RU2845941C1 - Versions of adeno-associated viral vector - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are a modified capsid protein of adeno-associated virus (AAV) for packaging a viral vector with formation of an AAV particle, wherein the modified AAV capsid protein comprises a targeting peptide that contains SEQ ID NO: 110, nucleic acid including a sequence coding the modified capsid protein, and a recombinant adeno-associated virus (rAAV) particle containing the rAAV vector packed by the modified capsid protein. In addition, an rAAV vector containing a nucleic acid coding a modified capsid protein, and a cell containing the rAAV vector obtained by transduction with an rAAV particle to obtain AAV particles are provided.

EFFECT: invention can be used to package a viral vector to form an AAV particle, wherein the targeting peptide targets the AAV particle to the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, pale ball, hippocampus, meninges, optic nerve, shell, spinal cord, substantia nigra, subthalamic nuclei or thalamus.

26 cl, 11 dwg, 6 tbl, 1 ex

Description

LINK TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62/939,315, filed November 22, 2019, and U.S. Provisional Application Serial No. 63/084,709, filed September 29, 2020, the entire contents of both applications being hereby incorporated by reference in their entireties.

LINK TO SEQUENCE LIST

[0002] This application contains a sequence listing that was submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The above ASCII copy, created on November 19, 2020, is named CHOPP0038WO_ST25.txt and is 63.8 kilobytes in size.

PREREQUISITES FOR THE CREATION OF THE INVENTION

1. Field of technology

[0003] The present invention relates generally to the fields of medicine, virology and neurology. More specifically, it relates to targeting peptides that direct the delivery of viral vectors to specific structures of the brain.

2. Description of the prior art

[0004] Various strategies have been developed to generate AAV-based vector variants, including rational design and directed evolution. The rational design approach uses knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, such as tyrosine mutations on the capsid surface to increase transduction efficiency. The directed evolution approach does not require any knowledge of the capsid structure and is accomplished through random mutagenesis, capsid shuffling, or random peptide insertions. These strategies typically use in vitro or mouse systems, which are ideal for cell or mouse studies but do not imply translation to the clinic. In fact, no AAV variants specifically or efficiently target specific brain structures. As such, AAV variants that are capable of targeting specific brain structures in primates are needed.

SUMMARY OF THE INVENTION

[0005] 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 a particular structure of the brain.

[0006] In one embodiment, modified adeno-associated virus (AAV) capsid proteins are provided, comprising a targeting peptide that targets a viral vector comprising a modified AAV capsid protein to a specific organ or structure of the brain, wherein the targeting peptide is from three to ten amino acids in length. In some aspects, the modified AAV capsid proteins are modified AAV1 capsid proteins, modified AAV2 capsid proteins, or modified AAV9 capsid proteins.

[0007] In some aspects, the modified AAV capsid proteins are derived from an AAV1 capsid protein (see SEQ ID NO: 138), and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, 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 aspects, the modified AAV1 capsid proteins have a sequence that is at least 95% identical to SEQ ID NO: 141.

[0008] In some aspects, the modified AAV capsid proteins are derived from an AAV2 capsid protein (see SEQ ID NO: 139), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, 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 aspects, the modified AAV2 capsid proteins have a sequence that is at least 95% identical to SEQ ID NO: 142.

[0009] In some aspects, the modified AAV capsid proteins are derived from an 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 flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. 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 proteins have a sequence that is at least 95% identical to SEQ ID NO:143.

[0010] In some aspects, the targeting peptide comprises a sequence of up to ten amino acids in length comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-137 and 144. In some aspects, the targeting peptide is seven amino acids in length.

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

[0012] In some aspects, the defined organ is the brain, kidney, heart, liver, gonad, spleen, or liver. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from the peptides listed in Table 4 to target the corresponding organ. In some aspects, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from the peptides listed in Table 5 to target the corresponding organ. In some aspects, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from the peptides listed in Table 6 to target the corresponding organ.

[0013] In one embodiment, provided herein are nucleic acids comprising a sequence encoding a modified capsid protein according to any of the embodiments of the present invention.

[0014] In one embodiment, provided herein are recombinant adeno-associated viruses (rAAV) comprising a modified capsid protein according to any of the embodiments of the present invention. In some aspects, provided are combinations of rAAV. For example, provided is a combination of an rAAV comprising a modified AAV1 capsid protein and the targeting peptide of SEQ ID NO:21, an rAAV comprising a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO:53, an rAAV comprising a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO:80, and an rAAV comprising a modified AAV9 capsid protein and the targeting peptide of SEQ ID NO:113.

[0015] In one embodiment, viral vectors are provided comprising a nucleic acid encoding a modified capsid protein according to any of the embodiments of the present invention. In some aspects, the viral vectors further comprise 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 embodiment, provided herein are cells comprising a viral vector according to any of the embodiments of the present invention. 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 embodiment, provided herein are pharmaceutical compositions comprising a viral vector according to embodiments of the present invention and a pharmaceutically acceptable carrier.

[0018] In one embodiment, provided herein are methods of delivering an agent to a specific brain structure of a subject comprising administering to the subject a virus in accordance with embodiments of the present invention. In some aspects, the specific brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nuclei, or thalamus. In some aspects, an rAAV comprising a modified AAV1 capsid protein is used and the targeting peptide is selected from the peptides listed in Table 1 to target the corresponding brain structure. In some aspects, an rAAV comprising a modified AAV2 capsid protein is used and the targeting peptide is selected from the peptides listed in Table 2 to target the corresponding brain structure. In some aspects, an rAAV comprising a modified AAV9 capsid protein is used, and the targeting peptide is selected from the peptides listed in Table 3 to target a relevant brain structure. In various aspects, combinations of any rAAVs are used. For example, a combination of an rAAV comprising a modified AAV capsid protein 1 and the targeting peptide of SEQ ID NO:21, an rAAV comprising a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO:53, an rAAV comprising a modified AAV2 capsid protein and the targeting peptide of SEQ ID NO:80, and an rAAV comprising a modified AAV9 capsid protein and the targeting peptide of SEQ ID NO:113 is used.

[0019] In one embodiment, provided herein are methods of delivering an agent to a specific organ of a subject comprising administering to the subject a virus in accordance with embodiments of the present invention. In some aspects, the organ is the brain, kidney, heart, liver, gonad, spleen, or liver. In some aspects, an rAAV comprising a modified AAV1 capsid protein is used and the targeting peptide is selected from the peptides listed in Table 4 to target the corresponding organ. In some aspects, an rAAV comprising a modified AAV2 capsid protein is used and the targeting peptide is selected from the peptides listed in Table 5 to target the corresponding organ. In some aspects, an rAAV comprising a modified AAV9 capsid protein is used and the targeting peptide is selected from the peptides listed in Table 6 to target the corresponding organ. In various aspects, combinations of any rAAVs are used.

[0020] In some aspects, the agent is a siRNA (short interfering RNA), shRNA (short hairpin RNA), microRNA, non-coding RNA, long non-coding RNA (lncRNA), therapeutic protein, or CRISPR system. In some aspects, administration is into the central nervous system. In some aspects, administration is into a large (cerebellar) cyst, an intraventricular space, an ependyma, a cerebral ventricle, a subarachnoid space, and/or an intrathecal space. In some aspects, a cerebral ventricle is a rostral part of a lateral ventricle and/or a caudal part of a lateral ventricle and/or a right lateral ventricle and/or a left lateral ventricle and/or a rostral part of a right lateral ventricle and/or a rostral part of a left lateral ventricle and/or a caudal part of a right lateral ventricle and/or a caudal part of a left lateral ventricle.

[0021] In some aspects, a plurality of viral particles are administered. In some aspects, the virus is administered at a dose of from about 1×10 ⁶ to about 1×10 ¹⁸ vector genomes per kilogram (vector genomes/kg). In some aspects, 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 -x10 ¹³ , or about 1×10 ¹³ -1X10 ¹⁴ vector genomes/kg of the patient. In some aspects, the subject is a human.

[0022] In one embodiment, provided herein are methods of treating a disease in a mammal comprising administering to the mammal viruses in accordance with embodiments of the present invention. In some aspects, the disease is a neurodegenerative disease. In some aspects, the neurodegenerative disease is Huntington's disease, ALS (amyotrophic lateral sclerosis), hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, CAG trinucleotide repeat expansion disorder, or Parkinson's disease. In some aspects, the mammal is a human.

[0023] The term "substantially free" with respect to said component is used herein to mean that none of said components has been intentionally included in the composition and/or is present only as an impurity or in trace amounts. Therefore, the total amount of said component as a result of any unintentional contamination of the composition is significantly below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amounts of said component can be detected using standard analytical methods.

[0024] As used herein, the words "a" or "an" may mean one or more. As used herein in the claim(s), when used in combination with the word "comprising", the words "a" or "an" may mean one or more than one.

[0025] The term "or" in the claims is used to mean "and/or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although the present description supports a definition that refers only to alternatives and "and/or". As used herein, the term "another" may mean at least a second or more.

[0026] Throughout this application, the term "approximately" is used to mean that a value includes the variation in error inherent in the device, the method used to determine the value, the variation that exists among study subjects, or a value that is within 10% of the determined value.

[0027] 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 showing preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF DRAWINGS

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

[0029] Fig. 1. Schematic of the AAV peptide display library.

[0030] Fig. 2 Schematic of the in vivo screening strategy.

[0031] Fig. 3. Graphical representation of input library diversity. Input viral library diversity measured on AAV1, AAV2, and AAV9 viral vector aliquots prior to cycle 1 ICV injection.

[0032] Fig. 4. Graphical representation of cycle-by-cycle barcode enrichment. Total number of unique barcodes obtained after cycle 1 and 2 enrichment in rhesus macaques for each tissue collected. Values for DNA and RNA after cycle 2 are shown.

[0033] Fig. 5. Illustration of cycle-by-cycle enrichment of barcodes in AAV1, AAV2, and AAV9 serotypes for the cerebellar cortex.

[0034] Fig. 6. Illustration of enrichment for AAV9 1999. The heat map depicts the enrichment of barcodes from AAV9, cells are colored according to the percentage of barcodes found in the indicated tissue. DNA-derived barcodes are shown on the left, while RNA-derived barcodes are shown on the right.

[0035] Fig. 7A-C. Heat map depicts enrichment of the opool barcodes from AAV1 (Fig. 7A), AAV2 (Fig. 7B), and AAV9 (Fig. 7C).

[0036] Fig. 8. In vivo validation of AAV9 1999 in rhesus macaques. The eGFP expression construct was packaged into AAV9 1999 under the control of the CAG promoter. 1.5E13 AAV9 1999 vector genomes were administered to a 5-year-old female rhesus macaque via ICV injection. Representative images of H&E-stained cerebellum are shown, demonstrating the transduction pattern of AAV9 1999.

[0037] Fig. 9A-D. In vivo validation of 1999 AAV9 in mice. The eGFP expression construct was packaged into 1999 AAV9 under the control of the CAG promoter. AAV9 1999 and AAV9 capsids containing the eGFP construct were delivered to C57BL/6 p0 mouse pups by ICV injection at 1E10 vector genomes. Representative images showing the fluorescent signal from eGFP include the whole brain (Fig. 9A), a sagittal section of the whole brain (Fig. 9B), sections of the cerebral cortex (Fig. 9C, left), sections of the hippocampus (Fig. 9C, middle), a sagittal section of the cerebellum (Fig. 9C, right), and a coronal section of the lumbar spinal cord (Fig. 9D).

[0038] Fig. 10A-C: Fluorescence images of the lateral ventricle (A), fourth ventricle (B), and meninges (C) of a rhesus macaque following administration of an AAV mixture in vivo.

[0039] Fig. 11A-C. Fluorescence images of cochlear whorls (A), inner hair cells (B), organ of Corti (C), and distal cochlear shaft (C) following injection of AAV9 1999 capsids containing the eGFP construct into the cochlea of mice.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0040] 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 a particular brain structure. In some embodiments, the brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nuclei, or thalamus. Targeting peptides for each brain structure are provided in Tables 1-3.

[0041] In some embodiments, the viral vector is an adeno-associated viral vector (AAV). In some embodiments, the AAV is AAV1, AAV2, or AAV9. An exemplary reference sequence of a wild-type AAV1 capsid protein is set forth in SEQ ID NO:138. An exemplary reference sequence of a wild-type AAV2 capsid protein is set forth in SEQ ID NO:139. An exemplary reference sequence of a wild-type AAV9 capsid protein is set forth in SEQ ID NO:140. In some 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. An exemplary modified AAV1 capsid protein sequence is shown in SEQ ID NO:141, which shows an insertion of a targeting peptide after position 590 as SSAX ₇ AS, wherein the leading SSA and trailing AS are linker sequences and X ₇ is a targeting peptide. An exemplary modified AAV2 capsid protein sequence is shown in SEQ ID NO:142, which shows an insertion of a targeting peptide after position 587 as AAAX ₇ AA, wherein the leading AAA and trailing AA are linker sequences and X ₇ is a targeting peptide. An exemplary modified AAV9 capsid protein sequence is shown in SEQ ID NO:143, which shows an insertion of a targeting peptide after position 588 as AAAX ₇ AS, where the leading AAA and trailing AS are linker sequences and X ₇ is the targeting peptide.

