Recombinant adeno-associated virus vector

Abstract

AAV capsid proteins containing modifications in their amino acid sequences and viral vectors containing the modified AAV capsid proteins are described, as are methods for administering the viral vectors and viral capsids to cells or to a subject in vivo.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 821,710, filed Mar. 21, 2019, which is hereby incorporated by reference in its entirety.

[0002] Description of Electronically Submitted Text File The content of the text file submitted electronically with this specification is a copy of the Sequence Listing in computer - readable format (filename: STRD_013_01WO_SeqList_ST25.txt, recording date, Mar. 20, 2020, file size, approximately 350 kilobytes), which is hereby incorporated by reference in its entirety.

[0003] This application relates to adeno - associated virus (AAV) vectors comprising recombinant capsid proteins. In some embodiments, the recombinant AAV vectors avoid neutralizing antibodies without reducing transduction efficiency.

Background Art

[0004] The first and repeated administrations of AAV vectors as vaccines and / or for gene therapy are prevented by pre - existing host - derived antibodies generated upon natural encounter with AAV or recombinant AAV vectors. Serological studies have revealed a high antibody prevalence in the human population worldwide, with approximately 67% of people having antibodies against AAV1, 72% having antibodies against AAV2, and approximately 40% having antibodies against AAV5 - AAV9.

[0005] In gene therapy, certain clinical scenarios involving gene silencing or tissue degeneration may require multiple AAV vector administrations to maintain long-term expression of the transgene. Thus, there is a need in the art for recombinant AAV vectors that avoid antibody recognition. Such vectors serve to a) expand the eligible cohort of subjects suitable for AAV-based gene therapy and b) enable multiple repeated administrations of AAV-based gene therapy vectors.

Summary of the Invention

[0006] Recombinant AAV vectors that avoid antibody recognition and / or selectively target tissues of the CNS are provided herein.

[0007] In some embodiments, an adeno-associated virus (AAV) vector comprises (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises a peptide having any one of the sequences of SEQ ID NOs: 12-20.

[0008] In some embodiments, an adeno-associated virus (AAV) vector comprises (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein has the sequence of X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 at amino acids 451-458 of the native AAV9 capsid protein sequence (SEQ ID NO: 158), and the peptide is not present in the native AAV9 capsid protein sequence.

[0009] In some embodiments, the adeno-associated virus (AAV) vector comprises (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein has X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 in the amino acids 587-594 of the native AAV9 capsid protein sequence (SEQ ID NO: 158), and the peptide is not present in the native AAV9 capsid protein sequence.

[0010] In some embodiments, the adeno-associated virus (AAV) vector comprises (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 165-187.

[0011] Also provided herein are nucleic acid sequences encoding the recombinant capsid protein and expression vectors containing the same.

[0012] Also provided are cells containing the nucleic acids, expression vectors, AAV vectors, or AAV capsids described herein.

[0013] Also provided are pharmaceutical compositions containing the nucleic acids, expression vectors, AAV vectors, AAV capsids, or cells described herein.

[0014] Also provided is a method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of the AAV vector described herein.

[0015] An in vitro method for introducing a nucleic acid molecule into a cell is also provided, the method comprising contacting the cell with an AAV vector as described herein.

[0016] An AAV vector as described herein is also provided for use as a medicament.

[0017] An AAV vector as described herein is also provided for use in a method of treatment of a subject in need thereof.

[0018] An AAV vector as described herein is also provided for use in a method of performing it in a subject in need of treatment or prevention of a CNS disease or disorder.

[0019] These and other embodiments are described in more detail below.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0022] All publications, patent applications, patents, GenBank or other accession numbers, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

[0023] The designations of amino acid positions in the AAV capsid proteins in the present disclosure and the appended claims are with respect to the VP1 capsid subunit numbering. One of ordinary skill in the art will understand that the modifications described herein may result in modifications in the VP1, VP2, and / or VP3 capsid subunits when inserted into the AAV cap gene. Alternatively, the capsid subunits may be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3).

[0024] Definitions The following terms are used in the description of this specification and the appended claims.

[0025] The singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0026] Furthermore, as used herein, the term "about", when referring to a measurable value such as the length of a polynucleotide or polypeptide sequence, dosage, time, temperature, etc., means a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0027] Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0028] It is specifically intended that, unless the context dictates otherwise, the various features described herein can be used in any combination. Further, in some embodiments, any feature or combination of features shown herein may be excluded or omitted. To further illustrate, for example, if this specification shows that a particular amino acid can be selected from A, G, I, L, and / or V, this term also means that the amino acid can be selected from any subset of these amino acids (s), e.g., A, G, I, or L; A, G, I, or V; A or G; only L, etc., as if each such partial combination were explicitly shown herein. Further, such terms also indicate that one or more of the particular amino acids may be excluded. For example, in some embodiments, the amino acid is not A, G, or I; not A; not G or V, etc., as if each such possible exclusion were explicitly shown herein.

[0029] As used herein, the terms "reduce", "reduces", "reduction", and similar terms mean a reduction of at least about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% or more.

[0030] As used herein, the terms "increase", "improve", "enhance", "enhances", "enhancement", and similar terms indicate an increase of at least about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more.

[0031] As used herein, the term "parvovirus" encompasses the family Parvoviridae, including parvoviruses and dependoviruses that replicate autonomously. Autonomous parvoviruses include members of the genera Protoparvovirus, Erythroparvovirus, Bocaparvovirus, and the subfamily Densovirus. Exemplary autonomous parvoviruses include, but are not limited to, murine minute virus, bovine parvovirus, canine parvovirus, avian parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, varicella-zoster parvovirus, B19 virus, and any other autonomous parvovirus known or later discovered. Other autonomous parvoviruses are known to those of skill in the art. See, for example, BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers; Cotmore et al. Archives of Virology DOI 10.1007 / s00705-013-1914-I).

[0032] As used herein, the term "adeno-associated virus" (AAV) includes, without limitation, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, lizard AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Some AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388, Moris et al., (2004) Virology 33-:375-383, and Table 2). Exemplary AAV capsid sequences for AAV1-9, AAVrh.10, and AAV11 are provided in SEQ ID NOs: 1-11.

[0033] As used herein, the term "chimeric AAV" refers to an AAV that includes a capsid protein having regions, domains, or individual amino acids derived from two or more different serotypes of AAV. In some embodiments, the chimeric AAV includes a capsid protein consisting of a first region derived from a first AAV serotype and a second region derived from a second AAV serotype. In some embodiments, the chimeric AAV includes a capsid protein consisting of a first region derived from a first AAV serotype, a second region derived from a second AAV serotype, and a third region derived from a third AAV serotype. In some embodiments, the chimeric AAV may include regions, domains, or individual amino acids derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. For example, the chimeric AAV may include regions, domains, and / or individual amino acids from the first and second AAV serotypes shown below (Table 1), where in the table, AAVX+Y indicates a chimeric AAV that includes sequences derived from AAVX and AAVY.

Table 1

[0034] By including individual amino acids or regions from multiple AAV serotypes in a single capsid protein, a capsid protein having multiple desired properties separately derived from multiple AAV serotypes can be obtained.

[0035] The genomic sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of the native terminal repeats (TR), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579. These disclosures are incorporated herein by reference to teach parvovirus as well as AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al., (1983) J. Virology 45:555, Chiorini et al, (1998) J Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309, Bantel-Schaal et al., (1999) J Virology 73:939, Xiao et al, (1999) J Virology 73:3994, Muramatsu et al., (1996) Virology 221:208, Shade et al, (1986) J. Virol.58:921, Gao et al, (2002) Proc. Nat. Acad. Sci. USA 99:11854, Moris et al, (2004) Virology 33:375-383, International Patent Publications WO00 / 28061, WO99 / 61601, WO98 / 11244, and U.S. Patent No. 6,156,303. These disclosures are incorporated herein by reference to teach parvovirus as well as AAV nucleic acid and amino acid sequences. See also Table 2.The capsid structures of autonomous parvoviruses and AAVs are described in more detail by BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also the descriptions of the crystal structures of AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99:10405-10), AAV9 (DiMattia et al., (2012) J. Virol. 86:6947-6958), AAV8 (Nam et al, (2007) J. Virol. 81:12260-12271), AAV6 (Ng et al., (2010) J. Virol. 84:12945-12957), AAV5 (Govindasamy et al.(2013) J. Virol. 87,11187-11199), AAV4 (Govindasamy et al.(2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al., (2010) Virology 403:26-36), BPV (Kailasan et al., (2015) J. Virol. 89:2603-2614), and CPV (Xie et al,(1996) J. Mol. Biol. 6:497-520 and Tsao et al,(1991) Science 251:1456-64).

Table 2-1

Table 2-2

[0036] The term "self-complementary AAV" or "scAAV" refers to a recombinant AAV vector that forms a dimer of inverted repeat DNA molecules that anneal spontaneously and results in faster and stronger transgene expression compared to a conventional single-stranded (ss) AAV genome. See, for example, McCarty, D.M., et al. Gene Therapy, 8, 1248-1254 (2001). Unlike conventional ssAAV, scAAV can bypass second-strand synthesis, which is the rate-limiting step of gene expression. Further, double-stranded scAAV is less prone to DNA degradation after viral transduction, thereby increasing the copy number of stable episomes. In particular, scAAV can typically carry only a genome of about 2.4 kb, which is half the size of a conventional AAV vector. In some embodiments, the AAV vectors described herein are self-complementary AAVs.

[0037] As used herein, the term "peptide" refers to a short amino acid sequence. The term peptide may be used to refer to a portion or region of an AAV capsid amino acid sequence. A peptide can be a peptide that naturally occurs within a native AAV capsid, or a peptide that does not naturally occur within a native AAV capsid. Naturally occurring AAV peptides within an AAV capsid can be replaced by non-naturally occurring peptides. For example, a modified capsid may be provided in which a non-naturally occurring peptide is substituted within the AAV capsid such that a naturally occurring peptide is replaced by the non-naturally occurring peptide.

