Adeno-associated virus vector variants

Abstract

The invention provides an adeno-associated virus vector variant. Provided herein are modified adeno-associated virus (AAV) capsid proteins comprising a targeting peptide that targets a viral vector comprising the modified AAV capsid protein to a unique organ or brain structure wherein the targeting peptide has a length of from 3 to 10 amino acids. Also provided herein are vectors containing a sequence encoding the targeting peptide, nucleic acids encoding the modified AAV capsid protein, viruses comprising the modified AAV capsid protein, and uses thereof.

Description

[0001] This application is a divisional application with the same invention name as the parent case. The Chinese application number of the parent case is 202080094086.2, the international application number is PCT / US2020 / 061464, and the application date is November 20, 2020.

[0002] Citation of Related Applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 939,315 filed on November 22, 2019 and U.S. Provisional Application Serial No. 63 / 084,709 filed on September 29, 2020, the entire contents of both applications are hereby incorporated by reference in their entirety.

[0004] Reference to a sequence listing

[0005] This application contains a sequence listing, which has been submitted in ASCII format by EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on November 19, 2020 is named CHOPP0038WO_ST25.txt and is 63.8 kilobytes in size. Technical Field

[0006] The present invention relates generally to the fields of medicine, virology and neurology. More specifically, the present invention relates to targeting peptides that target the delivery of viral vectors to unique structures in the brain. Background Art

[0007] Different strategies have been developed to generate AAV vector variants, including rational design and directed evolution. Rational design methods use the knowledge of AAV capsids to target changes to the capsid to change transduction efficiency or specificity, such as tyrosine mutations on the capsid surface for increasing transduction efficiency. Directed evolution methods do not require any knowledge of capsid structure, but are completed by random mutagenesis, capsid shuffling or random peptide insertion. These strategies usually use in vitro systems or mice, which are ideal for cell-based or mouse-based research, but do not imply clinical transformation. In fact, no AAV variant specifically or effectively targets unique brain structures. Therefore, AAV variants that can target unique primate brain structures are needed. Summary of the invention

[0008] Provided herein are viral vectors that each comprise a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a unique brain structure.

[0009] In one embodiment, a modified adeno-associated virus (AAV) capsid protein is provided, comprising a targeting peptide that targets a viral vector comprising a modified AAV capsid protein to a unique organ or brain structure, and the targeting peptide is 3 to 10 amino acids in length. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0010] In some aspects, the modified AAV capsid protein is 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 flanking of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is an SSA on the N-terminal side of the targeting peptide and an AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV1 capsid protein has a sequence at least 95% identical to SEQ ID NO: 141.

[0011] In some aspects, the modified AAV capsid protein is 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 flank of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. In some aspects, the modified AAV2 capsid protein has a sequence at least 95% identical to SEQ ID NO: 142.

[0012] In some aspects, the modified AAV capsid protein is derived from the AAV9 capsid protein (see SEQ ID NO: 140), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the flank of the targeting peptide is a linker sequence, and the linker sequence on each side of the targeting peptide is two or three amino acids long. In some aspects, the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV9 capsid protein has a sequence at least 95% identical to SEQ ID NO: 143.

[0013] In some aspects, the targeting peptide comprises a sequence up to 10 amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-137 and 144. In some aspects, the targeting peptide is 7 amino acids in length.

[0014] In some aspects, the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus or thalamus. In some aspects, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from those listed in Table 1 to target the corresponding brain structure. In some aspects, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from those listed in Table 2 to target the corresponding brain structure. In some aspects, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from those listed in Table 3 to target the corresponding brain structure.

[0015] In some aspects, the unique 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 those 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 those 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 those listed in Table 6 to target the corresponding organ.

[0016] In one embodiment, provided herein is a nucleic acid comprising a sequence encoding the modified capsid protein of any one of the present embodiments.

[0017] In one embodiment, provided herein is a recombinant adeno-associated virus (rAAV) comprising a modified capsid protein of any one of the present embodiments. In some aspects, a combination of rAAVs is provided. For example, there is provided an rAAV with a modified AAV1 capsid protein and a targeting peptide of SEQ ID NO: 21, an rAAV with a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO: 53, an rAAV with a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO: 80, and a combination of rAAV with a modified AAV9 capsid protein and a targeting peptide of SEQ ID NO: 113.

[0018] In one embodiment, provided herein is a viral vector comprising a nucleic acid encoding any one of the modified capsid proteins of the present embodiment. In some aspects, the viral vector further comprises a nucleic acid sequence encoding a target nucleic acid. In some aspects, the target nucleic acid is a therapeutic agent. In some aspects, the therapeutic agent is an enzyme or an RNAi molecule.

[0019] In one embodiment, provided herein is a cell comprising a viral vector of any one of the present embodiments. 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.

[0020] In one embodiment, provided herein is a pharmaceutical composition comprising the viral vector of the present embodiments and a pharmaceutically acceptable carrier.

[0021] In one embodiment, provided herein is a method of delivering a medicament to a unique brain structure of a subject, comprising administering a virus of the present embodiment to the subject. In some aspects, the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus or thalamus. In some aspects, rAAV with modified AAV1 capsid protein is used, and the targeting peptide is selected from those listed in Table 1 to target the corresponding brain structure. In some aspects, rAAV with modified AAV2 capsid protein is used, and the targeting peptide is selected from those listed in Table 2 to target the corresponding brain structure. In some aspects, rAAV with modified AAV9 capsid protein is used, and the targeting peptide is selected from those listed in Table 3 to target the corresponding brain structure. In various aspects, any combination of rAAV is used. For example, a combination of rAAV having a modified AAV1 capsid protein and a targeting peptide of SEQ ID NO:21, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO:53, rAAV having a modified AAV2 capsid protein and a targeting peptide of SEQ ID NO:80, and rAAV having a modified AAV9 capsid protein and a targeting peptide of SEQ ID NO:113 is used.

[0022] In one embodiment, provided herein is a method for delivering an agent to a unique organ of a subject, comprising administering a virus of the present embodiment to the subject. In some aspects, the organ is the brain, kidney, heart, liver, gonad, spleen or liver. In some aspects, rAAV with a modified AAV1 capsid protein is used, and the targeting peptide is selected from those listed in Table 4 to target the corresponding organ. In some aspects, rAAV with a modified AAV2 capsid protein is used, and the targeting peptide is selected from those listed in Table 5 to target the corresponding organ. In some aspects, rAAV with a modified AAV9 capsid protein is used, and the targeting peptide is selected from those listed in Table 6 to target the corresponding organ. In various aspects, any combination of rAAV is used.

[0023] In some aspects, the medicament is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein or CRISPR system. In some aspects, administration is for the central nervous system. In some aspects, administration is for the cerebellomedullary cistern, intraventricular space, ependyma, ventricle, subarachnoid space and / or intrathecal space. In some aspects, the ventricle is the anterior lateral ventricle, and / or the posterior lateral ventricle, and / or the right lateral ventricle, and / or the left lateral ventricle, and / or the right anterior lateral ventricle, and / or the left anterior lateral ventricle, and / or the right posterior lateral ventricle, and / or the left posterior lateral ventricle.

[0024] In some aspects, a plurality of viral particles are administered. In some aspects, about 1×10 6 To about 1×10 18 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 -1×10 13 or about 1×10 13 -1×10 14 The virus is administered at a dose of 100 vg / kg patient. In some aspects, the subject is a human.

[0025] In one embodiment, provided herein is a method for treating a disease in a mammal, comprising administering a virus of the present embodiment to the mammal. In some aspects, the disease is a neurodegenerative disease. In some aspects, the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease. In some aspects, the mammal is a human.

[0026] As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified component is purposely formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferably, a composition is one in which standard analytical methods cannot detect any amount of the specified component.

[0027] As used in the specification herein, "a" or "an" may mean one or more than one. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one.

[0028] In the claims, unless explicitly indicated to mean only one alternative or the two alternatives are mutually exclusive, the use of the term "or" is used to mean "and / or", but the present disclosure supports definitions that mean only one alternative and "and / or". As used herein, "another" may mean at least a second or more.

[0029] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error for the device or method being employed to determine the value, the variation that exists between study subjects, or is within 10% of the stated value.

[0030] Other objects, features and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that although the specific embodiments and specific examples show preferred embodiments of the present invention, they are given by way of example only, because those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present invention from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] Figure 1 .Schematic diagram of AAV peptide display library.

[0033] Figure 2 Schematic representation of the in vivo screening strategy.

[0034] Figure 3 Graphical representation of input library diversity. The diversity of the input viral library was measured from aliquots of AAV1, AAV2, and AAV9 viral vectors prior to the first round of ICV injection.

[0035] Figure 4. Graphical representation of round-over-round barcode enrichment. Total number of unique barcodes recovered after round 1 and round 2 enrichment in rhesus macaques for each tissue collected. Round 2 values ​​are shown for DNA and RNA.

[0036] Figure 5 . Schematic representation of round-by-round enrichment of barcodes in AAV1, AAV2, and AAV9 serotypes in the cerebellar cortex.

[0037] Figure 6 . Enrichment illustration of AAV9 1999. Heat map depiction of barcode enrichment from AAV9, with cells colored by the percentage of barcodes detected from the indicated tissues. Barcodes recovered from DNA are shown on the left, while those recovered from RNA are on the right.

[0038] Figures 7A-7C .From AAV1( Fig. 7A )、AAV2( Figure 7B ) and AAV9( Figure 7C )'s heat map depicting opool barcode enrichment.

[0039] Figure 8 .AAV9 1999 in vivo rhesus macaque validation. eGFP expression constructs were packaged into AAV9 1999 driven by the CAG promoter. 1.5E13 vg of AAV9 1999 were delivered to 5-year-old female rhesus macaques by ICV injection. Representative images of H&E-stained cerebellum depicting the transduction pattern of AAV9 1999 are shown.

[0040] Figure 9A-9D. AAV9 1999 in vivo mouse validation. The eGFP expression construct was packaged into AAV9 1999 driven by the CAG promoter. AAV9-1999 and AAV9 capsid containing the eGFP construct were delivered to C57BL / 6p0 mouse pups by ICV injection at 1E10 vg. Representative images of eGFP fluorescence signals are whole brain (Figure 9A), whole brain sagittal section (Figure 9B), S1 cortical section (Figure 9C, left), hippocampal section (Figure 9C, middle), cerebellar sagittal section (Figure 9C, right) and lumbar spinal cord coronal section (Figure 9D).

[0041] Figures 10A-10C .In vivo Rhesus macaque lateral ventricle ( Fig. 10A )、The fourth ventricle( Fig. 10B ) and meninges ( Fig. 10C ) Fluorescence image of the AAV mixture.

[0042] Figures 11A-11CAfter administration of AAV9 1999 capsids containing the eGFP construct into the mouse cochlea, the cochlear turn ( Fig.11A ), inner hair cells ( Fig. 11B ), cortical organs ( Fig. 11C ) and distal modiolus ( Fig. 11C ) fluorescence image. DETAILED DESCRIPTION

[0043] Provided herein are viral vectors that each comprise a modified capsid, wherein the modified capsid comprises at least one amino acid sequence that targets the viral vector to a unique brain structure. In certain embodiments, the brain structure is the brain stem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus or thalamus. Targeting peptides for each brain structure are provided in Tables 1-3.

[0044] In certain embodiments, the viral vector is an adeno-associated viral vector (AAV). In certain embodiments, the AAV is AAV1, AAV2, or AAV9. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 138. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO: 139. An exemplary wild-type reference AAV9 capsid protein sequence is provided in SEQ ID NO: 140. In certain aspects, the targeting peptide is inserted into position 590 of the AAV1 capsid, position 587 of the AAV2 capsid, or position 588 of the AAV9 capsid. An exemplary modified AAV1 capsid protein sequence is provided in SEQ ID NO: 141, which is shown as SSAX 7 Targeting peptide insertion after AS at position 590, where the leading SSA and trailing AS are linker sequences and X 7 represents a targeting peptide. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO: 142, which is shown as AAAX 7 AA is inserted after position 587, where the leading AAA and trailing AA are linker sequences and X 7 represents a targeting peptide. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO: 143, which is shown as AAAX 7 Targeting peptide insertion after AS at position 588, where the leading AAA and trailing AS are linker sequences and X 7 stands for targeting peptide.

