceutical compositions typically contain pharmaceutically acceptable excipients. Such excipients include any pharmaceutically active substance that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered and can be administered without excessive toxicity. Examples of pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline solution, glycerol, and ethanol. These may include pharmaceutically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate, and salts of organic acids such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances, such as surfactants, wetting agents or emulsifiers, and pH buffers, may also be present in such media.

[0119] Pharmaceutical compositions can be formulated to conform to specific routes of administration or delivery described herein or known to those skilled in the art. Accordingly, pharmaceutical compositions may include carriers, diluents, or excipients suitable for administration or delivery via various routes.

[0120] Suitable pharmaceutical forms for injecting viral particles include sterile aqueous solutions or dispersions adapted for immediate preparation of sterile injectable or injectable solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be a sterile fluid and stable under manufacturing, use, and storage conditions. Liquid carriers or media can be solvents or liquid dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by liposome formation, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Isotonic agents, such as sugars, buffers, or salts (e.g., sodium chloride), may be included. Sustained absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0121] A solution or suspension of virus particles may optionally contain one or more of the following components: sterile diluents, e.g., water for injection; saline solutions, e.g., phosphate-buffered saline (PBS); artificial CSF; surfactants; fixatives; polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol); glycerin; or other synthetic solvents; antimicrobial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose.

[0122] The compositions, methods, and pharmaceutical formulations, compositions, and delivery systems suitable for use of the present invention are known in the art (for example, Remington: The Science and Practice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., See Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253–315.

[0123] Viral particles and their compositions may be formulated into dosage unit forms to facilitate administration and ensure uniformity of dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable as a unit dose for the treated individual, each unit containing a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, along with the necessary pharmaceutical carrier. The dosage unit form depends on the number of viral particles deemed necessary to produce the desired effect. The required amount may be prescribed as a single dose or in multiple dosage units. The dose may be adjusted to an appropriate viral particle concentration and optionally combined with an anti-inflammatory agent, and packaged for use.

[0124] In one embodiment, the pharmaceutical composition would contain sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or improve the symptoms or adverse effects of the disease condition in question, or an amount sufficient to provide a desired benefit.

[0125] As used herein, "unit dosage form" refers to a physically discrete unit suitable as a unit dose to be administered to a subject to be treated, each unit containing a predetermined quantity calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered one or more times, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle or filler). The unit dosage form may be, for example, in ampoules and vials containing a liquid composition, or a composition in a lyophilized or freeze-dried state, and a sterile liquid carrier can be added thereto, for example, prior to in vivo administration or in vivo delivery. Individual unit dosage forms can be included in a multi-dose kit or container. Thus, for example, virus particles, and their pharmaceutical compositions, can be packaged in single or multiple unit dosage forms to facilitate administration and to provide uniform dosage.

[0126] Formulations containing virus particles typically contain an effective amount, which can be readily determined by one of ordinary skill in the art. The virus particles can typically be in the range of about 1% to about 95% (w / w) of the composition, or higher if appropriate. The amount to be administered depends on factors such as the age, weight and health status of the mammalian or human subject to which the treatment is contemplated. One of ordinary skill in the art can establish an effective dosage by routine testing to establish a dose-response curve.

[0127] V. Definitions The terms "polynucleotide", "nucleic acid" and "transgene" are used interchangeably herein to refer to any form of nucleic acid, oligonucleotide, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or inhibitory RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides can include natural, synthetic, and intentionally modified or engineered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded or triple-stranded, linear or circular, and can be of any suitable length. In discussions of polynucleotides, the sequence or structure of a particular polynucleotide can be described herein according to the convention of describing sequences in the 5' to 3' direction.

[0128] Nucleic acids encoding polypeptides often contain a reading frame encoding the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0129] A nucleic acid can include one or more expression control elements or expression regulatory elements operably linked to a reading frame, and the one or more regulatory elements are configured to direct transcription and translation of the polypeptide encoded by the reading frame in mammalian cells. Non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, TATA box, etc.), translation initiation sequences, mRNA stability sequences, polyA sequences, secretion sequences, etc. Expression control / regulatory sequences can be obtained from the genome of any suitable organism.

[0130] A "promoter" is a nucleotide sequence that is usually located upstream (5') of the coding sequence and directs and / or controls the expression of the coding sequence by providing a recognition site to RNA polymerase and other factors necessary for proper transcription. A pol II promoter includes a minimal promoter, which is a short DNA sequence consisting of a TATA box and optionally other sequences that specify the transcription start site, to which regulatory elements are added to control expression. A type 1 pol III promoter includes three cis-acting sequence elements downstream of the transcription start site: a) 5' sequence element (A block); b) intermediate sequence element (I block); c) 3' sequence element (C block). A type 2 pol III promoter includes two essential cis-acting sequence elements downstream of the transcription start site: a) A box (5' sequence element); and b) B box (3' sequence element). A type 3 pol III promoter includes several cis-acting promoter elements upstream of the transcription start site, such as the conventional TATA box, proximal sequence element (PSE), and distal sequence element (DSE).