Table 1. AAV1 targeting peptides for each brain structure Region Peptide SEQ ID NO Brain stem RPGREQA 1 RGVLVTT 2 RPGRESA 3 NESLKKK 4 DKTRAGS 5 TAKSKQA 6 PVKKKDA 7 GRETLKG 8 PIPAGKK 9 Caudate nucleus RPGRESA 3 PVKKKDA 7 RPGREQA 1 NVVRAGT 10 KATANTR 11 RDATRSS 12 VPTKSPK 13 AGVARSK 14 RSRSEVL 15 EVKGKGK 16 RPGREAS 144 RDSTRQL 26 SGVLVQR 32 DKTRAGS 5 TAPKSLK 25 Cerebellar cortex AKLNKSS 17 NESLKKK 4 NSVHNTA 18 NVVRGGA 19 NRLVAGG 20 RPGRESA 3 ERDRTRG 21 PIPAGKK 9 RPGREQA 1 Cerebellar cortex; cerebellar vermis RPGREAS 144 DKTRAGS 5 NSVRPLT 23 RDSTRQL 26 RDATRSS 12 Cerebellum; upper part of the right cerebellar vermis NSVRPLT 23 RDATRSS 12 DKTRAGS 5 TAPKSLK 25 RPGREAS 144 Cerebellum; lateral to the brainstem (right) NSVRPLT 23 TAPKSLK 25 RDSTRQL 26 RPGREAS 144 PIPAGKK 9 RDATRSS 12 Cerebellum; left hemisphere, near the upper part of the cerebellar vermis RPGREAS 144 DKTRAGS 5 RDATRSS 12 RDSTRQL 26 TAPKSLK 25 Cerebellum; left hemisphere, most lateral point RPGREAS 144 RDSTRQL 26 DKTRAGS 5 TAPKSLK 25 NSVRPLT 23 RDATRSS 12 Cerebellum; left hemisphere, near the brainstem NSVRPLT 23 RPGREAS 144 RDATRSS 12 DKTRAGS 5 NSVHNTA 18 TAPKSLK 25 Cerebral cortex VQGSKMK 22 RDATRSS 12 NSVRPLT 23 RPGRESA 3 NKIHANP 24 ERDRTRG 21 DKTRAGS 5 TAPKSLK 25 RDSTRQL 26 RPGREQA 1 Snail (ear) RDATRSS 12 RPGREAS 144 TAPKSLK 25 DKTRAGS 5 NSVRPLT 23 TATPRKG 49 Deep nuclei of the cerebellum RPGREAS 144 TAPKSLK 25 RDATRSS 12 RDSTRQL 26 DKTRAGS 5 Dorsal root ganglia, lumbar region RPGREAS 144 RDATRSS 12 NSVRPLT 23 DKTRAGS 5 TAPKSLK 25 Dorsal root ganglia, cervical region RPGREAS 144 DKTRAGS 5 TAPKSLK 25 RDATRSS 12 NSVHNTA 18 NSVRPLT 23 Dorsal root ganglia, thoracic region RDATRSS 12 RPGREAS 144 DKTRAGS 5 TAPKSLK 25 RDSTRQL 26 Ependyma ERDRTRG 21 RGVLVTT 2 RPGRESA 3 PIPAGKK 9 NESLKKK 4 NSVKSVL 27 PVKKKDA 7 VQGSKMK 22 NVTIKSK 28 Ependyma of the 3rd ventricle DKTRAGS 5 TAPKSLK 25 RPGREAS 144 RDATRSS 12 DRLKGIV 31 Ependyma of the 4th ventricle TAPKSLK 25 NSVRPLT 23 RPGREAS 144 KAAGRTV 46 RDATRSS 12 DKTRAGS 5 Ependyma of the left ventricle RPGREAS 144 NSVRPLT 23 RDATRSS 12 TAPKSLK 25 DETSRLV 30 DKTRAGS 5 Pallid globus RPGRESA 3 ERDRTRG 21 VSLKERV 29 VQGSKMK 22 DKTRAGS 5 DETSRLV 30 RDATRSS 12 AGVARSK 14 EVKGKGK 16 DRLKGIV 31 External globus pallidus RGVLVTT 2 SGTFVKA 33 EVKGKGK 16 SVASAKK 48 VSLKERV 29 TATPRKG 49 Internal globus pallidus RGVLVTT 2 TAPKSLK 25 TKTGLKL 50 ERDRTRG 21 SGVLVQR 32 TATPRKG 49 RPGREAS 144 Hippocampus RGVLVTT 2 RPGRESA 3 SGVLVQR 32 PVKKKDA 7 NESLKKK 4 SGTFVKA 33 RPGREQA 1 NSIARPV 34 Hippocampus; CA1 RGVLVTT 2 SGVLVQR 32 SVASAKK 48 RDATRSS 12 DKTRAGS 5 TAPKSLK 25 Hippocampus; CA3 RPGREAS 144 DETSRLV 30 RDATRSS 12 DKTRAGS 5 ERDRTRG 21 RDSTRQL 26 Hippocampus; DG NSVRPLT 23 RDATRSS 12 TAPKSLK 25 RDSTRQL 26 RPGREAS 144 Lower olive RPGREAS 144 DKTRAGS 5 TAPKSLK 25 NDVRAKG 40 RDATRSS 12 NSVRPLT 23 Lateral geniculate nuclei NSVRPLT 23 TAPKSLK 25 RPGREAS 144 DKTRAGS 5 RDATRSS 12 Meninges of the brain RPGRESA 3 ERDRTRG 21 RDATRSS 12 DKTRAGS 5 PIPAGKK 9 NRARAGE 35 ARHALGG 36 HSSRPVA 37 PVKKKDA 7 RPGREAS 144 TAPKSLK 25 NSVRPLT 23 Motor area of the cerebral cortex RPGREAS 144 RGVLVTT 2 NSVRPLT 23 TAPKSLK 25 DKTRAGS 5 RDATRSS 12 Optic nerve AGVARSK 14 RSRSEVL 15 EVKGKGK 16 DRLKGIV 31 KTGTARL 38 RPGRESA 3 PVKKKDA 7 RGVLVTT 2 ERDRTRG 21 RPGREAS 144 NSVRPLT 23 TAPKSLK 25 DKTRAGS 5 NDVRAKG 40 RDATRSS 12 Prefrontal cortex RPGREAS 144 NSVRPLT 23 TAPKSLK 25 DKTRAGS 5 RDATRSS 12 Pons RPGREAS 144 NSVRPLT 23 TAPKSLK 25 RDATRSS 12 DKTRAGS 5 Shell (putamen) DETSRLV 30 VKALGRP 39 NDVRAKG 40 NESLKKK 4 ERDRTRG 21 RPGREAS 144 QGVLVVR 41 KQYAGSQ 42 RDATRSS 12 VPTKSPK 13 DKTRAGS 5 KAAGRTV 46 TAPKSLK 25 Reticular formation RPGREAS 144 RDSTRQL 26 KGLRTPT 51 RDATRSS 12 NSVRPLT 23 DKTRAGS 5 Spinal cord PVKKKDA 7 RGVLVTT 2 PIPAGKK 9 SGVLVQR 32 NESLKKK 4 RPGRESA 3 TNRMALS 43 ERDRTRG 21 SGTFVKA 33 Spinal cord, thoracic region NSVRPLT 23 SGTFVKA 33 RPGREAS 144 RGVLVTT 2 RDATRSS 12 TAPKSLK 25 DKTRAGS 5 Spinal cord, lumbar region RPGREAS 144 TATPRKG 49 TAPKSLK 25 RDATRSS 12 KAAGRTV 46 Spinal cord, cervical region SGVLVQR 32 NSVRPLT 23 RPGREAS 144 RGVLVTT 2 SGTFVKA 33 TAPKSLK 25 RDATRSS 12 DKTRAGS 5 Substance Black RGVLVTT 2 GITTLGRL 44 RPGRESA 3 PIPAGKK 9 AGIMVRV 45 SGVLVQR 32 VSLKERV 29 RDSTRQL 26 RPGREAS 144 RDATRSS 12 Subthalamic nuclei KAAGRTV 46 RDATRSS 12 RGVLVTT 2 RPGRESA 3 HVIRLPS 47 EVKGKGK 16 NESLKKK 4 DETSRLV 30 NSVRPLT 23 SGTFVKA 33 PIPAGKK 9 VKALGRP 39 Temporal cortex NSVRPLT 23 RPGREAS 144 TAPKSLK 25 RDATRSS 12 DKTRAGS 5 Thalamus GRETLKG 8 RPGREQA 1 ERDRTRG 21 RGVLVTT 2 SVASAKK 48 TATPRKG 49 RDATRSS 12 NVTIKSK 28 TKTGLKL 50 KGLRTPT 51 Thalamus, anterior part SGVLVQR 32 NSVHNTA 18 PIPAGKK 9 DETSRLV 30 AKLNKSS 17 RPGREAS 144 Nuclei VA/VL of the thalamus RGVLVTT 2 DKTRAGS 5 DETSRLV 30 RDSTRQL 26 NKIHANP 24 TAPKSLK 25 Visual cortex RPGREAS 144 NSVRPLT 23 TAPKSLK 25 RDATRSS 12 NSVHNTA 18 DKTRAGS 5

Table 2. AAV2 targeting peptides for each brain structure Region Peptide SEQ ID NO Brain stem LTSRTSP 52 DDPSARR 53 GEQDLRR 54 VSTALPR 55 RDDVPLR 56 TRVGTAG 57 SSSKTGS 58 SLSTGPK 59 VQGRQGG 60 Caudate nucleus RGASGAV 61 NARAQGV 62 TSNRGQV 63 AVRGGMA 64 RGLDKGT 65 KGVDLKP 66 TAVREER 67 GNAGITK 68 SLSTGPK 59 SARAGAP 69 GSRAGVG 105 NARPVSA 76 HDGGASR 103 VTQSKGA 74 KAQGVGG 79 ESTGRER 73 Cerebellar cortex SGEFVGR 70 SGRKLEV 71 SARSGSV 72 ESTGRER 73 SSSKTGS 58 RDDVPLR 56 VQGRQGG 60 VTQSKGA 74 RGSGSAV 75 Cerebellar cortex; cerebellar vermis HDGGASR 103 EAQSHPR 91 ESTGRER 73 GRSTGMT 95 GSRAGVG 105 Cerebellum; upper part of the right cerebellar vermis HDGGASR 103 RAVPAGG 84 RSNAPQT 90 TKSLSSG 92 ESTGRER 73 RGSTQVG 94 Cerebellum; lateral to the brainstem (right) HDGGASR 103 ESTGRER 73 KAQGVGG 79 EAQSHPR 91 RAVPAGG 84 RDDVPLR 56 Cerebellum; left hemisphere, near the upper part of the cerebellar vermis HDGGASR 103 ESTGRER 73 EAQSHPR 91 NARAQGV 62 LTSRTSP 52 KAQGVGG 79 Cerebellum; left hemisphere, most lateral point HDGGASR 103 ESTGRER 73 EAQSHPR 91 KAQGVGG 79 GSRAGVG 105 Cerebellum; left hemisphere, near the brainstem ESTGRER 73 HDGGASR 103 EAQSHPR 91 GSRAGVG 105 KAQGVGG 79 GRGGAAL 100 Cerebral cortex DDPSARR 53 NARPVSA 76 TSNRGQV 63 NARAQGV 62 TARGGGG 77 KGVDLKP 66 GRSASGS 78 SSSKTGS 58 KAQGVGG 79 VQGRQGG 60 Snail (ear) HDGGASR 103 KAQGVGG 79 RSNAPQT 90 LTSRTSP 52 EAQSHPR 91 Deep nuclei of the cerebellum GSRAGVG 105 RAVPAGG 84 EAQSHPR 91 AVRGGMA 64 VPGRTAG 81 VTQSKGA 74 Dorsal root ganglia, lumbar region HDGGASR 103 TKSLSSG 92 KAQGVGG 79 GSRAGVG 105 VPGRTAG 81 Dorsal root ganglia, cervical region GRSTGMT 95 AVRGGMA 64 HDGGASR 103 TAAAGGQR 99 KAQGVGG 79 Dorsal root ganglia, thoracic region HDGGASR 103 GSRAGVG 105 ARGSGVN 82 RAVPAGG 84 KAQGVGG 79 Ependyma GRGAPGG 80 DDPSARR 53 TSNRGQV 63 RGSGSAV 75 VQGRQGG 60 VTQSKGA 74 NARPVSA 76 KGVDLKP 66 NARAQGV 62 TARGGGG 77 Ependyma of the 4th ventricle GSRAGVG 105 RDDVPLR 56 RGSTQVG 94 KAQGVGG 79 GRGGAAL 100 Ependyma of the left ventricle HDGGASR 103 ESTGRER 73 EAQSHPR 91 NARPVSA 76 KAQGVGG 79 Ependyma of the 3rd ventricle GRSTGMT 95 LTSRTSP 52 KAQGVGG 79 GRGGAAL 100 EAQSHPR 91 ARGSGVN 82 Pallid globus VQGRQGG 60 VPGRTAG 81 ARGSGVN 82 SVRVGGQ 83 RGSGSAV 75 RAVPAGG 84 VMSSGKP 85 STPAPKS 86 RGGAQVV 87 External globus pallidus EAQSHPR 91 GSRAGVG 105 VQGRQGG 60 ESTGRER 73 GRGGAAL 100 KASGAGG 88 Internal globus pallidus RDDVPLR 56 GSRAGVG 105 GAVGGVK 107 DDPSARR 53 RGSTQVG 94 KAQGVGG 79 Hippocampus DDPSARR 53 KASGAGG 88 KAQGVGG 79 TSNRGQV 63 VQGRQGG 60 VSTALPR 55 TGTAGLK 89 NARPVSA 76 SSSKTGS 58 Hippocampus; CA1 VAPISKS 101 HDGGASR 103 LTSRTSP 52 ESTGRER 73 KAQGVGG 79 Hippocampus; CA3 VQGRQGG 60 GRSTGMT 95 GRGGAAL 100 KAQGVGG 79 RGSTQVG 94 TSNRGQV 63 Hippocampus; DG HDGGASR 103 ESTGRER 73 RGSTQVG 94 EAQSHPR 91 KAQGVGG 79 GSRAGVG 105 Lower olive VPGRTAG 81 HDGGASR 103 VQGRQGG 60 GSRAGVG 105 RDDVPLR 56 KAQGVGG 79 Lateral geniculate nuclei HDGGASR 103 ESTGRER 73 GSRAGVG 105 KAQGVGG 79 EAQSHPR 91 VPGRTAG 81 Meninges of the brain SSSKTGS 58 NARPVSA 76 RSNAPQT 90 EAQSHPR 91 TKSLSSG 92 GRGAPGG 80 AAGAKVM 93 ESTGRER 73 KGVDLKP 66 VQGRQGG 60 HDGGASR 103 KAQGVGG 79 RGSTQVG 94 VPGRTAG 81 GSRAGVG 105 Optic nerve DDPSARR 53 RGGAQVV 87 KASGAGG 88 RGSGSAV 75 VQGRQGG 60 KAQGVGG 79 TRVGTAG 57 GEQDLRR 54 RGSTQVG 94 SSSKTGS 58 GSRAGVG 105 ESTGRER 73 HDGGASR 103 RDDVPLR 56 Pons ESTGRER 73 GSRAGVG 105 HDGGASR 103 KAQGVGG 79 RDDVPLR 56 GRGGAAL 100 Prefrontal cortex HDGGASR 103 GSRAGVG 105 ESTGRER 73 KAQGVGG 79 RGSTQVG 94 Primary motor area of the cerebral cortex GSRAGVG 105 VPGRTAG 81 ESTGRER 73 RGSTQVG 94 GRGGAAL 100 KAQGVGG 79 Primary visual cortex HDGGASR 103 ESTGRER 73 EAQSHPR 91 TKSLSSG 92 GSRAGVG 105 KAQGVGG 79 Shell GRSTGMT 95 RATSQST 96 VGRSVGA 97 VQGRQGG 60 GEGGGGR 98 VSTALPR 55 RGASGAV 61 TAAAGGQR 99 SLSTGPK 59 GRGGAAL 100 RAVPAGG 84 KAQGVGG 79 GSRAGVG 105 VPGRTAG 81 HDGGASR 103 RDDVPLR 56 Reticular formation VTQSKGA 74 DDPSARR 53 TGTAGLK 89 ARGSGVN 82 RGSTQVG 94 RDDVPLR 56 Spinal cord SLSTGPK 59 GRSTGMT 95 RGASGAV 61 TARGGGG 77 KASGAGG 88 VAPISKS 101 DDPSARR 53 TSNRGQV 63 SSSKTGS 58 VQGRQGG 60 Spinal cord, thoracic region HDGGASR 103 ESTGRER 73 EAQSHPR 91 GSRAGVG 105 VQGRQGG 60 KAQGVGG 79 Spinal cord, lumbar region HDGGASR 103 EAQSHPR 91 DDPSARR 53 KLSISGN 106 VMSSGKP 85 KAQGVGG 79 TSNRGQV 63 Spinal cord, cervical region ESTGRER 73 HDGGASR 103 EAQSHPR 91 GSRAGVG 105 RGSTQVG 94 KAQGVGG 79 Substance Black LTSRTSP 52 NARPVSA 76 SSSKTGS 58 RGGAQVV 87 APPVKLS 102 DDPSARR 53 RGSGSAV 75 HDGGASR 103 TRVGTAG 57 GSRAGVG 105 KLSISGN 106 KAQGVGG 79 TGTAGLK 89 ARGSGVN 82 Subthalamic nuclei VQGRQGG 60 RSGGAAV 104 KASGAGG 88 SSSKTGS 58 KAQGVGG 79 GSRAGVG 105 RGGAQVV 87 APPVKLS 102 RGSGSAV 75 TRVGTAG 57 RGSTQVG 94 KLSISGN 106 RDDVPLR 56 GRGGAAL 100 TSNRGQV 63 Temporal cortex ESTGRER 73 HDGGASR 103 GSRAGVG 105 KAQGVGG 79 RDDVPLR 56 Thalamus KASGAGG 88 RDDVPLR 56 LTSRTSP 52 KLSISGN 106 VSTALPR 55 VMSSGKP 85 GAVGGVK 107 KNESGKV 108 VTQSKGA 74 AGQLAGR 109 Thalamus, anterior part RGSTQVG 94 KAQGVGG 79 TKSLSSG 92 RAVPAGG 84 TSNRGQV 63 RDDVPLR 56 Nuclei VA/VL of the thalamus TRVGTAG 57 ARGSGVN 82 GRGGAAL 100 NARPVSA 76 VQGRQGG 60 GSRAGVG 105