[0045] Table 1. AAV1 targeting peptides used for each brain structure.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] Table 2. AAV2 targeting peptides used for each brain structure.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Table 3. AAV9 targeting peptides used for each brain structure.

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Table 4. AAV1 targeting peptides for various organs.

[0076]

[0077] Table 5. AAV2 targeting peptides for various organs.

[0078]

[0079]

[0080] Table 6. AAV9 targeting peptides for various organs.

[0081]

[0082]

[0083] I. Adeno-associated virus (AAV) vector

[0084] Adeno-associated virus (AAV) is a small, non-pathogenic virus of the parvoviridae family. To date, many serologically distinct AAVs have been identified, and more than a dozen AAVs have been isolated from humans or primates. AAV is distinguished from other members of the family because it relies on a helper virus for replication.

[0085] The AAV genome can exist in an extrachromosomal state without integrating into the host cell genome; has a wide host range; transduces both dividing and non-dividing cells in vitro and in vivo, and maintains high levels of expression of transduced genes. AAV virus particles are thermostable; resistant to solvents, detergents, pH and temperature changes; and can be column purified and / or concentrated on a CsCl gradient or by other means. The AAV genome contains positive or negative sense single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb genome of AAV consists of a single-stranded DNA of positive or negative polarity. At the end of the genome are short inverted terminal repeats (ITRs), which can fold into a hairpin structure and serve as the starting point for viral DNA replication.

[0086] AAV "genome" refers to the recombinant nucleic acid sequence that is finally packaged or encapsulated to form AAV particles. AAV particles often contain an AAV genome packaged with AAV capsid proteins. In the case of using a recombinant plasmid to construct or manufacture a recombinant vector, the AAV vector genome does not include a "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone", which is important for cloning and amplification of the plasmid (the process required for plasmid propagation and production), but it itself is not packaged or encapsulated into viral particles. Therefore, the AAV vector "genome" refers to the nucleic acid packaged or encapsulated by the AAV capsid protein.

[0087] AAV virions (particles) are non-enveloped icosahedral particles containing AAV capsids with a diameter of about 25 nm. AAV particles include icosahedral symmetry, which includes three related capsid proteins VP1, VP2 and VP3, which interact together to form capsids. The genomes of most natural AAVs often contain two open reading frames (ORFs), sometimes referred to as left ORFs and right ORFs. The right ORF often encodes capsid proteins VP1, VP2 and VP3. These proteins are often found in a ratio of 1:1:10, respectively, but the ratio may vary and all come from the right-hand ORF. The difference between VP1, VP2 and VP3 capsid proteins is that alternative splicing and unusual start codons are used. Deletion analysis has shown that the removal or change of VP1 translated from the information of alternative splicing can lead to a reduced yield of infectious particles. Mutations in the VP3 coding region result in the inability to produce any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particle encodes one, two or all three VP1, VP2 and VP3 polypeptides.

[0088] The left ORF often encodes nonstructural Rep proteins (Rep 40, Rep 52, Rep 68 and Rep78), which, in addition to participating in the production of single-stranded progeny genomes, are also involved in the regulation of replication and transcription. Two of the Rep proteins have been associated with the preferential integration of the AAV genome into the q arm region of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity and DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and several potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more Rep proteins can be delivered in trans and are therefore not included in the AAV particles comprising nucleic acids encoding polypeptides.

[0089] The end of the AAV genome comprises short reverse terminal repeats (ITRs), which have the potential to be folded into a T-shaped hairpin structure used as a viral DNA replication origin. Therefore, the genome of AAV comprises one or more (e.g., a pair) ITR sequences located on both sides of the single-stranded viral DNA genome. The ITR sequence often has a length of about 145 bases each. In the ITR region, two elements have been described, which are considered to be the core of ITR function, i.e., GAGC repeat motif and terminal melting site (trs). It has been shown that when ITR is in a linear or hairpin conformation, the repeat motif binds Rep. The combination is considered to locate Rep68 / 78 as the cutting that occurs in a site and strand-specific manner at trs. In addition to the role in replication, these two elements seem to be the core of viral integration. Containing a Rep binding site with adjacent trs in chromosome 19 integration loci. It has been shown that these elements are functional, and are essential for locus-specific integration.

[0090] The term "recombinant", as a modifier of vector (such as a recombinant viral vector, e.g., a lentiviral or parvoviral (e.g., AAV) vector), and modifiers of sequence (such as recombinant nucleic acid sequence and polypeptide), means that the composition has been manipulated (i.e., engineered) in a manner that does not normally occur in nature. A specific example of a recombinant vector (such as an AAV viral vector, a retroviral vector, or a lentiviral vector) would be the insertion of a nucleic acid sequence that is not normally present in the wild-type viral genome into the viral genome. An example of a recombinant nucleic acid sequence would be the case where a nucleic acid (e.g., a gene) encodes an inhibitory RNA cloned into a vector with or without the 5', 3', and / or intron regions of the gene that are normally associated within the viral genome. Although the term "recombinant" is not always used herein with reference to vectors (such as viral vectors) and sequences (such as polynucleotides), "recombinant" forms including nucleic acid sequences, polynucleotides, transgenes, etc. are explicitly included despite any such omissions.

[0091] Recombinant virus "vector" is derived from the wild-type genome of the virus in the following manner: a portion of the wild-type genome is removed from the virus using a molecular method and replaced with a non-natural nucleic acid (such as a nucleic acid sequence). Typically, for example, for AAV, one or two reverse terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome because a portion of the viral genome has been replaced with a non-natural sequence relative to the viral genomic nucleic acid (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 incorporation of such non-natural nucleic acid sequences defines a viral vector as a "recombinant" vector, which may be referred to as a "rAAV vector" in the case of AAV.

[0092] In certain embodiments, AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, AAV (e.g., rAAV) comprises a pair of ITRs flanking (i.e., at each 5′ and 3′ end of) a nucleic acid sequence encoding at least a polypeptide having a function or activity.

[0093] AAV vectors (e.g., rAAV vectors) can be packaged and referred to herein as "AAV particles" for subsequent ex vivo, in vitro or in vivo infection (transduction) of cells. In the case where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle may also be referred to as a "rAAV particle". In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle often comprises an rAAV vector or a portion thereof. The rAAV particle may be one or more rAAV particles (e.g., a plurality of AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. It is noteworthy that reference to rAAV vectors may also be used to refer to rAAV particles.

[0094] Any suitable AAV particles (e.g., rAAV particles) can be used for the methods or purposes herein. rAAV particles and / or the genomes contained therein can be derived from any suitable AAV serotype or strain. rAAV particles and / or the genomes contained therein can be derived from two or more AAV serotypes or strains. Therefore, rAAV can include proteins and / or nucleic acids or parts thereof of any serotype or strain of AAV, wherein the AAV particles are suitable for infection and / or transduction of mammalian cells. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0095] In certain embodiments, a plurality of rAAV particles include particles of the same strain or serotype (or subtype or variant), or particles derived from the same strain or serotype. In certain embodiments, a plurality of rAAV particles include a mixture of two or more different rAAV particles (e.g., different serotypes and / or strains).

[0096] As used herein, the term "serotype" is used to refer to the distinction of AAVs with capsids that are serologically distinguishable from other AAV serotypes. Serological distinction is determined based on the lack of cross-reactivity between antibodies against one AAV and another AAV. Such cross-reactivity differences are generally attributed to differences in capsid protein sequences / antigenic determinants (e.g., differences in VP1, VP2, and / or VP3 sequences due to AAV serotypes). Although there is the possibility that AAV variants, including capsid variants, may not be serologically distinguishable from reference AAV or other AAV serotypes, they differ from reference AAV serotypes or other AAV serotypes by at least one nucleotide or amino acid residue.

[0097] In certain embodiments, the rAAV vector based on the first serotype genome 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., ITRs) comprising the AAV vector genome corresponds to the serotype of the capsid comprising the rAAV particle.

[0098] In certain embodiments, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome that is distinct from the serotype of one or more AAV capsid proteins of the packaging vector. For example, the rAAV vector genome may comprise an AAV2-derived nucleic acid (e.g., ITR), while at least one or more of the three capsid proteins are derived from a different serotype, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype or variants thereof.

[0099] In certain embodiments, the polynucleotides, polypeptides, or subsequences of rAAV particles or their vector genomes associated with a reference serotype comprise or consist 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 particle. In certain embodiments, the capsid or ITR sequence of an rAAV particle or its vector genome associated with a reference serotype 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 the capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0100] In certain embodiments, the methods herein comprise the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0101] In certain embodiments, the method herein includes using, applying or delivering rAAV2 particles. In certain embodiments, rAAV2 particles include AAV2 capsids. In certain embodiments, rAAV2 particles include at least 60%, 65%, 70%, 75% or higher identical (e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) capsid protein (e.g., VP1, VP2 and / or VP3) with the corresponding capsid protein of natural or wild-type AAV2 particles. In certain embodiments, the rAAV2 particles comprise VP1, VP2, and VP3 capsid proteins that are at least 75% identical (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding capsid proteins of native or wild-type AAV2 particles. In certain embodiments, the rAAV2 particles are variants of native or wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variants have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of native or wild-type AAV2 particles.

[0102] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles comprise one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75%, or more identical (e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding capsid proteins of native or wild-type AAV9 particles. In certain embodiments, the rAAV9 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% identical or higher (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding capsid proteins of natural or wild-type AAV9 particles. In certain embodiments, the rAAV9 particles are variants of natural or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variants have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to the capsid proteins of natural or wild-type AAV9 particles.

[0103] In certain embodiments, the rAAV particle comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical (e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin that allows DNA replication; the ability to bind AAV DNA is integrated into the host cell genome; and / or, if desired, packaged).

[0104] In certain embodiments, the rAAV2 particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical (e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding ITRs of native or wild-type AAV2 particles, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin that allows DNA replication; to integrate the AAV DNA into the host cell genome; and / or, if desired, packaging).

[0105] In certain embodiments, the rAAV9 particles comprise one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical (e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical) to the corresponding ITRs of native or wild-type AAV2 particles, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin that allows DNA replication; to integrate the AAV DNA into the host cell genome; and / or, if desired, packaging).

[0106] rAAV particles may comprise ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of the AAV2 particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs comprising three "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs having less than four "GAGC" repeats. In certain embodiments, the rAAV2 particles comprise ITRs having more than four "GAGC" repeats. In certain embodiments, the ITRs of the rAAV2 particles comprise a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0107] Exemplary suitable lengths of DNA that can be incorporated into rAAV vectors for packaging / encapsidation into rAAV particles can be about 5 kilobases (kb) or less. In specific 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.

[0108] The rAAV vectors (e.g., see Sambrook et al., 1989) containing nucleic acid sequences for guiding RNAi or polypeptide expression can be generated using suitable recombinant techniques known in the art. Recombinant AAV vectors are usually packaged into AAV particles with transduction ability and propagated using the AAV virus packaging system. AAV particles with transduction ability can be incorporated into mammalian cells and enter therein, and then nucleic acid cargo (e.g., heterologous genes) are delivered to the nucleus. Therefore, complete rAAV particles with transduction ability are configured to transduce mammalian cells. The rAAV particles configured to transduce mammalian cells often do not have replication ability, and require additional protein machinery to replicate themselves. Therefore, the rAAV particles configured to transduce mammalian cells are engineered to bind to mammalian cells and enter therein and deliver nucleic acid to the cell, wherein the nucleic acid for delivery is often located between a pair of AAV ITRs in the rAAV genome.

[0109] Suitable host cells for producing AAV particles with transduction ability include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as receptors for heterologous rAAV vectors. Cells from a stable human cell line HEK293 (easily available through, for example, the American Type Culture Collection with accession number ATCC CRL1573) can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) transformed with adenovirus type 5 DNA fragments and expressing adenovirus E1a and E1b genes is used to generate recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles. Methods for generating high-titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be prepared as described in Wright, 2008 and Wright, 2009.