[0131] An "enhancer" is a DNA sequence that can stimulate transcriptional activity and enhance the level of expression or the tissue specificity of expression. It can be a promoter-specific or heterogeneous element. It can act in either direction (5'→3' or 3'→5') and has the ability to function whether it is placed upstream or downstream of the promoter.

[0132] Promoters and / or enhancers may be entirely derived from native genes, composed of different elements derived from different elements found in nature, or even composed of synthetic DNA segments. Promoters or enhancers may include DNA sequences involved in the binding of protein factors that modulate / regulate the effectiveness of transcription initiation in response to stimuli, physiological conditions, or developmental conditions.

[0133] Non-limiting examples of promoters include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV pre-early promoter region (CMVIE), the Roussarcoma virus (RSV) promoter, the pol II promoter, the pol III promoter, synthetic promoters, and hybrid promoters. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, are also considered useful here. Exemplary constitutive promoters include promoters of the following genes that encode certain constitutive or "housekeeping" functions, and other constitutive promoters known to those skilled in the art: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, and U6. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, long-terminal repeats (LTRs) of Moloney's leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In addition, sequences derived from intronic miRNA promoters, such as the miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566, and miR128 promoters, are also expected to find applications in this specification (see, for example, Monteys et al., 2010). Thus, any of the above-mentioned constitutive promoters can be used to control the transcription of heterologous gene insertion fragments.

[0134] In this specification, "transgene" is used for convenience to mean a nucleic acid sequence / polynucleotide that is introduced into or has been introduced into a cell or organism. Transgenes include genes encoding any nucleic acid, such as inhibitory RNA or polypeptide or protein, which are generally heterologous to the natural AAV genome sequence.

[0135] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by a viral particle can be called "transduction" of a cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or recipient organism, it can be stably maintained in that cell or organism and further transmitted or inherited by the progeny cells or progeny organisms of the recipient cell or recipient organism. Finally, the introduced transgene may exist extrachromosomally in the recipient cell or recipient host organism, or may exist only transiently. Therefore, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a transgene has been introduced or its progeny. Transduced cells can be grown, the transgene transcribed, and the inhibitory RNA or protein that encodes it can be expressed. In the case of use in gene therapy and gene therapy methods, transduced cells can exist in mammals.

[0136] Transgenes regulated by inductive promoters are expressed only in the presence of an inducer, or more strongly in the presence of an inducer (for example, transcription regulated by a metallothionein promoter is significantly increased in the presence of certain metal ions). Inductive promoters contain a responsive element (RE) that stimulates transcription when bound to a particular inducer. Examples include REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing a specific RE can be selected to obtain an inductive response, and in some cases, the RE itself may be bound to a different promoter to confer inductivity to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive or inductive promoter, strong or weak promoter), it is possible to control both the presence and expression level of polypeptides in genetically modified cells. When a polypeptide-encoding gene is regulated by an inductive promoter, in-situ delivery of the polypeptide is triggered by exposing the genetically modified cell in situ to conditions that allow polypeptide transcription, for example, by intraperitoneal injection of a specific inducer of the inductive promoter that controls the transcription of the active ingredient. For example, in-situ expression of polypeptides encoded by genes controlled by the metallothionein promoter in genetically modified cells can be enhanced by contacting the genetically modified cells in situ with a solution containing appropriate (i.e., inducible) metal ions.

[0137] A nucleic acid / transgene is "functionally linked" when it is placed in a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding an RNAi or polypeptide, or a nucleic acid that directs the expression of a polypeptide, may contain an inducible promoter or a tissue-specific promoter to control the transcription of the encoded polypeptide. A nucleic acid functionally linked to an expression regulatory element can also be called an expression cassette.

[0138] In certain embodiments, the methods and uses described herein utilize CNS-specific or inducible promoters, enhancers, etc. Non-limiting examples of CNS-specific promoters include those isolated from myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) genes. Non-limiting examples of inducible promoters include DNA-responsive elements to ecdysone, tetracycline, hypoxia, and IFN.

[0139] In certain embodiments, the expression regulatory element includes a CMV enhancer. In certain embodiments, the expression regulatory element includes a β-actin promoter. In certain embodiments, the expression regulatory element includes a chicken β-actin promoter. In certain embodiments, the expression regulatory element includes a CMV enhancer and a chicken β-actin promoter.

[0140] As used herein, the terms “modified” or “variant” and their grammatical variants mean that a nucleic acid, polypeptide, or subsequence thereof deviates from a reference sequence. Therefore, modified and variant sequences may have substantially the same, greater, or less than the reference sequence in terms of expression level, activity, or function, but retain at least partially the activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene that has mutations such as, for example, missense or nonsense mutations.

