Adeno-associated virus vector variant

Abstract

Provided herein are vectors comprising targeting peptides and sequences encoding the targeting peptides, wherein the targeting peptides deliver active substances to specific substructures in the brain. Provided herein are viral vectors, each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that allows the viral vector to target different brain structures. TIFF2023503455000034.tif49133

Description

Technical Field

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

[0002] Reference to Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS-Web, the entire contents of which are incorporated herein by reference. The ASCII copy created on November 19, 2020, is named CHOPP0038WO_ST25.txt and is 63.8 kilobytes in size.

[0003] 1. Field The present invention generally relates to the fields of medicine, virology, and neurology. More specifically, the present invention relates to targeting peptides that target the delivery of viral vectors to different structures within the brain.

Background Art

[0004] 2. Description of Related Art To generate AAV vector variants, various strategies have been developed, including rational design and directed evolution. In the rational design approach, knowledge of the AAV capsid is used to introduce targeted changes to the capsid, such as tyrosine mutations on the capsid surface to increase transduction efficiency, which can alter transduction efficiency or specificity. The directed evolution approach does not require knowledge of the capsid structure and is performed by random mutagenesis, capsid shuffling, or random peptide insertion. These strategies generally use in vitro systems or mice, which are ideal for cell-based or mouse studies but do not represent a bridge to the clinic. In fact, 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.

Summary of the Invention

[0005] Summary Provided herein are viral vectors each comprising a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to different brain structures.

[0006] In one aspect, provided is a modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide targets a viral vector comprising the modified AAV capsid protein to different organs or different brain structures and is 3 to 10 amino acids in length. 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 up to 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, shell, 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 aspects, the different organs are the brain, kidney, heart, liver, gonad, spleen, or liver. 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 4 to target the corresponding organ. 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 5 to target the corresponding organ. 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 6 to target the corresponding organ.

[0013] In one aspect, a nucleic acid comprising a sequence encoding any one of the modified capsid proteins of this aspect is provided herein.

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

[0015] In one aspect, a viral vector comprising a nucleic acid encoding any one of the modified capsid proteins of this aspect 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 agent. In some aspects, the therapeutic agent is an enzyme or an RNAi molecule.

[0016] In one aspect, a cell comprising any one of the viral vectors of this aspect is provided herein. In some aspects, the cell is a mammalian cell, such as a human cell. In some aspects, the cell is in vitro or in vivo.

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

[0018] In one aspect, provided herein is a method for delivering an agent to different brain structures of a subject, the method including the step of administering a virus of the present aspect to the subject. 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, an rAAV having a modified AAV1 capsid protein is used and the 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 the 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 the 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 an rAAV having a modified AAV1 capsid protein and the targeting peptide of SEQ ID NO: 21, an rAAV having a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO: 53, an rAAV having a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO: 80, and an rAAV having a modified AAV9 capsid protein and the targeting peptide of SEQ ID NO: 113 are used.

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

[0020] In some aspects, the agent is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system. In some aspects, the administration is to the central nervous system. In some aspects, the administration is to the cisterna magna, the intracerebroventricular space, the ependyma, the ventricle, the subarachnoid space, and / or the intrathecal space. In some aspects, the ventricle is the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right ventricle, and / or the left ventricle, and / or the right rostral lateral ventricle, and / or the left rostral lateral ventricle, and / or the right caudal lateral ventricle, and / or the left caudal lateral ventricle.

[0021] In some aspects, multiple virus particles are administered. In some aspects, the virus is administered at a dose of about 1×10 6 ~about 1×10 18 vector genomes per kilogram (vg / kg). In some aspects, the virus is about 1×10 7 ~1×10 17 vg per kg of patient, 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] In one aspect, provided herein is a method of treating a disease in a mammal, comprising the step of administering the virus of this aspect to the mammal. 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's 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 no particular component is intentionally formulated in the composition and / or is present only as an impurity or in trace amounts. The total amount of a particular component resulting from unintentional composition contamination is, therefore, significantly lower than 0.05%, preferably lower than 0.01%. Most preferably, the composition is one in which the amount of such a particular component cannot be detected by standard analytical methods.

[0024] As used herein, "a" or "an" can mean one or more. The word "a" or "an" as used in the claims herein can mean one or two or more when used in conjunction with the word "comprising".

[0025] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to mean only alternatives or mutually exclusive alternatives, but the present disclosure supports a definition of only alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0026] Throughout this application, the term "about" is used to indicate that a value includes a value that is within 10% of the recited value, due to the inherent error variability of the device, method used to determine that value, the variability that exists between the subjects of study, or the value itself.