Table 3. AAV9 targeting peptides for each brain structure Region Peptide SEQ ID NO Brain stem KGGGFHG 110 RAKPGME 111 GRDVTRS 112 RGDLQWV 113 GGDRTRG 114 RGDLASV 115 RDTRNL 116 KGGGVHG 117 Caudate nucleus KGGGFHG 110 RGDLQWV 113 RGDMYRV 118 RGDRPVS 119 RSDVGSL 120 RGDLASV 115 RDTRNL 116 AGVKPGR 121 KGGGVHG 117 RAKPGME 111 GADRTRG 127 GRDVTRS 112 ARGDGWR 132 R.G.G.V.Y.G. 126 GGDRTRG 114 GRDYTRL 133 Cerebellar cortex KGGGFHG 110 RGDLQWV 113 RAKPGME 111 RGDWPRG 122 RGDRPVS 119 GGRPGSW 123 RGDYPRS 124 RGDLRF 125 Cerebral cortex KGGGFHG 110 RAKPGME 111 RDTRNL 116 R.G.G.V.Y.G. 126 GGDRTRG 114 GADRTRG 127 RGDLQWV 113 RGDLRF 125 Prefrontal cortex KGGGFHG 110 MMGRPGR 136 AGVKPGR 121 R.G.G.V.Y.G. 126 RGDLRF 125 RGDWPRG 122 RGDLQWV 113 RGDRPVS 119 Cerebral cortex, motor area MMGRPGR 136 KGGGFHG 110 AGVKPGR 121 R.G.G.V.Y.G. 126 RGDLRF 125 RGDWPRG 122 RGDLQWV 113 Cerebral cortex, temporal KGGGFHG 110 R.G.G.V.Y.G. 126 MMGRPGR 136 RGDLRF 125 RGDLQWV 113 RGDWPRG 122 Cerebral cortex, visual area AGVKPGR 121 MMGRPGR 136 RGDLRF 125 RGDLQWV 113 RGDFMGL 128 R.G.G.V.Y.G. 126 KGGGFHG 110 AWDGTRV 131 Snail (ear) MMGRPGR 136 KGGGFHG 110 RGDLASV 115 AGVKPGR 121 GGRPGSW 123 R.G.G.V.Y.G. 126 GGDRTRG 114 Deep nuclei of the cerebellum RSDVGSL 120 KGGGFHG 110 RGDLQWV 113 GRDVTRS 112 RGDLRF 125 R.G.G.V.Y.G. 126 RAKPGME 111 Dorsal root ganglia, lumbar region MMGRPGR 136 AGVKPGR 121 GGRPGSW 123 GRDYTRL 133 R.G.G.V.Y.G. 126 GGDRTRG 114 Dorsal root ganglia, cervical region RGDLQWV 113 KGGGFHG 110 RRDETRT 129 R.G.G.V.Y.G. 126 GADRTRG 127 RGDRPVS 119 GGDRTRG 114 Dorsal root ganglia, thoracic region MMGRPGR 136 AGVKPGR 121 GGRPGSW 123 GRDYTRL 133 RGDFMGL 128 R.G.G.V.Y.G. 126 GGDRTRG 114 RAKPGME 111 AWDGTRV 131 Ependyma KGGGFHG 110 RGDLQWV 113 RAKPGME 111 RGDMYRV 118 RSDVGSL 120 RGDFMGL 128 RGDRPVS 119 Ependyma of the 4th ventricle RGDLQWV 113 RGDLASV 115 RSDVGSL 120 RGDLRF 125 MDLTKAV 135 R.G.G.V.Y.G. 126 RAKPGME 111 RGDRPVS 119 GGDRTRG 114 Ependyma of the left ventricle MMGRPGR 136 RSDVGSL 120 AGVKPGR 121 GGRPGSW 123 RAKPGME 111 R.G.G.V.Y.G. 126 GRDVTRS 112 RGDLQWV 113 Ependyma of the 3rd ventricle KGGGVHG 117 GADRTRG 127 GGDRTRG 114 MMGRPGR 136 RGDLQWV 113 R.G.G.V.Y.G. 126 GRDVTRS 112 Pallid globus KGGGFHG 110 RRDETRT 129 RAKPGME 111 GGDRTRG 114 GRDVTRS 112 RSDVGSL 120 RGDRPVS 119 External globus pallidus RAKPGME 111 RRGDAWS 134 RGDMYRV 118 RGDLQWV 113 GRDVTRS 112 R.G.G.V.Y.G. 126 Internal globus pallidus MDLTKAV 135 RDTRNL 116 AWDGTRV 131 R.G.G.V.Y.G. 126 KGGGVHG 117 RGDLQWV 113 Hippocampus KGGGFHG 110 AESPWER 130 RGDLQWV 113 RAKPGME 111 RRDETRT 129 RGDLRF 125 GGRPGSW 123 RDTRNL 116 Hippocampus CA1 KGGGFHG 110 ARGDGWR 132 R.G.G.V.Y.G. 126 AWDGTRV 131 GADRTRG 127 RAKPGME 111 Hippocampus CA3 RAKPGME 111 RRDETRT 129 MDLTKAV 135 AWDGTRV 131 RDTRNL 116 GGDRTRG 114 GRDVTRS 112 R.G.G.V.Y.G. 126 Hippocampus; DG KGGGFHG 110 RGDFMGL 128 R.G.G.V.Y.G. 126 ARGDGWR 132 RGDLRF 125 RGDLQWV 113 GRDYTRL 133 Lower olive RRGDAWS 134 GGRPGSW 123 KGGGFHG 110 KGGGVHG 117 RGDWPRG 122 R.G.G.V.Y.G. 126 GRDYTRL 133 RGDRPVS 119 Meninges of the brain KGGGFHG 110 RGDLQWV 113 RAKPGME 111 RGDWPRG 122 GGDRTRG 114 AWDGTRV 131 RGDMYRV 118 RGDRPVS 119 RSDVGSL 120 KGGGVHG 117 GRDVTRS 112 AGVKPGR 121 R.G.G.V.Y.G. 126 Optic nerve KGGGFHG 110 RAKPGME 111 RRDETRT 129 RDTRNL 116 GGDRTRG 114 ARGDGWR 132 KGGGVHG 117 RGDLRF 125 R.G.G.V.Y.G. 126 AGVKPGR 121 RGDLQWV 113 Pons KGGGFHG 110 R.G.G.V.Y.G. 126 RRDETRT 129 AGVKPGR 121 KGGGVHG 117 RAKPGME 111 RGDLQWV 113 GRDYTRL 133 Shell KGGGFHG 110 GRDVTRS 112 GADRTRG 127 GRDYTRL 133 RDTRNL 116 RGDLQWV 113 RRGDAWS 134 GGRPGSW 123 KGGGVHG 117 RGDMYRV 118 RAKPGME 111 R.G.G.V.Y.G. 126 GGDRTRG 114 RGDRPVS 119 Reticular formation MMGRPGR 136 GRDVTRS 112 GADRTRG 127 RAKPGME 111 RSDVGSL 120 R.G.G.V.Y.G. 126 AWDGTRV 131 Spinal cord KGGGFHG 110 RGDLQWV 113 RGDRPVS 119 RGDWPRG 122 RSDVGSL 120 GGRPGSW 123 RRGDAWS 134 RGDFMGL 128 Spinal cord, thoracic region KGGGFHG 110 R.G.G.V.Y.G. 126 RGDLQWV 113 RGDLRF 125 RRGDAWS 134 RGDRPVS 119 GRDYTRL 133 Spinal cord, lumbar region RGDLASV 115 KGGGFHG 110 R.G.G.V.Y.G. 126 MMGRPGR 136 RGDLQWV 113 AWDGTRV 131 RAKPGME 111 Spinal cord, cervical region KGGGFHG 110 RGDLRF 125 R.G.G.V.Y.G. 126 RRGDAWS 134 RGDLQWV 113 RGDWPRG 122 Substance Black KGGGFHG 110 RAKPGME 111 GRDVTRS 112 GGDRTRG 114 RRDETRT 129 RGDLQWV 113 KGGGVHG 117 MDLTKAV 135 RRGDAWS 134 GGRPGSW 123 GADRTRG 127 R.G.G.V.Y.G. 126 Subthalamic nuclei KGGGFHG 110 RAKPGME 111 MDLTKAV 135 RGDRPVS 119 RGDWPRG 122 ARGDGWR 132 RGDLQWV 113 RSDVGSL 120 RGDLASV 115 R.G.G.V.Y.G. 126 Thalamus KGGGFHG 110 GRDVTRS 112 GGDRTRG 114 RGDLQWV 113 GRDYTRL 133 RGDLRF 125 MMGRPGR 136 TGRPGVW 137 Thalamus, anterior part MDLTKAV 135 GGRPGSW 123 RGDLRF 125 RDTRNL 116 RAKPGME 111 R.G.G.V.Y.G. 126 GRDYTRL 133 RGDWPRG 122 Nuclei VA/VL of the thalamus RGDWPRG 122 GGDRTRG 114 RRGDAWS 134 RGDLRF 125 RGDYPRS 124 RGDRPVS 119 R.G.G.V.Y.G. 126

Table 4. AAV1 targeting peptides for different organs Organ Peptide SEQ ID NO Brain RPGREAS 144 TAPKSLK 25 RDATRSS 12 DKTRAGS 5 NSVRPLT 23 RDSTRQL 26 RGVLVTT 2

Table 5. AAV2 targeting peptides for different organs. Organ Peptide SEQ ID NO Brain HDGGASR 103 KAQGVGG 79 GSRAGVG 105 ESTGRER 73 RDDVPLR 56 RGSTQVG 94 EAQSHPR 91 Bud GSRAGVG 105 TSNRGQV 63 GRSTGMT 95 GRGAPGG 80 GAVGGVK 107 KAQGVGG 79 RDDVPLR 56 Heart HDGGASR 103 SLSTGPK 59 GSRAGVG 105 RGSTQVG 94 NARAQGV 62 ESTGRER 73 Liver KAQGVGG 79 GSRAGVG 105 RDDVPLR 56 HDGGASR 103 NARPVSA 76 Sex gland VPGRTAG 81 KAQGVGG 79 RGSTQVG 94 HDGGASR 103 GAVGGVK 107 RDDVPLR 56 Spleen KAQGVGG 79 GSRAGVG 105 GRGGAAL 100 VPGRTAG 81 ESTGRER 73 Lung ESTGRER 73 TKSLSSG 92 RGSTQVG 94 KAQGVGG 79 NARPVSA 76

Table 6. AAV9 targeting peptides for different organs. Organ Peptide SEQ ID NO Brain R.G.G.V.Y.G. 126 RAKPGME 111 KGGGFHG 110 GGDRTRG 114 MMGRPGR 136 RGDLQWV 113 MDLTKAV 135 GGRPGSW 123 Bud MMGRPGR 136 RGDLQWV 113 RAKPGME 111 RSDVGSL 120 KGGGFHG 110 R.G.G.V.Y.G. 126 Heart GGRPGSW 123 RSDVGSL 120 RGDLASV 115 KGGGFHG 110 GRDYTRL 133 R.G.G.V.Y.G. 126 RAKPGME 111 Liver GGDRTRG 114 GRDVTRS 112 RDTRNL 116 RRDETRT 129 RSDVGSL 120 R.G.G.V.Y.G. 126 RAKPGME 111 GRDYTRL 133 Sex gland KGGGVHG 117 KGGGFHG 110 RSDVGSL 120 GGRPGSW 123 RRDETRT 129 R.G.G.V.Y.G. 126 GGDRTRG 114 RGDRPVS 119 RAKPGME 111

I. Adeno-associated virus (AAV) vectors

[0042] Adeno-associated virus (AAV) is a small, nonpathogenic virus of the parvovirus family. To date, numerous serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV differs from other members of the family in its dependence on a helper virus for replication.