[0141] A "nucleic acid" variant or "polynucleotide" variant refers to a modified sequence in which the gene is altered compared to the wild type. A sequence can be genetically modified without altering the protein sequence it codes for. Alternatively, a sequence can be genetically modified to code for a variant protein. A nucleic acid variant or polynucleotide variant can also refer to a combination sequence that has been codon-modified to code for a protein that still maintains at least partial sequence identity with respect to a reference sequence such as a wild-type protein sequence, and also has been codon-modified to code for a variant protein. For example, some codons in such a nucleic acid variant may be altered without altering the amino acids of the protein they code for, while other codons in the nucleic acid variant may be altered, resulting in alterations of the amino acids of the protein they code for.

[0142] The terms “protein” and “polypeptide” are used interchangeably herein. “Polypeptides” encoded by “nucleic acids” or “polynucleotides” or “transgenes” disclosed herein include partial or full-length native sequences, as well as naturally occurring wild-type and functional polymorphic proteins, their functional subsequences (fragments), and their sequence variants, insofar as the polypeptide retains some degree of function or activity. Therefore, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences do not need to be identical to deficient endogenous proteins, or endogenous proteins whose activity, function, or expression is insufficient, deficient, or absent in the mammal being treated.

[0143] Non-limiting examples of modifications include the substitution of one or more nucleotides or amino acids (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000 or more nucleotides or residues).

[0144] Examples of amino acid modifications include conservative amino acid substitutions or deletions. In certain embodiments, the modified or variant sequence retains at least some of the function or activity of the unmodified sequence (e.g., the wild-type sequence).

[0145] Another example of amino acid modification is targeting peptides introduced into the capsid protein of viral particles. Peptides have been identified that allow recombinant viral vectors to target the central nervous system, for example, different brain regions.

[0146] Such modified recombinant viruses may preferentially bind to certain types of tissue (e.g., CNS tissue) over other types of tissue (e.g., liver tissue). In certain embodiments, recombinant viruses possessing modified capsid proteins can "target" cerebral vascular epithelial tissue by binding at higher levels than comparable unmodified capsid proteins. For example, recombinant viruses with modified capsid proteins may bind to cerebral vascular epithelial tissue at 50% to 100% higher levels than unmodified recombinant viruses.

[0147] A “nucleic acid fragment” is a portion of a given nucleic acid molecule. In most organisms, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA to proteins. Fragments and variants of nucleotide sequences disclosed, as well as the proteins or partial-length proteins encoded thereby, are also included in the present invention. A “fragment” or “part” means a full-length or less-than-full-length nucleotide sequence or amino acid sequence of a polypeptide or protein that encodes a polypeptide or protein. In certain embodiments, a fragment or part is biologically functional (i.e., retaining 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0148] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. In the case of nucleotide sequences, a variant includes sequences that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Such naturally occurring allelic variants can be identified using molecular biology techniques such as polymerase chain reaction (PCR) and hybridization. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those encoding native proteins and polypeptides with amino acid substitutions, which are produced using targeted mutagenesis. Generally, the nucleotide sequence variants of the present invention have sequence identity of at least 40%, 50%, 60%, to 70%, for example, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, and generally at least 80%, for example, 81% to 84%, and at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% with respect to the native (endogenous) nucleotide sequence. In a particular mode, the variant is biologically functional (i.e., retaining 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the wild-type activity or function).

[0149] "Conservative substitution" of a particular nucleic acid sequence refers to a nucleic acid sequence that encodes an amino acid sequence that is identical or essentially identical. Because the genetic code is degenerate, any given polypeptide is encoded by a number of functionally identical nucleic acids. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, anywhere the codon specifies arginine, the codon can be changed to any of the described corresponding codons without changing the encoded protein. Such nucleic acid variations are "silent variations" and are a type of "conservatively modified variation." Each of the nucleic acid sequences described herein that encode a polypeptide represents all possible silent variations, unless otherwise noted. It will be apparent to one of ordinary skill in the art that each codon in a nucleic acid (except for the ATG, which is usually the only methionine codon) can be modified by standard techniques to yield a functionally identical molecule. Thus, each "silent variation" of a nucleic acid encoding a polypeptide is implicitly represented by each described sequence.

[0150] The term "substantial identity" of a polynucleotide sequence means that the polynucleotide contains sequences that have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% sequence identity when compared to a reference sequence using one of the alignment programs described with standard parameters, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, and even at least 95%, 96%, 97%, 98%, or 99% sequence identity. It will be understood by those skilled in the art that these values ​​can be appropriately adapted by considering codon degeneracy, amino acid similarity, reading frame position, etc., to determine the corresponding identity of proteins encoded by two nucleotide sequences. For these purposes, substantial amino acid sequence identity typically means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%.

[0151] With respect to polypeptides, the term "substantial identity" indicates that a polypeptide contains a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% sequence identity with respect to a reference sequence within a specified comparison window, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, and even 95%, 96%, 97%, 98%, or 99% sequence identity. An indicator that two polypeptide sequences are identical is that one polypeptide reacts immunologically with an antibody produced against the other polypeptide. Therefore, for example, if the only difference between two polypeptides is a conservative substitution, one polypeptide is identical to the other.