[0027] [Invention 1001] A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide targets a virus vector comprising the modified AAV capsid protein to a different organ or a different brain structure and has a length of 3 to 10 amino acids, the modified AAV capsid protein. [Invention 1002] 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, putamen, globus pallidus, hippocampus, meninges, motor cortex, optic nerve, prefrontal cortex, putamen, spinal cord, substantia nigra, subthalamic nucleus, temporal cortex, thalamus, or visual cortex, the modified AAV capsid protein of Invention 1001. [Invention 1003] The modified AAV capsid protein of Invention 1001 is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein. [Invention 1004] 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, the modified AAV capsid protein of Invention 1001. [Invention 1005] The targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide have a length of 2 or 3 amino acids, the modified AAV capsid protein of Invention 1004. [Invention 1006] The linker sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide, the modified AAV capsid protein of Invention 1005. [Invention 1007] The modified AAV1 capsid protein has a sequence at least 95% identical to SEQ ID NO: 141, the modified AAV capsid protein of Invention 1006. [Invention 1008] 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. The modified AAV capsid protein of the present invention 1001. [The present invention 1009] The modified AAV capsid protein of the present 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 acids in length. [The present invention 1010] The modified AAV capsid protein of the present invention 1009, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. [The present invention 1011] The modified AAV 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. [The present 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 588 of the AAV9 capsid protein. [The present invention 1013] The modified AAV capsid protein of 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 acids in length. [The present invention 1014] The modified AAV capsid protein of the present invention 1013, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. [The present 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. [The present invention 1016] The modified AAV capsid protein of the present invention 1001, wherein the target peptide contains a sequence with a maximum length of 10 amino acids, and the sequence has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 137 or 144. [The present invention 1017] The modified AAV capsid protein of the present invention 1016, wherein the targeting peptide is 7 amino acids in length. [The present invention 1018] The modified AAV capsid protein according to any one of the present inventions 1001 to 1017, wherein the different brain structures are the brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, putamen, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. [The present invention 1019] The modified AAV capsid protein of the present invention 1018, wherein the different brain structure is the brain stem, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from SEQ ID NOs: 1 to 9. [The present 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 NOs: 1, 3, 5, 7, 10 to 16, 25, 26, 32, and 144. [The present 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 NOs: 1, 3, 4, 9, and 17 to 21. [The present 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 NOs: 1, 3, 5, 12, and 21 to 26. [The present invention 1023] 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 NOs: 2 to 4, 7, 9, 21, 22, 27, and 28. [The present invention 1024] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1025] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1026] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1027] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1028] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1029] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1030] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1031] The different brain structure is the hypothalamic 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1032] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1033] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1034] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1035] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1036] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1037] The different brain structures are the putamen, 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1038] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1039] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1040] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1041] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1042] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1043] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1044] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1045] The different brain structure is the hypothalamic 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1046] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1047] 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, the modified AAV capsid protein of the present invention 1018. [The present invention 1048] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1049] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1050] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1051] 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, 111, 113, 118-120, and 128. The modified AAV capsid protein of the present invention 1018. [The present invention 1052] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1053] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1054] The different brain structure is the 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1055] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1056] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1057] 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1058] 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 to 114, 117, and 129. The modified AAV capsid protein of the present invention 1018. [The present invention 1059] The different brain structure is the hypothalamus, 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1060] The different brain structures are 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. The modified AAV capsid protein of the present invention 1018. [The present invention 1061] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 1, and the different brain structures are the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, hippocampus, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1062] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 2, and the different brain structures are the brainstem, putamen, hippocampus, optic nerve, spinal cord, substantia nigra, hypothalamus, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1063] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 3, and 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, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1064] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 4, and the different brain structures are the brainstem, cerebellar cortex, putamen, hippocampus, putamen, spinal cord, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1065] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 5, and the different brain structures are the brainstem, cerebral cortex, globus pallidus, or meninges. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1066] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1067] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 7, and the different brain structures are the brainstem, caudate nucleus, putamen, hippocampus, meninges, optic nerve, or spinal cord. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1068] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 8, and the different brain structures are the brainstem or thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1069] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 9, and the different brain structures are the brainstem, cerebellar cortex, putamen, meninges, spinal cord, or substantia nigra. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1070] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1071] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 12, and the different brain structures are the caudate nucleus, cerebral cortex, globus pallidus, meninges, putamen, subthalamic nucleus, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1072] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1073] 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, the globus pallidus, or the optic nerve. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1074] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1075] 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, the globus pallidus, the optic nerve, or the hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1076] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 17 to 20, and the different brain structure is the cerebellar cortex. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1077] 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, the cerebral cortex, the ependyma, the globus pallidus, the meninges, the optic nerve, the putamen, the spinal cord, or the thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1078] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 22, and the different brain structures are the cerebral cortex, the putamen, or the globus pallidus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1079] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 23 to 26, and the different brain structures are the cerebral cortex. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1080] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 27, and the different brain structures are the putamen. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1081] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 28, and the different brain structures are the putamen or the thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1082] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 29, and the different brain structures are the globus pallidus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1083] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 30, and the different brain structures are the globus pallidus, the putamen, or the subthalamic nucleus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1084] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is SEQ ID NO: 31, and the different brain structures are the globus pallidus or the optic nerve. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1085] 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 spinal cord. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1086] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1087] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 35 to 37, and the different brain structure is the meninges. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1088] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1089] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 39 to 42, and the different brain structure is the putamen. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1090] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1091] 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. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1092] 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 hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1093] The modified AAV capsid protein is a modified AAV1 capsid protein, the targeting peptide is any one of SEQ ID NOs: 48 to 51, and the different brain structure is the thalamus. The modified AAV capsid protein of the present invention 1001 or 1007. [The present invention 1094] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 52, and the different brain structures are the brainstem, substantia nigra, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1095] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 53, and the different brain structures are the brainstem, cerebral cortex, putamen, hippocampus, meninges, optic nerve, spinal cord, or substantia nigra. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1096] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 54, and the different brain structures are the brainstem or optic nerve. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1097] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 55, and the different brain structures are the brainstem, hippocampus, putamen, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1098] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 56, and the different brain structures are the brainstem, cerebellar cortex, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1099] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 57, and the different brain structures are the brainstem, optic nerve, substantia nigra, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1100] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 58, and the different brain structures are the brainstem, cerebellar cortex, cerebral cortex, hippocampus, meninges, optic nerve, spinal cord, substantia nigra, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1101] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 59, and the different brain structures are the brainstem, caudate nucleus, putamen, or spinal cord. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1102] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 60, and the different brain structures are the brainstem, cerebellar cortex, cerebral cortex, superior vestibular nucleus, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1103] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 61, and the different brain structures are the caudate nucleus, putamen, or spinal cord. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1104] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 62, and the different brain structures are the caudate nucleus, cerebral cortex, or putamen, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1105] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 63, and the different brain structures are the caudate nucleus, cerebral cortex, putamen, hippocampus, or spinal cord, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1106] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NO: 64, 65, and 67 - 69, and the different brain structure is the caudate nucleus, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1107] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 66, and the different brain structures are the caudate nucleus, cerebral cortex, putamen, or meninges, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1108] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NO: 70 - 72, and the different brain structure is the cerebellar cortex, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1109] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 73, and the different brain structures are the cerebellar cortex or meninges, the modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1110] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 74, and the different brain structures are the cerebellar cortex, the putamen, or the thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1111] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 75, and the different brain structures are the cerebellar cortex, the putamen, the globus pallidus, the optic nerve, the substantia nigra, or the hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1112] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 76, and the different brain structures are the cerebral cortex, the putamen, the hippocampus, the meninges, or the substantia nigra. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1113] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 77, and the different brain structures are the cerebral cortex, the putamen, or the spinal cord. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1114] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 78, and the different brain structures are the cerebral cortex. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1115] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 79, and the different brain structures are the cerebral cortex, the hippocampus, the optic nerve, or the hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1116] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 80, and the different brain structures are the ependyma, hippocampus, or meninges. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1117] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 81 to 84 and 86, and the different brain structure is the globus pallidus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1118] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 85, and the different brain structures are the globus pallidus or the thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1119] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 87, and the different brain structures are the globus pallidus, optic nerve, substantia nigra, or hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1120] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is SEQ ID NO: 88, and the different brain structures are the hippocampus, optic nerve, spinal cord, hypothalamus, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1121] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1122] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 90 to 93, and the different brain structure is the meninges. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1123] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1124] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1125] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 96 to 100, and the different brain structure is the putamen. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1126] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1127] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1128] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1129] 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. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1130] The modified AAV capsid protein is a modified AAV2 capsid protein, the targeting peptide is any one of SEQ ID NOs: 106 to 109, and the different brain structure is the thalamus. The modified AAV capsid protein of the present invention 1001 or 1011. [The present invention 1131] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 110, and 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1132] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 111, and the different brain structures are the brainstem, cerebellar cortex, cerebral cortex, putamen, globus pallidus, hippocampus, meninges, optic nerve, substantia nigra, or subthalamic nucleus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1133] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 112, and the different brain structures are the brainstem, globus pallidus, putamen, substantia nigra, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1134] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 113, and the different brain structures are the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, putamen, hippocampus, meninges, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1135] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 114, and the different brain structures are the brainstem, cerebral cortex, globus pallidus, meninges, optic nerve, substantia nigra, or thalamus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1136] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 115, and the different brain structures are the brainstem or caudate nucleus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1137] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 116, and the different brain structures are the brainstem, caudate nucleus, cerebral cortex, hippocampus, optic nerve, or putamen. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1138] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 117, and the different brain structures are the brainstem, optic nerve, or substantia nigra. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1139] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 118, and the different brain structures are the caudate nucleus, putamen, or meninges. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1140] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 119, and the different brain structures are the caudate nucleus, cerebellar cortex, putamen, globus pallidus, meninges, spinal cord, or subthalamic nucleus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1141] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 120, and the different brain structures are the caudate nucleus, putamen, globus pallidus, meninges, spinal cord, or subthalamic nucleus, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1142] 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, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1143] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 122, and the different brain structures are the cerebellar cortex, meninges, spinal cord, or subthalamic nucleus, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1144] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 123, and the different brain structures are the cerebellar cortex, hippocampus, putamen, or spinal cord, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1145] 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, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1146] The modified AAV capsid protein is a modified AAV9 capsid protein, the targeting peptide is SEQ ID NO: 125, and the different brain structures are the cerebellar cortex, cerebral cortex, hippocampus, or thalamus, the modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1147] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1148] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1149] 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 putamen or the spinal cord. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1150] 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, the hippocampus, the optic nerve, or the substantia nigra. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1151] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1152] 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 the meninges. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1153] 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 hypothalamus. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1154] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1155] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1156] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1157] 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. The modified AAV capsid protein of the present invention 1001 or 1015. [The present invention 1158] A nucleic acid comprising a sequence encoding a modified capsid protein according to any one of the present inventions 1001 to 1157. [The present invention 1159] A recombinant adeno-associated virus (rAAV) virus comprising a modified capsid protein according to any one of the present inventions 1001 to 1157. [The present invention 1160] A viral vector comprising a nucleic acid encoding a modified capsid protein according to any one of the present inventions 1001 to 1157. [The present invention 1161] The viral vector of the present invention 1160, further comprising a nucleic acid sequence encoding a nucleic acid of interest. [The present invention 1162] The viral vector of the present invention 1161, wherein the nucleic acid of interest is a therapeutic substance. [The present invention 1163] The viral vector of the present invention 1162, wherein the therapeutic substance is an enzyme or an RNAi molecule. [The present invention 1164] A cell comprising a viral vector according to any one of the present inventions 1160 to 1163. [The present invention 1165] The cell of the present invention 1164, which is a mammalian cell. [The present invention 1166] The cell of the present invention 1164, which is a human cell. [The present invention 1167] The cells of the present invention 1164, which are in vitro. [The present invention 1168] The cells of the present invention 1164, which are in vivo. [The present invention 1169] A pharmaceutical composition comprising the viral vector of the present invention 1159 and a pharmaceutically acceptable carrier. [The present invention 1170] A method for delivering an agent to different brain structures of a subject, comprising the step of administering the virus of the present invention 1159 to the subject. [The present invention 1171] A method for delivering an agent to the brainstem of a subject, the method comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1-9, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 52-60, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110-117, the method of the present invention 1170. [The present invention 1172] A method for delivering an agent to the caudate nucleus of a subject, the method comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 7, and 10-16, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 59 and 61-69, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 113, 115, 116, and 118-121, the method of the present invention 1170. [The present invention 1173] A method for delivering an agent to the cerebellar cortex of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 4, 9, and 17-21, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 56, 58, 60, and 70-75, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 119, and 122-125, the method of the present invention 1170. [The present invention 1174] A method for delivering an agent to the cerebral cortex of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1, 3, 5, 12, and 21-26, an AAV2 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 114, 116, and 125-127, the method of the present invention 1170. [The present invention 1175] A method for delivering an agent to the upper garment of a subject, comprising administering an AAV1 virus comprising 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 comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 118-120, and 128, the method of the present invention 1170. [The present invention 1176] A method for delivering an agent to the target globus pallidus, comprising administering an AAV1 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 60, 75, and 81-87, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110-112, 114, 119, 120, and 129, the method of the present invention 1170. [The present invention 1177] A method for delivering an agent to the hippocampus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 1-4, 7, and 32-34, and 28, an AAV2 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 116, 123, 125, 129, and 130, the method of the present invention 1170. [The present invention 1178] A method for delivering an agent to the meninges of a subject, comprising administering an AAV1 virus comprising 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 comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 114, 118, 119, 122, and 131, the method of the present invention 1170. [The present invention 1179] A method for delivering an agent to a target optic nerve, comprising administering an AAV1 virus comprising 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 comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 114, 115, 117, 129, and 132, the method of the present invention 1170. [The present invention 1180] A method for delivering an agent to a target capsid, comprising administering an AAV1 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 55, 59, 60, 61, and 95-100, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 112, 113, 116, 123, 127, 133, and 134, the method of the present invention 1170. [The present invention 1181] A method for delivering an agent to a target spinal cord, comprising administering an AAV1 virus comprising 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 comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 113, 119, 120, 122, 123, 128, and 134, the method of the present invention 1170. [The present invention 1182] A method for delivering an agent to the substantia nigra of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2, 3, 9, 44, and 45, and 28, an AAV2 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110-114, 117, and 129, the method of the present invention 1170. [The present invention 1183] A method for delivering an agent to the subthalamic nucleus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 2-4, 12, 16, 30, 46, and 47, an AAV2 virus comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 111, 113, 119, 120, 122, 132, and 135, the method of the present invention 1170. [The present invention 1184] A method for delivering an agent to the thalamus of a subject, comprising administering an AAV1 virus comprising 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 comprising 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 comprising a modified capsid protein having a targeting peptide selected from SEQ ID NO: 110, 112-114, 125, 133, 136, and 137, the method of the present invention 1170. [The present invention 1185] The method according to any one of claims 1170 to 1184, wherein the agent is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, a therapeutic protein, or a CRISPR system. [Claim 1186] The method according to any one of claims 1170 to 1184, wherein the administration is to the central nervous system. [Claim 1187] The method of claim 1186, wherein the administration is to the cisterna magna, the intracerebroventricular space, the leptomeninges, the ventricle, the subarachnoid space, the cochlea, and / or the intrathecal space. [Claim 1188] The method of claim 1187, wherein the ventricle is the rostral lateral ventricle, and / or the caudal lateral ventricle, and / or the right ventricle, and / or the left ventricle, and / or the right rostral lateral ventricle, and / or the left rostral lateral ventricle, and / or the right caudal lateral ventricle, and / or the left caudal lateral ventricle. [Claim 1189] The method according to any one of claims 1170 to 1188, wherein a plurality of virus particles are administered. [Claim 1190] The method of claim 1189, wherein the virus is administered at a dose of about 1×10 6 to about 1×10 18 vector genomes per kilogram (vg / kg). [Claim 1191] The method of claim 1189, wherein the virus is administered at a dose of about 1×10 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 vector genomes per kilogram of the patient. [Claim 1192] The method according to any one of claims 1170 to 1191, wherein the subject is a human. [Claim 1193] A method for treating a disease in a mammal, comprising administering the virus of claim 1159 to the mammal. [Claim 1194] The method of claim 1193, wherein the disease is a neurodegenerative disease. [Claim 1195] The method of claim 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. [Claim 1196] The method of claim 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 indicating preferred embodiments of the invention, are given by way of illustration only, since 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 Description of the Drawings