[0038] As used herein, the term "tropism" refers to the preferential entry of a virus into a particular cell or tissue, optionally followed by, for example, expression (e.g., transcription, and optionally translation) of a sequence(s) carried by the viral genome within the cell for a recombinant virus, expression of a transgene of interest.

[0039] As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) indicate that the viral capsids or viral vectors described herein exhibit tropism for, or are transduced into, tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas). In some embodiments, systemic transduction of muscle tissues (e.g., skeletal muscle, diaphragm, and cardiac muscle) is achieved. In some embodiments, systemic transduction of skeletal muscle tissue is achieved. For example, in some embodiments, substantially all skeletal muscles throughout the body are transduced (however, the efficiency of transduction may vary depending on the type of muscle). In some embodiments, systemic transduction of limb muscles, cardiac muscle, and diaphragm muscle is achieved. Optionally, the viral capsid or viral vector is administered via a systemic route (e.g., a systemic route such as intravenous, intra-articular, or intralymphatic).

[0040] Alternatively, in some embodiments, the capsid or viral vector is delivered locally (e.g., to the footpad, intramuscularly, intradermally, subcutaneously, locally). In some embodiments, the capsid or viral vector is delivered locally to tissues of the central nervous system (CNS) such as the brain or spinal cord. In some embodiments, the capsid or viral vector is administered by intrathecal, intracerebral, or intraventricular injection.

[0041] Unless otherwise indicated, "efficient transduction" or "efficient tropism", or similar terms, can be determined by reference to a suitable control (e.g., at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or more of the control transduction or tropism, respectively). In some embodiments, a viral vector (e.g., an AVV vector) is efficiently transduced into or has efficient tropism for skeletal muscle, cardiac muscle, diaphragm muscle, pancreas (including β-islet cells), spleen, gastrointestinal tract (e.g., epithelium and / or smooth muscle), cells of the central nervous system, lung, joint cells, and / or kidney. Suitable controls depend on various factors including the desired tropism profile. For example, AAV8 and AAV9 are very efficient for transduction into skeletal muscle, cardiac muscle, and diaphragm muscle, but also have the drawback of transducing the liver with high efficiency. Thus, an AAV8 or AAV9 can be identified as a viral vector that shows efficient transduction into skeletal muscle, cardiac muscle, and / or diaphragm muscle but much lower transduction efficiency for the liver. Further, it will be appreciated that since the desired tropism profile can reflect tropism for multiple target tissues, a suitable vector may represent some trade-offs. By way of illustration, a viral vector may not be as efficient as AAV8 or AAV9 in transduction into skeletal muscle, cardiac muscle, and / or diaphragm muscle, but may still be highly desirable due to low levels of transduction into the liver.

[0042] Similarly, by reference to a suitable control, it can be determined whether a virus "does not efficiently transduce" or "does not have efficient tropism" for a target tissue or similar terms. In some embodiments, a viral vector does not efficiently transduce (i.e., does not have efficient tropism for) the liver, kidney, gonads, and / or germ cells. In some embodiments, unwanted transduction of a tissue(s) (e.g., the liver) is about 20% or less, about 10% or less, about 5% or less, about 1% or less, about 0.1% or less of the level of transduction of the desired target tissue(s) (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and / or cells of the central nervous system).

[0043] As used herein in connection with an AAV vector (or its capsid protein or peptide), the terms "selectively binds", "selective binding", and similar terms refer to the binding of an AAV vector (or its capsid protein or peptide) to a target in a manner that depends on the presence of a particular molecular structure. In some embodiments, selective binding refers primarily to the binding of AAV to a particular target without substantial or significant binding to other targets. In some embodiments, an AAV vector (or its capsid protein or peptide) specifically binds to a receptor within a cell or tissue of interest but does not exhibit substantial or significant binding to other receptors.

[0044] A "polynucleotide" is a sequence of nucleotide bases and can be an RNA, DNA, or DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides). In some embodiments, the polynucleotide is either a single-stranded or double-stranded DNA sequence.

[0045] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some of the other components of a naturally occurring organism or virus, such as cell or virus structural components, or other polypeptides or nucleic acids commonly found in association with the polynucleotide. In some embodiments, an "isolated" nucleotide is at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more enriched compared to the starting material.

[0046] Similarly, an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of a naturally occurring organism or virus, such as cellular or viral structural components, or other polypeptides or nucleic acids commonly found in association with the polypeptide. In some embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more compared to the starting material.

[0047] As used herein, to "isolate" or "purify" a viral vector (or grammatical equivalents) means that the viral vector is at least partially separated from at least some of the other components in the starting material. In some embodiments, an "isolated" or "purified" viral vector is enriched by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more compared to the starting material.

[0048] A "therapeutic" polypeptide or protein is one that can reduce, mitigate, prevent, delay, and / or stabilize symptoms resulting from the absence or deficiency of the protein in a cell or subject, and / or otherwise provide a benefit to the subject, such as an anti-cancer effect or an improvement in transplant survival rate.

[0049] The terms "treat", "treating", or "treatment of" (and their grammatical variations) mean that the severity of a subject's condition is reduced and at least partially improved or stabilized, and / or that a degree of alleviation, mitigation, reduction, or stabilization in at least one clinical symptom is achieved, and / or that there is a delay in the progression of a disease or disorder.

[0050] The terms "prevent", "preventing", and "prevention" (and their grammatical variations) refer to the prevention and / or delay of the onset of a disease, disorder, and / or clinical symptom(s) in a subject, and / or the reduction of the severity of the onset of a disease, disorder, and / or clinical symptom(s), compared to what would occur in the absence of the compositions and / or methods described herein. Prevention can be complete, e.g., a complete absence of a disease, disorder, and / or clinical symptom(s). Prevention can also be partial, such that the occurrence and / or the severity of the onset of a disease, disorder, and / or clinical symptom(s) in a subject is less than what would occur in the absence of the compositions and / or methods described herein.

[0051] As used herein, "therapeutically effective amount" refers to an amount sufficient to affect the treatment of a disease or at least one of the clinical symptoms of a disease when administered to a subject for treating the disease or the symptom thereof. A "therapeutically effective amount" can vary, for example, depending on the disease and / or symptoms of the disease, the severity of the disease and / or the symptoms of the disease or disorder, the age, weight, and / or health status of the patient being treated, and the judgment of the prescribing physician. The appropriate amount in any given case can be ascertained by one of ordinary skill in the art or may be determined by routine experimentation.

[0052] As used herein, the terms "viral vector", "vector", or "gene delivery vector" refer to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within the virion. Alternatively, in some contexts, the term "vector" can be used to refer to the vector genome / vDNA alone. In some embodiments, the vector genome (e.g., an AAV vector genome) can be included in a "cargo nucleic acid". In some embodiments, the vector genome is self-complementary (i.e., double-stranded). In some embodiments, the vector genome is not self-complementary (i.e., single-stranded).

[0053] An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that contains one or more heterologous nucleic acid sequences. rAAV vectors generally require only the inverted terminal repeats (ITRs) in cis to produce a virus. All other viral sequences are dispensable and can be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAV vector genome retains only one or two ITR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. The structural and non-structural protein coding sequences can be provided in trans (e.g., from a vector such as a plasmid or by stably integrating the sequences into the packaging cells). In some embodiments, the rAAV vector genome comprises at least one ITR sequence (e.g., an AAV ITR sequence), optionally two ITRs (e.g., two AAV ITRs), which are typically at the 5' and 3' termini of the vector genome (i.e., 5' ITR and 3' ITR), adjacent to but not necessarily contiguous with the heterologous nucleic acid.

[0054] The term "inverted terminal repeat" or "ITR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates desired functions such as replication, viral packaging, integration, and / or proviral rescue). The ITR can be an AAV ITR or a non-AAV ITR. For example, non-AAV ITR sequences such as those of other parvoviruses (e.g., canine parvovirus (CPV), murine parvovirus (MVM), human parvovirus B-19) or any other suitable viral sequence (e.g., an SV40 hairpin that functions as an SV40 origin of replication) can be used as an ITR and further modified by cleavage, substitution, deletion, insertion, and / or addition. Furthermore, the ITR can be partially or fully synthetic, such as the "double D sequence" described in U.S. Patent No. 5,478,745.

[0055] The "AAV inverted terminal repeat" or "AAV ITR" can be from any AAV including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or any other AAV now known or later discovered (see, e.g., Table 2). The AAV inverted terminal repeat need not have a native terminal repeat sequence (e.g., the native AAV ITR sequence can be modified by insertions, deletions, truncations, and / or missense mutations) so long as the terminal repeat mediates the desired function, such as replication, viral packaging, integration, and / or proviral rescue.

[0056] The viral vectors described herein can further be "targeted" viral vectors (e.g., having a directed tropism) and / or "hybrid" viral vectors (i.e., the viral ITR and viral capsid are from different viruses), as described in International Patent Publication WO00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619. In some embodiments, the viral vector targets cells and / or tissues of the CNS.

[0057] The viral vectors described herein can further be double-stranded viral particles, as described in International Patent Publication No. WO01 / 92551, the disclosure of which is incorporated herein by reference in its entirety. Thus, in some embodiments, a double-stranded (duplex) genome can be packaged into the viral capsids described herein. Further, the viral capsid or genomic element can contain other modifications including insertions, deletions, and / or substitutions.

[0058] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Naturally occurring left-handed (L-) amino acids are shown in Table 3.

Table 3

[0059] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 4), and / or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). Methods for chemically modifying amino acids are known in the art (see, e.g., Greg T. Hermanson, Bioconjugate Techniques, 1 st edition, Academic Press, 1996).

Table 4-1

Table 4-2

[0060] Furthermore, non-naturally occurring amino acids can be "unnatural" amino acids (as described in Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)). These unnatural amino acids can be advantageously used to chemically link the molecule of interest to the AAV capsid protein.