[0110] In certain embodiments, by transfecting the host cell with an AAV helper construct before or simultaneously with the transfection of the AAV expression vector, the AAV helper function is introduced into the host cell. Therefore, the AAV helper construct is sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the missing AAV functions required for productive AAV transduction. The AAV helper construct often lacks AAV ITR and can neither self-replicate nor self-package. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known.

[0111] An "expression vector" is a specialized vector containing 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 a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), introns, one or more ITRs, and a polyadenylation signal.

[0112] II. Therapeutic Agents

[0113] 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 administer nucleic acids encoding inhibitory RNA, noncoding RNA and / or therapeutic proteins to cells in culture or in a host organism.

[0114] A. Inhibitory RNA

[0115] "RNA interference (RNAi)" is a sequence-specific post-transcriptional gene silencing process initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA, resulting in sequence-specific inhibition of gene expression.

[0116] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecules, "short hairpin RNA" or "shRNA" molecules or "miRNA" are RNA duplexes of nucleotides that target a nucleic acid sequence of interest. As used herein, the term "siRNA" is a general term that covers a subset of shRNA and miRNA. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA is "targeted" to a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In certain embodiments, the siRNA targets a sequence encoding huntingtin. In some embodiments, the length of the siRNA duplex is less than 30 base pairs. In some embodiments, the length of the duplex 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 length of the duplex is 19 to 25 base pairs long. In certain embodiments, the length of the duplex is 19 or 21 base pairs long. The RNA duplex portion of the siRNA can be a part of a hairpin structure. In addition to the duplex portion, the hairpin structure can also contain a loop portion between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In certain embodiments, the length of the loop is 18 nucleotides. The hairpin structure can also contain 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.

[0117] shRNA comprises a stem-loop structure, which is designed to contain a 5' flanking region, a siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies utilize short hairpin RNA (shRNA) driven by a strong promoter based on polIII. Many shRNAs show effective knockdown of target sequences in vitro and in vivo, however, some shRNAs that show effective knockdown of target genes are found to be toxic in vivo.

[0118] miRNA is a small cellular RNA (about 22nt) processed from a precursor stem-loop transcript. Known miRNA stem-loops can be modified to contain RNAi sequences specific to the target gene. MiRNA molecules may be superior to shRNA molecules because miRNA is endogenously expressed. Therefore, miRNA molecules are less likely to induce dsRNA-responsive interferon pathways, they are more efficiently processed than shRNA, and they have been shown to have 80% higher silencing efficiency.

[0119] A recently discovered alternative is to use artificial miRNA (the original-miRNA scaffold of the shuttle siRNA sequence) as an RNAi vector. Artificial miRNA is more naturally similar to endogenous RNAi substrates and is more suitable for 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.

[0120] The transcription unit of "shRNA" comprises a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin-5, and once in the cytoplasm, it is processed by Dicer to generate functional siRNA. "miRNA" stem-loops comprise sense and antisense sequences connected by unpaired nucleotide loops, and are usually expressed as part of a larger original transcript (original-miRNA), which is excised by the Drosha-DGCR8 complex to generate an intermediate called a precursor miRNA, which is then exported from the nucleus by exportin-5, and once in the cytoplasm, it is processed by Dicer to generate functional siRNA. "Artificial miRNA" or "artificial miRNA shuttle vector" as used interchangeably herein refers to an original miRNA transcript having a region (at least about 9-20 nucleotides) of a duplex stem-loop excised via Drosha and Dicer processing, which is replaced by an siRNA sequence for a target gene, while retaining the structural elements required for effective Drosha processing within the stem-loop. The term "artificial" originates from the fact that the flanking sequences (upstream about 35 nucleotides and downstream about 40 nucleotides) are generated from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially generated miRNA shuttle vectors.

[0121] The siRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may further comprise a promoter. The nucleic acid sequence may further comprise a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.

[0122] When designing RNAi, there are several factors to consider, such as the property of siRNA, the durability of silencing effect and the selection of delivery system. In order to produce RNAi effect, the siRNA introduced in the organism will contain exon sequence usually. In addition, the RNAi process depends on homology, and therefore the sequence must be carefully selected to maximize gene specificity, while simultaneously reducing the possibility of cross interference between homology but not gene specific sequences. Preferably, siRNA shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity between siRNA sequence and gene to be inhibited. It is not too effective to be less than about 80% identical sequence with target gene. Therefore, the greater the homology between siRNA and gene to be inhibited, the less likely the expression of unrelated genes is to be affected.

[0123] In addition, the size of siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules, which comprise at least about 19-25 nucleotides and can regulate gene expression. In the context of the present invention, the length of siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides. More preferably, the length of siRNA is about 19 nucleotides to about 25 nucleotides.

[0124] siRNA target generally refers to a polynucleotide comprising a region encoding a polypeptide, or a polynucleotide region regulating replication, transcription or translation or other processes important to polypeptide expression, or a polynucleotide comprising a region encoding a polypeptide and a region regulating expression operably linked thereto. Any gene expressed in a cell can be targeted. Preferably, the target gene is a gene involved in the progression of or associated with a cell activity that is important for a disease or that is particularly interesting as a subject of study.

[0125] B. Non-coding RNA

[0126] As demonstrated by cDNA cloning projects and genomic chimeric arrays, more than 90% of the human genome undergoes transcription but does not encode proteins. These transcripts are referred to as non-protein coding RNA (ncRNA). A variety of ncRNA transcripts (such as ribosomal RNA, transfer RNA, competitive endogenous RNA (ceRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA)) are essential for cell function. Similarly, a large number of short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI interacting RNA (piRNA) and small nucleolar RNA (snoRNA) are also known to play important regulatory roles in eukaryotic cells. Recent studies have confirmed that a group of long ncRNA (lncRNA) transcripts showing cell type-specific expression and localization to specific subcellular compartments. It is also known that lncRNA plays an important role during cell development and differentiation, supporting the view that they are selected during the evolutionary process.

[0127] LncRNAs appear to have many different functions. In many cases, they appear to play a role in regulating the activity or localization of proteins, or act as an organizing framework for subcellular structures. In other cases, lncRNAs are processed to produce multiple small RNAs, or they may regulate the way other RNAs are processed. The latest version of data produced by the public research consortium GenCode (version 27) catalogued just under 16,000 lncRNAs in the human genome, yielding nearly 28,000 transcripts; when other databases are included, more than 40,000 lncRNAs are known.

[0128] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, regulate the activity of protein-binding partners, direct chromatin-modifying complexes to their sites of action, and undergo post-transcriptional processing to generate a large number of 5′-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0129] There is also growing evidence that aberrantly expressed lncRNAs play important roles in normal physiological processes as well as in a variety of disease states. lncRNAs are misregulated in a variety of diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation is also manifested in various types of cancer, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Several lncRNAs (e.g., gadd74 and lncRNA-RoR5) regulate cell cycle regulators, such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thereby providing an additional layer of flexibility and robustness to cell cycle progression. In addition, some lncRNAs are associated with mitotic processes, such as centromeric satellite RNA, which is essential for kinetochore formation and is therefore essential for chromosome segregation during mitosis in humans and Drosophila. Another nuclear lncRNA (MA-lincl) regulates M phase exit by acting in cis to repress the expression of its neighboring gene Pura, a regulator of cell proliferation.

[0130] lncRNA is a group of transcripts generally defined as those of more than 200 nucleotides (e.g., about 200 to about 1200 nt, about 2500 nt, or more) lacking an extended open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes lncRNA as well as shorter transcripts, such as those of less than about 200 nt (e.g., about 30 to 200 nt).

[0131] Therefore, in some embodiments, ncRNA is delivered to a specific brain structure such as a target to correct abnormal RNA expression levels or regulate the level of pathogenic lncRNA. Therefore, in some embodiments, the present invention provides rAAV, wherein the viral genome is engineered to encode therapeutic non-coding RNA (ncRNA). In some embodiments, ncRNA is a long non-coding RNA (lncRNA) of about 200 nucleotides (nt) or more in length. In some embodiments, the therapeutic agent is an ncRNA of about 25nt or about 30nt to about 200nt in length. In some embodiments, the length of lncRNA is about 200nt to about 1,200nt. In some embodiments, the length of lncRNA is about 200nt to about 1,100nt, about 1,000nt, about 900nt, about 800nt, about 700nt, about 600nt, about 500nt, about 400nt or about 300nt.

[0132] C. CRISPR system

[0133] Gene editing is a technology that allows the modification of targeted genes within living cells. Recently, on-demand gene editing using the bacterial immune system of CRISPR has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, which is an RNA-guided DNA endonuclease, can be engineered to target new sites relatively easily by changing its guide RNA sequence. This discovery makes sequence-specific gene editing functionally effective.

[0134] In general, a "CRISPR system" refers collectively to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") gene activity or direct its activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (encompassing "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0135] CRISPR / Cas nuclease or CRISPR / Cas nuclease system can comprise a non-coding RNA molecule (guide) RNA that binds sequence-specifically to DNA and a Cas protein (e.g., Cas9) having nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., from a specific organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.

[0136] The CRISPR system can induce double-strand breaks (DSBs) at the target site, followed by fragmentation as discussed herein. In other embodiments, a Cas9 variant considered to be a "nickase" is used to cut a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each nickase is guided by a pair of different gRNA targeting sequences so that after the nicks are introduced at the same time, 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 inactive Cas9 further comprises a transcriptional repressor or activator fused to a ribosome binding protein.

[0137] In some aspects, Cas nuclease and gRNA (including fusions of crRNA and fixed tracrRNA specific to target sequence) are introduced into cells. In general, the target site at the 5' end of gRNA uses complementary base pairing to target site, such as gene, by targeting Cas nuclease. The target site can be selected based on the 5' position of its adjacent protospacer adjacent motif (PAM) sequence (such as usually NGG or NAG). In this regard, by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11 or 10 nucleotides of the guide RNA to correspond to the target DNA sequence, the gRNA is targeted to the desired sequence. Generally, the CRISPR system is characterized in that it promotes the formation of CRISPR complex elements at the target sequence site. Generally, "target sequence" generally refers to a sequence designed to have complementarity in the guide sequence, wherein the hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. Complete complementarity is not necessarily required, provided that there is enough complementarity to cause hybridization and promote the formation of CRISPR complexes.

[0138] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in an organelle of a cell. Typically, a sequence or template that can be used to recombine into a targeting locus comprising a target sequence 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, recombination is homologous recombination.

[0139] Typically, in the context 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 in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence. A 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 a wild-type tracr sequence)) may also form part of a CRISPR complex, such as by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr partner sequence operably linked to the guide sequence. The tracr sequence has sufficient complementarity to the tracr partner sequence to hybridize and participate in the formation of the CRISPR complex, such as having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr partner sequence when optimally aligned.

[0140] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into the cell so that the expression of the elements of the CRISPR system directly forms a CRISPR complex at one or more target sites. The components can also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence connected to the tracr-partner sequence, and the tracr sequence can each be operably connected to a separate regulatory element on a separate vector. The Cas enzyme can be a target gene under the control of a regulated alternative splicing event as disclosed herein, as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. The gRNA may be under the control of a constitutive promoter.

[0141] Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, wherein one or more additional vectors provide any components of the CRISPR system not included in the first vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0142] The vector may comprise 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, Csf1, Csf2, Csf3, Csf4, their homologs or their modified forms. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0143] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S.pneumonia)). The CRISPR enzyme can guide the cutting of one or both chains at the location of the 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, so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts both chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with one or more guide sequences (e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively). This combination allows both chains to be nicked and used to induce NHEJ or HDR.

[0144] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a specific cell, such as a eukaryotic cell. Eukaryotic cells can be those of a specific organism or derived from a specific organism, such as a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon of a native sequence with a more frequent or most frequently used codon in the genes of the host cell while maintaining the native amino acid sequence. Various species exhibit specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is then considered to depend on factors such as the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of the selected tRNA in the cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized for optimal gene expression in a given organism based on codon optimization.

[0145] Typically, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0146] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0147] The CRISPR enzyme can be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may contain any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription 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-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, which is incorporated herein by reference.