[0043] AAV genomes can exist extrachromosomally without integration into host cell genomes; have a broad host range; transduce both dividing and non-dividing cells in vitro and in vivo, and maintain high levels of expression of transduced genes. AAV virus particles are thermostable; resistant to solvents, detergents, changes in pH, and temperature; and can be column purified and/or concentrated on CsCl gradients or other methods. The AAV genome consists of single-stranded deoxyribonucleic acid (ssDNA), either positive or negative sense. The approximately 4.7 kilobase AAV genome consists of a single segment of single-stranded DNA of either positive or negative polarity. The ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as origins of viral DNA replication.

[0044] The AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsidated to form an AAV particle. An AAV particle often contains the AAV genome packaged by AAV capsid proteins. In cases where recombinant plasmids are used to construct or produce recombinant vectors, the AAV-based vector genome does not include the portion of the "plasmid" that does not correspond to the vector genomic sequence of the recombinant plasmid. This non-vector genomic portion of the recombinant plasmid is called the "plasmid backbone," which is important for plasmid cloning and amplification, a process necessary for plasmid propagation and production, but is not itself packaged or encapsidated into viral particles. Thus, the "genome" of an AAV-based vector refers to the nucleic acid that is packaged or encapsidated by the AAV capsid proteins.

[0045] The AAV virion (particle) is an icosahedral, non-enveloped particle approximately 25 nm in diameter that contains the AAV capsid. The AAV particle has icosahedral symmetry, consisting of three related capsid proteins, VP1, VP2, and VP3, that interact together to form the capsid. The genome of most naturally occurring AAVs often contains two open reading frames (ORFs), sometimes referred to as the left ORF and right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins often occur in a 1:1:10 ratio, respectively, but can occur in other ratios, and all are derived from the right ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by their use of alternative splicing and an unusual initiation codon. Deletion analysis has shown that deletion or alteration of VP1 translated from alternatively spliced messenger RNA results in decreased yield of infectious particles. Mutations in the coding region of VP3 result in failure to produce any progeny single-stranded DNA or infectious particles. In some embodiments, the AAV particle genome encodes one, two, or all three VP1, VP2, and VP3 polypeptides.

[0046] The left ORF often encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in the regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two Rep proteins have been associated with the preferential integration of AAV genomes into the q arm region of human chromosome 19. Rep68/78 has been shown to have NTP binding activity, as well as DNA and RNA helicase activity. Some Rep proteins have a nuclear localization signal, as well as several potential phosphorylation sites. In some embodiments, an AAV (e.g., rAAV) genome encodes some or all of the Rep proteins. In some embodiments, an AAV (e.g., rAAV) genome does not encode Rep proteins. In some embodiments, one or more Rep proteins may be delivered in trans and therefore not included in an AAV particle containing a nucleic acid encoding a polypeptide.

[0047] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the ability to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Accordingly, the AAV genome contains one or more (e.g., a pair) ITR sequences that flank the viral genome as single-stranded DNA. Each ITR sequence is often approximately 145 bases in length. Within the ITR region, two elements have been described that are believed to play a central role in ITR function, the GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is believed to position Rep68/78 for cleavage at trs, which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to play a central role in viral integration. The chromosome 19 integration locus contains a Rep binding site together with the adjacent trs. These elements have been shown to be functional and required for locus-specific integration.

[0048] The term "recombinant" as a vector modifier, such as a recombinant viral, such as a lenti- or parvovirus (e.g., AAV) vector, and as a sequence modifier, such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been altered (i.e., engineered) in a manner that is not typically found in nature. A specific example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector, would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted into the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., a gene) that encodes an inhibitory RNA is cloned into a vector, with or without the 5', 3', and/or intronic regions with which the gene is normally associated within a viral genome. Although the term "recombinant" is not always used herein to refer to vectors, such as viral vectors, and sequences, such as polynucleotides, "recombinant" forms comprising nucleic acid sequences, polynucleotides, transgenes, etc., are expressly included despite any such omission.

[0049] A recombinant viral "vector" is produced from a wild-type viral genome using molecular techniques to remove a portion of the wild-type genome from the virus and replace it with a non-naturally occurring 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 a recombinant AAV-based vector. A "recombinant" viral vector (e.g., rAAV) differs from a viral genome (e.g., AAV) because a portion of the viral genome has been replaced with a non-naturally occurring sequence relative to a nucleic acid of the viral genome, such as a nucleic acid encoding a transactivator, or a nucleic acid encoding an inhibitory RNA, or a nucleic acid encoding a therapeutic protein. Therefore, the inclusion of such non-natural nucleic acid sequences defines the viral vector as a "recombinant" vector, which in the case of AAV can be called an "rAAV vector".

[0050] In some embodiments, an AAV (e.g., rAAV) comprises two ITRs. In some embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs. In some embodiments, an AAV (e.g., rAAV) comprises a pair of ITRs that flank (i.e., are at each 5' and 3' end of) a nucleic acid sequence that at least encodes a polypeptide that has a function or activity.

[0051] An AAV-based vector (e.g., a rAAV vector) can be packaged and referred to herein as an "AAV particle" for subsequent infection (transduction) of a cell ex vivo, in vitro, or in vivo. If a recombinant AAV-based vector is encapsulated or packaged within an AAV particle, the particle may also be referred to as an "rAAV particle." In some embodiments, the AAV particle is an rAAV particle. An rAAV particle often comprises an rAAV vector or a portion thereof. An rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles). rAAV particles typically comprise proteins that encapsulate or package the rAAV vector genome (e.g., capsid proteins). It should be noted that reference to an rAAV vector can also be used to refer to an rAAV particle.

[0052] Any suitable AAV particle (e.g., an rAAV particle) can be used for the method or use described herein. The rAAV particle and/or the genome contained therein can be from any suitable AAV serotype or strain. The rAAV particle and/or the genome contained therein can be from two or more AAV serotypes or strains. Accordingly, the rAAV can comprise proteins and/or nucleic acids or portions thereof of any AAV serotype or strain, wherein the AAV particle is suitable for infecting and/or transducing a mammalian cell. 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.

[0053] In some embodiments, the plurality of rAAV particles include particles of the same strain or serotype (or subgroup or variant) or particles derived therefrom. In some embodiments, the plurality of rAAV particles include a mixture of two or more different rAAV particles (e.g., different serotypes and/or strains).

[0054] As used herein, the term "serotype" is a distinction used to refer to an AAV that has a capsid that is serologically distinct from other AAV serotypes. A serological distinction is determined based on the lack of cross-reactivity between antibodies against one AAV compared to another AAV. Such differences in cross-reactivity are typically due to differences in capsid protein/antigenic determinant sequences (e.g., due to differences in VP1, VP2, and/or VP3 sequences of AAV serotypes). While it is possible that AAV variants, including capsid variants, may not be serologically distinct from a reference AAV or another AAV serotype, they differ in at least one nucleotide or amino acid residue compared to a reference or another AAV serotype.

[0055] In some embodiments, an rAAV vector based on the genome of a first serotype corresponds to the serotype of one or more capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., an ITR) that includes the genome of an AAV-based vector corresponds to the serotype of a capsid that includes an rAAV particle.

[0056] In some embodiments, the rAAV vector genome may be based on the genome of an AAV serotype (e.g., AAV2) that is different from the serotype of one or more AAV capsid proteins that package the vector. For example, the rAAV vector genome may include nucleic acids derived from AAV2 (e.g., ITRs), while at least one or more of the three capsid proteins are derived from another serotype, such as serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8, or a variant thereof.

[0057] In some embodiments, the rAAV particle or vector genome thereof linked to a reference serotype comprises a polynucleotide, polypeptide, or subsequence thereof that comprises or consists of 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 an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8. In particular embodiments, the rAAV particle or vector genome thereof linked to a reference serotype comprises a capsid or ITR sequence that comprises or consists of a sequence that is at least 60% or more identical (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to the capsid or ITR sequence of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8.

[0058] In some embodiments, the method described herein comprises using, administering, or delivering a particle of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8.

[0059] In some embodiments, a method described herein comprises using, administering, or delivering an rAAV2 particle. In some embodiments, the rAAV2 particle comprises an AAV2 capsid. In some embodiments, the rAAV2 particle comprises one or more capsid proteins (e.g., VP1, VP2, and/or VP3) that are at least 60%, 65%, 70%, 75%, or more, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the corresponding capsid protein of a naturally occurring or wild-type AAV2 particle. In some embodiments, the rAAV2 particle comprises VP1, VP2, and VP3 capsid proteins that are at least 75% or more identical, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding capsid protein of a naturally occurring or wild-type AAV2 particle. In some embodiments, the rAAV2 particle is a variant of a naturally occurring or wild-type AAV2 particle. In some aspects, one or more AAV2 variant capsid proteins have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to the capsid protein(s) of a naturally occurring or wild-type AAV2 particle.

[0060] In some embodiments, the rAAV9 particle comprises an AAV9 capsid. In some embodiments, the rAAV9 particle comprises one or more capsid proteins (e.g., VP1, VP2, and/or VP3) that are at least 60%, 65%, 70%, 75%, or more, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to the corresponding capsid protein of a naturally occurring or wild-type AAV9 particle. In some embodiments, the rAAV9 particle comprises VP1, VP2, and VP3 capsid proteins that are at least 75% or more identical, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding capsid protein of a naturally occurring or wild-type AAV9 particle. In some embodiments, the rAAV9 particle is a variant of a naturally occurring or wild-type AAV9 particle. In some aspects, one or more AAV9 variant capsid proteins have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to the capsid protein(s) of a naturally occurring or wild-type AAV9 particle.

[0061] In some embodiments, the rAAV particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100%, to an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, naturally occurring or wild type, provided that they retain one or more desired ITR functions (e.g., the ability to form a hairpin that allows DNA replication, integration of AAV DNA into the host cell genome, and/or packaging, if necessary).

[0062] In some embodiments, the rAAV2 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100%, to an ITR of a naturally occurring or wild-type AAV2 particle, so long as they retain one or more desirable functions of the ITR (e.g., the ability to form a hairpin that allows for DNA replication; integration of AAV DNA into the host cell genome, and/or packaging, if necessary).

[0063] In some embodiments, the rAAV9 particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, to the corresponding ITR of a naturally occurring or wild-type AAV2 particle, so long as they retain one or more desirable functions of the ITR (e.g., the ability to form a hairpin that allows for DNA replication; integration of AAV DNA into the host cell genome, and/or packaging, if necessary).

[0064] The rAAV particle may comprise an ITR with any suitable number of "GAGC" repeats. In some embodiments, the ITR of the AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In some embodiments, the rAAV2 particle comprises an ITR with three "GAGC" repeats. In some embodiments, the rAAV2 particle comprises an ITR that comprises fewer than four "GAGC" repeats. In some embodiments, the rAAV2 particle comprises an ITR that comprises more than four "GAGC" repeats. In some embodiments, the ITR of the rAAV2 particle comprises a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is a C rather than a T.

[0065] A typical suitable length of DNA that can be included in rAAV vectors for packaging/encapsulation into an rAAV particle can be about 5 kilobases (kb) or less. In particular 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.

[0066] Recombinant AAV-based vectors that include a nucleic acid sequence that directs the expression of RNAi or a polypeptide can be produced using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV-based vectors are typically packaged into transducible AAV particles and propagated using an AAV viral packaging system. A transducible AAV particle is capable of binding to and entering a mammalian cell and subsequently delivering a nucleic acid cargo (e.g., a heterologous gene) into the cell nucleus. Thus, an intact rAAV particle that is transducible is configured to transduce a mammalian cell. An rAAV particle configured to transduce a mammalian cell is often replication incompetent and requires additional protein structures for self-replication. Thus, an rAAV particle configured to transduce a mammalian cell is designed to bind to and enter a mammalian cell and deliver nucleic acid into the cell, with the nucleic acid for delivery often located between a pair of AAV ITRs in the rAAV genome.

[0067] Host cells suitable for producing transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be used or have been used as recipients of heterologous recombinant AAV vectors. Cells from the stable human cell line, HEK293 (readily available, for example, through the American Type Culture Collection under ATCC accession number CRL1573), can be used. In some embodiments, a modified human embryonic kidney cell line (e.g., HEK293) that has been transformed with adenovirus type 5 DNA fragments and expresses adenoviral E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for producing rAAV particles. Methods for producing high titer AAV particles capable of transducing mammalian cells are known in the art. For example, an AAV particle can be produced as described in Wright, 2008 and Wright, 2009.

[0068] In some embodiments, AAV helper functions are introduced into a host cell by transfecting the host cell with an AAV helper construct either prior to or concurrently with transfection of an AAV expression vector. Thus, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and/or cap genes to supplement missing AAV functions required for efficient AAV transduction. AAV helper constructs often lack the AAV ITR and are incapable of replicating or packaging themselves. These constructs may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used pAAV/Ad and pIM29+45 plasmids, which encode both Rep and Cap expression products. A number of other vectors are known that encode Rep and/or Cap expression products.