[0061] Modified AAV capsid protein and AAV vector containing the same AAV vector Also provided herein is an adeno-associated virus (AAV) vector comprising (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein. In some embodiments, the recombinant capsid protein (VP1, VP2, and / or VP3) may comprise peptides of amino acid sequences that do not occur in any native AAV capsid sequence. The inventors have shown that capsid proteins comprising the peptides described herein can confer one or more desired properties to viral vectors, including but not limited to the ability to evade neutralizing antibodies. Accordingly, the AAV vectors described herein address limitations associated with conventional AAV vectors.

[0062] Accordingly, in some embodiments, the present disclosure provides an adeno-associated virus (AAV) vector comprising (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises a peptide having any one of the sequences of SEQ ID NOs: 12-20. In some embodiments, the cargo nucleic acid comprises 5' and 3' AAV inverted terminal repeats. In some embodiments, the cargo nucleic acid comprises a transgene. In some embodiments, the cargo nucleic acid is double-stranded. In some embodiments, the cargo nucleic acid is single-stranded. In some embodiments, the transgene encodes a therapeutic protein or RNA. In some embodiments, the recombinant capsid protein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the native sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV capsid. In some embodiments, the recombinant capsid protein has at least 90% sequence identity to the native sequence of AAV9 capsid.

[0063] In some embodiments, the peptide is located at amino acid positions corresponding to amino acids 451-458 of the native AAV9 capsid, or equivalent amino acid residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV, and the peptide is selected from any one of SEQ ID NOs: 12-18. In some embodiments, the peptide is located at amino acid positions corresponding to amino acids 587-594 of the native AAV9 capsid, or equivalent amino acid residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV, and the peptide is selected from SEQ ID NO: 19 or 20.

[0064] In some embodiments, the recombinant capsid protein comprises a) a first peptide having a sequence of any one of SEQ ID NOs: 12-18 and b) a second peptide having a sequence of any one of SEQ ID NOs: 19-20. In some embodiments, the first peptide is at amino acid positions 451-458 and the second peptide is at amino acid 587-594, with amino acid numbering based on the native AAV9 capsid, or equivalent amino acid residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV.

[0065] In some embodiments, the peptide inhibits the binding of at least one antibody to the capsid protein. In some embodiments, the peptide inhibits the neutralization of the infectivity of the AAV vector by the antibody.

[0066] In some embodiments, the peptide selectively binds to a receptor expressed on the cell surface of the central nervous system (CNS). In some embodiments, the cell is in the premotor cortex, thalamus, cerebellar cortex, dentate nucleus, spinal cord, or dorsal root ganglion. In some embodiments, the peptide selectively binds to a receptor expressed on the cell surface of the heart.

[0067] In some embodiments, the adeno-associated virus (AAV) vector comprises (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence of X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 (SEQ ID NO: 158) that is not present in the native AAV9 capsid protein sequence. In some embodiments, X 1 is not I, X 2 is not N, X 3 is not G, X 4 is not S, X 5 is not G, X 6 is not Q, X 7 is not N, and / or X 8 is not Q. In some embodiments, X 1 is S, F, Q, G, K, or R. In some embodiments, X 2 is C, G, R, D, T, or Q. In some embodiments, X 3 is Q, V, G, Y, R, F, or D. In some embodiments, X 4 is P, Q, A, or R. In some embodiments, X 5 is T, N, A, P, or I. In some embodiments, X 6 is V, Q, A, or I. In some embodiments, X 7 is M, P, R, Q, or N. In some embodiments, X8 is N, L, F, E, H, or A. In some embodiments, X 1 is S, X 2 is C, X 3 is Q, X 4 is P, X 5 is T, X 6 is V, X 7 is M, X 8 is N. In some embodiments, X 1 is F, X 2 is G, X 3 is V, X 4 is P, X 5 is N, X 6 is Q, X 7 is P, X 8 is L. In some embodiments, X 1 is Q, X 2 is R, X 3 is G, X 4 is Q, X 5 is A, X 6 is A, X 7 is P, X 8 is F. In some embodiments, X 1 is G, X 2 is D, X 3 is Y, X 4 is A, X 5 is P, X 6 is I, X 7 is R, X 8 is E. In some embodiments, X 1 is K, X 2 is T, X 3 is R, X 4 is R, X 5 is I, X 6 is V, X 7 is Q, X 8 is H. In some embodiments, X 1 is F, X 2 is G, X 3 is F, X 4 is P, X 5is N, X 6 is Q, X 7 is P, X 8 is L. In some embodiments, X 1 is R, X 2 is Q, X 3 is D, X 4 is Q, X 5 is P, X 6 is I, X 7 is N, X 8 is A.

[0068] In some embodiments, an adeno-associated virus (AAV) vector comprises (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises a peptide having the sequence (SEQ ID NO: 158) of amino acids 587-594 of the native AAV9 capsid protein sequence, and the peptide is not present in the native AAV9 capsid protein sequence. In some embodiments, X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 In some embodiments, X 1 is not A, X 2 is not Q, X 3 is not A, X 4 is not Q, X 5 is not A, X 6 is not Q, X 7 is not T, and / or X 8 is not G. In some embodiments, X 1 is S. In some embodiments, X 2 is K or T. In some embodiments, X 3 is V. In some embodiments, X 4 is E or D. In some embodiments, X 5 is S. In some embodiments, X 6 is W or I. In some embodiments, X 7is T or A. In some embodiments, X 8 is E or I. In some embodiments, X 1 is S, and X 2 is K, and X 3 is V, and X 4 is E, and X 5 is S, and X 6 is W, and X 7 is T, and X 8 is E. In some embodiments, X 1 is S, and X 2 is T, and X 3 is V, and X 4 is D, and X 5 is S, and X 6 is I, and X 7 is A, and X 8 is I.

[0069] In some embodiments, an adeno-associated virus (AAV) vector comprises (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 165-187. In some embodiments, the capsid protein comprises an amino acid sequence of any one of SEQ ID NOs: 165-187. In some embodiments, the capsid protein comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the capsid protein comprises the amino acid sequence of SEQ ID NO: 180.

[0070] In some embodiments, the AAV vector selectively delivers the cargo nucleic acid to cells or tissues of the central nervous system. In some embodiments, the tissue of the central nervous system is the premotor cortex, thalamus, cerebellar cortex, dentate nucleus, spinal cord, or dorsal root ganglion. In some embodiments, the AAV vector delivers the cargo nucleic acid to the brain but does not deliver the AAV vector to the heart. In some embodiments, the AAV vector delivers the cargo nucleic acid to the brain and the heart. In some embodiments, the delivery of the cargo nucleic acid is greater to the brain than to the heart. In some embodiments, the delivery of the cargo nucleic acid is approximately equal in the brain and the heart.

[0071] AAV capsid protein In some embodiments, the disclosure provides an adeno-associated virus (AAV) capsid protein comprising one or more amino acid modifications (e.g., substitutions and / or deletions) compared to a native AAV capsid protein, wherein the one or more modifications modify one or more antigenic sites on the AAV capsid protein. Modification of the one or more antigenic sites results in inhibition of binding of one or more antibodies to the one or more antigenic sites and / or inhibition of neutralization of the infectivity of virus particles comprising the AAV capsid protein. The one or more amino acid modifications (e.g., substitutions and / or deletions) can be within one or more antigenic footprints identified by peptide epitope mapping and / or cryo-electron microscopy studies of AAV-antibody complexes comprising the AAV capsid protein. In some embodiments, the one or more antigenic sites are general antigenic motifs or CAMs as described in WO2017 / 058892, which is incorporated herein by reference in its entirety. In some embodiments, the antigenic site is in a variable region (VR) of the AAV capsid protein, such as VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, VR-IX. In some embodiments, the one or more antigenic sites are within the HI loop of the AAV capsid protein.

[0072] In some embodiments, the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAV10, AAV11, AAV12, AAVrh32.22, bovine AAV, or avian AAV capsid protein comprises an amino acid modification (e.g., substitution or deletion) in one or more of the regions specified in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0073] In some embodiments, the amino acid substitutions replace any eight amino acids in an AAV capsid protein from any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAV10, AAV11, AAV12, AAVrh32.22, bovine AAV, or avian AAV. For example, the amino acid substitutions can be the following amino acids (VP1 numbering) in any of the AAV serotypes listed above: 355-362, 363-370, 371-378, 379-386, 387-394, 395-402, 403-410, 411-418, 419-426, 427-434, 435-442, 443-450, 451-458, 459-466, 467-474, 475-482, 483-490, 491-498, 499-506, 507-514, 515-522, 523-530, 531-538, 539-546, 547-554, 555-562, 563-570, 571-578, 579-586, 587-594, 595-602, 603-610, 611-618, 619-626, 627-634, 635-642, 643-650, 651-658, 659-666, 667-674, 675-682, 683-690, 691-698, 699-706, 707-714, 715-722.

[0074] In some embodiments, the amino acid substitution is selected from any one of SEQ ID NOs: 19-20. In some embodiments, the amino acid substitution has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with any one of SEQ ID NOs: 12-18. In some embodiments, the substitution is at the amino acid corresponding to amino acids 587-594 of the wild-type AAV9 capsid. In some embodiments, the substitution is at the amino acid corresponding to amino acids 587-594 of the wild-type AAV1 capsid. In some embodiments, the substitution is at the amino acid corresponding to amino acids 587-594 of the wild-type AAV6 capsid. In some embodiments, the substitution is at the amino acid corresponding to amino acids 589-596 of the wild-type AAV8 capsid. In some embodiments, the substitution is at the amino acid corresponding to amino acids 587-594 of the wild-type AAVrh8 capsid. In some embodiments, the substitution is at the amino acid corresponding to amino acids 589-596 of the wild-type AAVrh10 capsid.

[0075] In some embodiments, the amino acid substitution is selected from any one of SEQ ID NOs: 18-20. In some embodiments, the amino acid substitution has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with any one of SEQ ID NOs: 18-20. In some embodiments, the substitution is at the amino acid corresponding to amino acids 451-458 of the wild-type AAV9 capsid.

[0076] In some embodiments, the amino acid deletion includes a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids compared to the wild-type capsid.