[0148] D. Therapeutic proteins

[0149] Some embodiments relate to the expression of recombinant proteins and polypeptides. In some aspects, the protein or polypeptide may be modified to increase serum stability. Therefore, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide", it will be understood by those of ordinary skill in the art that this includes, for example, proteins or polypeptides that have additional advantages over unmodified proteins or polypeptides. It is particularly contemplated that embodiments regarding "modified proteins" may be implemented relative to "modified polypeptides" and vice versa.

[0150] Recombinant proteins may have a deletion and / or substitution of amino acids; therefore, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further comprise inserted or added amino acids, such as, for example, fusion proteins or proteins with joints. "Modified deletion proteins" lack one or more residues of a native protein, but may have the specificity and / or activity of a native protein. "Modified deletion proteins" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is a protein with amino acid residues missing from at least one antigenic region (i.e., a protein region determined to be antigenic in a specific organism, such as an organism to which the modified protein is being administered).

[0151] Substitution or substitution variants generally exchange one amino acid at one or more sites within a protein for another amino acid, and can be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. Substitutions may or may not be conservative, that is, 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, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0152] In addition to deletions or substitutions, modified proteins may also have insertions of residues, which typically involve adding at least one residue in a polypeptide. This may include insertions of targeting peptides or polypeptides or only single residues. Terminal additions referred to as fusion proteins are discussed below.

[0153] The term "biological functional equivalent" is well known in the art and is further defined in detail herein. Thus, it includes about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids identical or functionally equivalent to the amino acids of the control polypeptide, provided that the biological activity of the protein is maintained. In some aspects, the recombinant protein can be biologically functionally equivalent to its natural counterpart.

[0154] It is also understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, but still be substantially as shown in one of the sequences disclosed herein, as long as the sequence meets the above criteria, including maintaining biological protein activity in the case of protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences that may, for example, include various non-coding sequences flanking the 5' or 3' portion of the coding region, or may include various internal sequences known to occur within genes, i.e., introns.

[0155] As used herein, protein or peptide generally refers to, but is not limited to, a protein greater than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide greater than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

[0156] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are continuous, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In specific embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0157] Thus, the term "protein or peptide" encompasses an amino acid sequence comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0158] Certain embodiments of the present invention relate to fusion proteins. These molecules may have therapeutic proteins connected to heterologous domains at the N-terminus or C-terminus. For example, fusions may also employ leader sequences from other species to allow recombinant expression of proteins in heterologous hosts. Another useful fusion includes adding protein affinity tags (such as serum albumin affinity tags or six histidine residues) or immunologically active domains (such as preferably cleavable antibody epitopes) 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).

[0159] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by synthesizing the entire fusion protein de novo, or by attaching a DNA sequence encoding a heterologous domain, followed by expression of the entire fusion protein.

[0160] The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by connecting the gene with a bridging DNA segment encoding a peptide linker that splices between the tandemly linked polypeptides. The linker will be of sufficient length to allow proper folding of the resulting fusion protein.

[0161] II. Method of application

[0162] In some aspects, viral vectors can be directly applied 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 carrier nucleic acid molecule, a plasmid, a virus (e.g., an AAV vector, a retroviral vector, a lentiviral vector), or other vectors that can be manipulated by inserting or incorporating nucleic acids. Vectors (such as viral vectors) can be used to introduce / transfer nucleic acid sequences into cells so that the nucleic acid sequences therein are transcribed and, if encoding proteins, subsequently translated by the cells.

[0163] Any suitable cell or mammal can be applied or processed by the methods or uses described herein. Generally, mammals that need the methods described herein are suspected of having or expressing abnormal or abnormal proteins associated with disease states. Alternatively, the mammalian receptor may suffer from a condition suitable for gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration of exogenous genetic material encoding a therapeutic agent to a receptor and the subsequent in situ expression of the administered genetic material. Therefore, the phrase "condition suitable for gene replacement therapy" covers conditions such as the following: genetic diseases (i.e., disease conditions attributable to one or more gene defects), acquired conditions (i.e., pathological conditions that are not attributable to congenital defects), cancer, and preventive processes (i.e., prevention of diseases or undesirable medical conditions). Therefore, as used herein, the term "therapeutic agent" refers to any agent or material that has a beneficial effect on a mammalian receptor. Therefore, "therapeutic agent" covers therapeutic molecules and preventive molecules with nucleic acid or protein components.

[0164] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In certain 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, teenager, child, infant, or mammal in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, for example, an animal model having or expressing an abnormal or abnormal protein associated with a disease state, or an animal model having insufficient protein expression that causes a disease state.

[0165] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years or older) and children (less than 18 years old). Adults include the elderly. Representative adults are 50 years or older. Children range in age from 1-2 years old, or 2-4, 4-6, 6-18, 8-10, 10-12, 12-15, and 15-18 years old. Children also include infants. The age of infants is generally in the range of 1-12 months.

[0166] In certain embodiments, the method comprises administering to a mammal a plurality of viral particles as described herein, wherein the severity, frequency, progression or onset of one or more symptoms of a disease state (such as a neurodegenerative disease) is reduced, reduced, prevented, inhibited or delayed. In certain embodiments, the method comprises administering to a mammal a plurality of viral particles to treat adverse symptoms of a disease state (such as a neurodegenerative disease). In certain embodiments, the method comprises administering to a mammal a plurality of viral particles to stabilize, delay or prevent deterioration or progression of a disease state (such as a neurodegenerative disease), or reversal and adverse symptoms.

[0167] In certain embodiments, the methods comprise administering a plurality of viral particles to the central nervous system of a mammal, or a portion thereof as described herein, and reducing, reducing, preventing, inhibiting, or delaying the severity, frequency, progression, or onset of one or more symptoms of a disease state (such as a neurodegenerative disease) by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days.

[0168] In certain embodiments, symptoms or adverse effects include early, intermediate, or late symptoms; behavioral, personality, or language symptoms; symptoms of swallowing, movement, seizures, tremors, or restlessness; ataxia; and / or cognitive symptoms, such as memory, organizational skills.

[0169] III. Pharmaceutical Compositions

[0170] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. 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 substantially undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical preparations and compositions can be used, for example, to administer viral particles to a subject.

[0171] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic agents, and absorption enhancers or delay agents that are compatible with drug administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the formulations and compositions.

[0172] Pharmaceutical compositions generally contain pharmaceutically acceptable excipients. Such excipients include any agent that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80 and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included, such as mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances (such as surfactants, wetting agents or emulsifiers, pH buffer substances, etc.) may be present in such vehicles.

[0173] As described herein or known to those skilled in the art, the pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery. Thus, the pharmaceutical composition comprises a carrier, diluent or excipient suitable for administration or delivery by various routes.

[0174] The pharmaceutical form suitable for injection or infusion of viral particles may include a sterile aqueous solution or dispersion suitable for preparing sterile injectable or infusible solutions or dispersions on the spot, which are optionally encapsulated in liposomes. In all cases, the final form should be a sterile fluid and is stable under manufacturing, use and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, which comprises, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and their suitable mixtures. Suitable fluidity can be maintained, for example, by forming liposomes, in the case of dispersions, by maintaining the required particle diameter or by using a surfactant. Isotonic agents may be included, such as sugar, buffer or salt (for example, sodium chloride). The extended absorption of injectable compositions may be produced by using a delayed absorption reagent (for example, aluminum monostearate and gelatin) in the composition.

[0175] The solution or suspension of viral particles may optionally contain one or more of the following components: a sterile diluent, such as water for injection, a saline solution (such as phosphate buffered saline (PBS)), an artificial CSF, a surfactant, a fixed oil, a 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.

[0176] Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the present invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th Edition, Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18th Edition, Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th Edition, Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technical Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th Edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (2003 ... Systems) (1980), RL Juliano, ed., Oxford, NY, pp. 253-315).

[0177] The viral particles and their compositions can be formulated into dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable for use as unit doses for individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The dosage unit form depends on the number of viral particles considered necessary to produce one or more desired effects. The necessary amount can be formulated into a single dose, or it can be formulated into multiple dosage units. The dosage can be adjusted to a suitable viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0178] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, ie, an amount sufficient to reduce or ameliorate the symptoms or adverse effects of the disease state in question or an amount sufficient to confer a desired benefit.

[0179] As used herein, "unit dosage form" refers to a physically discrete unit suitable for use as a unit dosage for a subject to be treated; each unit contains a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent, vehicle or filler), calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. Unit dosage forms can be in, for example, ampoules and vials, which can include liquid compositions, or compositions in a freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added prior to in vivo administration or delivery. A single unit dosage form can be contained in a multi-dose kit or container. Thus, for example, viral particles and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0180] The formulation containing the viral particles generally contains an effective amount, which is easily determined by those skilled in the art. The viral particles generally can range from about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount to be administered depends on factors such as the age, weight and physical condition of the mammal or human subject being treated. One of ordinary skill in the art can establish an effective dose by routine experiments establishing a dose response curve.

[0181] IV. Definitions

[0182] The terms "polynucleotide", "nucleic acid" and "transgene" are used interchangeably herein to refer to all forms of nucleic acids, 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, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides may include naturally occurring, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides may be single-stranded, double-stranded or triplicated, linear or circular, and may have any suitable length. When discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing a sequence in the 5' to 3' direction.

[0183] A nucleic acid encoding a polypeptide often comprises an open reading frame encoding the polypeptide. Unless otherwise indicated, a specific nucleic acid sequence also comprises degenerate codon substitutions.

[0184] The nucleic acid may comprise one or more expression control or regulatory elements operably linked to an open reading frame, wherein the one or more regulatory elements are configured to direct 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 boxes, etc.), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, etc. Expression control / regulatory elements may be obtained from the genome of any suitable organism.

[0185] "Promoter" refers to a nucleotide sequence usually located upstream (5') of a coding sequence that directs and / or controls the expression of a coding sequence by providing recognition for RNA polymerase and other factors required for proper transcription. Pol II promoters include minimal promoters, which are short DNA sequences that contain a TATA box and optionally other sequences that specify a transcription start site, to which regulatory elements are added to control expression. Type 1 pol III promoters contain three cis-acting sequence elements downstream of the transcription start site: a) 5' sequence element (A block); b) middle sequence element (I block); c) 3' sequence element (C block). Type 2 pol III promoters contain two essential cis-acting sequence elements downstream of the transcription start site: a) A box (5' sequence element); and b) B box (3' sequence element). Type 3 pol III promoters contain several cis-acting promoter elements upstream of the transcription start site, such as the traditional TATA box, proximal sequence element (PSE), and distal sequence element (DSE).

[0186] An "enhancer" is a DNA sequence that stimulates transcriptional activity and may be an intrinsic element of a promoter or a heterologous element that increases the level or tissue specificity of expression. It can operate in either orientation (5'->3' or 3'->5') and may be able to function even when located upstream or downstream of a promoter.

[0187] Promoters and / or enhancers may be derived entirely from natural genes, or may be composed of different elements derived from different elements found in nature, or may even comprise synthetic DNA segments. Promoters or enhancers may comprise DNA sequences that participate in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0188] Non-limiting examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter region (CMVIE)), Rous sarcoma virus (rous sarcoma virus) (RSV) promoter, polII promoter, polIII promoter, synthetic promoter, hybrid promoter, etc. In addition, sequences derived from non-viral genes (such as mouse metallothionein gene) can also be used herein. Exemplary constitutive promoters include promoters of the following genes encoding certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol 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, among others: the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus. In addition, sequences derived from intronic miRNA promoters (such as, for example, miR107, miR206, miR208b, miR548f-2, miR569, miR590, miR566, and miR128 promoters) can also be used herein (see, for example, Monteys et al., 2010). Thus, any of the constitutive promoters mentioned above can be used to control transcription of heterologous gene inserts.

[0189] "Transgene" is used herein for convenience to refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene encoding an inhibitory RNA or a polypeptide or protein, and is generally heterologous to the naturally occurring AAV genomic sequence.

[0190] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism, and further transmitted to or inherited by the daughter cells or organisms of the recipient cell or organism. Finally, the introduced transgene may be present in the recipient cell or host organism extrachromosomally, or only transiently. Therefore, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Therefore, a "transduced" cell is a cell or its progeny into which a transgene has been introduced. Transduced cells can be propagated, transgenes can be transcribed, and encoded inhibitory RNA or protein can be expressed. For gene therapy purposes and methods, transduced cells can be in mammals.