[0028] The following drawings form a part of this specification and are provided to further demonstrate certain specific aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

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Mode for Carrying Out the Invention

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

[0030] In certain embodiments, the viral vector is an adeno-associated virus vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, or AAV9. Exemplary wild-type reference AAV1 capsid protein sequences are provided in SEQ ID NO: 138. Exemplary wild-type reference AAV2 capsid protein sequences are provided in SEQ ID NO: 139. Exemplary wild-type reference AAV9 capsid protein sequences are provided in SEQ ID NO: 140. In certain aspects, 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. Exemplary modified AAV1 capsid protein sequences are provided in SEQ ID NO: 141, which shows the targeting peptide insertion after position 590 as SSAX7AS, where the leading SSA and trailing AS are linker sequences and X7 represents the targeting peptide. Exemplary modified AAV2 capsid protein sequences are provided in SEQ ID NO: 142, which shows the targeting peptide insertion after position 587 as AAAX7AA, where the leading AAA and trailing AA are linker sequences and X7 represents the targeting peptide. Exemplary modified AAV9 capsid protein sequences are provided in SEQ ID NO: 143, which shows the 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 TIFF0007712271000001.tif52154TIFF0007712271000002.tif227154TIFF0007712271000003.tif227154TIFF0007712271000004.tif227154TIFF0007712271000005.tif227154TIFF0007712271000006.tif227154TIFF0007712271000007.tif227154TIFF0007712271000008.tif227154TIFF0007712271000009.tif129154

[0032] (Table 2) AAV2 targeting peptides for each brain structure TIFF0007712271000010.tif83154TIFF0007712271000011.tif222154TIFF0007712271000012.tif232154TIFF0007712271000013.tif227154TIFF0007712271000014.tif227154TIFF0007712271000015.tif227154TIFF0007712271000016.tif227154TIFF0007712271000017.tif227154TIFF0007712271000018.tif170154

[0033] (Table 3) AAV9 targeting peptides for each brain structure TIFF0007712271000019.tif42154TIFF0007712271000020.tif227154TIFF0007712271000021.tif227154TIFF0007712271000022.tif227154TIFF0007712271000023.tif227154TIFF0007712271000024.tif227154TIFF0007712271000025.tif227154TIFF0007712271000026.tif227154TIFF0007712271000027.tif57154

[0034] (Table 4) AAV1 targeting peptides for various organs TIFF0007712271000028.tif42154

[0035] (Table 5) AAV2 targeting peptides for various organs TIFF0007712271000029.tif217154

[0036] (Table 6) AAV9 targeting peptides for various organs TIFF0007712271000030.tif201154

[0037] I. Adeno-associated virus (AAV) vector Adeno-associated virus (AAV) is a small non-pathogenic virus of the Parvoviridae family. To date, numerous serologically different AAVs have been identified, and more than 12 have been identified from humans or primates. AAV differs from other members of this family in that its replication depends on a helper virus.

[0038] The AAV genome exists in an episomal state without integrating into the host cell genome, has a broad host range, can transduce both dividing and non-dividing cells in vitro and in vivo, and can maintain high-level expression of the transduced gene. AAV virus particles have thermal stability, are resistant to changes in solvents, detergents, pH, and temperature, and can be purified by column chromatography and / or concentrated by CsCl gradient or other means. The AAV genome contains single-stranded deoxyribonucleic acid (ssDNA) of either the plus or minus strand. The approximately 4.7 kb genome of AAV consists of one segment of single-stranded DNA with a plus or minus polarity. Both ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and function as origins of viral DNA replication.

[0039] The AAV "genome" ultimately refers to the recombinant nucleic acid sequence that is finally packaged or encapsulated to form AAV particles. AAV particles often contain an AAV genome packaged with AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not include parts of the "plasmid" that do 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", which is important for plasmid cloning and amplification, processes necessary for plasmid propagation and production, but which itself is not packaged or encapsulated into viral particles. Thus, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by AAV capsid proteins.