[0152] The terms “to treat” and “treatment” refer to both therapeutic treatment and preventive or deterrent treatment, the purpose of which is to prevent, inhibit, reduce, or decrease undesirable physiological changes or disorders, such as the onset, progression, or exacerbation of the disorder. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction of the severity of the disease, stabilization of the symptoms or adverse effects of the disease (i.e., no exacerbation or progression), delay or slowing of disease progression, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” can also mean an extension of survival compared to the survival expected without treatment. Those requiring treatment include those who already have the condition or disorder, as well as those with a predisposition (e.g., determined by a gene assay).

[0153] VI. Kit The present invention provides a kit comprising packaging material and one or more components therein. The kit typically includes a label or accompanying document containing a description of the components contained therein or instructions for the in vitro, in vivo, or ex vivo use of the components contained therein. The kit may include collections of such components, such as nucleic acids, recombinant vectors, and / or viral particles.

[0154] A kit is a physical structure that contains one or more components of a kit. Packaging materials can be made of materials commonly used for such purposes, such as paper, cardboard, glass, plastic, foil, ampoules, vials, and tubes, that can keep the components sterile.

[0155] The label or package insert may include identification information for one or more components contained therein, dosage, and clinical pharmacology of the active ingredient, such as mechanism of action, pharmacokinetics, and pharmacodynamics. The label or package insert may include information confirming the manufacturer, lot number, place and date of manufacture, and expiration date. The label or package insert may include information confirming manufacturer information, lot number, place and date of manufacture. The label or package insert may include information about diseases for which the components of the kit may be used. The label or package insert may include instructions for clinicians or patients for using one or more of the components of the kit in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency or duration of administration, and instructions for carrying out any of the methods, use, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0156] Labels or package inserts may include information about the benefits that the components may provide, such as preventive or therapeutic benefits. Labels or package inserts may also include information about potentially adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which the use of a particular composition may be inappropriate. Adverse side effects or complications may also occur if the subject has previously taken, will take, or is currently taking one or more other medicines that may be incompatible with the composition, or if the subject has previously taken, will take, or is currently taking another treatment protocol or regimen that may be incompatible with the composition; therefore, instructions may also include information about such incompatibility.

[0157] Labels or accompanying documents include "printed materials," such as paper or cardboard, that are separate, attached to components, kits or packaging materials (e.g., boxes), or attached to ampoules, tubes or vials containing components of a kit. Labels or accompanying documents may further include computer-readable media, such as barcode-bearing printed labels, disks, optical disks, such as CD- or DVD-ROM / RAM, DVDs, MP3s, or electronic storage media, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash memory, hybrid and memory cards. [Examples]

[0158] VII. Examples The following embodiments are provided to demonstrate preferred embodiments of the present invention. As those skilled in the art will understand, the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in practice of the present invention and can therefore be considered to constitute preferred forms for its implementation. However, those skilled in the art will understand that many modifications can be made to the specific embodiments disclosed in light of this disclosure, and similar or comparable results can still be obtained without departing from the spirit and scope of the present invention.

[0159] Example 1 - Identification of AAV variants that target brain parenchyma We developed advanced barcoded AAV libraries using AAV1, AAV2, and AAV9 capsids as starting platforms. AAV1, AAV2, and AAV9 peptide display libraries were created by inserting random sequences at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, and position 588 of the AAV9 capsid (Figure 1). This library has a resolution of 1 × 10⁻⁶. 7 Each individual clone exhibited unique clonal diversity (Figure 3).

[0160] To test the usefulness of the library, a pilot study was conducted using bench-grade (low titer, low purity) capsid-modified AAV2. The AAV2 library was administered to two C57BL / 6 mice, with each animal receiving 8 × 10⁶ vector genome samples. 10 Individual mice were injected intravenously. 72 hours later, the cerebral cortex, cerebellum, and spinal cord were dissected. Notably, the heart, skeletal muscle, and diaphragm were collected separately to identify muscle tropism. Viral genomic DNA was isolated, and the recovered random oligonucleotide sequences were amplified by PCR. PCR products from the brain were pooled to create a second-round library, which was then used to create a vector genome of 4 × 10⁶ per animal in two mice. 10 Individual injections were administered. After the second injection, the vector genome was recovered in the same manner as before and subjected to NexGen sequencing along with the starting library and first-round tissue. To test whether sequences showing enrichment in brain tissue could actually deliver AAV2 to the brain, individual hits were cloned into AAV2 capsid packaging plasmids to construct AAV2 expressing eGFP. Bench-grade vectors were constructed, and the vector genome of the AAV2-based capsid-modified virus was 3 × 10⁶. 10 The individual molecules were injected into mice. After four weeks, even in the case of these low-titer variants, eGFP fluorescence was observed in the brain.