[0069] An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in the cell and optionally additional elements such as a heterologous nucleic acid sequence, an expression control element (e.g., a promoter, enhancer), an intron, ITR(s), and a polyadenylation signal.

II. Therapeutic agents

[0070] In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to introduce nucleic acids encoding inhibitory RNAs, non-coding RNAs, and/or therapeutic proteins into cells in culture or in a host organism.

A. Inhibitory RNAs

[0071] "RNA interference (RNAi)" is a process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces degradation of the target mRNA, followed by sequence-specific inhibition of gene expression.

[0072] An "inhibitory RNA," "RNAi," "small interfering RNA," or "short interfering RNA," or "siRNA" molecule, "short hairpin RNA," or "shRNA" molecule, or "microRNA" is an RNA duplex of nucleotides that targets a nucleic acid sequence of interest. As used herein, the term "siRNA" is a general term that encompasses a subset of shRNAs and microRNAs. An "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. An siRNA "targets" a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the siRNA targets a sequence encoding huntingtin. In some embodiments, the siRNA duplex is less than 30 base pairs long. In some embodiments, the duplex length may 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 some embodiments, the duplex length is from 19 to 25 base pairs. In some embodiments, the duplex length is 19 or 21 base pairs. The RNA duplex portion of the siRNA may be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may comprise a loop portion located between the two sequences that form the duplex. The loop may be of varying lengths. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some embodiments, the loop is 18 nucleotides long. The hairpin structure may also comprise 3' and/or 5' overhangs. In some embodiments, the overhang is a 3' and/or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0073] shRNAs consist of stem-loop structures that are engineered to contain a 5' flanking region, siRNA region segments, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies have used short hairpin RNAs (shRNAs) driven by strong polIII-based promoters. Many shRNAs have demonstrated efficient knockdown of target sequences both in vitro and in vivo, however, some shRNAs that have demonstrated efficient knockdown of a target gene have been found to be toxic in vivo.

[0074] microRNAs are small cellular RNAs (~22 nucleotides) that are processed from stem-loop precursor transcripts. Known stem-loop structures of microRNAs can be modified to contain RNAi sequences specific for genes of interest. MicroRNA molecules may be preferred over shRNA molecules because microRNAs are expressed endogenously. Therefore, microRNA molecules are unlikely to induce dsRNA-sensitive interferon activation pathways, they are processed more efficiently than shRNAs, and have been shown to induce silencing with 80% greater efficiency.

[0075] A recently discovered alternative approach is to use artificial microRNAs (pri-miRNA scaffolds that carry siRNA sequences) as RNAi vectors. Artificial microRNAs more naturally resemble endogenous RNAi substrates and are more amenable to Pol II transcription (e.g., allowing tissue-specific expression of RNAi) and polycistronic strategies (e.g., allowing delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, which is incorporated by reference.

[0076] The shRNA transcriptional unit consists of a sense and antisense sequence linked by an unpaired nucleotide loop. shRNAs are exported from the nucleus by Exportin-5 and, once in the cytoplasm, are processed by Dicer to form functional siRNAs. Stem-loop "miRNA" structures consist of a sense and antisense sequence linked by an unpaired nucleotide loop, typically expressed as part of larger primary transcripts (primary miRNAs), which are excised by the Drosha-DGCR8 complex to form intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5 and, once in the cytoplasm, are processed by Dicer to form functional siRNAs. "Artificial microRNA" or "artificial microRNA shuttle vector," as used interchangeably herein, refers to a primary microRNA transcript that contains a region of stem-loop duplex structure (at least approximately 9-20 nucleotides) that is excised by Drosha and Dicer processing, replaced by siRNA sequences for the target gene, while retaining the structural elements within the stem-loop required for efficient Drosha processing. The term "artificial" arises from the fact that the flanking sequences (~35 nucleotides 5' and ~40 nucleotides 3') are derived from restriction enzyme sites in the siRNA multiple cloning site. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially generated miRNA shuttle vectors.

[0077] The siRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a six-T sequence.

[0078] Several factors must be considered when designing RNAi, such as the nature of the siRNA, the robustness of the silencing effect, and the choice of delivery system. To induce an RNAi effect, the siRNA that is introduced into the organism will typically contain exonic sequences. In addition, the RNAi process is homology-dependent, so sequences must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, 85%, 90%, 95%, 98%, or even 100% sequence identity between the siRNA and the gene to be inhibited. Sequences that are less than approximately 80% identical to the target gene are significantly less effective. Thus, the higher the homology between the siRNA and the gene to be inhibited, the less likely it is that the expression of unrelated genes will be affected.

[0079] In addition, an important factor to consider is the size of the siRNA. In some embodiments, the present invention relates to siRNA molecules that include at least about 19-25 nucleotides and are capable of modulating gene expression. In the context 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.

[0080] The term "siRNA target" generally means a polynucleotide containing a region encoding a polypeptide, or a region of a polynucleotide that regulates replication, transcription, or translation, or other processes important for the expression of a polypeptide, or a polynucleotide containing both a region encoding a polypeptide and a region operably linked thereto that regulates expression. Any gene expressed in a cell can be a target. Preferably, the target gene is a gene involved in or associated with an increase in cellular activities that are important for a disease or that is of particular interest as a subject of study.

B. Non-coding RNAs

[0081] As demonstrated by cDNA cloning projects and tiled genome arrays, more than 90% of the human genome is transcribed but does not encode proteins. These transcription products are called non-protein-coding RNAs (ncRNAs). A number of ncRNA transcripts, such as ribosomal RNAs, transfer RNAs, competing endogenous RNAs (ceRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snRNAs), are essential for cellular function. A large number of short ncRNAs, such as microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (PIWI-RNAs), and small nucleolar RNAs (snRNAs), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long non-coding RNA transcripts (lncRNAs) that exhibit cell type-specific expression and localize to specific subcellular compartments. LncRNAs are also known to play important roles during cell development and differentiation, supporting the notion that they have been selected for by evolutionary processes.

[0082] Long noncoding RNAs appear to have a variety of different functions. In many cases, they appear to play a role in regulating the activity or localization of proteins, or to serve as organizational structures for subcellular structures. In other cases, long noncoding RNAs are processed to form multiple small RNAs, or they may modulate the processing of other RNAs. The latest data release from the public research consortium GenCode (version #27) catalogs just under 16,000 long noncoding RNAs in the human genome, representing nearly 28,000 transcripts; when other databases are included, over 40,000 long noncoding RNAs are known.

[0083] Interestingly, long noncoding RNAs can influence the expression of specific target proteins at specific genomic loci, modulate the activity of protein binding partners, target chromatin modifying complexes to their sites of action, and undergo post-transcriptional processing to generate numerous 5'-capped small RNAs. Epigenetic pathways can also regulate differential expression of long noncoding RNAs.

[0084] Growing evidence also suggests that aberrantly expressed long non-coding RNAs (lncRNAs) play important roles in normal physiological processes as well as in a variety of disease states. Long non-coding RNAs 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 established in a variety of cancers such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Several lncRNAs, such as gadd74 and lncRNA-RoR5, modulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, and thus provide an additional layer of flexibility and robustness to cell cycle progression. In addition, several lncRNAs are associated with mitotic processes, such as centromeric satellite RNA, which is required for kinetochore formation and is thus critical for chromosome segregation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, regulates M-phase exit by functioning in cis to repress expression of the adjacent gene Pura, a regulator of cell proliferation.

[0085] Long non-coding RNAs are a group that are generally defined as transcripts of more than 200 nucleotides (e.g., from about 200 to about 1200 nucleotides, about 2500 nucleotides or more) that lack a long open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes long non-coding RNAs as well as shorter transcripts, such as those of less than about 200 nucleotides, such as about 30 to 200 nucleotides.

[0086] Thus, in some embodiments, delivery of an ncRNA, for example, to a specific brain structure of interest, corrects aberrant RNA expression levels or modulates disease-causing long non-coding RNA levels. Accordingly, in some embodiments, the present invention provides a recombinant AAV whose genome is engineered to encode a therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA that is about 200 nucleotides or more in length. In some embodiments, the therapeutic agent is an ncRNA that is about 25 nucleotides in length, or about 30 nucleotides to about 200 nucleotides in length. In some embodiments, the long non-coding RNAs are about 200 nucleotides to about 1200 nucleotides in length. In some embodiments, the length of the long non-coding RNAs is from about 200 nucleotides to about 1100, about 1000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nucleotides.

C. CRISPR systems

[0087] Gene editing is a technology that allows modification of target genes in living cells. Recently, leveraging the bacterial immune system CRISPR to perform gene editing on demand has revolutionized the way scientists approach genome editing. The CRISPR protein Cas9, which is an RNA-guided DNA endonuclease, can be engineered to target new sites relatively easily by changing the sequence of its guide RNA. This discovery has made sequence-specific gene editing functionally efficient.

[0088] In general, a "CRISPR system" refers to transcripts and other elements involved in the expression or control of the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr- (trans-activating CRISPR) sequence (e.g., a tracrRNA or an active partial tracrRNA), a tracr-related sequence (including a "direct repeat" and a partial direct repeat resulting from tracrRNA processing in the case of an endogenous CRISPR system), a guide sequence (also called a "spacer" in the case of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

[0089] A CRISPR/Cas nuclease or a CRISPR/Cas nuclease system may include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a Type I, Type II, or Type III CRISPR system, such as derived from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes .

[0090] A CRISPR system can induce double-strand breaks (DSBs) at a target site, followed by destructions as described herein. In other embodiments, Cas9 variants, considered "nichases," are used to cause single-strand breaks at a target site. Paired nichases can be used, for example, to increase specificity, each nichasing a pair of different guide RNAs to target sequences such that when nicks are introduced simultaneously, a 5' overhang is introduced. In other embodiments, a catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor (e.g., KRAB) or activator, to affect gene expression. Alternatively, a CRISPR system with a catalytically inactivatable Cas9 further comprises a transcriptional repressor or activator fused to a ribosome binding protein.

[0091] In some aspects, a Cas nuclease and a guide RNA (comprising a fusion of a crRNA specific for a target sequence and an unchanged tracr-RNA) are introduced into a cell. Typically, target sites at the 5' end of the guide RNA target the Cas nuclease to a target site, such as 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, such as typically NGG or NAG. In this regard, the guide RNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to match the target DNA sequence. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, a “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, whereby hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required, as long as the complementarity is sufficient to induce hybridization and promote the formation of a CRISPR complex.

[0092] The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of the cell. Typically, a sequence or template that can be used to recombine at a target locus containing target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0093] Typically, in the case of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs thereof). The tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing at least a portion of the tracr sequence to all or a portion of a tracr-aligned sequence that is operably linked to a guide sequence. The tracr sequence has sufficient complementarity with the tracr-aligned sequence to hybridize and participate in the formation of a CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complementarity with the tracr-aligned sequence, over its entire length, when optimally aligned.

[0094] One or more vectors that direct the expression of one or more elements of the CRISPR system can be introduced into a cell such that expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cells as proteins and/or RNA. For example, each element selected from the Cas enzyme, the guide sequence associated with the tracr-related sequence, and the tracr sequence can be operably linked to separate regulatory elements in separate vectors. The Cas enzyme can be a target gene under the control of a regulated alternative splicing event as disclosed herein, either as a chimeric minigene based on the target gene or as a target gene for a transactivator of the chimeric minigene. The guide RNA can be under the control of a constitutive promoter.

[0095] Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may include one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, the one or more insertion sites are located 5' and/or 3' of one or more sequence elements of the one or more vectors. By using multiple different guide sequences, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0096] The vector may 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, homologues thereof, or modified variants thereof. These enzymes are known; For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0097] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia ). The CRISPR enzyme may direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR enzyme is unable to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate to alanine (D10A) substitution in the catalytic domain of RuvC I Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves one strand). In some embodiments, the Cas9 nickase may be used in combination with a guide sequence(s), such as two guide sequences that target the sense and antisense strands of a target DNA, respectively. This combination allows for breaks to be made in both strands and used to induce NHEJ or HDR.

[0098] In some embodiments, an enzyme encoding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells from or derived from a specific organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in host cells of interest by replacing at least one codon of a naturally occurring sequence with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the natural amino acid sequence. Different species exhibit a specific bias for certain codons for a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend on, among other things, the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on optimization of codon usage.

[0099] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and a corresponding target sequence when optimally aligned using a suitable algorithm for alignment is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[00100] The optimal alignment may be determined using any suitable algorithm for sequence alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., the Burrows-Wheeler alignment setup), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[00101] The CRISPR enzyme may be part of a fusion protein comprising one or more domains of a heterologous protein. The protein fused to the CRISPR enzyme may comprise any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release 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, but are not limited to, glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YEP), and autofluorescent proteins, including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds DNA molecules or binds other cellular molecules, including, but not limited to, fusion to maltose binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD), fusion to GAL4A DNA-binding domain, and fusion to herpes simplex virus (HSV) BP16 protein. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U.S. Patent Application No. 20110059502, incorporated herein by reference.

D. Therapeutic proteins

[00102] Some embodiments relate to the expression of recombinant proteins and polypeptides. In some aspects, a protein or polypeptide may be modified to improve stability in serum. Thus, when the present application refers to a function or activity of a "modified protein" or a "modified polypeptide," one of ordinary skill in the art will recognize that it includes, for example, a protein or polypeptide that has an additional advantage over an unmodified protein or polypeptide. In particular, it is contemplated that embodiments relating to a "modified protein" may be implemented with respect to a "modified polypeptide," and vice versa.

[00103] Recombinant proteins may have deletions and/or substitutions of amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments, these proteins may further include insertions or added amino acids, such as, for example, in the case of fusion proteins or proteins with linkers. A "deletion-modified protein" lacks one or more residues of a native protein, but may have the specificity and/or activity of a native protein. A "deletion-modified protein" may also have reduced immunogenicity or antigenicity. An example of a deletion-modified protein is a protein in which an amino acid residue is deleted from at least one antigenic region, i.e., a region of the protein recognized as antigenic in a particular organism, such as the organism to which the modified protein is administered.