[0040] An AAV virion (particle) is a non-enveloped icosahedral particle approximately 25 nm in diameter that contains an AAV capsid. AAV particles contain 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 open reading frames (ORFs), sometimes referred to as the left ORF and the right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10 respectively, although the ratio can vary, and all are derived from the right-hand ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by alternative splicing and the use of rare start codons. Deletion analysis has shown that the removal or modification of VP1 translated from a message undergoing alternative splicing results in a reduction in the yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of an AAV particle encodes one, two, or all three of the VP1, VP2, and VP3 polypeptides.

[0041] The left ORF often encodes the 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 in a region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity in addition to DNA helicase and RNA helicase activities. Some of the Rep proteins have nuclear localization signals along with several potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more of the Rep proteins can be delivered in trans and thus are not included in AAV particles containing a nucleic acid encoding a polypeptide.

[0042] The termini of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into T-shaped hairpin structures that serve as origins of viral DNA replication. Thus, the AAV genome contains one or more (e.g., a pair) of ITR sequences flanking the single-stranded viral DNA genome. The ITR sequences often each have a length of about 145 bases. Within the ITR region, two elements, the GAGC repeat motif and the terminal resolution site (trs), which are considered central to the function of the ITR, are 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 cleavage at the trs, which occurs site- and strand-specifically. These two elements also appear to be central to viral inclusion in addition to their role in replication. The Rep binding site adjacent to the trs is included in the chromosome 19 integration locus. These elements have been shown to be functional and necessary for site-specific inclusion.

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

[0044] A recombinant viral "vector" is derived from the wild-type genome of a virus by using molecular methods to remove a portion of the wild-type genome from the virus and replace it with a non-native nucleic acid such as a nucleic acid sequence. Typically, for example in the case of AAV, one or both of the inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome in that a portion of the viral genome has been replaced with a non-native sequence such as a nucleic acid encoding a transactivating factor or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein. Therefore, the incorporation of such a non-native nucleic acid sequence defines the viral vector as a "recombinant" vector and in the case of AAV it can be referred to as an "rAAV vector".

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

[0046] AAV vectors (e.g., rAAV vectors) can be packaged and are herein referred to as "AAV particles" for in vitro, ex vivo, or in vivo cell infection (transduction). When a recombinant AAV vector is encapsulated or packaged in an AAV particle, the particle can also be referred to as an "rAAV particle". In certain embodiments, the AAV particle is an rAAV particle. rAAV particles often contain an rAAV vector or a portion thereof. rAAV particles can be one or more rAAV particles (e.g., multiple AAV particles). rAAV particles typically contain proteins (e.g., capsid proteins) that encapsulate or package the rAAV vector genome. Note that reference to an rAAV vector can also be used to refer to an rAAV particle.

[0047] Any suitable AAV particles (e.g., rAAV particles) can be used in the methods or uses described herein. The rAAV particles and / or the genomes contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particles and / or the genomes contained therein can be derived from two or more serotypes or strains of AAV. Thus, rAAV can contain proteins and / or nucleic acids or portions thereof of any serotype or strain of AAV, and the AAV particles are suitable for infecting and / or transducing 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, a plurality of rAAV particles comprises 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, a plurality of rAAV particles comprises a mixture of two or more different (e.g., different serotypes and / or different strains of) rAAV particles.

[0049] As used herein, the term "serotype" is a characteristic used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are usually due to differences in the capsid protein sequence / epitope (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of an AAV serotype). Even though AAV variants containing capsid variants may not be serologically distinct from a reference AAV serotype or other AAV serotypes, they differ by at least one nucleotide residue or amino acid residue compared to the reference serotype or other AAV serotypes.

[0050] In certain embodiments, an rAAV vector based on a serotype 1 genome corresponds to one or more of the serotypes of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome corresponds to the serotype of the capsid that makes up the rAAV particle.

[0051] In certain embodiments, the rAAV vector genome can be based on an AAV (e.g., AAV2) serotype genome derived from one or more of the serotypes of the AAV capsid proteins that package the vector. For example, the rAAV vector genome can include nucleic acids (e.g., ITRs) 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 certain embodiments, the rAAV particles or their vector genomes related to a reference serotype comprise or consist of a polynucleotide, polypeptide, or a subsequence thereof having a 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 a polynucleotide, polypeptide, or subsequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particles. In certain embodiments, the rAAV particles or their vector genomes related to a reference serotype comprise or consist of a capsid or ITR sequence having a 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 a capsid or ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0053] In certain embodiments, the methods 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 include the use, administration, or delivery of rAAV2 particles. In certain embodiments, the rAAV2 particles include an AAV2 capsid. In certain embodiments, the rAAV2 particles are at least 60%, 65%, 70%, 75%, or more 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%, up to 100% identical, to the corresponding capsid protein of native AAV2 particles or wild-type AAV2 particles, and include one or more capsid proteins (e.g., VP1, VP2, and / or VP3). In certain embodiments, the rAAV2 particles are at least 75% or more identical, e.g., 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%, up to 100% identical, to the corresponding capsid protein of native AAV2 particles or wild-type AAV2 particles, and include VP1, VP2, and VP3 capsid proteins. In certain embodiments, the rAAV2 particles are variants of native AAV2 particles or wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variant 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 certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles are at least 60%, 65%, 70%, 75%, or more 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%, up to 100% identical to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles, and comprise one or more capsid proteins (e.g., VP1, VP2, and / or VP3). In certain embodiments, the rAAV9 particles are at least 75% or more identical, e.g., 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%, up to 100% identical to the corresponding capsid proteins of native AAV9 particles or wild-type AAV9 particles, and comprise VP1, VP2, and VP3 capsid proteins. In certain embodiments, the rAAV9 particles are variants of native AAV9 particles or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variant 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 certain embodiments, the rAAV particles include one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 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%, up to 100% identical, to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10 or AAV-2i8, so long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, the incorporation of AAV DNA into the host cell genome, and / or packaging if desired).

[0057] In certain embodiments, the rAAV2 particles include one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 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%, up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, so long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that enable DNA replication, the incorporation of AAV DNA into the host cell genome, and / or packaging if desired).

[0058] In certain embodiments, the rAAV9 particles include one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 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%, up to 100% identical, to the corresponding ITRs of native or wild-type AAV2 particles, as long as they retain one or more desired ITR functions (e.g., the ability to form hairpins that allow DNA replication, inclusion of AAV DNA into the host cell genome, and / or packaging if desired).

[0059] The rAAV particles can include ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of AAV2 particles include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having 3 "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having less than 4 "GAGC" repeats. In certain embodiments, the rAAV2 particles include ITRs having more than 4 "GAGC" repeats. In certain embodiments, the ITRs of the rAAV2 particles include a Rep binding site where the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0060] Examples of suitable lengths of DNA that can be incorporated into an rAAV vector for packaging / capsidation into rAAV particles can be about 5 kilobases (kb) or less. In certain embodiments, the length of the DNA is 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 direct the expression of RNAi or polypeptides can be produced using appropriate recombinant techniques known in the art (see, for example, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using an AAV virus packaging system. Transducible AAV particles have the ability to bind to and enter mammalian cells and subsequently deliver their nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, intact rAAV particles that are transducible are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often non-replicable and require additional protein machinery to self-replicate. Thus, rAAV particles configured to transduce mammalian cells are engineered to bind to and enter mammalian cells and deliver nucleic acids to those cells, and the nucleic acids delivered are often positioned 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 that have been used as recipients of heterologous rAAV vectors. The stable human cell line HEK293 (readily available, for example, under accession number ATCC CRL1573 from the American Type Culture Collection) can be used. In certain embodiments, a modified human fetal kidney cell line (e.g., HEK293) transformed with an adenovirus type 5 DNA fragment and expressing the adenovirus E1a and E1b genes is used to generate recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for producing rAAV particles. Methods for generating high-titer AAV particles with the ability to transduce mammalian cells are known in the art. For example, AAV particles can be made as described in Wright, 2008 and Wright, 2009.

[0063] In certain embodiments, AAV helper functions are introduced into host cells by transfecting an AAV helper construct into the host cells before or simultaneously with transfection of the AAV expression vector. Thus, in some cases, AAV helper constructs are used to at least transiently express the AAV rep gene and / or cap gene for the purpose of complementing the missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and cannot 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, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both the Rep expression product and the Cap expression product. Several other vectors encoding the Rep expression product and / or the Cap expression product are also known.