[0161] Using these advanced barcoded AAV libraries, we identified AAV variants capable of targeting different primate brain structures in non-human primates. AAV1, AAV2, and AAV9 libraries were delivered to a single non-human primate by intracerebroventricular injection (Figure 2). 72 hours after injection, brain regions were microdissected for viral DNA isolation, and AAV DNA was amplified by PCR. The products were pooled and used to package the second round library, which was then injected into further NHP. Brain regions were subsequently microdissected 12 days after injection. After two rounds of panning, the vector genome was recovered and subjected to next-generation sequencing. Specifically, genomic DNA extracted from tissues from rounds 1 and 2 was PCR-amplified to create an Illumina amplicon sequencing library at the vector barcode locations. The resulting library was pooled and executed on a single lane of an Illumina HiSeq 4000 using 100 bp single-end read chemistry. To illustrate the usefulness of this approach, several target regions—ependyma, meninges, and cerebellum—were tested as examples. Generally, the sequences directing AAVx to the ependyma, meninges, and cerebellum differed and varied across different serotypes.

[0162] Round-over-round enrichment graphs (Figure 4) and heatmaps (Figures 5 and 6) were created for the following tissues: brainstem, caudate nucleus, cerebellar cortex (Figure 5), cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, and thalamus. These illustrate the enrichment of the displayed barcodes at baseline (round 0) and after in vivo passage rounds 1 and 2 in rhesus monkeys. To create these, fastq result files for each tissue and round combination were processed using a custom Python script designed to extract and quantify unique barcode configurations observed at the DNA level. A custom R script was used to calculate the percentage of barcodes present in each sample and to convert DNA barcodes to amino acid barcodes. Table 1 corresponds to samples processed with AAV1-derived libraries; Table 2 represents tissues processed with AAV2-derived libraries; and Table 3 corresponds to samples processed with AAV9-derived libraries. Top hits were selected from these three libraries and assembled into a validation library containing barcodes derived from 50 (AAV1), 58 (AAV2), and 30 (AAV9). This validation library was delivered to additional rhesus monkeys by ICV injection. Tissues were collected and processed again to facilitate the recovery of barcode abundances by deep sequencing. Barcode abundances were evaluated in the recovered tissues and input virus libraries. The enrichment value of each barcode was calculated relative to their abundance in the input virus library. The resulting relative enrichment values ​​are a robust indicator of vector performance across the various tissues evaluated and facilitate the identification of broad and specific AAV vector variants (Figures 7A-C).

[0163] To validate the identified cell type specificity, AAV9-1999 (containing the targeting peptide sequence KGGGFHG; SEQ ID NO: 110) was selected for in vivo validation. The eGFP expression construct was packaged into AAV9-1999 driven by a CAG promoter. Five-year-old female rhesus monkeys were administered 1.5E13 vg of AAV9-1999 by ICV injection into the left ventricular cavity. Brains were collected 30 days after injection for histological analysis. Cerebellar slices were stained with H&E to depict the transduction pattern of AAV9-1999 (Figure 8). Cochlea were also collected from these animals and, surprisingly, showed strong transduction of hair cells. Furthermore, AAV9-1999 containing the eGFP construct and AAV9 capsid were delivered to C57BL / 6 p0 mouse pups by ICV injection at a dose of 1E10 vg per hemisphere. After 21 days, the mice were perfused. Whole-mount brain (Figure 9A), 40 μm whole-brain sagittal section (Figure 9B), 40 μm S1 cortical section (Figure 9C, left), 40 μm hippocampal section (Figure 9C, center), 40 μm cerebellar sagittal section (Figure 9C, right), and 40 μm lumbar coronal section (Figure 9D) were imaged for eGFP fluorescence signaling. AAV9-1999 injected into Bl / 6 neonatal mouse pups showed greater ubiquitous expression than dose-matched injections of AAV9.

[0164] One adult rhesus macaque was injected with a mixture of four modified AAVs: AAV9 with the RGDLQWV (SEQ ID NO: 113) targeting peptide sequence and mTAGBFP2 tag; AAV1 with the ERDRTRG (SEQ ID NO: 21) targeting peptide sequence as mTFP1; AAV2 with the GRGAPGG (SEQ ID NO: 80) targeting peptide sequence and mNG tag; and AAV2 with the DDPSARR (SEQ ID NO: 53) targeting peptide sequence and mRuby3 tag. The viruses were directly mixed in equal amounts to achieve the final total doses of each. AAV9.RGDL mTagBFP2 6.13E12 All vg AAV1.ERDR mTFP1 1.23E13 All vg AAV2.GRGA mNG 8.8E12 Full vg AAV2.DDPS mRuby3 1.32E13 All vg

[0165] Brain tissue was collected 30 days after injection for fluorescence imaging. Lateral ventricular sections (Figure 10A), fourth ventricular sections (Figure 10B), and meningeal sections (Figure 10C) were imaged for mTagBFP2, mTFP2, mNG, and mRuby3 fluorescence signals.

[0166] Additional experiments were conducted by injecting AAV9-1999 into the cochlea of ​​rhesus monkeys. Based on the results of cochlear transduction, AAV9-1999 was administered into the lateral ventricles of the animals. Individual animals received AAV9-1999 3E11 vg injected directly into their cochlear window via fenestration (Figures 11A-C).