[0191] Transgenes under the control of an inducible promoter are expressed only in the presence of an inducer or are expressed to a greater extent (e.g., transcription under the control of a metallothionein promoter is greatly increased in the presence of certain metal ions). Inducible promoters include response elements (REs) that stimulate transcription when binding to their inducing factors. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain inducible responses, and in some cases, the REs themselves can be attached to different promoters, thereby conferring inducibility to the recombinant gene. Therefore, by selecting a suitable promoter (constitutive versus inducible; strong versus weak), the presence and expression level of polypeptides in genetically modified cells can be controlled. If the gene encoding the polypeptide is under the control of an inducible promoter, the in situ delivery of the polypeptide is triggered by exposing the genetically modified cells in situ to conditions that allow transcription of the polypeptide, such as by intraperitoneal injection of a specific inducer of the inducible promoter that controls the transcription of the agent. For example, in situ expression of a polypeptide encoded by a gene under the control of a metallothionein promoter by a genetically modified cell is enhanced by contacting the genetically modified cell in situ with a solution containing the appropriate (ie, inducing) metal ion.

[0192] 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 expression of a polypeptide may include an inducible promoter, or a tissue-specific promoter for controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element may also be referred to as an expression cassette.

[0193] In certain embodiments, CNS-specific or inducible promoters, enhancers, etc. are employed in the methods and uses described herein. Non-limiting examples of CNS-specific promoters include those isolated from genes for myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron-specific enolase (NSE). Non-limiting examples of inducible promoters include DNA response elements for ecdysone, tetracycline, hypoxia, and IFN.

[0194] In certain embodiments, the expression control element comprises a CMV enhancer. In certain embodiments, the expression control element comprises a β-actin promoter. In certain embodiments, the expression control element comprises a chicken β-actin promoter. In certain embodiments, the expression control element comprises a CMV enhancer and a chicken β-actin promoter.

[0195] As used herein, the term "modification" or "variant" and grammatical variations thereof refer to a deviation of a nucleic acid, polypeptide, or subsequence thereof from a reference sequence. Thus, modified and variant sequences may have substantially the same, greater, or less expression, activity, or function as compared to a reference sequence, but retain at least a portion of the activity or function of the reference sequence. A specific type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation (e.g., a missense or nonsense mutation).

[0196] "Nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered compared to the wild type. A sequence may be genetically modified without changing the encoded protein sequence. Alternatively, a sequence may be genetically modified to encode a variant protein. Nucleic acid or polynucleotide variants may also refer to a combined sequence that has been codon-modified 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 codon-modified to encode a variant protein. For example, some codons of such nucleic acid variants will be changed without changing the amino acid of the protein encoded thereby, and some codons of the nucleic acid variant will be changed, which in turn changes the amino acid of the protein encoded thereby.

[0197] The terms "protein" and "polypeptide" are used interchangeably herein. A "polypeptide" encoded by a "nucleic acid" or "polynucleotide" or "transgene" disclosed herein includes a partial or full-length native sequence, just like naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, as long as the polypeptide retains a certain degree of function or activity. Therefore, in the methods and uses of the present invention, such polypeptides encoded by nucleic acid sequences need not be identical to endogenous proteins that are defective or active, under-functioned or under-expressed, lacking or absent in the mammal being treated.

[0198] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000, or more nucleotides or residues).

[0199] Examples of amino acid modifications are conservative amino acid substitutions or deletions.In certain embodiments, the modified sequence or variant sequence retains at least part of the function or activity of the unmodified sequence (eg, wild-type sequence).

[0200] Another example of an amino acid modification is a targeting peptide introduced into the capsid protein of the viral particle.Peptides have been identified that target recombinant viral vectors to the central nervous system, such as to distinct brain regions.

[0201] The recombinant virus thus modified may preferentially bind to one type of tissue (e.g., CNS tissue) relative to another type of tissue (e.g., liver tissue). In certain embodiments, a recombinant virus carrying a modified capsid protein may "target" cerebrovascular epithelial tissue by binding at a level higher than that of a comparable unmodified capsid protein. For example, a recombinant virus having a modified capsid protein may bind to cerebrovascular epithelial tissue at a level 50% to 100% greater than that of an unmodified recombinant virus.

[0202] "Nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in most organisms is the genetic material, while ribonucleic acid (RNA) is involved in transferring the information contained in DNA to proteins. The present invention also encompasses fragments and variants of the disclosed nucleotide sequences and proteins encoded therefrom or proteins of partial length. "Fragment" or "portion" means a nucleotide sequence of a full-length or less-than-full-length coding polypeptide or protein or its amino acid sequence. In certain embodiments, a fragment or portion has a biological function (i.e., retaining 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% activity or function of the wild type).

[0203] The "variant" of a molecule is a sequence that is substantially similar to the sequence of a natural molecule. For nucleotide sequences, variants include those sequences that encode the same amino acid sequence of a natural protein due to the degeneracy of the genetic code. Molecular biological techniques (such as, for example, using polymerase chain reaction (PCR) and hybridization techniques) can be used to identify naturally occurring allele variants such as these. Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as, for example, nucleotide sequences encoding natural proteins generated using site-directed mutagenesis, and nucleotide sequences encoding polypeptides with amino acid substitutions. Typically, the nucleotide sequence variants of the invention will have at least 40%, 50%, 60% to 70% (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, typically at least 80%, for example, 81%-84%, at least 85%, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%) sequence identity with the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the activity or function of the wild-type).

[0204] "Conservative variations" of a particular nucleic acid sequence refer to nucleic acid sequences that encode identical or substantially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Therefore, at each position where arginine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded protein. Such nucleic acid variations are "silent variations," which are one of the "conservatively modified variations." Unless otherwise indicated, each nucleic acid sequence encoding a polypeptide described herein also describes each possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is typically the only codon for methionine) can be modified by standard techniques to produce functionally identical molecules. Therefore, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each of the described sequences.

[0205] The term "substantially identical" to a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, or at least 90%, 91%, 92%, 93% or 94%, or even at least 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. Substantially identical amino acid sequences for these purposes generally mean at least 70%, at least 80%, 90% or even at least 95% sequence identity.

[0206] The term "substantially identical" in the context of polypeptides 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%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% identical to a reference sequence over a specified comparison window. Two polypeptide sequences are identical in that one polypeptide is immunoreactive with an antibody raised against a second polypeptide. Thus, a polypeptide is identical to a second polypeptide where, for example, the two peptides differ only in conservative substitutions.

[0207] The term "treat" or "treatment" refers to therapeutic treatment and preventive or preventive measures, wherein the purpose is to prevent, inhibit, reduce or reduce undesirable physiological changes or conditions, such as the development, progression or deterioration of a condition. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of symptoms or adverse effects of the disease (i.e., no deterioration or progression), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to expected survival when not receiving treatment. Those in need of treatment include those already suffering from a condition or disorder and those susceptible to the disease (e.g., as determined by genetic assays).

[0208] V. Kit

[0209] The present invention provides a kit having packaging material and one or more components therein. The kit typically includes a label or package insert that includes a description of the components or instructions for in vitro, in vivo or ex vivo use of the components therein. The kit may contain a batch of such components, such as nucleic acids, recombinant vectors and / or viral particles.

[0210] Kit refers to a physical structure that houses one or more components of the kit. The packaging material can maintain the components sterilely, and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0211] The label or insert may include identification information of one or more components, dosage amounts, clinical pharmacology (including mechanism of action, pharmacokinetics and pharmacodynamics) of one or more active ingredients. The label or insert may include information identifying the manufacturer, batch number, place of manufacture and date, and expiration date. The label or insert may include information identifying manufacturer information, batch number, manufacturer location and date. The label or insert may include information about diseases for which the kit components can be used. The label or insert may include instructions for clinicians or subjects to use one or more kit components in methods, uses, treatment protocols or treatment regimens. The instructions may include dosage amounts, frequency or duration, and instructions for implementing any method, use, treatment protocol or prevention or treatment regimen described herein.

[0212] The label or insert may include information about any benefits (such as preventive or therapeutic benefits) that the components may provide. The label or insert may include information about potential adverse side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which the particular composition is not suitable for use. Adverse side effects or complications may also occur when the subject has, will, or is currently taking one or more other drugs that may be incompatible with the composition, or when the subject has, will, or is currently receiving another treatment protocol or treatment regimen that will be incompatible with the composition, and therefore, the instructions may include information about such incompatibilities.

[0213] Labels or inserts include "printed matter", e.g., paper or cardboard, either alone or affixed to a component, kit or packaging material (e.g., box), or attached to an ampoule, tube or vial containing a kit component. Labels or inserts may additionally include computer-readable media, such as barcode printed labels, magnetic disks, optical disks (such as CD- or DVD-ROM / RAM, DVD, MP3), or electrical storage media (such as RAM and ROM) or hybrids of these, such as magnetic / optical storage media, flash memory, hybrids, and memory-type cards.

[0214] This article includes the following implementations:

[0215] 1. A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide that targets a viral vector comprising the modified AAV capsid protein to a unique organ or brain structure, wherein the targeting peptide is 3 to 10 amino acids in length.

[0216] 2. The modified AAV capsid protein of embodiment 1, wherein the modified AAV capsid protein is a modified AAV9 capsid protein having a sequence at least 95% identical to SEQ ID NO: 143, wherein the targeting peptide is SEQ ID NO: 110, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellum, cochlea (ear), cortex, cerebral cortex, deep cerebellar nucleus, ependyma, globus pallidus, hippocampus, meninges, motor cortex, optic nerve, prefrontal cortex, putamen, spinal cord, substantia nigra, subthalamic nucleus, temporal cortex, thalamus or visual cortex.

[0217] 3. The modified AAV capsid protein according to embodiment 1, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein or a modified AAV9 capsid protein.

[0218] 4. The modified AAV capsid protein according to embodiment 1, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein (see SEQ ID NO: 138), wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

[0219] 5. A modified AAV capsid protein according to embodiment 4, wherein the targeting peptide is flanked by a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long.

[0220] 6. A modified AAV capsid protein according to embodiment 5, wherein the linker sequence is SSA at the N-terminal side of the targeting peptide and AS at the C-terminal side of the targeting peptide.

[0221] 7. The modified AAV capsid protein according to embodiment 6, wherein the modified AAV1 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 141.

[0222] 8. A modified AAV capsid protein according to embodiment 1, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein (see SEQ ID NO: 139), wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

[0223] 9. A modified AAV capsid protein according to embodiment 8, wherein the targeting peptide is flanked by a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long.

[0224] 10. A modified AAV capsid protein according to embodiment 9, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

[0225] 11. The modified AAV capsid protein of embodiment 10, wherein the modified AAV2 capsid protein has a sequence that is at least 95% identical to SEQ ID NO: 142.

[0226] 12. A modified AAV capsid protein according to embodiment 1, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein (see SEQ ID NO: 140), wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

[0227] 13. A modified AAV capsid protein according to embodiment 12, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

[0228] 14. A modified AAV capsid protein according to embodiment 13, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0229] 15. The modified AAV capsid protein of any one of Embodiments 14, wherein the modified AAV9 capsid protein has a sequence at least 95% identical to SEQ ID NO: 143.

[0230] 16. A modified AAV capsid protein according to embodiment 1, wherein the targeting peptide comprises a sequence of up to 10 amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-137 or 144.

[0231] 17. A modified AAV capsid protein according to embodiment 16, wherein the targeting peptide is 7 amino acids in length.

[0232] 18. The modified AAV capsid protein of any one of embodiments 1-17, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus.

[0233] 19. The modified AAV capsid protein of embodiment 18, wherein the unique brain structure is the brainstem, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 1-9.

[0234] 20. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the caudate nucleus, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 1, 3, 5, 7, 10-16, 25, 26, 32 and 144.

[0235] 21. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebellar cortex, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 1, 3, 4, 9 and 17-21.

[0236] 22. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebral cortex, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 1, 3, 5, 12 and 21-26.

[0237] 23. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the ependyma, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 2-4, 7, 9, 21, 22, 27 and 28.