[0064] An "expression vector" is a special vector that contains a gene or nucleic acid sequence together with the essential regulatory regions necessary for expression in a host cell. Expression vectors can contain at least an origin of replication for propagation in the cell and optionally additional elements such as heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

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

[0066] A. Inhibitory RNAs "RNA interference (RNAi)" is a process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces the 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" molecules, "short hairpin RNA" or "shRNA" molecules, or "miRNA" are RNA duplexes of nucleotides that target nucleic acid sequences of interest. As used herein, the term "siRNA" is a generic name that encompasses subsets of shRNA and miRNA. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. Since the nucleotide sequence of the duplex 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 huntingtin. In some embodiments, the length of the siRNA duplex is less than 30 base pairs. In some embodiments, the duplex 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 in length. In some embodiments, the duplex length is 19-25 base pairs in length. In certain embodiments, the duplex length is 19 base pairs or 21 base pairs in length. The RNA duplex portion of siRNA can be part of a hairpin structure. The hairpin structure contains, in addition to the duplex portion, a loop portion located between the two sequences that form the duplex. 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 in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure can also contain 3' overhangs and / or 5' overhangs. In some embodiments, the overhang is a 3' overhang and / or 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0068] shRNA is composed of a stem-loop structure designed to contain a 5' adjacent region, an siRNA region segment, a loop region, a 3' siRNA region, and a 3' adjacent region. In most RNAi expression strategies, short hairpin RNAs (shRNAs) driven by strong polIII promoters have been used. Although many shRNAs show effective knockdown of target sequences both in vitro and in vivo, some shRNAs that show effective knockdown of 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 miRNAs are endogenously expressed. Therefore, miRNA molecules are less likely to induce the dsRNA-responsive interferon pathway, are processed more efficiently than shRNAs, and have been shown to silence 80% more effectively than shRNAs.

[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 natural-like with endogenous RNAi substrates and are also more suitable for 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, which is incorporated herein by reference.

[0071] The transcription unit of 「shRNA」 consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin-5 and, upon entering the cytoplasm, undergoes processing by Dicer to generate functional siRNA. The stem-loop of 「miRNA」 consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, which is typically expressed as part of a larger primary transcript (pri-miRNA), which is excised by the Drosha-DGCR8 complex to generate an intermediate known as pre-miRNA, which is then exported from the nucleus by exportin-5 and, upon entering the cytoplasm, undergoes processing by Dicer to generate functional siRNA. As used interchangeably herein, 「artificial miRNA」 or 「artificial miRNA shuttle vector」 refers to a primary miRNA transcript in which the region of the double-stranded stem loop (at least about 9 to 20 nucleotides) excised by Drosha and Dicer processing has been replaced with an siRNA sequence for the target gene while maintaining the structural elements within the stem loop necessary for efficient Drosha processing. The term 「artificial」 derives from the fact that the flanking sequences (approximately 35 nucleotides upstream and approximately 40 nucleotides downstream) are derived from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term 「miRNA」 encompasses both natural miRNA sequences and artificially created miRNA shuttle vectors.

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

[0073] In the design of RNAi, there are several factors that need to be considered, such as the properties of siRNA, the persistence of the silencing effect, and the selection of the delivery system. To produce the RNAi effect, the siRNA introduced into an 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 potential for cross-interference between sequences that are homologous but not gene-specific. Preferably, the siRNA exhibits an identity greater than 80%, 85%, 90%, 95%, or 98%, and even 100% identity between the siRNA sequence and the gene to be inhibited. Sequences with an identity of less than about 80% to the target gene are considerably less effective. Thus, the higher the homology between the siRNA and the gene to be inhibited, the lower the likelihood 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 siRNA molecules that contain at least about 19-25 nucleotides and are capable of modulating gene expression. For the purposes of the present invention, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in length.

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

[0076] B. Non-coding RNA As demonstrated by cDNA cloning projects and genomic tiling arrays, over 90% of the human genome is transcribed but does not encode proteins. These transcripts are referred to as non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNA, transfer RNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are essential for cellular functions. Similarly, numerous short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI-interacting RNA (piRNA), and small nucleolar RNA (snoRNA), are known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long non-coding RNA (lncRNA) transcripts that exhibit cell-type-specific expression and are localized to specific intracellular compartments. LncRNAs are also known to play important roles during cell development and differentiation, supporting the view that they have been selected during the process of evolution.

[0077] LncRNAs appear to have many different functions. In many cases, they serve to regulate the activity or localization of proteins or function as an organizational framework for intracellular structures. In other cases, lncRNAs can be processed to generate multiple small RNAs or can modulate the processing of other RNAs. The most recent version of data generated by the public research consortium GenCode (version number 27) catalogs just under 16,000 lncRNAs in the human genome, generating nearly 28,000 transcripts; when other databases are included, over 40,000 lncRNAs are known.

[0078] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, regulate the activity of protein-binding partners, direct chromatin-modifying complexes to their sites of action, and be post-transcriptionally processed to generate multiple 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0079] Evidence is also accumulating to suggest that aberrantly expressed lncRNAs play important roles in normal physiological processes and in 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 shown in various types of cancer, such as breast, colon, prostate, hepatocellular, and leukemia cancers. Some lncRNAs, such as gadd74 and lncRNA-RoR5, regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thus providing additional flexibility and robustness to cell cycle progression. Furthermore, some lncRNAs are essential for kinetochore formation and are thus involved in mitotic processes, such as centromere satellite RNAs, which are important for chromosome segregation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, functions in cis to regulate M-phase exit by suppressing the expression of the adjacent gene Pura, a regulator of cell proliferation.

[0080] LncRNAs are generally defined as a group of transcripts greater than 200 nucleotides lacking an extended open reading frame (ORF) (e.g., about 200 - about 1200 nt, about 2500 nt, or more). The term "non-coding RNA" (ncRNA) includes lncRNAs and short transcripts less than about 200 nt, such as about 30 - 200 nt.

[0081] Thus, in some embodiments, delivery of an ncRNA to a particular brain structure of interest, for example, corrects abnormal RNA expression levels or modulates levels of lncRNAs that cause disease. Thus, in some embodiments, the invention provides an rAAV in which the viral genome has been engineered to encode a therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) having a length of about 200 nucleotides (nt) or more. In some embodiments, the therapeutic agent is an ncRNA having a length of about 25 nt or about 30 nt to about 200 nt. In some embodiments, the lncRNA has a length of about 200 nt to about 1,200 nt. In some embodiments, the lncRNA has a length 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.

[0082] C. CRISPR System Gene editing is a technique that enables modification of target genes in living cells. In recent years, the implementation of on-demand gene editing using the bacterial CRISPR immune system has brought about a major transformation in the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be engineered to target new sites relatively easily by changing its guide RNA sequence. This discovery has made sequence-specific gene editing functionally effective.

[0083] Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in the expression or direction of activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active partial tracrRNA), tracr-mate sequences (including "direct repeats" and, in the case of endogenous CRISPR systems, partial direct repeats that have undergone tracrRNA processing), guide sequences (also referred to as "spacers" 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 can include a non-coding RNA molecule (guide RNA) that binds sequence-specifically to DNA and a Cas protein (e.g., Cas9) having nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, and can, for example, be derived from a particular organism, including an endogenous CRISPR system, such as Streptococcus pyogenes.

[0085] As discussed herein, the CRISPR system can induce double stranded breaks (DSBs) at target sites, followed by fragmentation. In another aspect, Cas9 variants considered to be "nickases" are used to nick one strand at the target site. For example, for purposes such as improving specificity, a pair of nickases can be used, each directed by a different pair of gRNAs that target the sequence such that a 5' overhang is introduced when the nicks are introduced simultaneously. In another aspect, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor (e.g., KRAB) or transcriptional activator to affect gene expression. Alternatively, the CRISPR system with catalytically inactive Cas9 further includes a transcriptional repressor or transcriptional activator fused to a ribosome-binding protein.

[0086] In some instances, a Cas nuclease and a gRNA (including a fusion of a crRNA specific for the target sequence and an invariant tracrRNA) are introduced into a cell. Generally, the target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, e.g., 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 promote the formation of the CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence designed such that the guide sequence has complementarity thereto, and hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex.

[0087] The target sequence can include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located within the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Generally, a sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide can be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0088] Typically, in the case of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes to the target sequence and forms a complex with one or more Cas proteins) results in 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 base pairs from the target sequence). A tracr sequence that can include or consist of all or part of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence, or more nucleotides than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more) can also form part of the CRISPR complex, for example, by hybridization to all or part of a tracr mate sequence operably linked to the 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, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity over the full length of the tracr mate sequence when optimally aligned.