[0077] In some embodiments, the AAV capsid comprises one or more amino acid substitutions and one or more amino acid deletions. In some embodiments, the capsid comprises at least one amino acid substitution and at least one amino acid deletion. In some embodiments, the capsid comprises at least one amino acid substitution and at least one amino acid deletion, and the at least one amino acid substitution and the at least one amino acid deletion are directly adjacent to each other in the capsid amino acid sequence.

[0078] In some embodiments, the capsid protein is modified to produce an AAV capsid that, when present within an AAV viral particle or AAV viral vector, has a phenotype that selectively targets the CNS (e.g., the brain, spinal cord). In some embodiments, the capsid protein is modified to produce an AAV capsid that, when present within an AAV viral particle or AAV viral vector, has a phenotype that avoids neutralizing antibodies. The AAV viral particle or vector can also have a phenotype of enhanced or maintained transduction efficiency in addition to a phenotype that avoids neutralizing antibodies and / or targets the CNS.

[0079] In some embodiments, one or more of the substitutions can introduce one or more sequences from the capsid protein of a first AAV serotype into the capsid protein of a second AAV serotype that is different from the first AAV serotype.

[0080] The base AAV capsid protein to which the modification is added can be a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh.32.33, AAVrh74, bovine AAV, avian AAV, or any other AAV that is currently known or later identified. In some embodiments, the base AAV capsid protein is of the AAV9 serotype. In some embodiments, the base AAV capsid protein is chimeric. In some embodiments, the base AAV capsid protein is an AAV8 / 9 chimera.

[0081] Some examples of modified AAV capsid proteins are provided herein. In the following examples, the capsid protein can include the specific substitutions described, and in some embodiments, can include fewer or more substitutions than those described. As used herein, "substitution" can refer to a single amino acid substitution, or substitution of two or more adjacent amino acids. For example, in some embodiments, the capsid protein can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. single amino acid substitutions. In some embodiments, the capsid protein can include one or more substitutions of multiple adjacent amino acids, such as one or more substitutions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 adjacent amino acids.

[0082] Furthermore, in some embodiments described herein where an amino acid residue is substituted by any amino acid residue other than the amino acid residue present in the wild-type or native amino acid sequence, any other amino acid residue can be any natural or non-natural amino acid residue known in the art (see, for example, Tables 2 and 3). In some embodiments, the substitution may be a conservative substitution, and in some embodiments, the substitution may be a non-conservative substitution. In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions, each of which is individually selected from SEQ ID NOs: 12-18 as shown in Table 6.1. [Table 6]

[0083] In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions, each of which is selected from SEQ ID NOs: 19-20 as shown in Table 6.2. [Table 7]

[0084] In some embodiments, the AAV capsid protein can comprise a first substitution selected from the sequences listed in Table 6.1 and a second substitution selected from the sequences listed in Table 6.2. In some embodiments, the AAV capsid protein can comprise a first substitution and a second substitution as shown in Tables 6.3 and 6.4. [Table 8] [Table 9]

[0085] In some embodiments, the AAV capsid protein comprises amino acid modifications (e.g., substitutions and / or deletions) that modify one or more surface-exposed regions on the AAV capsid protein, such as antigenic regions.

[0086] In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions, and at least one of the amino acid substitutions comprises one of SEQ ID NOs: 19-20. In some embodiments, the substitution replaces the amino acid corresponding to amino acids 587-594 of the wild-type AAV9 capsid.

[0087] In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions, and at least one of the amino acid substitutions comprises one of SEQ ID NOs: 12-18. In some embodiments, the substitution replaces the amino acid corresponding to amino acids 451-458 of the wild-type AAV9 capsid.

[0088] In some embodiments, the AAV capsid protein comprises substitutions that include the sequence of eight amino acids (X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 )(SEQ ID NO: 158) that do not occur in the native capsid protein sequence. In some embodiments, X 1 is not I, X 2 is not N, X 3 is not G, X 4 is not S, X 5 is not G, X 6 is not Q, X 7 is not N, and / or X 8 is not Q. In some embodiments, X 1 is S, F, Q, G, K, or R. In some embodiments, X 2 is C, G, R, D, T, or Q. In some embodiments, X 3 is Q, V, G, Y, R, F, or D. In some embodiments, X 4 is P, Q, A, or R. In some embodiments, X 5 is T, N, A, P, or I. In some embodiments, X6 is V, Q, A, or I. In some embodiments, X 7 is M, P, R, Q, or N. In some embodiments, X 8 is N, L, F, E, H, or A. In some embodiments, X 1 is S, and X 2 is C, and X 3 is Q, and X 4 is P, and X 5 is T, and X 6 is V, and X 7 is M, and X 8 is N. In some embodiments, X 1 is F, and X 2 is G, and X 3 is V, and X 4 is P, and X 5 is N, and X 6 is Q, and X 7 is P, and X 8 is L. In some embodiments, X 1 is Q, and X 2 is R, and X 3 is G, and X 4 is Q, and X 5 is A, and X 6 is A, and X 7 is P, and X 8 is F. In some embodiments, X 1 is G, and X 2 is D, and X 3 is Y, and X 4 is A, and X 5 is P, and X 6 is I, and X 7 is R, and X 8 is E. In some embodiments, X 1 is K, and X 2 is T, and X 3 is R, and X 4 is R, and X 5 is I, and X 6 is V, and X 7 is Q, and X 8 is H. In some embodiments, X 1is F, X 2 is G, X 3 is F, X 4 is P, X 5 is N, X 6 is Q, X 7 is P, X 8 is L. In some embodiments, X 1 is R, X 2 is Q, X 3 is D, X 4 is Q, X 5 is P, X 6 is I, X 7 is N, X 8 is A.

[0089] In some embodiments, X 1 is not A, X 2 is not Q, X 3 is not A, X 4 is not Q, X 5 is not A, X 6 is not Q, X 7 is not T, and / or X 8 is not G. In some embodiments, X 1 is S. In some embodiments, X 2 is K or T. In some embodiments, X 3 is V. In some embodiments, X 4 is E or D. In some embodiments, X 5 is S. In some embodiments, X 6 is W or I. In some embodiments, X 7 is T or A. In some embodiments, X 8 is E or I. In some embodiments, X 1 is S, X 2 is K, X 3 is V, X 4 is E, X 5 is S, X 6 is W, X 7 is T, X 8is E. In some embodiments, X 1 is S, and X 2 is T, and X 3 is V, and X 4 is D, and X 5 is S, and X 6 is I, and X 7 is A, and X 8 is I.

[0090] In some embodiments, the AAV capsid protein comprises one or more amino acid deletions, and the amino acid deletions comprise deletions of at least 6 or at least 8 amino acids compared to the wild-type AAV capsid. In some embodiments, the AAV capsid protein comprises a deletion of 8 consecutive amino acids compared to the native capsid protein sequence. In some embodiments, the AAV capsid protein comprises a deletion of 6 consecutive amino acids compared to the native capsid protein sequence.

[0091] In some embodiments, the AAV capsid protein comprises the sequence LSKTQTLK (SEQ ID NO: 1374) or the sequence LSKTDPQTLK (SEQ ID NO: 1375). In some embodiments, the AAV capsid protein comprising SEQ ID NO: 1374 or 1375 is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV.

[0092] In some embodiments, the AAV capsid protein comprises a first substitution comprising a sequence selected from SEQ ID NOs: 12-18 and a second substitution comprising a sequence selected from SEQ ID NOs: 19-20.

[0093] In some embodiments, the AAV capsid protein comprises amino acid deletions and substitutions, and the substitutions comprise sequences selected from SEQ ID NOs: 12-20.

[0094] In some embodiments, the recombinant capsid protein has a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9 (AAV9), and includes one or more of the following amino acid substitutions: I451S, I451F, I451Q, I451G, I451K, I451R, N452C, N452G, N452R, N452D, N452T, N452Q, G453Q, G453V, G453Y, G453R, G453F, G453D, S454P, S454Q, S454A, S454R, G455T, G455N, G455A, G455P, G455I, Q456V, Q456A, Q456I, N457M, N457P, N457R, N457Q, Q458N, Q458L, Q458F, Q458E, Q458H, Q458A, A587S, Q588K, Q588T, A589V, Q590E, Q590D, A591S, Q592W, Q592I, T593A, G594E, G594I.

[0095] Any of the AAV capsids described herein may further include a modification (e.g., a substitution or deletion) within the HI loop. The HI loop is a prominent domain on the AAV capsid surface that extends from each VP subunit that overlaps adjacent pentameric viral proteins (VPs) between β-strands βH and βI. In some embodiments, the AAV capsid includes 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions within the HI loop. In some embodiments, the AAV capsid includes one or more of the following substitutions within the HI loop: P661R, T662S, Q666G, S667D, numbered corresponding to the wild-type AAV8 capsid (SEQ ID NO: 8). In some embodiments, the AAV capsid includes one or more of the following substitutions within the HI loop: P659R, T660S, A661T, K664G, numbered corresponding to the wild-type AAV9 capsid (SEQ ID NO: 9).

[0096] In some embodiments, the AAV capsid protein comprises one, two, three, or four amino acid substitutions, each substitution modifying a different antigenic site on the AAV capsid protein, and at least one of the amino acid substitutions modifies the HI loop of the capsid protein.

[0097] In some embodiments, the AAV capsid protein comprises first, second, third, and fourth amino acid substitutions. In some embodiments, at least one of the substitutions modifies the HI loop of the capsid protein. In some embodiments, the AAV capsid comprises one or more of the following substitutions within the HI loop: P661R, T662S, Q666G, S667D (numbering corresponds to wild-type AAV8 capsid (SEQ ID NO: 8)), or P659R, T660S, A661T, K664G (numbering corresponds to wild-type AAV9 capsid (SEQ ID NO: 9)). In some embodiments, the AAV capsid protein comprises an amino acid sequence of any one of SEQ ID NOs: 185-187. In some embodiments, the AAV capsid protein comprises an amino acid sequence that shares at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 165-187.