[0238] 24. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the globus pallidus, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 3, 5, 12, 14, 16, 21, 22 and 29-31.

[0239] 25. The modified AAV capsid protein of embodiment 18, wherein the unique brain structure is the hippocampus, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from the group consisting of SEQ ID NOs: 1-4, 7, and 32-34.

[0240] 26. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the meninges, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 3, 5, 7, 9, 12, 21 and 35-37.

[0241] 27. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the optic nerve, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 2, 3, 7, 14-16, 21, 31 and 38.

[0242] 28. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the putamen, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 3, 4, 12, 13, 21, 30 and 39-42.

[0243] 29. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the spinal cord, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 2-4, 7, 9, 21, 32, 33 and 43.

[0244] 30. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the substantia nigra, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 2, 3, 9, 44 and 45.

[0245] 31. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the subthalamic nucleus, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 2-4, 12, 16, 30, 46 and 47.

[0246] 32. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the thalamus, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 1, 2, 8, 12, 21, 28 and 48-51.

[0247] 33. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the brainstem, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 52-60.

[0248] 34. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the caudate nucleus, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 59 and 61-69.

[0249] 35. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebellar cortex, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 56, 58, 60 and 70-75.

[0250] 36. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebral cortex, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 53, 58, 60, 62, 63, 66 and 76-79.

[0251] 37. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the ependyma, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 53, 60, 62, 63, 66, 74-77 and 80.

[0252] 38. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the globus pallidus, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 60, 75 and 81-87.

[0253] 39. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the hippocampus, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 53, 55, 58, 60, 63, 76, 79, 88 and 89.

[0254] 40. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the meninges, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 58, 60, 66, 73, 76, 80 and 90-93.

[0255] 41. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the optic nerve, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 53, 54, 57, 58, 60, 75, 79, 87, 88 and 94.

[0256] 42. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the putamen, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 55, 59, 60, 61 and 95-100.

[0257] 43. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the spinal cord, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 53, 58-61, 63, 77, 88, 95 and 101.

[0258] 44. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the substantia nigra, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 52, 53, 57, 58, 75, 76, 87, 102 and 103.

[0259] 45. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the subthalamic nucleus, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 57, 58, 60, 75, 79, 87, 88, 102, 104 and 105.

[0260] 46. ​​A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the thalamus, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 52, 55, 56, 74, 85, 88 and 106-109.

[0261] 47. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the brainstem, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110-117.

[0262] 48. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the caudate nucleus, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 113, 115, 116 and 118-121.

[0263] 49. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebellar cortex, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 119 and 122-125.

[0264] 50. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the cerebral cortex, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 114, 116 and 125-127.

[0265] 51. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the ependyma, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 118-120 and 128.

[0266] 52. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the globus pallidus, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110-112, 114, 119, 120 and 129.

[0267] 53. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the hippocampus, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 116, 123, 125, 129 and 130.

[0268] 54. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the meninges, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 114, 118, 119, 122 and 131.

[0269] 55. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the optic nerve, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 114, 115, 117, 129 and 132.

[0270] 56. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the putamen, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 112, 113, 116, 123, 127, 133 and 134.

[0271] 57. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the spinal cord, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 113, 119, 120, 122, 123, 128 and 134.

[0272] 58. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the substantia nigra, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110-114, 117 and 129.

[0273] 59. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the subthalamic nucleus, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 111, 113, 119, 120, 122, 132 and 135.

[0274] 60. A modified AAV capsid protein according to embodiment 18, wherein the unique brain structure is the thalamus, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, and wherein the targeting peptide is selected from SEQ ID NO: 110, 112-114, 125, 133, 136 and 137.

[0275] 61. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 1, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, hippocampus, or thalamus.

[0276] 62. The modified AAV capsid protein of embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 2, wherein the unique brain structure is the brainstem, ependyma, hippocampus, optic nerve, spinal cord, substantia nigra, subthalamic nucleus, or thalamus.

[0277] 63. The modified AAV capsid protein of embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 3, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, or subthalamic nucleus.

[0278] 64. The modified AAV capsid protein of embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 4, wherein the unique brain structure is the brainstem, cerebellar cortex, ependyma, hippocampus, putamen, spinal cord, or subthalamic nucleus.

[0279] 65. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO:5, wherein the unique brain structure is the brainstem, cerebral cortex, globus pallidus, or meninges.

[0280] 66. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 6, wherein the unique brain structure is the brainstem.

[0281] 67. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO:7, wherein the unique brain structure is the brainstem, caudate nucleus, ependyma, hippocampus, meninges, optic nerve or spinal cord.

[0282] 68. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 8, wherein the unique brain structure is the brainstem or thalamus.

[0283] 69. The modified AAV capsid protein of embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 9, wherein the unique brain structure is the brainstem, cerebellar cortex, ependyma, meninges, spinal cord, or substantia nigra.

[0284] 70. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 10 or 11, wherein the unique brain structure is the caudate nucleus.

[0285] 71. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 12, wherein the unique brain structure is the caudate nucleus, cerebral cortex, globus pallidus, meninges, putamen, subthalamic nucleus, or thalamus.

[0286] 72. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 13, wherein the unique brain structure is the caudate nucleus or putamen.

[0287] 73. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 14, wherein the unique brain structure is the caudate nucleus, globus pallidus, or optic nerve.

[0288] 74. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 15, wherein the unique brain structure is the caudate nucleus or the optic nerve.

[0289] 75. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 16, wherein the unique brain structure is the caudate nucleus, globus pallidus, optic nerve, or subthalamic nucleus.

[0290] 76. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 17-20, and wherein the unique brain structure is the cerebellar cortex.

[0291] 77. The modified AAV capsid protein of embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 21, wherein the unique brain structure is the cerebellar cortex, cerebral cortex, ependyma, globus pallidus, meninges, optic nerve, putamen, spinal cord or thalamus.

[0292] 78. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 22, wherein the unique brain structure is the cerebral cortex, ependyma, or globus pallidus.

[0293] 79. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 23-26, wherein the unique brain structure is the cerebral cortex.

[0294] 80. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 27, wherein the unique brain structure is the ependyma.

[0295] 81. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 28, wherein the unique brain structure is the ependyma or the thalamus.

[0296] 82. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 29, wherein the unique brain structure is the globus pallidus.

[0297] 83. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO:30, and wherein the unique brain structure is the globus pallidus, putamen, or subthalamic nucleus.

[0298] 84. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 31, wherein the unique brain structure is the globus pallidus or the optic nerve.

[0299] 85. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 32 or 33, wherein the unique brain structure is the hippocampus or the spinal cord.

[0300] 86. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 34, wherein the unique brain structure is the hippocampus.

[0301] 87. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 35-37, and wherein the unique brain structure is the meninges.

[0302] 88. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 38, wherein the unique brain structure is the optic nerve.

[0303] 89. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is any one of SEQ ID NO: 39-42, and wherein the unique brain structure is the putamen.

[0304] 90. The modified AAV capsid protein of embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 43, wherein the unique brain structure is the spinal cord.

[0305] 91. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO:44 or 45, and wherein the unique brain structure is the substantia nigra.

[0306] 92. A modified AAV capsid protein according to embodiments 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is SEQ ID NO: 46 or 47, and wherein the unique brain structure is the subthalamic nucleus.

[0307] 93. A modified AAV capsid protein according to embodiment 1 or 7, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 48-51, and wherein the unique brain structure is the thalamus.

[0308] 94. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 52, wherein the unique brain structure is the brainstem, substantia nigra, or thalamus.

[0309] 95. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 53, wherein the unique brain structure is the brainstem, cerebral cortex, ependyma, hippocampus, meninges, optic nerve, spinal cord, or substantia nigra.

[0310] 96. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 54, wherein the unique brain structure is the brainstem or the optic nerve.

[0311] 97. A modified AAV capsid protein according to embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:55, and wherein the unique brain structure is the brainstem, hippocampus, putamen, or thalamus.

[0312] 98. A modified AAV capsid protein according to embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:56, and wherein the unique brain structure is the brainstem, cerebellar cortex, or thalamus.

[0313] 99. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 57, wherein the unique brain structure is the brainstem, optic nerve, substantia nigra, or subthalamic nucleus.

[0314] 100. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 58, wherein the unique brain structure is the brainstem, cerebellar cortex, cerebral cortex, hippocampus, meninges, optic nerve, spinal cord, substantia nigra, or subthalamic nucleus.

[0315] 101. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 59, wherein the unique brain structure is the brainstem, caudate nucleus, putamen, or spinal cord.

[0316] 102. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 60, wherein the unique brain structure is the brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, or subthalamic nucleus.

[0317] 103. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 61, wherein the unique brain structure is the caudate nucleus, putamen, or spinal cord.

[0318] 104. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 62, wherein the unique brain structure is the caudate nucleus, cerebral cortex, or ependyma.

[0319] 105. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 63, wherein the unique brain structure is the caudate nucleus, cerebral cortex, ependyma, hippocampus, or spinal cord.

[0320] 106. A modified AAV capsid protein according to embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 64, 65, and 67-69, wherein the unique brain structure is the caudate nucleus.

[0321] 107. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 66, wherein the unique brain structure is the caudate nucleus, cerebral cortex, ependyma, or meninges.

[0322] 108. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 70-72, wherein the unique brain structure is the cerebellar cortex.

[0323] 109. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 73, wherein the unique brain structure is the cerebellar cortex or the meninges.

[0324] 110. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 74, wherein the unique brain structure is the cerebellar cortex, ependyma, or thalamus.

[0325] 111. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:75, and wherein the unique brain structure is the cerebellar cortex, ependyma, globus pallidus, optic nerve, substantia nigra, or subthalamic nucleus.

[0326] 112. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 76, wherein the unique brain structure is the cerebral cortex, ependyma, hippocampus, meninges, or substantia nigra.

[0327] 113. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:77, and wherein the unique brain structure is the cerebral cortex, ependyma or spinal cord.

[0328] 114. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:78, and wherein the unique brain structure is the cerebral cortex.

[0329] 115. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 79, wherein the unique brain structure is the cerebral cortex, hippocampus, optic nerve, or subthalamic nucleus.

[0330] 116. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:80, and wherein the unique brain structure is the ependyma, hippocampus or meninges.

[0331] 117. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 81-84 and 86, and wherein the unique brain structure is the globus pallidus.

[0332] 118. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:85, and wherein the unique brain structure is the globus pallidus or thalamus.

[0333] 119. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:87, and wherein the unique brain structure is the globus pallidus, optic nerve, substantia nigra, or subthalamic nucleus.

[0334] 120. The modified AAV capsid protein of embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 88, wherein the unique brain structure is the hippocampus, optic nerve, spinal cord, subthalamic nucleus, or thalamus.

[0335] 121. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 89, and wherein the unique brain structure is the hippocampus.

[0336] 122. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 90-93, and wherein the unique brain structure is the meninges.

[0337] 123. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO:94, and wherein the unique brain structure is the optic nerve.

[0338] 124. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 95, wherein the unique brain structure is the putamen or the spinal cord.

[0339] 125. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 96-100, wherein the unique brain structure is the putamen.

[0340] 126. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 101, wherein the unique brain structure is the spinal cord.

[0341] 127. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 102, and wherein the unique brain structure is the substantia nigra or the subthalamic nucleus.

[0342] 128. A modified AAV capsid protein according to embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 103, and wherein the unique brain structure is the substantia nigra.

[0343] 129. A modified AAV capsid protein according to embodiments 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is SEQ ID NO: 104 or 105, wherein the unique brain structure is the subthalamic nucleus.

[0344] 130. The modified AAV capsid protein of embodiment 1 or 11, wherein the modified AAV capsid protein is a modified AAV2 capsid protein, wherein the targeting peptide is any one of SEQ ID NOs: 106-109, wherein the unique brain structure is the thalamus.

[0345] 131. The modified AAV capsid protein of embodiments 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 110, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus.

[0346] 132. The modified AAV capsid protein of embodiments 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 111, wherein the unique brain structure is the brainstem, cerebellar cortex, cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, substantia nigra, or subthalamic nucleus.

[0347] 133. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 112, wherein the unique brain structure is the brainstem, globus pallidus, putamen, substantia nigra, or thalamus.