[0089] One or more vectors that drive the expression of one or more elements of the CRISPR system can be introduced into cells such that the expression of those elements of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. The components can also be delivered to the cell 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 can be a target gene that is subject to the control of the regulatory alternative splicing events disclosed herein as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA can be under the control of a constitutive promoter.

[0090] Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in one vector, and one or more additional vectors can provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of the 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 the cell.

[0091] A vector can include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), 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 forms thereof. These enzymes are known. For example, the amino acid sequence of Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0092] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme mutated such that, compared to the corresponding wild-type enzyme, it lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, a substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (one that cleaves one strand). In some embodiments, Cas9 nickase can be used in combination with a guide sequence, e.g., in combination with two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination makes it possible to nick both strands and use them for the induction of NHEJ or HDR.

[0093] In some embodiments, the enzyme-encoding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be of a particular organism, such as, but not limited to, a mammal including human, mouse, rat, rabbit, dog, or non-human primate, or can be derived from such particular organisms. Generally, codon optimization refers to a process of modifying a nucleic acid sequence by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage frequency among organisms) often correlates with the translation efficiency of messenger RNA (mRNA), and it is generally thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of a selected tRNA in a cell is generally a reflection of the codons that are most frequently used in peptide synthesis. Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization.

[0094] Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence sufficient to hybridize with the target sequence and direct 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 about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using an appropriate alignment algorithm.

[0095] The optimal alignment can be determined using any suitable algorithm for aligning sequences, and non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (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] The CRISPR enzyme may be part of a fusion protein that includes one or more heterologous protein domains. The CRISPR enzyme fusion protein can include any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to the 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-limiting 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 glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), but are not limited thereto. The CRISPR enzyme can be fused to a gene sequence encoding a protein or a fragment of such a protein that binds to a DNA molecule or to other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can 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 aspects relate to the expression of recombinant proteins and polypeptides. In some instances, the protein or polypeptide can be modified to increase stability in serum. Thus, when the present 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 a protein or polypeptide that has, for example, additional advantages compared to an unmodified protein or polypeptide. It is particularly contemplated that aspects regarding "modified proteins" can also be practiced with respect to "modified polypeptides", and vice versa.

[0098] Recombinant proteins can possess amino acid deletions and / or substitutions. Thus, a protein having a deletion, a protein having a substitution, and a protein having both a deletion and a substitution are modified proteins. In some aspects, these proteins can further include inserted or added amino acids, such as, for example, a fusion protein or a protein having a linker. A "modified deletion protein" lacks one or more residues of the native protein but can have the specificity and / or activity of the native protein. A "modified deletion protein" can also have reduced immunogenicity or antigenicity. Examples of modified deletion proteins are those in which amino acid residues are deleted from at least one antigenic region, i.e., a region of the protein determined to be antigenic in a particular organism, such as the organism to which the modified protein is administered.

[0099] A substitution variant or replacement variant typically contains an exchange of one amino acid for another at one or more sites within a protein and can be designed to modulate one or more properties of the 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 by one having a similar shape and charge, or it may not be. Conservative substitutions are well known in the art and include, for example, the change from alanine to serine, from arginine to lysine, from asparagine to glutamine or histidine, from aspartic acid to glutamic acid, from cysteine to serine, from glutamine to asparagine, from glutamic acid to aspartic acid, from glycine to proline, from histidine to asparagine or glutamine, from isoleucine to leucine or valine, from leucine to valine or isoleucine, from lysine to arginine, from methionine to leucine or isoleucine, from phenylalanine to tyrosine, leucine or methionine, from serine to threonine, from threonine to serine, from tryptophan to tyrosine, from tyrosine to tryptophan or phenylalanine, and from valine to isoleucine or leucine.

[0100] In addition to deletions or substitutions, the modified protein may possess an insertion of a residue. This typically involves the addition of at least one residue in the polypeptide. This can include the insertion of a targeting peptide or targeting polypeptide or simply a single residue insertion. Terminal additions, which are called fusion proteins, will be described later.

[0101] The term "biologically functional equivalent" is well understood in the art and will be further detailed herein. Accordingly, sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of a control polypeptide are included as long as the biological activity of the protein is maintained. A recombinant protein can be a biologically functional equivalent to the corresponding native protein in certain aspects.

[0102] It will also be understood that amino acid and nucleic acid sequences can include additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' sequences, and still be essentially as described in one of the sequences disclosed herein as long as the sequence meets the above criteria, including maintenance of biological protein activity where protein expression is involved. The addition of terminal sequences is particularly applicable to nucleic acid sequences that can include various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences known to be present within the gene, i.e., introns.

[0103] As used herein, a protein or peptide generally refers to a protein translated from a gene that is more than about 200 amino acids up to the full-length sequence at most; a polypeptide having more than about 100 amino acids; and / or a peptide having about 3 to about 100 amino acids, but is not limited thereto. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

[0104] As used herein, "amino acid residue" refers to 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 have no non-amino acid sequences interrupting the sequence of amino acid residues. In another embodiment, the sequence may contain 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] Thus, 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 unusual amino acid.

[0106] Certain embodiments of the invention relate to fusion proteins. These molecules can have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, a leader sequence from another species may be used in the fusion 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, preferably cleavable, or an immunologically active domain such as an antibody epitope, to facilitate purification of the protein. Non-limiting examples of affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).

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

[0108] The production of fusion proteins that restore the functional activity of a parent protein can be facilitated by linking genes with a cross - bridging DNA segment encoding a peptide linker that is joined between tandemly linked polypeptides. The linker is of sufficient length to allow proper folding of the resulting fusion protein.

[0109] III. Methods of Administration Viral vectors may, in some instances, be administered directly (in vivo) to a patient or used to treat cells in vitro or ex vivo and then 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 vehicle that can be manipulated by insertion or incorporation of nucleic acid. Vectors such as viral vectors can be used to introduce / transfer nucleic acid into cells such that the nucleic acid sequence within the nucleic acid is transcribed by the cell and, if it encodes a protein, subsequently translated.

[0110] Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein is suspected of having or expressing an abnormal protein or an aberrant protein associated with a disease state. Alternatively, a mammalian recipient may have a condition suitable for gene replacement therapy. As used herein, "gene replacement therapy" refers to 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 genetic diseases (i.e., disease states resulting from one or more gene deficiencies), acquired pathologies (i.e., pathological states not resulting from congenital deficiencies), cancer, and preventive processes (i.e., prevention of disease or an undesirable medical condition). Thus, as used herein, the term "therapeutic substance" refers to any agent 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 (such as apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (such as dogs and cats), agricultural animals (such as horses, cows, goats, sheep, pigs), and laboratory animals (such as mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (such as humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or any stage of development (such as an adult, a teenager, a child, an infant, or a mammalian fetus in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, such as an animal model having or expressing an abnormal protein or an abnormal protein associated with a disease state, or an animal model in which the expression of a protein is insufficient and causes a disease.

[0112] Mammals (subjects) treated with the methods or compositions described herein include adults (18 years of age or older) and children (under 18 years of age). Adults include the elderly. A representative adult is 50 years of age or older. The age of children ranges 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 include infants. Infants typically range from 1 - 12 months of age.

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

[0114] In certain embodiments, the method includes administering a plurality of viral particles to the central nervous system or a portion thereof, as described herein, to reduce, mitigate, prevent, inhibit, or delay the severity, frequency, progression, or time of onset of one or more symptoms of a disease state, such as a neurodegenerative disease, 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.

[0115] In certain embodiments, the symptoms or detrimental effects include early, intermediate, or late symptoms, behavioral, personality, or speech symptoms, swallowing, movement, seizures, tremors, or restlessness symptoms, ataxia, and / or cognitive symptoms, such as memory, the ability to organize.

[0116] IV. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable 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 "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without substantially causing undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical formulations and pharmaceutical compositions can be used, for example, when administering virus 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, dispersion media and suspending media, coatings, isotonicity and absorption promoting or absorption delaying agents, which are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions can include suspending and thickening agents. Auxiliary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) can also be incorporated into the formulations and compositions.

[0118] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of harmful antibodies in the individual to whom the composition is administered and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol, and ethanol. It can include pharmaceutically acceptable salts, such as mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate, and salts of organic acids such as acetate, propionate, malonate, benzoate. Additionally, auxiliary substances such as surfactants, wetting or emulsifying agents, pH buffering substances, etc. can be present in such media.