[0167] All methods disclosed and asserted herein can be constructed and implemented in light of this disclosure without excessive experimentation. While the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that various modifications can be applied to the methods and steps or sequences of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant active substances can be used in place of the active substances described herein, and the same or similar results can be achieved. All such similar substitutions and modifications, which will be apparent to those skilled in the art, are considered to fall within the spirit, scope, and concept of the invention as defined by the appended claims.

[0168] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement what is described herein. TIFF0007888221000031.tif189150TIFF0007888221000032.tif32150

[0169] Array information SEQUENCE LISTING <110> The Children's Hospital of Philadelphia <120> ADENO-ASSOCIATED VIRAL VECTOR VARIANTS <150> US 63 / 084,709 <151> 2020-09-29 <150> US 62 / 939,315 <151> 2019-11-22 <160> 144 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 1 Arg Pro Gly Arg Glu Gln Ala 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 2 Arg Gly Val Leu Val Thr Thr 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 3 Arg Pro Gly Arg Glu Ser Ala 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 4 Asn Glu Ser Leu Lys Lys Lys 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 5 Asp Lys Thr Arg Ala Gly Ser 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 6 Thr Ala Lys Ser Lys Gln Ala 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 7 Pro Val Lys Lys Lys Asp Ala 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Gly Arg Glu Thr Leu Lys Gly 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 9 Pro Ile Pro Ala Gly Lys Lys 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 10 Asn Val Val Arg Ala Gly Thr 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 11 Lys Ala Thr Ala Asn Thr Arg 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 12 Arg Asp Ala Thr Arg Ser Ser 1 5 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 13 Val Pro Thr Lys Ser Pro Lys 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 14 Ala Gly Val Ala Arg Ser Lys 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 15 Arg Ser Arg Ser Glu Val Leu 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 16 Glu Val Lys Gly Lys Gly Lys 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 17 Ala Lys Leu Asn Lys Ser Ser 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 18 Asn Ser Val His Asn Thr Ala 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 19 Asn Val Val Arg Gly Gly Ala 1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 20 Asn Arg Leu Val Ala Gly Gly 1 5 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 21 Glu Arg Asp Arg Thr Arg Gly 1 5 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 22 Val Gln Gly Ser Lys Met Lys 1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Asn Ser Val Arg Pro Leu Thr 1 5 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Asn Lys Ile His Ala Asn Pro 1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 25 Thr Ala Pro Lys Ser Leu Lys 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 26 Arg Asp Ser Thr Arg Gln Leu 1 5 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 27 Asn Ser Val Lys Ser Val Leu 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 28 Asn Val Thr Ile Lys Ser Lys 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 29 Val Ser Leu Lys Glu Arg Val 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 30 Asp Glu Thr Ser Arg Leu Val 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 31 Asp Arg Leu Lys Gly Ile Val 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 32 Ser Gly Val Leu Val Gln Arg 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 33 Ser Gly Thr Phe Val Lys Ala 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 34 Asn Ser Ile Ala Arg Pro Val 1 5 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 35 Asn Arg Ala Arg Ala Gly Glu 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 36 Ala Arg His Ala Leu Gly Gly 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 37 His Ser Ser Arg Pro Val Ala 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 38 Lys Thr Gly Thr Ala Arg Leu 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 39 Val Lys Ala Leu Gly Arg Pro 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 40 Asn Asp Val Arg Ala Lys Gly 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 41 Gln Gly Val Leu Val Val Arg 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 42 Lys Gln Tyr Ala Gly Ser Gln 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 43 Thr Asn Arg Met Ala Leu Ser 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 44 Gly Ile Thr Leu Gly Arg Leu 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 45 Ala Gly Ile Met Val Arg Val 1 5 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 46 Lys Ala Ala Gly Arg Thr Val 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 47 His Val Ile Arg Leu Pro Ser 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 48 Ser Val Ala Ser Ala Lys Lys 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 49 Thr Ala Thr Pro Arg Lys Gly 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 50 Thr Lys Thr Gly Leu Lys Leu 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 51 Lys Gly Leu Arg Thr Pro Thr 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 52 Leu Thr Ser Arg Thr Ser Pro 1 5 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 53 Asp Asp Pro Ser Ala Arg Arg 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 54 Gly Glu Gln Asp Leu Arg Arg 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 55 Val Ser Thr Ala Leu Pro Arg 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 56 Arg Asp Asp Val Pro Leu Arg 1 5 <210> 57 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 57 Thr Arg Val Gly Thr Ala Gly 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 58 Ser Ser Ser Lys Thr Gly Ser 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 59 Ser Leu Ser Thr Gly Pro Lys 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 60 Val Gln Gly Arg Gln Gly Gly 1 5 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 61 Arg Gly Ala Ser Gly Ala Val 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 62 Asn Ala Arg Ala Gln Gly Val 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 63 Thr Ser Asn Arg Gly Gln Val 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 64 Ala Val Arg Gly Gly Met Ala 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 65 Arg Gly Leu Asp Lys Gly Thr 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 66 Lys Gly Val Asp Leu Lys Pro 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 67 Thr Ala Val Arg Glu Glu Arg 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 68 Gly Asn Ala Gly Ile Thr Lys 1 5 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 69 Ser Ala Arg Ala Gly Ala Pro 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 70 Ser Gly Glu Phe Val Gly Arg 1 5 <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 71 Ser Gly Arg Lys Leu Glu Val 1 5 <210> 72 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 72 Ser Ala Arg Ser Gly Ser Val 1 5 <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 73 Glu Ser Thr Gly Arg Glu Arg 1 5 <210> 74 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 74 Val Thr Gln Ser Lys Gly Ala 1 5 <210> 75 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 75 Arg Gly Ser Gly Ser Ala Val 1 5 <210> 76 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 76 Asn Ala Arg Pro Val Ser Ala 1 5 <210> 77 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 77 Thr Ala Arg Gly Gly Gly Gly 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 78 Gly Arg Ser Ala Ser Gly Ser 1 5 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 79 Lys Ala Gln Gly Val Gly Gly 1 5 <210> 80 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 80 Gly Arg Gly Ala Pro Gly Gly 1 5 <210> 81 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 81 Val Pro Gly Arg Thr Ala Gly 1 5 <210> 82 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 82 Ala Arg Gly Ser Gly Val Asn 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 83 Ser Val Arg Val Gly Gly Gln 1 5 <210> 84 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 84 Arg Ala Val Pro Ala Gly Gly 1 5 <210> 85 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 85 Val Met Ser Ser Gly Lys