[0348] 134. The modified AAV capsid protein of embodiments 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 113, wherein the unique brain structure is the brainstem, caudate nucleus, cerebellar cortex, cerebral cortex, ependyma, hippocampus, meninges, putamen, spinal cord, substantia nigra, subthalamic nucleus, or thalamus.

[0349] 135. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 114, wherein the unique brain structure is the brainstem, cerebral cortex, globus pallidus, meninges, optic nerve, substantia nigra, or thalamus.

[0350] 136. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 115, and wherein the unique brain structure is the brainstem or caudate nucleus.

[0351] 137. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 116, wherein the unique brain structure is the brainstem, caudate nucleus, cerebral cortex, hippocampus, optic nerve, or putamen.

[0352] 138. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 117, wherein the unique brain structure is the brainstem, optic nerve, or substantia nigra.

[0353] 139. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 118, and wherein the unique brain structure is the caudate nucleus, ependyma, or meninges.

[0354] 140. The modified AAV capsid protein of embodiments 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 119, wherein the unique brain structure is the caudate nucleus, cerebellar cortex, ependyma, globus pallidus, meninges, spinal cord, or subthalamic nucleus.

[0355] 141. A modified AAV capsid protein according to embodiments 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 120, wherein the unique brain structure is the caudate nucleus, ependyma, globus pallidus, meninges, spinal cord, or subthalamic nucleus.

[0356] 142. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 121, and wherein the unique brain structure is the caudate nucleus.

[0357] 143. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 122, and wherein the unique brain structure is the cerebellar cortex, meninges, spinal cord, or subthalamic nucleus.

[0358] 144. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 123, wherein the unique brain structure is the cerebellar cortex, hippocampus, putamen, or spinal cord.

[0359] 145. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 124, wherein the unique brain structure is the cerebellar cortex.

[0360] 146. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 125, and wherein the unique brain structure is the cerebellar cortex, cerebral cortex, hippocampus or thalamus.

[0361] 147. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 126, and wherein the unique brain structure is the cerebral cortex.

[0362] 148. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 127, and wherein the unique brain structure is the cerebral cortex or putamen.

[0363] 149. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 128, wherein the unique brain structure is the ependyma or the spinal cord.

[0364] 150. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 129, wherein the unique brain structure is the globus pallidus, hippocampus, optic nerve, or substantia nigra.

[0365] 151. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 130, wherein the unique brain structure is the hippocampus.

[0366] 152. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 131, wherein the unique brain structure is the meninges.

[0367] 153. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 132, and wherein the unique brain structure is the optic nerve or the subthalamic nucleus.

[0368] 154. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 133, and wherein the unique brain structure is the putamen or thalamus.

[0369] 155. The modified AAV capsid protein of embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 134, wherein the unique brain structure is the putamen or the spinal cord.

[0370] 156. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 135, and wherein the unique brain structure is the subthalamic nucleus.

[0371] 157. A modified AAV capsid protein according to embodiment 1 or 15, wherein the modified AAV capsid protein is a modified AAV9 capsid protein, wherein the targeting peptide is SEQ ID NO: 136 or 137, and wherein the unique brain structure is the thalamus.

[0372] 158. A nucleic acid comprising a sequence encoding the modified capsid protein of any one of embodiments 1-157.

[0373] 159. A recombinant adeno-associated virus (rAAV) virus comprising a modified capsid protein according to any one of embodiments 1-157.

[0374] 160. A viral vector comprising a nucleic acid encoding the modified capsid protein of any one of embodiments 1-157.

[0375] 161. The viral vector of embodiment 160, further comprising a nucleic acid sequence encoding a target nucleic acid.

[0376] 162. A viral vector according to embodiment 161, wherein the target nucleic acid is a therapeutic agent.

[0377] 163. The viral vector of embodiment 162, wherein the therapeutic agent is an enzyme or an RNAi molecule.

[0378] 164. A cell comprising the viral vector of any one of embodiments 160-163.

[0379] 165. A cell according to embodiment 164, wherein the cell is a mammalian cell.

[0380] 166. A cell according to embodiment 164, wherein the cell is a human cell.

[0381] 167. A cell according to embodiment 164, wherein the cell is in vitro.

[0382] 168. The cell of embodiment 164, wherein the cell is in vivo.

[0383] 169. A pharmaceutical composition comprising the viral vector according to embodiment 159 and a pharmaceutically acceptable carrier.

[0384] 170. A method of delivering an agent to a unique brain structure in a subject, comprising administering to the subject the virus of embodiment 159.

[0385] 171. A method according to embodiment 170, wherein the method is a method of delivering an agent to the brainstem of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1-9, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 52-60, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110-117.

[0386] 172. A method according to embodiment 170, wherein the method is a method of delivering an agent to the caudate nucleus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1, 3, 7 and 10-16, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 59 and 61-69, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 113, 115, 116 and 118-121.

[0387] 173. A method according to embodiment 170, wherein the method is a method of delivering an agent to the cerebellar cortex of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1, 3, 4, 9 and 17-21, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 56, 58, 60 and 70-75, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 119 and 122-125.

[0388] 174. A method according to embodiment 170, wherein the method is a method of delivering an agent to the cerebral cortex of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1, 3, 5, 12 and 21-26, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 58, 60, 62, 63, 66 and 76-79, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 114, 116 and 125-127.

[0389] 175. A method according to embodiment 170, wherein the method is a method of delivering an agent to the ependyma of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 7, 9, 21, 22, 27 and 28, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 60, 62, 63, 66, 74-77 and 80, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 118-120 and 128.

[0390] 176. A method according to embodiment 170, wherein the method is a method of delivering an agent to the globus pallidus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 3, 5, 12, 14, 16, 21, 22 and 29-31, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 60, 75 and 81-87, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110-112, 114, 119, 120 and 129.

[0391] 177. A method according to embodiment 170, wherein the method is a method of delivering an agent to the hippocampus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1-4, 7 and 32-34 and 28, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 55, 58, 60, 63, 76, 79, 80, 88 and 89, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 116, 123, 125, 129 and 130.

[0392] 178. A method according to embodiment 170, wherein the method is a method of delivering an agent to the meninges of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 3, 5, 7, 9, 12, 21 and 35-37 and 28, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 58, 60, 66, 73, 76, 80 and 90-93, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 114, 118, 119, 122 and 131.

[0393] 179. A method according to embodiment 170, wherein the method is a method of delivering an agent to the optic nerve of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2, 3, 7, 14-16, 21, 31 and 38, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 54, 57, 58, 60, 75, 79, 87, 88 and 94, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 114, 115, 117, 129 and 132.

[0394] 180. A method according to embodiment 170, wherein the method is a method of delivering an agent to the putamen of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 3, 4, 12, 13, 21, 30 and 39-42, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 55, 59, 60, 61 and 95-100, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 112, 113, 116, 123, 127, 133 and 134.

[0395] 181. A method according to embodiment 170, wherein the method is a method of delivering an agent to the spinal cord of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 7, 9, 21, 32, 33 and 43, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 53, 58-61, 63, 77, 88, 95 and 101, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 113, 119, 120, 122, 123, 128 and 134.

[0396] 182. A method according to embodiment 170, wherein the method is a method of delivering an agent to the substantia nigra of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2, 3, 9, 44, 45 and 28, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 52, 53, 57, 58, 75, 76, 87, 102 and 103, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110-114, 117 and 129.

[0397] 183. A method according to embodiment 170, wherein the method is a method of delivering an agent to the subthalamic nucleus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 2-4, 12, 16, 30, 46 and 47, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 57, 58, 60, 75, 79, 87, 88, 102, 104 and 105, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 111, 113, 119, 120, 122, 132 and 135.

[0398] 184. A method according to embodiment 170, wherein the method is a method of delivering an agent to the thalamus of a subject, comprising administering an AAV1 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 1, 2, 8, 12, 21, 28 and 48-51, an AAV2 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 52, 55, 56, 74, 85, 88 and 106-109, or an AAV9 virus comprising a modified capsid protein having a targeting peptide selected from SEQ ID NOs: 110, 112-114, 125, 133, 136 and 137.

[0399] 185. A method according to any one of embodiments 170-184, wherein the agent is a siRNA system, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein or CRISPR system.

[0400] 186. The method of any one of embodiments 170-184, wherein the administration is to the central nervous system.

[0401] 187. The method of embodiment 186, wherein the administration is to the cisterna magna, the intraventricular space, the ependyma, the ventricles, the subarachnoid space, the cochlea, and / or the intrathecal space.

[0402] 188. A method according to embodiment 187, wherein the cerebral ventricle is the anterior lateral ventricle, and / or the posterior lateral ventricle, and / or the right lateral ventricle, and / or the left lateral ventricle, and / or the right anterior lateral ventricle, and / or the left anterior lateral ventricle, and / or the right posterior lateral ventricle, and / or the left posterior lateral ventricle.

[0403] 189. The method of any one of embodiments 170-188, wherein a plurality of viral particles are administered.

[0404] 190. The method according to embodiment 189, wherein about 1×10 6 To about 1×10 18 The virus is administered at a dose of 1 vector genome per kilogram (vg / kg).

[0405] 191. The method according to embodiment 189, wherein 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 -1×1014 , 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 -1×10 13 or about 1×10 13 -1×10 14 The virus is administered at a dose of vg / kg of the patient.

[0406] 192. The method of any one of embodiments 170-191, wherein the subject is human.

[0407] 193. A method of treating a disease in a mammal, comprising administering to the mammal a virus according to embodiment 159.

[0408] 194. A method according to embodiment 193, wherein the disease is a neurodegenerative disease.

[0409] 195. The method of embodiment 194, wherein the neurodegenerative disease is Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, polyglutamine repeat disease, or Parkinson's disease.

[0410] 196. The method of embodiment 193, wherein the mammal is a human.

[0411] VI. Examples

[0412] The following examples are included to demonstrate the preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to function well in the practice of the present invention and therefore can be considered to constitute preferred modes of implementation thereof. However, it will be appreciated by those skilled in the art in light of this disclosure that many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0413] Example 1 - Identification of AAV variants that target brain parenchyma

[0414] Advanced 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 ( Figure 1 ). The library has 1×10 7 The diversity of unique clones ( Figure 3 ).

[0415] To test the utility of the library, pilot studies were performed using bench-grade (low titer, low purity) capsid-modified AAV2. The AAV2 library was plated at 8 × 10 10 Each vector genome was injected intravenously into two C57BL / 6 mice. After 72 hours, the cerebral cortex, cerebellum, and spinal cord were dissected. Of note, the heart, skeletal muscle, and diaphragm were harvested separately to identify muscle tropism. Viral genomic DNA was isolated, and the recovered random oligonucleotide sequences were amplified by PCR. PCR products from the brain were pooled to generate a second round library, which was cloned at 4 × 10 per animal. 10 Each vector genome was injected into two mice. After the second injection, the vector genomes were recovered as described above and subjected to NexGen sequencing along with the starting library and round 1 tissue. To test whether the sequences shown to be enriched in brain tissue could indeed extend AAV2 to the brain, a single hit was cloned into an AAV2 capsid packaging plasmid and AAV2 expressing eGFP was generated. Bench-scale vectors were made and 3×10 of the AAV2-based capsid-modified virus were added to the 10 After 4 weeks, eGFP fluorescence could be seen in the brain even for these low titer variants.

[0416] Using these advanced barcoded AAV libraries, AAV variants were identified that can target unique primate brain structures in nonhuman primates. AAV1, AAV2, and AAV9 libraries were delivered via intracerebroventricular injection into a nonhuman primate ( Figure 2). 72 hours after infusion, the brain regions were microdissected for viral DNA isolation and AAV DNA was amplified by PCR. The products were pooled and used to package the second round of libraries, which were infused into additional NHPs. The brain regions were then microdissected 12 days after infusion. After two rounds of panning, the vector genome was recovered and subjected to next generation sequencing. Specifically, genomic DNA extracted from rounds 1 and 2 of tissue was PCR amplified to generate Illumina amplicon sequencing libraries at the vector barcode position. The resulting libraries were pooled and run on a single lane of Illumina HiSeq 4000 using a 100bp single-end read chemistry. To illustrate the practicality of the method, several target areas were tested as examples: ependyma, meninges, and cerebellum. In general, the sequences that direct AAVx to the ependyma, meninges, and cerebellum are different and are different for different serotypes.