[0098] Also provided herein is a nucleotide sequence encoding one or more of the AAV capsid proteins described herein, or an expression vector comprising the same. The nucleotide sequence can be a DNA sequence or an RNA sequence. In some embodiments, a cell comprises one or more of the nucleotide sequences or expression vectors described herein.

[0099] In some embodiments, the AAV capsid comprises an AAV capsid protein described herein. Further provided herein are viral vectors comprising the AAV capsid, and compositions comprising an AAV capsid protein, an AAV capsid, and / or a viral vector in a pharmaceutically acceptable carrier.

[0100] In some embodiments, modification of one or more antigenic sites results in inhibition of binding of the antibody to one or more antigenic sites. In some embodiments, modification of one or more antigenic sites results in inhibition of neutralization of the infectivity of virus particles comprising the AAV capsid protein.

[0101] As described herein, the nucleic acid and amino acid sequences of the capsid proteins from some AAVs are known in the art. Accordingly, the "corresponding" amino acids at the amino acid positions of the native AAV capsid protein can be readily determined for any other AAV (e.g., by using sequence alignment).

[0102] The modified capsid protein can be produced by modifying the capsid protein of any AAV that is currently known or later discovered. Further, the base AAV capsid protein to be modified can be a naturally occurring AAV capsid protein (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or any of the AAVs shown in Table 2), but is not so limited. Those skilled in the art will understand that various manipulations of AAV capsid proteins are known in the art, and the present disclosure is not limited to the modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may already have modifications compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV that is currently known or later discovered). In some embodiments, the capsid protein can be a chimeric capsid protein. In some embodiments, the capsid protein can be an engineered AAV such as AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, etc.

[0103] Thus, in some embodiments, the AAV capsid protein to be modified can be derived from a naturally occurring AAV, but further comprises one or more foreign sequences (e.g., exogenous to the native virus) that are inserted into and / or substituted for and / or modified by deletion of one or more amino acids of the capsid protein.

[0104] Accordingly, when referring herein to a particular AAV capsid protein (e.g., an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a capsid protein from any of the AAVs shown in Table 2, etc.), it is intended to encompass the native capsid protein, as well as capsid proteins having modifications other than those described herein. Such modifications include substitutions, insertions, and / or deletions. In some embodiments, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids inserted (excluding the insertions described herein) compared to the native AAV capsid protein sequence. In some embodiments, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acid substitutions (excluding the amino acid substitutions described herein) compared to the native AAV capsid protein sequence, and in some embodiments, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acid deletions compared to the native AAV capsid protein sequence.

[0105] In some embodiments, the AAV capsid protein has an amino acid sequence that is at least about 90%, about 95%, about 97%, about 98%, or about 99% similar or identical to the native AAV capsid protein sequence.

[0106] Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined using standard techniques including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48, 443 (1970), the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the best fit sequence program described by Devereux et al, Nucl. Acid Res. 12, 387-395 (1984), or by inspection.

[0107] Another preferred algorithm is the BLAST algorithm described in Altschul et al., J Mol. Biol. 215, 403-410, (1990), and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http: / / blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters and is set to default values optionally. The parameters are dynamic values and are established by the program itself depending on the composition of the particular sequences and the composition of the particular database against which the sequences of interest are being searched. However, the values may be adjusted to increase sensitivity.

[0108] Further, a useful additional algorithm is gap BLAST, as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402.

[0109] In some embodiments, the viral capsid comprises a modified AAV capsid protein as described herein. In some embodiments, the viral capsid is a parvovirus capsid, which may further be an autonomous parvovirus capsid or a dependovirus capsid. Optionally, the viral capsid is an AAV capsid. In some embodiments, the AAV capsid is AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV capsid, avian AAV capsid, or any other AAV currently known or later identified. A non-limiting list of AAV serotypes is shown in Table 2. The AAV capsid can be any of the AAV serotypes listed in Table 2 or can be derived from any of the foregoing by one or more insertions, substitutions, and / or deletions. Molecules that can be packaged by the modified viral capsid and transferred to cells include cargo nucleic acids (e.g., heterologous DNA or RNA), polypeptides, small organic molecules, metals, or combinations thereof.

[0110] Heterologous molecules are defined as those not naturally found in AAV infection, e.g., those not encoded by the wild-type AAV genome. Further, therapeutically useful molecules can associate with the outside of the chimeric viral capsid for transfer of the molecule to the host target cell. Such associating molecules can include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and / or polypeptides. In some embodiments, the therapeutically useful molecule is covalently attached (i.e., conjugated or chemically bonded) to the capsid protein. Methods of covalently attaching molecules are known to those of skill in the art.

[0111] The modified viral capsid is also used for the production of antibodies against the novel capsid structure. As a further alternative, an exogenous amino acid sequence can be inserted into the modified viral capsid for antigen presentation to cells, for example, to administer to a subject to generate an immune response against the exogenous amino acid sequence.

[0112] In some embodiments, the viral capsid can be administered to block a particular cell site before and / or simultaneously with (e.g., within minutes or hours of each other) the administration of a viral vector that delivers a nucleic acid encoding a polypeptide or functional RNA of interest. For example, the capsids of the present invention can be delivered to block cell receptors on hepatocytes, and the delivery vector can be administered later or simultaneously, which can reduce hepatocyte transduction and enhance transduction of other targets (e.g., skeletal muscle, cardiac muscle, and / or diaphragmatic muscle).

[0113] According to some embodiments, the modified viral capsid can be administered to a subject before and / or simultaneously with the modified viral vector described herein. Further, the present disclosure provides compositions and pharmaceutical formulations comprising the modified viral capsids of the present invention, and optionally, the compositions also comprise the modified viral vectors described herein.

[0114] In some embodiments, a nucleic acid (optionally, an isolated nucleic acid) encodes the modified viral capsid and capsid protein described herein. Further provided are vectors comprising the nucleic acid, and cells (in vivo or in culture) comprising the nucleic acid and / or vector described herein. As an example, a viral vector comprises (a) a modified AAV capsid described herein and (b) a nucleic acid comprising at least one terminal repeat sequence, and the nucleic acid is encapsidated by the AAV capsid.

[0115] Other suitable vectors include, but are not limited to, viral vectors (e.g., adenovirus, AAV, herpesvirus, vaccinia, poxvirus, baculovirus, lentivirus, coronavirus, etc.), plasmids, phages, YACs, BACs, etc. Such nucleic acids, vectors, and cells can be used, for example, as reagents (e.g., helper packaging constructs or packaging cells) for the production of the modified viral capsids or viral vectors described herein.

[0116] The viral capsids described herein can be produced using any method known in the art, for example, by using a baculovirus system (Brown et al., (1994) Virology 198:477-488).

[0117] The modifications to the AAV capsid proteins described herein are "selective" modifications. This approach is in contrast to previous studies on full subunit or global swaps between AAV serotypes (see, for example, International Patent Publication No. WO00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774). In some embodiments, "selective" modifications result in the insertion, and / or substitution, and / or deletion of about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer adjacent amino acids.

[0118] The modified capsid proteins and capsids described herein can further include any other modifications currently known or later identified. For example, AAV capsid proteins and viral capsids can be chimeric in that they can include all or part of a capsid subunit from another virus, optionally another parvovirus or AAV, as described in International Patent Publication No. WO00 / 28004.

[0119] In some embodiments, the viral capsid can be a targeted viral capsid that includes a targeting sequence (e.g., substituted or inserted into the viral capsid) that directs the viral capsid to interact with cell surface molecules present on the desired target tissue(s) (see, e.g., International Patent Publication No. WO00 / 28004, and Hauck et al., (2003) J. Virology 77:2768-2774), Shi et al., Human Gene Therapy 17:353-361 (2006) [describing the insertion of the integrin receptor-binding motif RGD at positions 520 and / or 584 of the AAV capsid subunit], and U.S. Patent No. 7,314,912 [describing the insertion of a PI peptide containing an RGD motif following amino acids 447, 534, 573, and 587 of the AAV2 capsid subunit]). Other positions within the AAV capsid subunit that tolerate insertion are known in the art (e.g., positions 449 and 588 as described by Grifman et al., Molecular Therapy 3:964-975 (2001)).

[0120] For example, the viral capsids described herein may have a relatively inefficient tropism for certain target tissues of interest (e.g., liver, skeletal muscle, myocardium, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lung). Target sequences can be advantageously incorporated into these low transduction vectors, thereby conferring the desired tropism on the viral capsid and, optionally, a selective tropism for specific tissue(s). AAV capsid proteins, capsids, and vectors containing the target sequences are described, for example, in International Patent Publication No. WO00 / 28004. As another example, one or more non-naturally occurring amino acids, such as those described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006), can be incorporated at orthogonal sites into the AAV capsid subunits described herein as a means of redirecting the low transduction vector to the desired target tissue(s). These non-natural amino acids include, but are not limited to, glycans (mannose - dendritic cell targeting), RGD, bombesin, or neuropeptides for targeted delivery to specific cancer cell types, RNA aptamers or peptides selected from phage display that target specific cell surface receptors such as growth factor receptors, integrins, etc., and can be advantageously used to chemically link the molecule of interest to the AAV capsid protein.

[0121] In some embodiments, the target sequence may be a viral capsid sequence (e.g., an autonomous parvovirus capsid sequence, an AAV capsid sequence, or any other viral capsid sequence) that directs infection to specific cell type(s).

[0122] As another non-limiting example, a heparin or heparan sulfate binding domain (e.g., the respiratory syncytial virus heparin binding domain) can be inserted or substituted into a capsid subunit that does not typically bind to an HS receptor (e.g., AAV4, AAV5) to confer heparin and / or heparan sulfate binding on the resulting variant.

[0123] B19 uses globoside as its receptor and infects primary erythroid progenitor cells (Brown et al., (1993) Science 262:114). The structure of B19 has been determined at 8 Å resolution (Agbandje-McKenna et al., (1994) Virology 203:106). The region of the B19 capsid that binds to globoside has been mapped to amino acids 399 - 406 (Chapman et al., (1993) Virology 194:419), and to a loop-out region between β-barrel structures E and F (Chipman et al., (1996) Proc. Nat. Acad. Sci. USA 93:7502). Thus, the globoside receptor-binding domain of the B19 capsid can be replaced with an AAV capsid protein to target the viral capsid or a viral vector containing it to erythroid cells.