[0119] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery described herein or known to those skilled in the art. Thus, the pharmaceutical composition includes carriers, diluents or excipients suitable for administration or delivery by various routes.

[0120] Pharmaceutical forms suitable for injection of virus particles are adapted for the immediate preparation of sterile injectable or infusible solutions or dispersions, and can include sterile aqueous solutions or aqueous dispersions, optionally encapsulated in liposomes. In either case, the final dosage form should be a sterile fluid and stable under the conditions of manufacture, use and storage. Liquid carriers or media can be solvents or liquid dispersion media including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Isotonic agents, such as sugars, buffers or salts (such as sodium chloride) can be included. Sustained absorption of the injectable composition can be brought about by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0121] The solution or suspension of virus particles can optionally contain one or more of the following components: a sterile diluent, such as water for injection, a saline solution, such as phosphate buffered saline (PBS), artificial CSF, a surfactant, a fixed oil, a polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents, antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate, and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0122] Pharmaceutical formulations, compositions, and delivery systems suitable for the compositions, methods, and uses of the present invention are known in the art (e.g., 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), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

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

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

[0125] As used herein, the term "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, for example, can be added before 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. 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 administered depends on factors such as the age, weight and health status of the mammalian or human subject for which the treatment is contemplated. One of ordinary skill in the art can establish the 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 their polymers. 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 the 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 or 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" generally refers to 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 recognition sites for RNA polymerase and other factors necessary for proper transcription. A pol II promoter includes a minimal promoter, which is a short DNA sequence composed of a TATA box and other sequences that optionally function to specify the transcription start site, to which regulatory elements for controlling expression are added. A type 1 pol III promoter contains three cis-acting sequence elements downstream of the transcription start site: a) a 5' sequence element (A block); b) an intermediate sequence element (I block); c) a 3' sequence element (C block). A type 2 pol III promoter contains two essential cis-acting sequence elements downstream of the transcription start site: a) an A box (5' sequence element); and b) a B box (3' sequence element). A type 3 pol III promoter contains several cis-acting promoter elements upstream of the transcription start site, such as a conventional TATA box, a proximal sequence element (PSE), and a distal sequence element (DSE).

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

[0132] A promoter and / or enhancer may be wholly derived from a native gene, or may be composed of different elements derived from different elements found in nature, or may even be composed of synthetic DNA segments. A promoter or enhancer may include a DNA sequence involved in the binding of protein factors that regulate / control 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, such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, are also considered useful here. Exemplary constitutive promoters include the promoters of the following genes encoding certain constitutive or "housekeeping" functions, and other constitutive promoters known to those skilled in the art: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6. In addition, many viral promoters function constitutively in eukaryotic cells. These include, among others, the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In addition, sequences derived from intronic miRNA promoters, such as, for example, the miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566, and miR128 promoters, are also considered to find use herein (see, for example, Monteys et al., 2010). Thus, any of the above-described constitutive promoters can be used to control the transcription of heterologous gene inserts.

[0134] "Transgene" is used herein for convenience to refer to a nucleic acid sequence / polynucleotide that is or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as an inhibitory RNA or a gene encoding a polypeptide or protein, which are generally heterologous to the native AAV genomic 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). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. When 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 episomally or only transiently in the recipient cell or recipient host organism. 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 can be transcribed, and the encoded inhibitory RNA or protein can be expressed. In the case of use in gene therapy and methods of gene therapy, transduced cells can be present in a mammal.

[0136] A transgene under the control of an inducible promoter is expressed only in the presence of an inducer or is expressed more strongly in the presence of an inducer (for example, transcription under the control of the metallothionein promoter is significantly increased in the presence of certain metal ions). Inducible promoters contain responsive elements (REs) that stimulate transcription when the respective inducer binds. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. A promoter containing a specific RE can be selected to obtain an inducible response, and in some cases, the RE itself can be ligated to a different promoter to confer inducibility on the recombinant gene. Therefore, by selecting an appropriate promoter (constitutive or inducible, strong or weak), it is possible to control both the presence and the expression level of a polypeptide in genetically modified cells. When a gene encoding a polypeptide is under the control of an inducible promoter, in situ delivery of the polypeptide is triggered by exposing the in situ genetically modified cells to conditions that permit transcription of the polypeptide, for example, by intraperitoneal injection of a specific inducer of the inducible promoter that controls transcription of the agent. For example, in situ expression of a polypeptide encoded by a gene under the control of the metallothionein promoter is enhanced by contacting the genetically modified cells in situ with a solution containing the 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 a polypeptide or a nucleic acid that directs the expression of a polypeptide may include an inducible promoter or a tissue-specific promoter to control the transcription of the encoded polypeptide. A nucleic acid functionally linked to an expression control element can also be referred to as an expression cassette.

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

[0139] In certain embodiments, the expression control element comprises a CMV enhancer. In certain embodiments, the expression control element comprises a β-actin promoter. In certain embodiments, the expression control element comprises a chicken β-actin promoter. In certain embodiments, the expression control element comprises 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 a subsequence thereof deviates from a reference sequence. Therefore, a modified sequence and a variant sequence may have an expression level, activity, or function that is substantially the same as, greater than, or less than that of the reference sequence, but retains 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 having a mutation such as a missense mutation or a nonsense mutation.

[0141] A "nucleic acid" variant or "polynucleotide" variant refers to a modified sequence in which the gene has changed as compared to the wild type. The sequence can be genetically modified without changing the encoded protein sequence. Alternatively, the sequence can be genetically modified to encode a variant protein. A nucleic acid variant or polynucleotide variant also refers to a combined sequence that is codon-modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild-type protein sequence, and is also codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant change so as not to change the amino acid of the protein encoded thereby, and some codons of the nucleic acid variant change, resulting in a change in the amino acid of the protein it encodes.

[0142] The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptides" encoded by the "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial-length or full-length native sequences, as well as native wild-type and functional polymorphic proteins, their functional partial sequences (fragments), and their sequence variants, as long as the polypeptide retains some function or activity. Thus, in the methods and uses of the present invention, such polypeptides encoded by a nucleic acid sequence need not be identical to an endogenous protein that is defective or whose activity, function or expression is insufficient, lacking or absent in the mammal being treated.

[0143] Non-limiting examples of modifications include 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 are conservative amino acid substitutions or deletions. In certain embodiments, the modified sequence or variant sequence retains at least a portion of the function or activity of the unmodified sequence (e.g., wild-type sequence).

[0145] Another example of an amino acid modification is a targeting peptide introduced into the capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors to the central nervous system, e.g., different brain regions.

[0146] Such modified recombinant viruses may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, recombinant viruses that retain the modified capsid protein can "target" cerebrovascular epithelial tissue by binding at a higher level than a comparable unmodified capsid protein. For example, a recombinant virus having a modified capsid protein can bind to cerebrovascular epithelial tissue at a level 50% to 100% higher than an unmodified recombinant virus.

[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 the disclosed nucleotide sequences, as well as the proteins or partial-length proteins encoded thereby, are also encompassed by the present invention. "Fragment" or "portion" means a nucleotide sequence or amino acid sequence of a polypeptide or protein that encodes a full-length or less than full-length polypeptide or protein. In certain embodiments, the fragment or portion is biologically functional (i.e., retains 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 an array substantially similar to the sequence of the native molecule. In the case of a nucleotide sequence, variants include sequences that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Such natural allelic variants can be identified using molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences include those of synthetic origin, such as those encoding native proteins produced using site-directed mutagenesis, as well as those encoding polypeptides having amino acid substitutions. In general, nucleotide sequence variants of the present invention have at least 40%, 50%, 60%, or 70%, for example 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, and generally at least 80%, for example 81% - 84%, at least 85%, for example 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the activity or function of the wild type).

[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, at any position where arginine is specified by a codon, 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 appreciated by those skilled 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 obtain a functionally identical molecule. Thus, each "silent variation" of a nucleic acid encoding a polypeptide is implicitly represented by each of the described sequences.

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

[0151] The term "substantial identity" with respect to a polypeptide means that the polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99% sequence identity to a reference sequence in a specified comparison window. A measure that two polypeptide sequences are identical is that one polypeptide immunoreacts with an antibody raised against the other polypeptide. Thus, for example, if the differences between two polypeptides are only conservative substitutions, one polypeptide is identical to the other polypeptide.