[0417] Generate round-by-round enrichment plots for the following tissues: Figure 4 ) and heatmaps ( Figure 5 and Figure 6 ): Brainstem, caudate nucleus, cerebellar cortex ( Figure 5 ), cerebral cortex, ependyma, globus pallidus, hippocampus, meninges, optic nerve, putamen, spinal cord, substantia nigra, subthalamic nucleus, and thalamus. These illustrate the enrichment of the indicated barcodes at baseline (round 0) and after the first and second rounds of in vivo passage in rhesus macaques. To generate these, the fastq result files for each tissue and round combination were processed using a custom Python script designed to extract and quantify the unique barcode configurations observed at the DNA level. A custom R script was used to calculate the percentage of barcodes present in each sample and to convert DNA barcodes to amino acid barcodes. Table 1 corresponds to samples treated with AAV1-derived libraries; Table 2 represents tissues treated with AAV2-derived libraries; and Table 3 corresponds to samples treated with AAV9-derived libraries. The top hits were selected from these three libraries and assembled and generated as validation libraries containing 50 (AAV1), 58 (AAV2), and 30 (AAV9) derived barcodes. The validation library was delivered to additional rhesus macaques by ICV injection. Tissues were collected and processed again to help recover barcode abundance by deep sequencing. Barcode abundance was evaluated in recovered tissues and input viral libraries. The enrichment value of each barcode was calculated relative to their abundance in the input viral library. The resulting relative enrichment value is a robust indicator of vector performance in various tissues evaluated, helping to identify broad and specific AAV vector variants ( Figures 7A-7C ).

[0418] To verify the identified cell type specificity, AAV9-1999 (with a targeting peptide sequence of KGGGFHG SEQ ID NO: 110) was selected for in vivo validation. The eGFP expression construct was packaged into AAV9-1999 driven by the CAG promoter. 1.5E13 vg of AAV9-1999 was administered to 5-year-old female rhesus macaques by ICV injection into the left lateral ventricle. Brains were collected 30 days after injection for histological analysis. H&E staining was performed on cerebellar sections to depict the transduction pattern of AAV9-1999 ( Figure 8 ). The cochlea was also collected from the animal, and it surprisingly had strong hair cell transduction. In addition, AAV9-1999 and AAV9 capsid containing the eGFP construct were delivered to C57BL / 6p0 mouse pups by ICV injection of 1E10 vg per hemisphere. After 21 days, the mice were perfused. The eGFP fluorescence signal was imaged in the sealed whole brain (Figure 9A), 40μm whole brain sagittal section (Figure 9B), 40μm S1 cortical section (Figure 9C, left), 40μm hippocampal section (Figure 9C, middle), 40μm cerebellar sagittal section (Figure 9C, right) and 40μm lumbar spinal cord coronal section (Figure 9D). AAV9-1999 injected into Bl / 6 newborn mouse pups showed ubiquitous expression greater than that of AAV9 injected with a dose-matched injection.

[0419] An adult rhesus macaque was injected with a mixture of four modified AAVs: AAV9 with the RGDLQWV (SEQ ID NO: 113) targeting peptide sequence and the mTAGBFP2 tag; AAV1 with the ERDRTRG (SEQ ID NO: 21) targeting peptide sequence and the mTFP1 tag; AAV2 with the GRGAPGG (SEQ ID NO: 80) targeting peptide sequence and the mNG tag; and AAV2 with the DDPSARR (SEQ ID NO: 53) targeting peptide sequence and the mRuby3 tag. The viruses were directly mixed in equal volumes to achieve the following total final doses of each virus:

[0420] AAV9.RGDL mTagBFP2 6.13E12 total vg

[0421] AAV1.ERDR mTFP1 1.23E13 total vg

[0422] AAV2.GRGA mNG 8.8E12 total vg

[0423] AAV2.DDPS mRuby3 1.32E13 total vg

[0424] Brains were collected 30 days after injection for fluorescence imaging. The fluorescence signals of mTagBFP2, mTFP2, mNG, and mRuby3 were analyzed on the lateral ventricle sections ( Fig. 10A )、Fourth ventricle section ( Fig. 10B ) and meningeal sections ( Fig. 10C ) for imaging.

[0425] Additional experiments were performed by injecting AAV9-1999 into the cochlea of ​​rhesus macaques. Based on the results of cochlear transduction, animals received AAV9-1999 in the lateral ventricle. Single animals received 3E11 vg of AAV9-1999 injected directly into their round window ( Figures 11A-11C ).

[0426] In light of the present disclosure, all methods disclosed and claimed herein can be prepared and implemented without undue experimentation. Although the compositions and methods of the present invention have been described in accordance with preferred embodiments, it will be apparent to those skilled in the art that the steps or the order of steps of the methods and methods described herein may be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents both chemically and physiologically related may replace the agents described herein, while still achieving the same or similar results. All such similar substitutes 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 attached embodiments.

[0427] References

[0428] The following references are expressly incorporated herein by reference, to the extent they provide exemplary procedural or other details supplementary to the disclosure herein.

[0429] U.S. Patent No. 8,299,215

[0430] U.S. Patent No. 8,691,948

[0431] U.S. Patent Application Publication No. 2018 / 0142259

[0432] 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.

[0433] Chen et al., Overcoming Limitations Inherent in Sulfamidase to Improve Mucopolysaccharidosis IIIA Gene Therapy. Mol Ther, 2018. 26(4): pp. 1118-1126.

[0434] Deverman et al., Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol, 2016. 34(2): 204-9.

[0435] Hartz et al., Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J Vis Exp, 2018(139).

[0436] Hordeaux et al., The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL / 6J Mice. Mol Ther, 2018. 26(3): 664-668.

[0437] 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.

[0438] Keiser et al., Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy. Brain, 2015. 138(Pt 12): 3555-66.

[0439] Koerber et al., DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Ther, 2008. 16(10): p. 1703-9.

[0440] 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: 182-188.

[0441] 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.

[0442] Monteys et al., CRISPR / Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo. Mol Ther, 2017. 25(1): 12-23.

[0443] 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.

[0444] Schaffer and Maheshri. Directed evolution of AAV mutants for enhanced gene delivery. Conf Proc IEEE Eng Med Biol Soc, 2004. 5: pp. 3520-3.

[0445] Zhong et al., Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci USA, 2008. 105(22): pp. 7827-32.

Claims

1. A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide that targets a viral vector comprising the modified AAV capsid protein to a unique organ or brain structure, wherein the targeting peptide is 3 to 10 amino acids in length.

2. The modified AAV capsid protein according to claim 1, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein or a modified AAV9 capsid protein.

3. The modified AAV capsid protein of claim 1, wherein the targeting peptide comprises a sequence of up to 10 amino acids in length having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-109, 111-137 and 144.

4. The modified AAV capsid protein of claim 1, wherein the targeting peptide is 7 amino acids in length.

5. The modified AAV capsid protein according to claim 1, wherein the modified AAV capsid protein is a modified AAV1 capsid protein derived from an AAV1 capsid protein having an amino acid sequence of SEQ ID NO: 138, and wherein the targeting peptide is selected from SEQ ID NO: 2, 12, 21, 23-36 and 144 and is inserted after residue 590 of the AAV1 capsid protein.

6. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is a modified AAV2 capsid protein derived from an AAV2 capsid protein having an amino acid sequence of SEQ ID NO: 139, and wherein the targeting peptide is selected from SEQ ID NOs: 73 and 103-105 and is inserted after residue 587 of the AAV2 capsid protein.

7. The modified AAV capsid protein of claim 1, wherein the modified AAV capsid protein is a modified AAV9 capsid protein derived from an AAV9 capsid protein having an amino acid sequence of SEQ ID NO: 140, and wherein the targeting peptide is SEQ ID NO: 113, SEQ ID NO: 136 or SEQ ID NO: 137 and is inserted after residue 588 of the AAV9 capsid protein.

8. The modified AAV capsid protein according to claim 1: wherein the unique brain structure is the brainstem, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1-9; wherein the unique brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1, 3, 5, 7, 10-16, 25, 26, 32, and 144; wherein the unique brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1, 3, 4, 9, and 17-21; wherein the unique brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1, 3, 5, 12, and 21-26; wherein the unique brain structure is the ependyma, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 2-4, 7, 9, 21, 22, 27, and 28; wherein the unique brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 3, 5, 12, 14, 16, 21, 22, and 29-31; wherein the unique brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1-4, 7, and 32-34; wherein the unique brain structure is the meninges, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 3, 5, 7, 9, 12, 21, and 35-37; wherein the unique brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 2, 3, 7, 14-16, 21, 31, and 38; wherein the unique brain structure is the putamen, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 3, 4, 12, 13, 21, 30, and 39-42; wherein the unique brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 2-4, 7, 9, 21, 32, 33, and 43; wherein the unique brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 2, 3, 9, 44, and 45; wherein the unique brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 2-4, 12, 16, 30, 46, and 47; wherein the unique brain structure is the thalamus, the modified AAV capsid protein is a modified AAV1 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 1, 2, 8, 12, 21, 28, and 48-51; wherein the unique brain structure is the brainstem, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 52-60; wherein the unique brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 59 and 61-69; wherein the unique brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 56, 58, 60, and 70-75; wherein the unique brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 53, 58, 60, 62, 63, 66, and 76-79; wherein the unique brain structure is the ependyma, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 53, 60, 62, 63, 66, 74-77, and 80; wherein the unique brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 60, 75, and 81-87; wherein the unique brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 53, 55, 58, 60, 63, 76, 79, 88, and 89; wherein the unique brain structure is the meninges, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 58, 60, 66, 73, 76, 80, and 90-93; wherein the unique brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 53, 54, 57, 58, 60, 75, 79, 87, 88, and 94; wherein the unique brain structure is the putamen, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 55, 59, 60, 61, and 95-100; wherein the unique brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 53, 58-61, 63, 77, 88, 95, and 101; wherein the unique brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 52, 53, 57, 58, 75, 76, 87, 102, and 103; wherein the unique brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 57, 58, 60, 75, 79, 87, 88, 102, 104, and 105; wherein the unique brain structure is the thalamus, the modified AAV capsid protein is a modified AAV2 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 52, 55, 56, 74, 85, 88, and 106-109; wherein the unique brain structure is the brainstem, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111-117; wherein the unique brain structure is the caudate nucleus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 113, 115, 116, and 118-121; wherein the unique brain structure is the cerebellar cortex, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 119, and 122-125; wherein the unique brain structure is the cerebral cortex, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 114, 116, and 125-127; wherein the unique brain structure is the ependyma, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 118-120, and 128; wherein the unique brain structure is the globus pallidus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 112, 114, 119, 120, and 129; wherein the unique brain structure is the hippocampus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 116, 123, 125, 129 and 130; wherein the unique brain structure is the meninges, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 114, 118, 119, 122, and 131; wherein the unique brain structure is the optic nerve, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 114, 115, 117, 129 and 132; wherein the unique brain structure is the putamen, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 112, 113, 116, 123, 127, 133, and 134; wherein the unique brain structure is the spinal cord, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 113, 119, 120, 122, 123, 128, and 134; wherein the unique brain structure is the substantia nigra, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111-114, 117, and 129; wherein the unique brain structure is the subthalamic nucleus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 111, 113, 119, 120, 122, 132, and 135; or wherein the unique brain structure is the thalamus, the modified AAV capsid protein is a modified AAV9 capsid protein, and the targeting peptide is selected from any one of SEQ ID NOs: 112-114, 125, 133, 136 and 137.

9. A nucleic acid comprising a sequence encoding the modified AAV capsid protein according to any one of claims 1-8.

10. A recombinant adeno-associated virus (rAAV) comprising the modified AAV capsid protein according to any one of claims 1-8.

11. A pharmaceutical composition comprising the rAAV according to claim 10 and a pharmaceutically acceptable carrier.

12. Use of the rAAV of claim 10 in the manufacture of a medicament for delivering an agent to a unique brain structure of a subject.

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