Pro 1 5 <210> 86 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 86 Ser Thr Pro Ala Pro Lys Ser 1 5 <210> 87 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 87 Arg Gly Gly Ala Gln Val Val 1 5 <210> 88 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 88 Lys Ala Ser Gly Ala Gly Gly 1 5 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 89 Thr Gly Thr Ala Gly Leu Lys 1 5 <210> 90 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 90 Arg Ser Asn Ala Pro Gln Thr 1 5 <210> 91 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 91 Glu Ala Gln Ser His Pro Arg 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 92 Thr Lys Ser Leu Ser Ser Gly 1 5 <210> 93 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 93 Ala Ala Gly Ala Lys Val Met 1 5 <210> 94 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 94 Arg Gly Ser Thr Gln Val Gly 1 5 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 95 Gly Arg Ser Thr Gly Met Thr 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 96 Arg Ala Thr Ser Gln Ser Thr 1 5 <210> 97 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 97 Val Gly Arg Ser Val Gly Ala 1 5 <210> 98 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 98 Gly Glu Gly Gly Gly Gly Arg 1 5 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 99 Thr Ala Ala Gly Gly Gln Arg 1 5 <210> 100 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 100 Gly Arg Gly Gly Ala Ala Leu 1 5 <210> 101 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 101 Val Ala Pro Ile Ser Lys Ser 1 5 <210> 102 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 102 Ala Pro Pro Val Lys Leu Ser 1 5 <210> 103 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 103 His Asp Gly Gly Ala Ser Arg 1 5 <210> 104 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 104 Arg Ser Gly Gly Ala Ala Val 1 5 <210> 105 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 105 Gly Ser Arg Ala Gly Val Gly 1 5 <210> 106 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 106 Lys Leu Ser Ile Ser Gly Asn 1 5 <210> 107 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 107 Gly Ala Val Gly Gly Val Lys 1 5 <210> 108 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 108 Lys Asn Glu Ser Gly Lys Val 1 5 <210> 109 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 109 Ala Gly Gln Leu Ala Gly Arg 1 5 <210> 110 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 110 Lys Gly Gly Gly Phe His Gly 1 5 <210> 111 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 111 Arg Ala Lys Pro Gly Met Glu 1 5 <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 112 Gly Arg Asp Val Thr Arg Ser 1 5 <210> 113 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 113 Arg Gly Asp Leu Gln Trp Val 1 5 <210> 114 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 114 Gly Gly Asp Arg Thr Arg Gly 1 5 <210> 115 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 115 Arg Gly Asp Leu Ala Ser Val 1 5 <210> 116 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 116 Arg Asp Thr Thr Arg Asn Leu 1 5 <210> 117 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 117 Lys Gly Gly Gly Val His Gly 1 5 <210> 118 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 118 Arg Gly Asp Met Tyr Arg Val 1 5 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 119 Arg Gly Asp Arg Pro Val Ser 1 5 <210> 120 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 120 Arg Ser Asp Val Gly Ser Leu 1 5 <210> 121 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 121 Ala Gly Val Lys Pro Gly Arg 1 5 <210> 122 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 122 Arg Gly Asp Trp Pro Arg Gly 1 5 <210> 123 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 123 Gly Gly Arg Pro Gly Ser Trp 1 5 <210> 124 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 124 Arg Gly Asp Tyr Pro Arg Ser 1 5 <210> 125 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 125 Arg Gly Asp Leu Arg Phe Ile 1 5 <210> 126 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 126 Arg Gly Gly Gly Val Tyr Gly 1 5 <210> 127 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 127 Gly Ala Asp Arg Thr Arg Gly 1 5 <210> 128 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 128 Arg Gly Asp Phe Met Gly Leu 1 5 <210> 129 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 129 Arg Arg Asp Glu Thr Arg Thr 1 5 <210> 130 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 130 Ala Glu Ser Pro Trp Glu Arg 1 5 <210> 131 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 131 Ala Trp Asp Gly Thr Arg Val 1 5 <210> 132 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 132 Ala Arg Gly Asp Gly Trp Arg 1 5 <210> 133 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 133 Gly Arg Asp Tyr Thr Arg Leu 1 5 <210> 134 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 134 Arg Arg Gly Asp Ala Trp Ser 1 5 <210> 135 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 135 Met Asp Leu Thr Lys Ala Val 1 5 <210> 136 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 136 Met Met Gly Arg Pro Gly Arg 1 5 <210> 137 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 137 Thr Gly Arg Pro Gly Val Trp 1 5 <210> 138 <211> 736 <212> PRT <213> adeno-associated virus 1 <400> 138 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590 Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700 Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu 705 710 715 720 Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 <210> 139 <211> 735 <212> PRT <213> adeno-associated virus 2 <400> 139 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 140 <211> 736 <212> PRT <213> adeno-associated virus 9 <400> 140 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 141 <211> 748 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (594)..(600) <223> X is any amino acid <400> 141 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Ser Ser 580 585 590 Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Ser Pro Ala Thr Gly Asp Val 595 600 605 His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val 610 615 620 Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His 625 630 635 640 Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys Asn Pro Pro 645 650 655 Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Ala 660 665 670 Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr 675 680 685 Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser 690 695 700 Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser 705 710 715 720 Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro 725 730 735 Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 740 745 <210> 142 <211> 747 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (591)..(597) <223> X is any amino acid <400> 142 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Ala Ala Ala Xaa Xaa 580 585 590 Xaa Xaa Xaa Xaa Xaa Ala Ala Arg Gln Ala Ala Thr Ala Asp Val Asn 595 600 605 Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr 610 615 620 Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe 625 630 635 640 His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro 645 650 655 Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr 660 665 670 Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly 675 680 685 Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys 690 695 700 Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val 705 710 715 720 Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg 725 730 735 Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 <210> 143 <211> 748 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (592)..(598) <223> X is any amino acid <400> 143 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Ala Ala Xaa 580 585 590 Xaa Xaa Xaa Xaa Xaa Xaa Ala Ser Ala Gln Ala Gln Thr Gly Trp Val 595 600 605 Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val 610 615 620 Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn 625 630 635 640 Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met Lys His Pro Pro 645 650 655 Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr 660 665 670 Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr 675 680 685 Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser 690 695 700 Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser 705 710 715 720 Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro 725 730 735 Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 740 745 <210> 144 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 144 Arg Pro Gly Arg Glu Ala Ser 1 5