[0124] In some embodiments, the exogenous target sequence can be any amino acid sequence encoding a peptide that modifies the tropism of a viral capsid or viral vector containing a modified AAV capsid protein. In some embodiments, the target peptide or protein can be naturally occurring or, alternatively, can be fully or partially synthetic. Exemplary target sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as the ROD peptide sequence, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors I and II, etc.), cytokines, melanocyte-stimulating hormones (e.g., a, β, or γ), neuropeptides, and endorphins, etc., as well as fragments thereof that retain the ability to target cells to their cognate receptors. Other exemplary peptides and proteins include, as described above, substance P, keratinocyte growth factor, neuropeptide Y, gastrin-releasing peptide, interleukin 2, chicken egg white lysozyme, erythropoietin, gonadoliberin, cortistatin, β-endorphin, leu-enkephalin, rimorphin, alpha-neoenkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof. As yet a further alternative, the binding domain from a toxin (e.g., a snake toxin such as tetanus toxin or alpha-bungarotoxin) can be substituted for the capsid protein as the target sequence. In some embodiments, the AAV capsid protein can be modified by substitution of a "non-classical" import / export signal peptide (e.g., fibroblast growth factors -1 and -2, interleukin 1, HIV-1 Tat protein, herpesvirus VP22 protein, etc.) as described by Cleves (Current Biology 7:R318 (1997)). Also included are peptide motifs that direct uptake by specific cells, such as the FVFLP (SEQ ID NO: 22) peptide motif that causes uptake by hepatocytes.

[0125] Using phage display techniques, as well as other techniques known in the art, peptides that recognize any desired cell type can be identified.

[0126] The target sequence can encode any peptide that targets a cell surface binding site, including a receptor (e.g., a protein, carbohydrate, glycoprotein, or proteoglycan). Examples of cell surface binding sites include heparan sulfate, chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties found in mucins, glycoproteins, and carbohydrate constituents (including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, etc.) found in gangliosides, MHC1 glycoproteins, and membrane glycoproteins, but are not limited thereto.

[0127] In some embodiments, the heparan sulfate (HS) or heparin binding domain is substituted into the viral capsid (e.g., into an AAV capsid that would not otherwise bind to HS or heparin). It is known in the art that HS / heparin binding is mediated by a "basic patch" rich in arginine and / or lysine. In some embodiments, a sequence following the motif BXXB (SEQ ID NO: 23), where "B" is a basic residue and X is a neutral and / or hydrophobic residue, can be used. As a non-limiting example, BXXB can be RGNR (SEQ ID NO: 24). As another non-limiting example, BXXB is substituted at amino acid positions 262-265 of the native AAV2 capsid protein or at the corresponding position(s) of the capsid protein of another AAV serotype.

[0128] Table 7 shows other non-limiting examples of suitable target sequences.

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

[0129] In some embodiments, the targeting sequence may be a peptide that can be used for chemical conjugation to another molecule that targets entry into cells (e.g., may contain arginine and / or lysine residues that can be chemically conjugated via their R groups).

[0130] In some embodiments, the AAV capsid protein or viral capsid may contain mutations described in WO2006 / 066066. For example, the capsid protein may contain selective amino acid substitutions at amino acid positions 263, 705, 708, and / or 716 of the native AAV2 capsid protein, or corresponding changes (s) in the capsid protein from another AAV serotype.

[0131] Additionally or alternatively, in some embodiments, the capsid protein, viral capsid, or vector contains an insertion of a selective amino acid immediately following amino acid position 264 of the AAV2 capsid protein, or a corresponding change in the capsid protein from another AAV. "Immediately following amino acid position X" is intended to mean that the insertion follows immediately after the indicated amino acid position (e.g., "after amino acid position 264" indicates an insertion at position 265, or a larger insertion, e.g., positions 265-268, etc.).

[0132] Furthermore, in some embodiments, the capsid protein, viral capsid, or vector may include amino acid modifications as described in PCT Publication No. WO2010 / 093784 (e.g., 2i8) and / or PCT Publication No. WO2014 / 144229 (e.g., dual glycan).

[0133] In some embodiments, the capsid protein, viral capsid, or vector may have transduction efficiency equal to, or enhanced relative to, the transduction efficiency of the AAV serotype from which the capsid protein, viral capsid, or vector is derived. In some embodiments, the capsid protein, viral capsid, or vector may have reduced transduction efficiency relative to the transduction efficiency of the AAV serotype from which the capsid protein, viral capsid, or vector is derived. In some embodiments, the capsid protein, viral capsid, or vector may have tropism equal to, or enhanced relative to, the tropism of the AAV serotype from which the capsid protein, viral capsid, or vector is derived. In some embodiments, the capsid protein, viral capsid, or vector may have a modified or different tropism relative to the tropism of the AAV serotype from which the capsid protein, viral capsid, or vector is derived. In some embodiments, the capsid protein, viral capsid, or vector may have tropism for, or be engineered to have tropism for, brain tissue. In some embodiments, the capsid protein, viral capsid, or vector may have tropism for, or be engineered to have tropism for, liver tissue.

[0134] Using the AAV vectors described herein, heterologous nucleic acids can be delivered to cells or a subject. For example, the modified vectors can be used to treat mucopolysaccharidosis disorders (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [alpha-L-iduronidase], Scheie syndrome [alpha-L-iduronidase], Hurler-Scheie syndrome [alpha-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilippo syndrome (A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:alpha-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase]), Morquio syndrome (A [galactose-6-sulfatase], B [β-galactosidase]), Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (e.g., Pompe disease, lysosomal acid alpha-glucosidase), etc.) of glycosphingolipid storage disorders as described herein.

[0135] One of ordinary skill in the art will understand that for some AAV capsid proteins, the corresponding modification is an insertion and / or substitution depending on whether the corresponding amino acid position is partially or fully present in the virus or alternatively completely absent.

[0136] In some embodiments, the viral vector comprises the modified capsid protein and capsid described herein. In some embodiments, the viral vector is a parvovirus vector (e.g., comprising a parvovirus capsid and / or vector genome), e.g., an AAV vector (e.g., comprising an AAV capsid and / or vector genome). In some embodiments, the viral vector comprises a modified AAV capsid comprising the modified capsid and vector genome described herein.

[0137] For example, in some embodiments, the viral vector comprises (a) a modified viral capsid (e.g., a modified AAV capsid) comprising a modified capsid protein described herein, and (b) a nucleic acid comprising terminal repeats (e.g., AAV TR), wherein the nucleic acid comprising terminal repeats is encapsidated by the modified viral capsid. The nucleic acid may optionally comprise two terminal repeats (e.g., two AAV TR).

[0138] In some embodiments, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant viral vectors are described in more detail below.

[0139] In some embodiments, the viral vector (i) has reduced transduction of the liver as compared to the level of transduction by a viral vector that does not comprise a modified capsid protein, (ii) exhibits enhanced systemic transduction by the viral vector in an animal subject as compared to the level observed by a viral vector that does not comprise a modified capsid protein, (iii) shows enhanced endothelial cell crossing as compared to the level of trafficking by a viral vector that does not comprise a modified capsid protein, and / or (iv) shows selective enhancement in transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle, and / or diaphragm muscle), (v) exhibits selective enhancement in transduction of liver tissue, and / or (vi) has reduced transduction of brain tissue (e.g., neurons) as compared to the level of transduction by a viral vector that does not comprise a modified capsid protein. In some embodiments, the viral vector has systemic transduction with respect to the liver.

[0140] One of ordinary skill in the art will understand that the modified capsid proteins, viral capsids, and viral vectors described herein exclude those capsid proteins, capsids, and viral vectors having the amino acids indicated at specific positions in their native state (i.e., are not variants).

[0141] Method for producing viral vectors This specification also provides a method for producing viral vectors. In some embodiments, a method for producing an AAV vector that avoids neutralizing antibodies comprises: a) identifying contacting amino acid residues that form a three-dimensional antigen footprint on the AAV capsid protein; b) generating a library of AAV capsid proteins comprising amino acid substitutions of the contacting amino acid residues identified in (a); c) producing AAV particles comprising the capsid protein from the library of AAV capsid proteins of (b); d) contacting the AAV particles of (c) with cells under conditions where infection and replication can occur; e) completing at least one infection cycle and selecting AAV particles that can replicate to a titer similar to that of control AAV particles; f) contacting the AAV particles selected in (e) with neutralizing antibodies and cells under conditions where infection and replication can occur; and g) selecting AAV particles that are not neutralized by the neutralizing antibodies of (f). Non-limiting examples of methods for identifying contacting amino acid residues include peptide epitope mapping and / or cryo-electron microscopy.

[0142] Resolution and identification of antibody contact residues within the three-dimensional antigen footprint enable their subsequent modification through random, rational, and / or mutagenic mutagenesis to generate AAV capsids that avoid antibodies, which can be identified through further selection and / or screening.

[0143] Thus, in some embodiments, a method of producing an AAV vector that evades neutralizing antibodies includes: a) identifying contacting amino acid residues that form a three-dimensional antigen footprint on the AAV capsid protein; b) generating an AAV capsid protein that includes amino acid substitutions of the contacting amino acid residues identified in (a) by random, rational, and / or mutagenic mutagenesis; c) producing AAV particles that include the capsid protein from the AAV capsid protein of (b); d) contacting the AAV particles of (c) with cells under conditions where infection and replication can occur; e) completing at least one infection cycle and selecting AAV particles that can replicate to a titer similar to that of control AAV particles; f) contacting the AAV particles selected in (e) with neutralizing antibodies and cells under conditions where infection and replication can occur; and g) selecting AAV particles that are not neutralized by the neutralizing antibodies of (f).