[0152] The terms "treating" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, inhibit, reduce, or decrease an undesirable physiological change or disorder, e.g., the development, progression, or worsening of a disorder. For purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, diminishment of the degree of a disease, stabilization of a disease state or symptoms or adverse effects thereof (i.e., absence of worsening or progression), delay or slowing of disease progression, improvement or alleviation of a disease state, and remission (whether partial or complete). "Treatment" can also mean an extension of survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already having a condition or disorder, as well as those having a predisposition (e.g., as determined by a genetic assay).

[0153] VI. Kit The present invention provides a kit that includes a packaging material and one or more components therein. The kit typically includes a label or package insert that contains a description of the components therein or instructions regarding the in vitro, in vivo, or ex vivo use of the components contained therein. The kit can include a collection of such components, e.g., nucleic acids, recombinant vectors, and / or viral particles.

[0154] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can be made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.) that can maintain the components in a sterile manner.

[0155] The label or package insert can include identification information of one or more components contained therein, dosage, clinical pharmacology of the active ingredient, such as mechanism of action, pharmacokinetics and pharmacodynamics. The label or package insert can include information for verifying the manufacturer, lot number, manufacturing location and date of manufacture, and expiration date. The label or package insert can include information for verifying the manufacturer information, lot number, manufacturing location and date of manufacture. The label or package insert can include information regarding the diseases for which the components of the kit can be used. The label or package insert can include instructions for clinicians or subjects for using one or more of the components of the kit in a method, use or treatment protocol or therapeutic regimen. The instructions can include dosage, dosing frequency or duration, and instructions for implementing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimens described herein.

[0156] The label or package insert can include information regarding the benefits that the components can provide, such as prophylactic or therapeutic benefits. The label or package insert can include information regarding potentially harmful side effects, complications or reactions, such as warnings to subjects or clinicians regarding situations in which it is considered inappropriate to use a particular composition. Since harmful side effects or complications can occur if the subject has taken or will take or is taking one or more other medicaments that may be incompatible with the composition, or if the subject has undergone or will undergo or is undergoing another treatment protocol or therapeutic regimen that is considered incompatible with the composition, the instructions can also include information regarding such incompatibilities.

[0157] Labels or accompanying documents include "printed matter", such as paper or cardboard, which are independent or attached to components, kits or packaging materials (such as boxes), or attached to ampoules, tubes or vials containing components of the kit. Labels or accompanying documents can further include computer-readable media, such as printed labels with barcodes, 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-type cards.

Examples

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

[0159] Example 1 - Identification of AAV Variants Targeting Brain Parenchyma Using AAV1, AAV2, and AAV9 capsids as starting platforms, an advanced barcoded AAV library was developed. By inserting random sequences at position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, and position 588 of the AAV9 capsid, respectively, AAV1, AAV2, and AAV9 peptide display libraries were generated (Figure 1). This library had a diversity of 1×10 7 individual unique clones (Figure 3).

[0160] To test the utility of the library, a pilot study was conducted using bench-grade (low titer, low purity) capsid-modified AAV2. The AAV2 library was intravenously injected into two C57BL / 6 mice at 8×10 10 vector genomes per animal. After 72 hours, 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 generate a second-round library, which was injected into two mice at 4×10 10 vector genomes per animal. After the second injection, the vector genome was recovered as before and subjected to NexGen sequencing together with the starting library and the first-round tissues. To test whether sequences indicative of enrichment in brain tissue could actually reach the brain with AAV2, individual hits were cloned into an AAV2 capsid packaging plasmid to generate AAV2 expressing eGFP. Bench-grade vectors were generated and 3×10 10 vector genomes of the capsid-modified virus based on AAV2 were injected into mice. After 4 weeks, fluorescence of eGFP was seen in the brain even for these low-titer variants.

[0161] Using these highly barcoded AAV libraries, AAV variants that can target different primate brain structures were identified in non-human primates. The AAV1, AAV2, and AAV9 libraries were delivered by intracerebroventricular injection to one non-human primate (Figure 2). Seventy-two hours after injection, brain regions were microdissected for viral DNA isolation, and AAV DNA was amplified by PCR. The products were pooled and used for packaging of the second-round library, which was then injected into additional NHPs. Brain regions were then microdissected 12 days after injection. After two rounds of panning, the vector genomes were recovered and subjected to next-generation sequencing. Specifically, genomic DNA extracted from round 1 and round 2 tissues was PCR amplified to generate an Illumina amplicon sequencing library at the location of the vector barcode. The resulting libraries were pooled and run on a single lane of an Illumina HiSeq 4000 using 100 bp single-end read chemistry. To illustrate the utility of this approach, several target regions: the cortex, meninges, and cerebellum were tested as examples. In general, the sequences directing AAVx to the cortex, meninges, and cerebellum were different and differed among the various serotypes.

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

[0163] To verify the identified cell type specificity, AAV9-1999 (having the targeting peptide sequence of KGGGFHG; SEQ ID NO: 110) was selected for in vivo verification. An eGFP expression construct was packaged into AAV9-1999 driven by the CAG promoter. Five-year-old female rhesus monkeys were administered 1.5E13 vg of AAV9-1999 by ICV injection into the left ventricle. 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). The cochleae were also collected from this animal and, surprisingly, had strong transduction of hair cells. Furthermore, AAV9-1999 and AAV9 capsids containing the eGFP construct were delivered to C57BL / 6 p0 mouse pups by ICV injection at 1E10 vg per hemisphere. After 21 days, the mice were perfused. Whole-mount brains (Figure 9A), 40 μm whole-brain sagittal sections (Figure 9B), 40 μm S1 cortical sections (Figure 9C, left), 40 μm hippocampal sections (Figure 9C, middle), 40 μm cerebellar sagittal sections (Figure 9C, right), and 40 μm lumbar spinal cord coronal sections (Figure 9D) were imaged for eGFP fluorescence signal. AAV9-1999 injected into Bl / 6 neonatal mouse pups showed more ubiquitous expression than injections with a matched dose of AAV9.

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

[0165] For fluorescence imaging, the brain was collected 30 days after injection. Lateral ventricle sections (Figure 10A), fourth ventricle sections (Figure 10B), and meninges sections (Figure 10C) were imaged for mTagBFP2, mTFP2, mNG, and mRuby3 fluorescence signals.

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

[0167] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been 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 of the methods described herein or to the order of the steps without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents can be used in place of the agents described herein, but that the same or similar results can be achieved. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0168] References The following references are hereby specifically incorporated by reference herein to the extent that they provide exemplary procedures or other details that supplement those described herein. TIFF0007712271000031.tif189150TIFF0007712271000032.tif32150

Claims

**Claim 1** A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide, wherein the targeting peptide targets a virus vector comprising the modified AAV capsid protein to different brain structures, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 110, the targeting peptide is at most 10 amino acids in length, and the modified AAV capsid protein is a modified AAV9 capsid protein. **Claim 2** The modified AAV9 capsid protein according to claim 1, which is derived from an AAV9 capsid protein having the amino acid sequence of SEQ ID NO: 140, and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. **Claim 3** The modified AAV capsid protein according to claim 2, 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 acids in length. **Claim 4** The modified AAV capsid protein according to claim 3, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. **Claim 5** The modified AAV capsid protein according to claim 4, wherein the modified AAV9 capsid protein has a sequence that is at least 95% identical to SEQ ID NO:

143. **Claim 6** The modified AAV capsid protein according to claim 5, wherein the targeting peptide is at positions 592-598 of the amino acid residues of SEQ ID NO:

143. **Claim 7** The modified AAV capsid protein according to claim 1, wherein the targeting peptide is 7 amino acids in length and consists of SEQ ID NO:

110. **Claim 8** The modified AAV capsid protein according to claim 1, wherein 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. **Claim 9** A nucleic acid comprising a sequence encoding the modified AAV capsid protein according to any one of claims 1-8. **Claim 10** A recombinant adeno-associated virus (rAAV) comprising the modified AAV capsid protein according to any one of claims 1 to 8.

11. A viral vector comprising a nucleic acid encoding the modified AAV capsid protein according to any one of claims 1 to 8.

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

13. An in vitro cell comprising the viral vector according to claim 11 or 12.

14. A pharmaceutical composition comprising the rAAV according to claim 10, or the viral 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 a disease in a mammal.

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 paraplegia, 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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