[00104] Substitution or substitution variants typically comprise the replacement of one amino acid with another at one or more locations in a protein and may be designed to modulate one or more properties of the polypeptide, in particular its effector functions and/or bioavailability. The substitutions may or may not be conservative, i.e., one amino acid is replaced by an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, substitutions of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[00105] In addition to a deletion or substitution, a modified protein may have a residue insertion, which typically involves the addition of at least one residue to a polypeptide. This may involve the insertion of a targeting peptide or polypeptide, or simply a single residue. Terminal additions, called fusion proteins, are discussed below.

[00106] The term "biologically functional equivalent" is well known in the art and is further defined in detail herein. Accordingly, sequences are included that contain from about 70% to about 80%, or from about 81% to about 90%, or even from about 91% to about 99% of amino acids that are identical or functionally equivalent to the amino acids of a reference polypeptide, so long as the biological activity of the protein is retained. A recombinant protein may be biologically functionally equivalent to its native counterpart in some aspects.

[00107] It will also be understood that amino acid sequences and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and still remain substantially as set forth in one of the sequences disclosed herein, provided that the sequence meets the criteria set forth above, including maintaining the biological activity of the protein when it comes to protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, include various non-coding sequences flanking either the 5' or 3' portions of the coding region, or may include various internal sequences, i.e., introns, which are known to occur within genes.

[00108] As used herein, the term "protein or peptide" generally refers to, but is not limited to, a protein of greater than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide containing from about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[00109] As used herein, the term "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art. In some embodiments, the residues of a protein or peptide are consecutive, without any non-amino acid moieties interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In particular embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[00110] Accordingly, the term "protein or peptide" encompasses amino acid sequences containing at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[00111] Some embodiments of the present invention relate to fusion proteins. These molecules may comprise a therapeutic protein linked at the N- or C-terminus to a heterologous domain. For example, fusions may also utilize leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Another useful fusion involves the addition of a protein affinity tag, such as a serum albumin or six histidine affinity tag, or an immunologically active domain, such as an antibody epitope, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione S-transferase (GST).

[00112] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of the entire fusion protein or by joining a DNA sequence encoding a heterologous domain, followed by expression of the intact fusion protein.

[00113] Production of fusion proteins that restore the functional activities of the original proteins can be facilitated by joining genes with a bridging segment of DNA encoding a peptide linker spliced between the polypeptides joined together. The linker should be of sufficient length to ensure proper folding of the resulting fusion protein.

III. Methods of administration

[00114] In some aspects, viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro or ex vivo and then administered to patients. The term "vector" refers to a small nucleic acid carrier molecule, plasmid, virus (e.g., an AAV-based vector, a retroviral vector, a lentiviral vector), or other carrier that can be manipulated by insertion or incorporation of nucleic acid. Vectors, such as viral vectors, can be used to introduce/transfer nucleic acid sequences into cells such that the nucleic acid sequence therein is transcribed and, if it encodes a protein, subsequently translated by the cells.

[00115] Any suitable cell or mammal can be administered or treated using the method or use described herein. Typically, a mammal in need of a method described herein is suspected of having or expressing an abnormal or aberrant protein that is associated with a disease state. Alternatively, the recipient mammal may have a condition that is amenable to gene replacement therapy. As used herein, the term "gene replacement therapy" refers to the administration to a recipient of exogenous genetic material encoding a therapeutic agent and the subsequent expression of the introduced genetic material in situ. Thus, the term "condition amenable to gene replacement therapy" includes conditions such as genetic diseases (i.e., a disease state associated with one or more gene defects), acquired pathologies (i.e., a pathological condition associated with a congenital defect), cancers, and prophylactic measures (i.e., prevention of a disease or an undesirable medical condition). Accordingly, the term "therapeutic agent" as used herein refers to any agent or material that has a beneficial effect on a mammalian recipient. Thus, "therapeutic agent" includes both therapeutic and prophylactic molecules containing nucleic acid or protein components.

[00116] Non-limiting examples of mammals include humans, non-human primates (e.g., monkeys, gibbons, chimpanzees, orangutans, apes, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In some embodiments, the non-rodent mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult mammal, an adolescent, a child, an infant, or an in utero mammal). The mammal can be male or female. In some embodiments, the mammal may be an animal model of the disease, such as animal models that have or express an abnormal or aberrant protein that is associated with a disease state, or animal models that lack expression of a protein that causes a disease state.

[00117] Mammals (subjects) treated with the method or composition described herein include adult mammals (18 years of age and older) and children (under 18 years of age). Adults include elderly mammals. Representative adults are adults 50 years of age and older. Children range in age from 1 to 2 years, or from 2 to 4, 4 to 6, 6 to 18, 8 to 10, 10 to 12, 12 to 15, and 15 to 18 years of age. Children also include infants. Infants typically range in age from 1 to 12 months.

[00118] In some embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, wherein the severity, frequency, progression, or time of onset of one or more symptoms of a disease state, such as a neurodegenerative disease, is reduced, shortened, prevented, inhibited, or delayed. In some embodiments, the method comprises administering a plurality of viral particles to a mammal to treat an adverse symptom of a disease state, such as a neurodegenerative disease. In some embodiments, the method comprises administering a plurality of viral particles to a mammal to stabilize, slow, or prevent worsening or progression, or reverse an adverse symptom of a disease state, such as a neurodegenerative disease.

[00119] In some embodiments, the method comprises administering a plurality of viral particles into the central nervous system or a portion thereof, as described herein, of a mammal, and the severity, frequency, progression, or time of onset of one or more symptoms of a disease state, such as a neurodegenerative disease, is reduced, shortened, prevented, inhibited, or delayed by at least about 5-10, about 10-25, about 25-50, or about 50-100 days.

[00120] In some embodiments, the symptom or adverse effect comprises an early, mid, or late stage symptom; a behavioral, personality, or language symptom; a swallowing, movement, seizure, tremor, or restlessness symptom; ataxia; and/or a cognitive symptom such as memory, organization ability.

IV. Pharmaceutical compositions

[00121] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, preparation, liquid or solid, or mixture thereof, which is suitable for one or more modes of administration, in vivo delivery, or contacting. A "pharmaceutically acceptable" 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 causing significant undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" preparations and compositions can be sterile. Such pharmaceutical preparations and compositions can be used, for example, to administer a viral particle to a subject.

[00122] Such preparations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic agents, and agents promoting or delaying absorption(s) compatible with pharmaceutical administration or in vivo contacting or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Additional active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) may also be included in the preparations and compositions.

[00123] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that 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. Pharmaceutically acceptable salts may be included, for example, salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances such as surfactants, wetting agents or emulsifiers, pH adjusting agents, and the like may be present in such carriers.

[00124] Pharmaceutical compositions can be formulated to be compatible with a particular mode of administration or delivery, as described herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by a variety of routes.

[00125] Pharmaceutical forms suitable for injection or infusion of viral particles may include sterile aqueous solutions or dispersions adapted for the extemporaneous preparation of sterile injectable or infusable solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final form must be a liquid, sterile and stable under the conditions of manufacture, use and storage. The liquid carrier or medium may be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycols, etc.), vegetable oils, non-toxic glycerol esters and suitable mixtures thereof. Proper fluidity can be maintained, for example, by forming liposomes, maintaining the required particle size in the case of dispersions, or by using surfactants. Agents for imparting isotonicity, such as sugars, buffers or salts (e.g. sodium chloride), may be included. Prolonged absorption of injectable compositions may be brought about by the use in the compositions of agents which delay absorption, such as aluminum monostearate and gelatin.

[00126] Solutions or suspensions of viral particles may optionally include 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 cerebrospinal fluid, surfactants, fixed oils, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), glycerol, 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 acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose.

[00127] Pharmaceutical preparations, compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2003) 20^th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18^th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12^th ed., Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11^thed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[00128] The viral particles and compositions thereof may be formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the individual to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Unit dosage forms depend on the quantity of viral particles believed to be necessary to produce the desired effect(s). The required quantity may be formulated as a single dose or may be formulated as multiple dosage units. The dose may be adjusted to a suitable concentration of viral particles, optionally combined with an anti-inflammatory agent, and packaged for use.

[00129] In one embodiment, the pharmaceutical compositions will include an amount of genetic material sufficient to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or alleviate the symptoms or adverse effect of the disease state in question, or an amount sufficient to provide the desired benefit.

[00130] "Unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity, optionally in association with a pharmaceutical carrier (vehicle, diluent, carrier, or excipient), calculated to produce the desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more dosages. Unit dosage forms can be contained within, for example, ampoules and vials that can include a liquid composition or a composition in a lyophilized or freeze-dried state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Thus, for example, viral particles and pharmaceutical compositions thereof can be packaged as unit dosage forms for single or multiple use for the sake of ease of administration and uniformity of dosage.

[00131] Preparations containing viral particles typically contain an effective amount that can be readily determined by one skilled in the art. Typically, viral particles can be in the range of about 1% to about 95% (weight/weight) of the composition, or even higher if appropriate. The amount administered depends on factors such as the age, weight, and physical condition of the mammal or human being considered for treatment. Effective dosages can be determined by one of ordinary skill in the art through routine testing to establish dose-response curves.

V. Definitions

[00132] The terms "polynucleotide," "nucleic acid," and "transgene" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and polymers thereof. Polynucleotides include genomic DNA, cDNA, and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA, and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-spliced RNA, or antisense RNA). Polynucleotides may include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., a nucleic acid variant). Polynucleotides may be single-stranded, double-stranded, or triple-stranded, linear or circular, and may be of any suitable length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of giving the sequence in the 5' to 3' direction.

[00133] A nucleic acid encoding a polypeptide often includes an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[00134] The nucleic acids may include one or more expression control or regulatory elements operably linked to the open reading frame, wherein the one or more regulatory elements are configured to control transcription and translation of a polypeptide encoded by the open reading frame in a mammalian cell. 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, poly-A sequences, secretory sequences, etc. The expression control/regulatory elements may be obtained from the genome of any suitable organism.

[00135] "Promoter" refers to a nucleotide sequence, typically upstream (5') of a coding sequence, that directs and/or controls the expression(s) of the coding sequence by providing recognition by RNA polymerase and other factors necessary for proper transcription. The promoter for pol II includes a minimal promoter, which is a short DNA sequence consisting of a TATA box and optionally other sequences that serve to define a transcription initiation site to which regulatory elements are added to control expression. The promoter for pol III type 1 includes three elements as cis-acting sequences 3' of the transcription start site: a) a 5' sequence element (box A); b) an intermediate sequence element (box I); c) a 3' sequence element (box C). The promoter for pol III type 2 includes two important cis-acting sequence elements 3' of the transcription start site: a) the A box (5' sequence element); and b) the B box (3' sequence element). The promoter for pol III type 3 includes several cis-acting promoter elements 5' of the transcription start site, such as the traditional TATA box, the proximal sequence element (PSE), and the distal sequence element (DSE).

[00136] An "enhancer" is a DNA sequence that can stimulate transcriptional activity and can be a natural promoter element or a heterologous element that increases the level or tissue specificity of expression. It can function in any orientation (5'->3' or 3'->5') and can function even when located either 5' or 3' from the promoter.

[00137] Promoters and/or enhancers may be derived entirely from a naturally occurring gene or may consist of different elements derived from different naturally occurring elements or may even consist of synthetic DNA segments. A promoter or enhancer may include DNA sequences that are involved in binding protein factors that modulate/control the efficiency of transcription initiation in response to stimuli, physiological conditions, or developmental conditions.

[00138] Non-limiting examples of promoters include the early promoter (early gene promoter) of SV40, the LTR promoter of mouse mammary tumor virus; the major late promoter of adenovirus (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, promoters for pol II, promoters for pol III, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene will also find use herein. Examples of constitutive promoters include the promoters of the following genes, which encode certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerate kinase (PGK), pyruvate kinase, phosphoglycerate mutase, actin promoter, U6, and other constitutive promoters known to those skilled in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: 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, among many others. In addition, sequences derived from intronic microRNA promoters, such as, for example, the miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566 and miR128 promoters, will also find use here (see, for example, Monteys et al., 2010). Accordingly, any of the above-mentioned constitutive promoters can be used to control the transcription of a heterologous gene insertion.

[00139] "Transgene" is used herein to conveniently refer to a nucleic acid/polynucleotide sequence that is intended to be or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene encoding an inhibitory RNA or a polypeptide or protein, and are typically heterologous to naturally occurring AAV genomic sequences.

[00140] The term "transduce" refers to the introduction of a nucleic acid sequence into a host cell or organism via a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell via a viral particle may be referred to as "transducing" the cell. The transgene may or may not integrate into the genomic nucleic acid of the transduced cell. If the introduced transgene integrates into the nucleic acid (genomic DNA) of the recipient cell or organism, it may be stably maintained in that cell or organism and subsequently transmitted to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extrachromosomally or only transiently. Therefore, a "transduced cell" is a cell into which the transgene has been introduced by transduction. Thus, a "transduced" cell is a cell or its progeny into which a transgene has been introduced. The transduced cell can be propagated, transcribed, and express the encoded inhibitory RNA or protein. For gene therapy applications and methods, the transduced cell may be in a mammal.

[00141] Transgenes under the control of inducible promoters are expressed only or to a greater extent in the presence of an inducing agent (e.g., transcription under the control of the metallothionein gene promoter is significantly increased in the presence of certain metal ions). Inducible promoters include response elements (REs) that stimulate transcription upon binding of factors that induce them. For example, REs exist for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing a particular RE can be selected to produce an inducible response, and in some cases the RE itself can be linked to another promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting an appropriate promoter (constitutive or inducible, strong or weak), both the presence and level of expression of a polypeptide in a genetically modified cell can be controlled. If the gene encoding the polypeptide is under the control of an inducible promoter, the delivery of the polypeptide in situ is initiated by exposing the genetically modified cell in situ to conditions that ensure transcription of the polypeptide, for example, by intraperitoneal injection of specific inducers of inducible promoters that control transcription of the agent. For example, in situ expression by genetically modified cells of a polypeptide encoded by a gene under the control of the metallothionein gene promoter is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ.