Claims

1. A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide causes a viral vector comprising the modified AAV capsid protein to target different brain structures, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide comprises a sequence up to 10 amino acids in length having SEQ ID NO:

21.

2. The modified AAV capsid protein according to claim 1, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid long.

3. The modified AAV capsid protein according to claim 2, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide.

4. The modified AAV capsid protein according to claim 1, wherein the modified AAV capsid protein is a modified AAV1 capsid protein derived from an AAV1 capsid protein having the amino acid sequence SEQ ID NO: 138, and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

5. The modified AAV capsid protein according to claim 1, wherein the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

141.

6. The modified AAV capsid protein according to claim 1, wherein the targeting peptide is SEQ ID NO:

21.

7. The modified AAV capsid protein according to claim 1, wherein the targeting peptide has a length of 7 amino acids.

8. The modified AAV capsid protein according to claim 1, wherein the different brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus.

9. A modified AAV capsid protein according to claim 8, The aforementioned different brain structure is the brainstem, and the aforementioned modified AAV capsid protein is the modified AAV1 capsid protein. The aforementioned different brain structure is the cerebellar cortex, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the cerebral cortex, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the ependymia, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the globus pallidus, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the meninges, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the optic nerve, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The aforementioned different brain structure is the putamen, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. The different brain structure is the spinal cord, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO: 21, or The aforementioned different brain structure is the thalamus, the modified AAV capsid protein is the modified AAV1 capsid protein, and the targeting peptide is SEQ ID NO:

21. Modified AAV capsid protein.

10. A nucleic acid comprising a sequence encoding a modified capsid protein according to any one of claims 1 to 9.

11. Recombinant adeno-associated virus (rAAV) comprising a modified capsid protein according to any one of claims 1 to 9.

12. The rAAV vector according to claim 11, further comprising a nucleic acid sequence encoding a nucleic acid of interest.

13. In vitro cells comprising the rAAV vector according to claim 11 or 12.

14. A pharmaceutical composition comprising the rAAV vector according to claim 11 or 12 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14 for use in the treatment of diseases in mammals.

16. The pharmaceutical composition according to claim 15, wherein the disease is a neurodegenerative disease.

17. The pharmaceutical composition according to claim 16, wherein the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease.

18. The pharmaceutical composition according to claim 15, wherein the mammal is a human.

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