[0144] Non-limiting examples of methods for identifying contacting amino acid residues include peptide epitope mapping and / or cryo-electron microscopy. Methods for generating AAV capsid proteins that include amino acid substitutions of contacting amino acid residues by random, rational, and / or mutagenic mutagenesis are known in the art.

[0145] This comprehensive approach presents a platform technology that can be applied to modify any AAV capsid. Application of this platform technology results in an AAV antigen variant derived from the original AAV capsid template without compromising transduction efficiency. One advantage and benefit is that application of this technology expands the cohort of patients who can be treated with gene therapy using AAV vectors.

[0146] In some embodiments, a method of producing a viral vector comprises providing to a cell: (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence); and (b) AAV sequences sufficient for replication of the nucleic acid template and capsid formation into an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding an AAV capsid). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In some embodiments, the nucleic acid template comprises two AAV ITR sequences located 5' and 3' of the heterologous nucleic acid sequence (if present), although they need not be directly contiguous thereto.

[0147] The nucleic acid template and the AAV rep and cap sequences are provided under conditions such that a viral vector comprising the nucleic acid template packaged within an AAV capsid is produced intracellularly. The method may further comprise the step of collecting the viral vector from the cell. The viral vector can be collected from the culture medium and / or by lysing the cells.

[0148] The cell can be a cell that permits AAV virus replication. Any suitable cell known in the art can be used. In some embodiments, the cell is a mammalian cell. Alternatively, the cell can be a trans-complementary packaging cell line that provides a function deleted from a replication-defective helper virus, e.g., a 293 cell or other E1a trans-complementary cell.

[0149] AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep / cap genes on a single plasmid. The AAV replication and packaging sequences need not be provided together, although it can be convenient to do so. The AAV rep and / or cap sequences can be provided by any viral or non-viral vector. For example, the rep / cap sequences can be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). The AAV cap and rep genes may be expressed using an EBV vector. One advantage of this method is that the EBV vector is episomal but maintains a high copy number through successive cell divisions (i.e., is stably integrated into the cell as an extrachromosomal element designated as an "EBV-based nuclear episome", see Margolski, (1992) Curr. Top. Microbiol. Immunol. 158:67).

[0150] As a further alternative, the rep / cap sequences can be stably integrated into the cell.

[0151] Typically, the AAV rep / cap sequences are not adjacent to the TR to prevent rescue and / or packaging of these sequences.

[0152] The nucleic acid template can be provided to cells using any method known in the art. For example, the template can be supplied by a non-viral (e.g., plasmid) or viral vector. In some embodiments, the nucleic acid template is supplied by a herpes virus or adenovirus vector (e.g., inserted into the deleted E1a or E3 region of adenovirus). As another example, Palombo et al., (1998) J. Virology 72:5025 describes a baculovirus vector carrying a reporter gene adjacent to the AAV TR. An EBV vector can be used to deliver the template as described above for the rep / cap genes.

[0153] In some embodiments, the nucleic acid template is provided by a replicating rAAV virus. In some embodiments, the AAV provirus containing the nucleic acid template is stably integrated into the chromosome of the cell.

[0154] To enhance virus titer, helper virus functions (e.g., adenovirus or herpes virus) that promote productive AAV infection can be provided to the cells. Helper virus sequences required for AAV replication are known in the art. Typically, these sequences are provided by a helper adenovirus or herpes virus vector. Alternatively, the adenovirus or herpes virus sequences can be provided as another non-viral or viral vector, e.g., Ferrari et al., (1997) Nature Med. 3:1295, and as a non-infectious adenovirus miniplasmid carrying all of the helper genes that promote efficient AAV production as described in U.S. Patent Nos. 6,040,183 and 6,093,570.

[0155] Furthermore, the helper virus function can be provided by packaging cells having helper sequences embedded in the chromosome or can be maintained as a stable extrachromosomal element. Generally, helper virus sequences cannot be packaged into AAV virions and, for example, are not adjacent to the TR.

[0156] One of ordinary skill in the art will appreciate that it may be advantageous to provide AAV replication and capsid sequences, as well as helper virus sequences (e.g., adenovirus sequences), on a single helper construct. This helper construct can be a non-viral or viral construct. As one non-limiting exemplary embodiment, the helper construct can be a hybrid adenovirus or hybrid herpesvirus that includes the AAV rep / cap genes.

[0157] In some embodiments, the AAV rep / cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. This vector can further include a nucleic acid template. The AAV rep / cap sequences and / or the rAAV template can be inserted into the deleted region of the adenovirus (e.g., the E1a or E3 region).

[0158] In some embodiments, the AAV rep / cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. According to this embodiment, the rAAV template can be provided as a plasmid template.

[0159] In some embodiments, the AAV rep / cap sequences and the adenovirus helper sequences are provided by a single adenovirus helper vector and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained intracellularly as an extrachromosomal element (e.g., as an EBV-based nuclear episome).

[0160] In some embodiments, the AAV rep / cap sequences and the adenovirus helper sequences are provided by a single adenovirus helper. The rAAV template can be provided as a separate replication viral vector. For example, the rAAV template can be provided by rAAV particles or a second recombinant adenovirus particle.

[0161] According to the foregoing method, the hybrid adenovirus vector typically includes adenovirus 5' and 3' cis sequences (i.e., adenovirus terminal repeats and the PAC sequence) sufficient for adenovirus replication and packaging. Since the AAV rep / cap sequences, and when present, the rAAV template, are embedded in the adenovirus backbone and adjacent to the 5' and 3' cis sequences, these sequences can be packaged into the adenovirus capsid. As described above, the adenovirus helper sequences and the AAV rep / cap sequences are generally not adjacent to the TR so that these sequences are not packaged into the AAV virion. Zhang et al., ((2001) Gene Ther. 18:704-12) describe a chimeric helper that includes both adenovirus and the AAV rep and cap genes.

[0162] Herpesvirus can also be used as a helper virus in the AAV packaging method. A hybrid herpesvirus encoding the AAV Rep protein(s) can advantageously facilitate an expandable AAV vector production scheme. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO00 / 17377).

[0163] As a further alternative, viral vectors can be produced in insect cells using a baculovirus vector to deliver the rep / cap genes and the rAAV template, for example, as described by Urabe et al., (2002) Human Gene Therapy 13:1935-43.

[0164] AAV vector stocks free of contaminating helper virus can be obtained by any method known in the art. For example, AAV and helper virus can be readily differentiated based on size. Also, AAV may be separated away from the helper virus based on its affinity for a heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973). A replication-deficient helper virus that has been deleted can be used so that any contaminating helper virus does not have the ability to replicate. As a further alternative, since only early gene expression of adenovirus is required to mediate the packaging of AAV virus, an adenovirus helper lacking late gene expression may be used. Adenovirus mutants lacking late gene expression (e.g., ts100K and ts149 adenovirus mutants) are known in the art.

[0165] Recombinant viral vector The viral vectors described herein are useful for the delivery of nucleic acids to cells in vitro, ex vivo, and in vivo. In particular, viral vectors can be advantageously used to deliver or transfer nucleic acids to animal cells, including mammalian cells. Thus, in some embodiments, the nucleic acid can be encapsidated by the capsid proteins described herein. In some embodiments, the nucleic acid is a cargo nucleic acid. In some embodiments, the cargo nucleic acid comprises a vector genome (e.g., 5' ITR, transgene, and 3' ITR).

[0166] The cargo nucleic acid sequence delivered by the viral vector can be any heterologous nucleic acid sequence(s) of interest. Nucleic acids of interest include nucleic acids encoding polypeptides, including therapeutic (e.g., for medical or veterinary use) or immunogenic (e.g., for vaccines) polypeptides or RNAs. In some embodiments, the cargo nucleic acid comprises a 5' ITR and a 3' ITR. In some embodiments, the cargo nucleic acid comprises a 5' ITR, a transgene, and a 3' ITR. In some embodiments, the transgene encodes a therapeutic protein or RNA.

[0167] Therapeutic polypeptides include cystic fibrosis transmembrane regulator (CFTR), dystrophin (including mini- and micro-dystrophin, for example, Vincent et al., (1993) Nature Genetics 5:130, US Patent Publication No. 2003 / 017131, International Publication No. WO / 2008 / 088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000), and Gregorevic et al., Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, apolipoproteins, for example, apo A (apo A1, apo A2, apo A4, apo A-V), apo B (apo B100, apo B48), apo C (apo CI, apo CII, apo CIII, apo CIV), apo D, apo E, apo H, apo L, apo(a), anti-inflammatory polypeptides, for example, Ikappa B dominant mutants, amyloid beta, tau, sarcospan, utrophin (Tinsley et al,(1996) Nature 384:349), mininutrophin, coagulation factors (e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, progranulin, ornithine transcarbamylase, β-globin, α-globin, spectrin, alpha-1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, β-glucosylceramidase, battenin, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, frataxin, RP65 protein, cytokines (e.g., alpha-interferon, beta-interferon, gamma-interferon, interleukin-2, interleukin-4, alpha-synuclein, parkin, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors -3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial-derived growth factor, transforming growth factors -α and -β, etc.), huntingin, lysosomal acid alpha-glucosidase, iduronate-2-sulfatase, N-sulfo-glucosamine sulfohydrolase, alpha-galactosidase A, receptors (e.g., tumor necrosis growth factor soluble receptor), S100A1, ubiquitin protein ligase E3, parvalbumin, adenylyl cyclase type 6, molecules that regulate calcium handling (e.g., SERCA, 2A, inhibitors of PP1 and fragments thereof (e.g., WO2006 / 029319 and WO2007 / 100465), molecules that result in knockdown of G protein-coupled receptor kinase type 2 (such as truncated constitutively active bARKct), anti-inflammatory factors (such as IRAP), anti-myostatin proteins, aspartoacylase, monoclonal antibodies (including single-chain monoclonal antibodies, an exemplary Mab is Herceptin® Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see U.S. 7,071,172)), angiogenesis inhibitors such as vasohibin and other VEGF inhibitors (e.g., vasohibin 2 [see WO JP2006 / 073052]), but are not limited thereto. Other exemplary heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that enhance or inhibit transcription of host factors (e.g., nuclease-dead Cas9 linked to a transcriptional enhancer or inhibitor element, zinc finger protein linked to a transcriptional enhancer or inhibitor element, transcriptional activator-like (TAL) effector linked to a transcriptional enhancer or inhibitor element), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide having a therapeutic effect in a subject in need of treatment. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, e.g., antibodies or antibody fragments directed against myostatin (see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005)). Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein, β-galactosidase, alkaline phosphatase, luciferase, and the chloramphenicol acetyltransferase gene.