[00142] A nucleic acid/transgene is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid/transgene encoding an RNAi or polypeptide, or a nucleic acid directing the expression of a polypeptide, may include an inducible promoter or a tissue-specific promoter to control transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element may also be referred to as an expression cassette.

[00143] In some embodiments, CNS-specific or inducible promoters, enhancers, and the like are used in the methods and uses described herein. Non-limiting examples of CNS-specific promoters include those derived from the myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and neuronal-specific enolase (NSE) genes. Non-limiting examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and IFN.

[00144] In some embodiments, the expression control element comprises a CMV enhancer. In some embodiments, the expression control element comprises a beta-actin promoter. In some embodiments, the expression control element comprises a chicken beta-actin promoter. In some embodiments, the expression control element comprises a CMV enhancer and a chicken beta-actin promoter.

[00145] As used herein, the terms "modify" or "variant" and grammatical variations thereof mean that a nucleic acid, polypeptide, or subsequence thereof deviates from a reference sequence. Accordingly, modified sequences and sequence variants may have substantially the same, greater, or lesser expression, activity, or function as (than) the reference sequence, but retain at least partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene that has a mutation, such as a missense or nonsense mutation.

[00146] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered compared to a wild type. The sequence may be genetically modified without altering the sequence of the encoded protein. Alternatively, the sequence may be genetically modified to encode a protein variant. A nucleic acid or polynucleotide variant may also refer to a combined sequence that has been modified in codon usage to encode a protein that still retains at least partial sequence identity with a reference sequence, such as a wild type protein sequence, and has also been modified in codon usage to encode a protein variant. For example, some codons of such a nucleic acid variant will be altered without altering the amino acids of the protein it encodes, and some codons of the nucleic acid variant will be altered, which in turn will alter the amino acids of the protein it encodes.

[00147] The terms "protein" and "polypeptide" are used interchangeably herein. "Polypeptides" encoded by a "nucleic acid" or "polynucleotide" or "transgene" disclosed herein include partial or full-length natural sequences, as in the case of naturally occurring wild-type proteins and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Accordingly, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences need not be identical to an endogenous protein that is defective or the activity, function, or expression of which is deficient, defective, or absent in the mammal being treated.

[00148] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., from about 1 to about 3, from about 3 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 100, from about 100 to about 150, from about 150 to about 200, from about 200 to about 250, from about 250 to about 500, from about 500 to about 750, from about 750 to about 1000 or more nucleotides or residues).

[00149] An example of an amino acid modification is a conservative amino acid substitution or deletion. In particular embodiments, the modified sequence or sequence variant retains at least a portion of the function or activity of the unmodified sequence (e.g., the wild-type sequence).

[00150] 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, such as specific areas of the brain.

[00151] A recombinant virus modified in this manner may preferentially bind to one tissue type (e.g., CNS tissue) over another tissue type (e.g., liver tissue). In some embodiments, a recombinant virus carrying a modified capsid protein may "target" cerebral vascular epithelial tissue by binding at a higher level than a comparable unmodified capsid protein. For example, a recombinant virus having a modified capsid protein may bind to cerebral vascular epithelial tissue at a level that is 50-100% higher than the level of an unmodified recombinant virus.

[00152] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) is the genetic material of most organisms, while ribonucleic acid (RNA) is involved in the transfer of information contained in DNA into proteins. Fragments and variants of the disclosed nucleotide sequences and the proteins or proteins encoded by them of a subset of their size are also encompassed by the present invention. By "fragment" or "portion" is meant the full or subset of the size of a nucleotide sequence encoding a polypeptide or protein, or the amino acid sequence of a polypeptide or protein. In some 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).

[00153] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a native molecule. With respect to nucleotide sequences, variants include those sequences that, due to the degeneracy of the genetic code, encode an amino acid sequence identical to the native protein. Naturally occurring allelic variants such as these can be identified using molecular biology techniques such as, for example, polymerase chain reaction (PCR) and hybridization techniques. Variants of nucleotide sequences also include synthetically produced nucleotide sequences, such as those created, for example, using site-directed mutagenesis, that encode a native protein, as well as sequences that encode a polypeptide having amino acid substitutions. Typically, the nucleotide sequence variants according to the present invention will be at least 40%, 50%, 60%, up to 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, up to 79%, typically at least 80%, such as 81-84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, up to 98%, identical in sequence to the native (endogenous) nucleotide sequence. In some 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).

[00154] "Conservative variations" of a particular nucleic acid sequence refer to those nucleic acid sequences that encode identical or substantially identical amino acid sequences. Due to the degeneracy of the genetic code, any given polypeptide is encoded by a large 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 each position where arginine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded protein. Such nucleic acid variations are "silent variations," which are one type of "conservatively modified variations." Each nucleic acid sequence described herein that encodes a polypeptide also describes every possible silent variation, unless otherwise noted. One skilled in the art will appreciate that each codon in a nucleic acid (except ATG, which is typically the only codon for methionine) can be modified to produce a functionally identical molecule by standard methods. Accordingly, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each sequence described.

[00155] The term "substantial identity" of polynucleotide sequences means that the polynucleotide includes a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% identical in sequence, 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% identical in sequence when compared to a reference sequence using one of the alignment programs described using standard parameters. It will be appreciated by those skilled in the art that these values can be appropriately adjusted to determine the respective identity of the proteins encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame position, etc. Substantial amino acid sequence identity for these purposes typically means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%.

[00156] The term "substantial identity" when applied to a polypeptide indicates that the polypeptide comprises a sequence that is 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% identical, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% identical to a reference sequence within a given comparison window. An indication that the sequences of two polypeptides are identical is that one polypeptide reacts immunologically with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to the second polypeptide, for example, if the two peptides differ only by a conservative substitution.

[00157] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventive measures that aim to prevent, inhibit, reduce, or lessen an undesirable physiological change or disorder, such as the development, progression, or worsening of a disorder. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the prevalence of a disease, stabilization (i.e., not worsening or progressing) of a symptom or adverse effect of a disease, delay or slowing of the progression of a disease, attenuation or temporary relief of a painful condition, and remission (either partial or complete), whether detectable or undetectable. "Treatment" may also mean an increase in survival time compared to the expected survival time if no treatment is administered. Those in need of treatment include those who already have the condition or disorder, as well as those who are predisposed to it (e.g., as determined by genetic analysis).

VI. Sets

[00158] The present invention provides kits together with packaging material and one or more components therein. A kit typically includes a label or package insert that includes a description of the components or instructions for using the components contained therein in vitro, in vivo, or ex vivo. A kit may contain a plurality of such components, such as a nucleic acid, a recombinant vector, and/or viral particles.

[00159] A kit refers to a physical structure containing one or more components of a kit. The packaging material may maintain the sterility of the components and may be made of a material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, test tubes, etc.).

[00160] The labels or inserts may include identifying information about one or more components therein, dosage amounts, clinical pharmacology of the active ingredient(s), including mechanism of action, pharmacokinetics, and pharmacodynamics. The labels or inserts may include information identifying the manufacturer, lot numbers, location and date of manufacture, and expiration dates. The labels or inserts may include information identifying information about the manufacturer, lot numbers, location of the manufacturer, and date. The labels or inserts may include information about the disease for which the component of the kit may be used to treat. The labels or inserts may include instructions for a clinician or subject for using one or more components of the kit in a method, application, treatment protocol, or therapeutic regimen. The instructions may include dosage, frequency, or duration, as well as instructions for practicing any methods, applications, treatment protocols, or prophylactic or therapeutic regimens described herein.

[00161] Labels or inserts may include information about any benefit that the component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which it would be inappropriate to use a particular composition. Adverse side effects or complications may also occur if the subject has taken, will take, or is currently taking one or more other medications that may be incompatible with the composition, or if the subject has taken, will take, or is currently undergoing treatment under another protocol or therapeutic regimen that will be incompatible with the composition, and therefore the instructions may include information about such incompatibilities.

[00162] Labels or inserts include "printed material" such as paper or cardboard, either separate from or attached to a component, kit, or packaging material (e.g., a box), or attached to an ampoule, tube, or vial containing a component of the kit. Labels or inserts may further include a machine-readable medium such as a printed barcode label, a disc, an optical disc such as a CD or DVD-ROM/RAM, DVD, MP3, or an electrical storage medium such as RAM and ROM, or hybrids thereof such as magnetic/optical storage media, flash memory, hybrids, and memory cards.

VII. Examples

[00163] The following examples are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventor has found to work well in practicing the present invention and thus may be considered to represent preferred modes for practicing it. However, it will be understood by those skilled in the art, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present invention.

Example 1: Identification of AAV variants targeting brain parenchyma

[00164] Enhanced barcoded AAV libraries were developed using AAV1, AAV2, and AAV9 capsids as starting platforms. AAV1, AAV2, and AAV9 peptide display libraries were generated 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 (Fig. 1). The library diversity was 1× ¹⁰⁷ unique clones (Fig. 3).

[00165] To test the utility of the libraries, preliminary studies were conducted using laboratory-grade (low titer, low purity) capsid-modified AAV2. The AAV2 library was injected intravenously into two C57BL/6 mice at 8 × 10 ¹⁰ vector genomes per animal. After 72 h, the cerebral cortex, cerebellum, and spinal cord were dissected. Of note, the heart, skeletal muscle, and diaphragm were collected separately to determine muscle tropism. Viral genomic DNA was isolated and the resulting random oligonucleotide sequences were amplified by PCR. The PCR products from the brain were pooled to create a second-round library, which was injected into two mice at 4 × 10 ¹⁰ vector genomes per animal. After the second injection, vector genomes were isolated as before and subjected to NexGen sequencing along with the original library and cycle 1 tissues. To test whether sequences enriched in brain tissues could increase AAV2 access to the brain, selected sequences identified were cloned into a plasmid for packaging into the AAV2 capsid, generating eGFP-expressing AAV2. Laboratory-grade vectors were constructed, and 3×10 ¹⁰ vector genomes of viruses with modified AAV2 capsids were injected into mice. After four weeks, eGFP fluorescence was observed in the brain, even for these low-titer variants.

[00166] Using these refined barcoded AAV libraries, AAV variants were identified that could target specific structures in the brain of non-human primates. AAV1, AAV2, and AAV9 libraries were delivered via intracerebral ventricular injection (ICV) into one non-human primate (Figure 2). Seventy-two hours post-infusion, brain sections were microdissected to isolate viral DNA, and AAV DNA was amplified by PCR. The products were pooled and used to package a round 2 library, which was infused into another non-human primate. Brain sections were then microdissected 12 days post-infusion. After two rounds of panning, the vector genomes were isolated and subjected to next-generation sequencing. Specifically, genomic DNA extracted from cycle 1 and cycle 2 tissues was amplified by PCR to generate Illumina amplicon sequencing libraries at the vector barcode position. The resulting libraries were pooled and run on a single lane of an Illumina HiSeq 4000 using 100 bp single-end chemistry. To illustrate the utility of the approach, several target regions were examined as examples: ependyma, meninges, and cerebellum. In general, sequences targeting AAVx to the ependyma, meninges, and cerebellum were distinct and varied across serotypes.

[00167] Cycle-by-cycle enrichment plots (Figure 4) and heat maps (Figures 5 and 6) were generated for the following tissues: brainstem, caudate nucleus, cerebellar cortex (Figure 5), cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nuclei, and thalamus. They illustrate the enrichment of the indicated barcodes at baseline (cycle 0) and after the first and second cycles of in vivo passage in rhesus macaques. To generate them, fastq results files for each tissue and cycle combination were processed using a custom Python script designed to extract and quantify unique barcode patterns observed at the DNA level. A custom R script was used to calculate the percentage of barcodes present in each sample and convert DNA barcodes to amino acid barcodes. Table 1 represents samples processed with the AAV1-derived library; Table 2 represents tissues processed with the AAV2-derived library; Table 3 represents samples processed with the AAV9-derived library. The best identified barcodes were selected from the three libraries and pooled and formed into a validation library containing barcodes from 50 (AAV1), 58 (AAV2), and 30 (AAV9) origins. This validation library was delivered into another rhesus macaque via intracerebral ventricular injection (ICV). Tissues were again pooled and processed to facilitate the generation of multiple barcodes by deep sequencing. The relative barcode abundance was assessed in the resulting tissues and in the input viral library. Enrichment values for each barcode were calculated relative to their abundance in the input viral library. The resulting relative enrichment values provide a robust indicator of vector performance across the various tissues evaluated, facilitating the identification of broad and specific AAV vector variants (Fig. 7A-C).

[00168] To confirm cell type specificity, AAV9-1999 (targeting peptide sequence KGGGFHG; SEQ ID NO:110) was selected for in vivo validation. An eGFP expression construct was packaged into AAV9-1999 under the control of the CAG promoter. Five-year-old female rhesus macaques were injected with 1.5E13 AAV9-1999 vector genomes by ICV injection into the left lateral ventricle. Brains were collected 30 days post-injection for histological analysis. Cerebellar sections were stained with hematoxylin and eosin to demonstrate the transduction pattern of AAV9-1999 (Figure 8). Cochleae were also collected from this animal and unexpectedly showed strong transduction of hair cells. In addition, AAV9-1999 and AAV9 capsids containing the eGFP construct were delivered to C57BL/6 p0 mouse pups by ICV injection at a dose of 1E10 vector genomes per hemisphere. After 21 days, the mice were perfused. Whole brains (Fig. 9A), sagittal sections of whole brain at 40 μm (Fig. 9B), sections of S1 cortex at 40 μm (Fig. 9C, left), sections of hippocampus at 40 μm (Fig. 9C, middle), sagittal sections of cerebellum at 40 μm (Fig. 9C, right), and coronal sections of lumbar spinal cord at 40 μm (Fig. 9D) were visualized for eGFP fluorescence signal. AAV9-1999 injected into neonatal Bl/6 mice showed ubiquitous expression exceeding that seen with dose-matched AAV9 injection.

[00169] A single adult rhesus macaque was injected with a mixture of four engineered AAVs: AAV9 with the RGDFQWV targeting peptide sequence (SEQ ID NO:113) and the mTAGBFP2 tag; AAV1 with the ERDRTRG targeting peptide sequence (SEQ ID NO:21) and the mTFP1 tag; AAV2 with the GRGAPGG targeting peptide sequence (SEQ ID NO:80) and the mNG tag; and AAV2 with the DDPSARR targeting peptide sequence (SEQ ID NO:53) and the mRuby3 tag. The viruses were mixed directly in equal volumes to yield final total doses for each as follows:

AAV9.RGDL mTagBFP2 6.13E12 of all vector genomes AAV1.ERDR mTFP1 1.23E13 of all vector genomes AAV2.GRGA mNG 8.8E12 of all vector genomes AAV2.DDPS mRuby3 1.32E13 of all vector genomes

[00170] Brains were collected 30 days post-injection for fluorescence imaging. Lateral ventricle sections (Fig. 10A), fourth ventricle sections (Fig. 10B), and meningeal sections (Fig. 10C) were visualized for mTagBFP2, mTFP2, mNG, and mRuby3 fluorescence signals.

[00171] Additional experiments were performed by injecting AAV9-1999 into the cochlea of rhesus macaques. Following cochlear transduction, the animal was injected with AAV9-1999 into the lateral ventricle. One animal was injected with 3E11 AAV9-1999 viral genomes directly into the cochlear window via duct fenestration (Fig. 11A-C).

[00172] All of the methods disclosed and claimed herein can be realized and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the methods and steps or sequence of steps of the method described herein without departing from the spirit, scope and scope of the present invention. More specifically, it will be apparent that certain agents that are related chemically and physiologically can be substituted for the agents described herein while achieving the same or similar results. 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 present invention as defined by the appended claims.

LIST OF CITED BIBLIOGRAPHY

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are expressly incorporated herein by reference.

US Patent No. 8299215

US Patent 8691948

US Patent Application Publication No. 2018/0142259

Chen et al. , Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy. Nat Med, 2009. 15(10): p. 1215-8.

Chen et al. , Overcoming Limitations Inherent in Sulfamidase to Improve Mucopolysaccharidosis IIIA Gene Therapy. Mol Ther, 2018. 26(4): p. 1118-1126.

Deverman et al. , Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol, 2016. 34(2): p. 204-9.

Hartz et al. , Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J Vis Exp, 2018(139).

Hordeaux et al. , The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. Mol Ther, 2018. 26(3): p. 664-668.

Katz et al. , AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease. Sci Transl Med, 2015. 7(313): p. 313ra180.

Keiser et al. , Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy. Brain, 2015. 138(Pt 12): p. 3555-66.

Koerber et al. , DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Ther, 2008. 16(10): p. 1703-9.

Matsuzaki et al. , Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain. Neurosci Lett, 2018. 665: p. 182-188.

McBride et al. , Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington's disease. Mol Ther, 2011. 19(12): p. 2152-62.

Monteys et al. , CRISPR/Cas9 Editing of the Mutant Hunting Allele In Vitro and In Vivo. Mol Ther, 2017. 25(1): p. 12-23.

Muller et al. , Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol, 2003. 21(9): p. 1040-6.

Schaffer & Maheshri, Directed evolution of AAV mutants for enhanced gene delivery. Conf Proc IEEE Eng Med Biol Soc, 2004. 5: p. 3520-3.

Zhong et al. , Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines leading to high-efficiency transduction at lower doses. Proc Natl Acad Sci USA, 2008. 105(22): p. 7827-32.

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LIST OF SEQUENCES

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<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 69

Ser Ala Arg Ala Gly Ala Pro

1 5

<210> 70

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 70

Ser Gly Glu Phe Val Gly Arg

1 5

<210> 71

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 71

Ser Gly Arg Lys Leu Glu Val

1 5

<210> 72

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 72

Ser Ala Arg Ser Gly Ser Val

1 5

<210> 73

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 73

Glu Ser Thr Gly Arg Glu Arg

1 5

<210> 74

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 74

Val Thr Gln Ser Lys Gly Ala

1 5

<210> 75

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 75

Arg Gly Ser Gly Ser Ala Val

1 5

<210> 76

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 76

Asn Ala Arg Pro Val Ser Ala

1 5

<210> 77

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 77

Thr Ala Arg Gly Gly Gly Gly

1 5

<210> 78

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 78

Gly Arg Ser Ala Ser Gly Ser

1 5

<210> 79

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 79

Lys Ala Gln Gly Val Gly Gly

1 5

<210> 80

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 80

Gly Arg Gly Ala Pro Gly Gly

1 5

<210> 81

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 81

Val Pro Gly Arg Thr Ala Gly

1 5

<210> 82

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 82

Ala Arg Gly Ser Gly Val Asn

1 5

<210> 83

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 83

Ser Val Arg Val Gly Gly Gln

1 5

<210> 84

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 84

Arg Ala Val Pro Ala Gly Gly

1 5

<210> 85

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 85

Val Met Ser Ser Gly Lys Pro

1 5

<210> 86

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 86

Ser Thr Pro Ala Pro Lys Ser

1 5

<210> 87

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 87

Arg Gly Gly Ala Gln Val Val

1 5

<210> 88

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 88

Lys Ala Ser Gly Ala Gly Gly

1 5

<210> 89

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 89

Thr Gly Thr Ala Gly Leu Lys

1 5

<210> 90

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 90

Arg Ser Asn Ala Pro Gln Thr

1 5

<210> 91

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 91

Glu Ala Gln Ser His Pro Arg

1 5

<210> 92

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 92

Thr Lys Ser Leu Ser Ser Gly

1 5

<210> 93

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 93

Ala Ala Gly Ala Lys Val Met

1 5

<210> 94

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 94

ArgGlySerThrGlnValGly

1 5

<210> 95

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 95

Gly Arg Ser Thr Gly Met Thr

1 5

<210> 96

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 96

Arg Ala Thr Ser Gln Ser Thr

1 5

<210> 97

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 97

Val Gly Arg Ser Val Gly Ala

1 5

<210> 98

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 98

Gly Glu Gly Gly Gly Gly Arg

1 5

<210> 99

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 99

Thr Ala Ala Gly Gly Gln Arg

1 5

<210> 100

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 100

Gly Arg Gly Gly Ala Ala Leu

1 5

<210> 101

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 101

Val Ala Pro Ile Ser Lys Ser

1 5

<210> 102

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 102

Ala Pro Pro Val Lys Leu Ser

1 5

<210> 103

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 103

His Asp Gly Gly Ala Ser Arg

1 5

<210> 104

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 104

Arg Ser Gly Gly Ala Ala Val

1 5

<210> 105

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 105

Gly Ser Arg Ala Gly Val Gly

1 5

<210> 106

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 106

Lys Leu Ser Ile Ser Gly Asn

1 5

<210> 107

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 107

Gly Ala Val Gly Gly Val Lys

1 5

<210> 108

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 108

Lys Asn Glu Ser Gly Lys Val

1 5

<210> 109

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 109

Ala Gly Gln Leu Ala Gly Arg

1 5

<210> 110

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 110

Lys Gly Gly Gly Phe His Gly

1 5

<210> 111

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 111

Arg Ala Lys Pro Gly Met Glu

1 5

<210> 112

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 112

Gly Arg Asp Val Thr Arg Ser

1 5

<210> 113

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 113

Arg Gly Asp Leu Gln Trp Val

1 5

<210> 114

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 114

Gly Gly Asp Arg Thr Arg Gly

1 5

<210> 115

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 115

Arg Gly Asp Leu Ala Ser Val

1 5

<210> 116

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 116

Arg Asp Thr Thr Arg Asn Leu

1 5

<210> 117

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 117

Lys Gly Gly Gly Val His Gly

1 5

<210> 118

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 118

Arg Gly Asp Met Tyr Arg Val

1 5

<210> 119

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 119

ArgGlyAspArgProValSer

1 5

<210> 120

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 120

Arg Ser Asp Val Gly Ser Leu

1 5

<210> 121

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 121

Ala Gly Val Lys Pro Gly Arg

1 5

<210> 122

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 122

ArgGly Asp Trp Pro ArgGly

1 5

<210> 123

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 123

Gly Gly Arg Pro Gly Ser Trp

1 5

<210> 124

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 124

ArgGlyAspTyrProArgSer

1 5

<210> 125

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 125

Arg Gly Asp Leu Arg Phe Ile

1 5

<210> 126

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 126

Arg Gly Gly Gly Val Tyr Gly

1 5

<210> 127

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 127

Gly Ala Asp Arg Thr Arg Gly

1 5

<210> 128

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 128

Arg Gly Asp Phe Met Gly Leu

1 5

<210> 129

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 129

Arg Arg Asp Glu Thr Arg Thr

1 5

<210> 130

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 130

Ala Glu Ser Pro Trp Glu Arg

1 5

<210> 131

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 131

Ala Trp Asp Gly Thr Arg Val

1 5

<210> 132

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 132

Ala Arg Gly Asp Gly Trp Arg

1 5

<210> 133

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 133

Gly Arg Asp Tyr Thr Arg Leu

1 5

<210> 134

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 134

Arg Arg Gly Asp Ala Trp Ser

1 5

<210> 135

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 135

Met Asp Leu Thr Lys Ala Val

1 5

<210> 136

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 136

Met Met Gly Arg Pro Gly Arg

1 5

<210> 137

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 137

Thr Gly Arg Pro Gly Val Trp

1 5

<210> 138

<211> 736

<212> Protein

<213> adeno-associated virus 1

<400> 138

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 139

<211> 735

<212> Protein

<213> adeno-associated virus 2

<400> 139

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 140

<211> 736

<212> Protein

<213> adeno-associated virus 9

<400> 140

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 141

<211> 748

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<220>

<221> Various sign

<222> (594)..(600)

<223> X represents any amino acid

<400> 141

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Ser Ser

580 585 590

Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Ser Pro Ala Thr Gly Asp Val

595 600 605

His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val

610 615 620

Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His

625 630 635 640

Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys Asn Pro Pro

645 650 655

Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Ala

660 665 670

Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr

675 680 685

Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser

690 695 700

Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser

705 710 715 720

Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro

725 730 735

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

740 745

<210> 142

<211> 747

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<220>

<221> Various sign

<222> (591)..(597)

<223> X represents any amino acid

<400> 142

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Ala Ala Ala Xaa Xaa

580 585 590

Xaa Xaa Xaa Xaa Xaa Ala Ala Arg Gln Ala Ala Thr Ala Asp Val Asn

595 600 605

Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr

610 615 620

Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe

625 630 635 640

His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro

645 650 655

Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr

660 665 670

Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly

675 680 685

Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys

690 695 700

Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val

705 710 715 720

Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg

725 730 735

Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 143

<211> 748

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<220>

<221> Various sign

<222> (592)..(598)

<223> X represents any amino acid

<400> 143

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Ala Ala Xaa

580 585 590

Xaa Xaa Xaa Xaa Xaa Xaa Ala Ser Ala Gln Ala Gln Thr Gly Trp Val

595 600 605

Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val

610 615 620

Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn

625 630 635 640

Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met Lys His Pro Pro

645 650 655

Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr

660 665 670

Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr

675 680 685

Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser

690 695 700

Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser

705 710 715 720

Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro

725 730 735

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 144

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 144

Arg Pro Gly Arg Glu Ala Ser

1 5

<---

Claims (26)

1. A modified adeno-associated virus (AAV) capsid protein for packaging a viral vector to form an AAV particle, wherein the modified AAV capsid protein comprises a targeting peptide that comprises SEQ ID NO: 110, wherein the targeting peptide is up to ten amino acids in length and wherein the targeting peptide targets the AAV particle to the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nuclei, or thalamus. 2. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the brainstem. 3. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the caudate nucleus. 4. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the cerebellar cortex. 5. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the cerebral cortex. 6. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the ependyma. 7. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the globus pallidus. 8. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the hippocampus. 9. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the meninges. 10. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the optic nerve. 11. The modified AAV capsid protein of claim 8, wherein said targeting peptide targets the AAV particle to the shell. 12. The modified AAV capsid protein of claim 1, wherein the targeting peptide targets the AAV particle to the spinal cord. 13. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the substantia nigra. 14. The modified AAV capsid protein of claim 12, wherein said targeting peptide targets the AAV particle to the subthalamic nuclei. 15. The modified AAV capsid protein of claim 1, wherein said targeting peptide targets the AAV particle to the thalamus. 16. The modified AAV capsid protein of any one of claims 1-15, wherein the modified AAV capsid protein is a modified AAV capsid protein derived from the AAV9 capsid protein having the amino acid sequence of SEQ ID NO: 140, and wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. 17. The modified AAV capsid protein of claim 16, wherein said targeting peptide is flanked by linker sequences, and the length of the linker sequences on each side of the targeting peptides is two or three amino acids. 18. The modified AAV capsid protein of claim 17, wherein the linker sequences are AAA at the N-terminal side of the targeting peptide and AS at the C-terminal side of the targeting peptide. 19. The modified AAV capsid protein of claim 18, wherein the modified AAV capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 143. 20. The modified AAV capsid protein of claim 19, wherein said targeting peptide is located at amino acid residues 592-598 of SEQ ID NO: 143 and consists of the amino acid sequence of SEQ ID NO: 110. 21. The modified AAV capsid protein of claim 16, wherein said targeting peptide is seven amino acids in length and consists of SEQ ID NO: 110. 22. A nucleic acid comprising a sequence encoding a modified capsid protein according to any one of claims 1-21, wherein the nucleic acid is capable of directing expression of the modified capsid protein in a host cell, thereby facilitating packaging of the viral vector to form an AAV particle. 23. A recombinant adeno-associated virus (rAAV) particle comprising an rAAV vector packaged via a modified capsid protein according to any one of claims 1-21, for infecting a cell. 24. An rAAV vector comprising a nucleic acid encoding a modified capsid protein according to any one of claims 1-21, for packaging the rAAV vector to form a rAAV particle. 25. The viral vector of claim 24, further comprising a nucleic acid sequence encoding a nucleic acid of interest. 26. A cell containing the rAAV vector of claim 24 or 25, obtained by transduction with an rAAV particle to produce AAV particles.