[0168] Optionally, the heterologous nucleic acid encodes a secreted polypeptide (e.g., a polypeptide that is secreted in its native state or a polypeptide that has been engineered to be secreted, e.g., by operable association with a secretion signal sequence known in the art).

[0169] Alternatively, in some embodiments, the heterologous nucleic acid may encode an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Patent No. 5,877,022), an RNA that effects spliceosome-mediated / ram splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246, U.S. Patent No. 6,013,487, U.S. Patent No. 6,083,702), interfering RNA (RNAi) including siRNA, shRNA, or miRNA that mediates gene silencing (see Sharp et al., (2000) Science 287:2431), and other non-translated RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929, see U.S. Patent No. 5,869,248 to Yuan et al.). Exemplary non-translated RNAs include RNAi against multiple drug resistance (MDR) gene products (e.g., for treating and / or preventing tumors and / or for administration to the heart to prevent damage by chemotherapy), RNAi against myostatin (e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., for treating and / or preventing tumors), RNAi against phospholamban (e.g., for treating cardiovascular disease, see, e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)), phospholamban inhibitory or dominant-negative molecules, such as phospholamban S16E (see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002) for treating cardiovascular disease), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0170] Furthermore, nucleic acid sequences targeting alternative splicing can be delivered. By way of example, an antisense sequence (or other inhibitory sequence) complementary to the 5’ and / or 3’ splice sites of dystrophin exon 51 can be delivered together with a U1 or U7 small nuclear (sn)RNA promoter to induce skipping of this exon. For example, a DNA sequence containing a U1 or U7 snRNA promoter located 5’ of the antisense / inhibitory sequence(s) can be packaged and delivered in a modified capsid.

[0171] In some embodiments, nucleic acid sequences targeting gene editing can be delivered. For example, the nucleic acid can encode a guide RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA) comprising a crRNA sequence and a tracrRNA sequence. In some embodiments, the nucleic acid may encode a nuclease. In some embodiments, the nuclease is a zinc finger nuclease, a homing endonuclease, a TALEN (transcription activator-like effector nuclease), NgAgo (Argonaute endonuclease), SGN (structure-guided endonuclease), RGN (RNA-guided nuclease), or a modified or cleaved variant thereof. In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, a Cas12(a) nuclease (Cpf1), a Cas12b nuclease, a Cas12c nuclease, a TrpB-like nuclease, a Cas13a nuclease (C2c2), a Cas13b nuclease, or a modified or cleaved variant thereof. In some embodiments, the Cas9 nuclease is isolated from or derived from S. pyogenes or S. aureus.

[0172] In some embodiments, nucleic acid sequences that direct gene knockdown can be delivered. For example, the nucleic acid sequence can encode siRNA, shRNA, microRNA, or antisense nucleic acid. The viral vector can also contain a heterologous nucleic acid that shares homology with and recombines with a locus on the host chromosome. This approach can be utilized, for example, to correct genetic deficiencies in host cells.

[0173] Also provided, for example, are viral vectors that express an immunogenic polypeptide for vaccination. The nucleic acid can encode any immunogen known in the art, including but not limited to immunogens from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigen, cancer antigen, bacterial antigen, viral antigen, etc.

[0174] The use of parvovirus as a vaccine vector is known in the art (see, for example, Miyamura el al, (1994) Proc. Nat. Acad. Sci USA 91:8507, U.S. Patent No. 5,916,563 to Young et al., U.S. Patent No. 5,905,040 to Mazzara et al., U.S. Patent No. 5,882,652, U.S. Patent No. 5,863,541 to Samulski et al.). The antigen can be presented in the parvovirus capsid.

[0175] Alternatively, the antigen can be expressed from a heterologous nucleic acid introduced into the recombinant vector genome. In some embodiments, any immunogen of interest described herein and / or known in the art can be provided by the viral vectors described herein.

[0176] An immunogenic polypeptide can be any polypeptide that is capable of inducing an immune response and / or protecting a subject from infections and / or diseases, including but not limited to microbial, bacterial, protozoal, parasitic, fungal, and / or viral infections and diseases. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen such as influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as the HIV or SIV envelope GP 160 protein, the HIV or SIV matrix / capsid protein, and the HIV or SIV gag, pol, and env gene products). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen such as the Lassa fever virus nucleocapsid protein and the Lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen such as the vaccinia virus LI or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as the NP and GP gene products), a bunyavirus immunogen (e.g., an RVFV, CCHF, and / or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen such as the human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens), a mumps immunogen, a measles immunogen, a rubella immunogen, diphtheria toxin, or other diphtheria immunogens, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or any other vaccine immunogen currently known in the art or later identified as an immunogen.

[0177] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of the cancer cell.

[0178] Exemplary cancers and tumor cell antigens are described in S. A. Rosenberg (Immunity 10:281 (1991)). Other exemplary cancers and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, ΚΙΑA0205, HPVE, SART-1, FRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515, Kawakami et al., (1994) J. Exp. Med., 180:347, Kawakami et al., (1994) Cancer Res. 54:3124), MART-1, gp100, MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J Exp. Med. 178:489), HER-2 / neu gene product (U.S. Patent No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG72, AFP, CA 19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623), mucin antigen (International Patent Publication No. WO90 / 05142), telomerase, nuclear matrix protein, prostate acid phosphatase, papillomavirus antigen, and / or antigens currently known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignancy or its metastasis currently known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

[0179] As a further alternative, the heterologous nucleic acid can desirably encode any polypeptide produced in cells in vitro, ex vivo, or in vivo. For example, a viral vector can be introduced into cultured cells and the expressed gene product isolated therefrom.

[0180] One of ordinary skill in the art will understand that the heterologous nucleic acid(s) of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid can be operably associated with expression control elements such as transcriptional / translational control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and / or enhancers.

[0181] Furthermore, regulated expression of the heterologous nucleic acid(s) of interest can be achieved at the post-transcriptional level, for example, by regulating alternative splicing of different introns by the presence or absence of oligonucleotides, small molecules, and / or other compounds that selectively block splicing activity at specific sites (e.g., as described in WO2006 / 119137).

[0182] One of ordinary skill in the art will understand that various promoter / enhancer elements can be used depending on the desired level and tissue-specific expression. The promoter / enhancer can be constitutive or inducible depending on the desired expression pattern. The promoter / enhancer can be native or foreign and can be a native or synthetic sequence. By foreign is intended that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced.

[0183] In some embodiments, the promoter / enhancer element may be native to the target cell or to the subject being treated. In some embodiments, the promoter / enhancer element may be native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally selected to function in the target cell(s) of interest. Further, in some embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element can be constitutive or inducible.

[0184] Inducible expression control elements are typically advantageous in those applications where it is desirable to provide regulation over the expression of the heterologous nucleic acid sequence(s). Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferential promoter / enhancer elements, including muscle-specific or preferential (including myocardial, skeletal muscle, and / or smooth muscle-specific or preferential), nerve tissue-specific or preferential (including brain-specific or preferential), eye-specific or preferential (including retina-specific and corneal-specific), liver-specific or preferential, bone marrow-specific or preferential, pancreas-specific or preferential, spleen-specific or preferential, and lung-specific or preferential promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, the Tet on / off element, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and metallothionein promoter.

[0185] In some embodiments where the heterologous nucleic acid sequence(s) is transcribed and then translated within the target cell, specific initiation signals are generally included for efficient translation of the inserted protein coding sequence. These exogenous translation control sequences, which can include the ATG start codon and adjacent sequences, can be of various origins, both natural and synthetic.

[0186] The viral vectors described herein provide a means for delivering heterologous nucleic acids to a wide range of cells, including dividing and non-dividing cells. The viral vectors can be used to deliver the nucleic acid of interest to cells in vitro, for example, to produce polypeptides in vitro, or for ex vivo gene therapy. The viral vectors are further useful in methods of performing such delivery to a subject in need thereof, for example, to express an immunogenic or therapeutic polypeptide or a functional RNA. Thus, the polypeptide or functional RNA can be produced in vivo in the subject. Since the subject has a deficiency in the polypeptide, the subject may be in need of the polypeptide. Further, the method can be carried out because the production of the polypeptide or functional RNA in the subject can result in some beneficial effect.

[0187] It is also possible to produce the polypeptide or functional RNA of interest using the viral vector, either in cultured cells or in a subject (for example, using the subject as a bioreactor to produce the polypeptide, or observing the effect of the functional RNA on the subject, for example, in the context of a screening method).

[0188] Generally, the viral vectors described herein can be used to deliver a heterologous nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent any disease state in which it is beneficial to deliver a therapeutic polypeptide or functional RNA. Exemplary disease states include cystic fibrosis (cystic fibrosis transmembrane regulator protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin), and other blood disorders, Alzheimer's disease (GDF, neprylisin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi for repeat removal), Canavan disease, amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferon, RNAi including RNAi against VEGF or multidrug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma]), diabetes mellitus type I (insulin), Duchenne (dystrophin, minidystrophin, insulin-like growth factor I, sarcoglycan [e.g., a, β, γ], RNAi against myostatin polypeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptide, e.g., Ikappa B dominant mutant, sarcospan, utrophin, miniutrophin, antisense or RNAi against splice sites of the dystrophin gene that induce exon skipping [see, e.g., WO / 2003 / 095647], antisense against U7 snRNA for inducing exon skipping [see, e.g., WO / 2006 / 021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker including muscular dystrophy, myotonic dystrophy 1 or 2, facioscapulohumeral muscular dystrophy (FSHD), Gaucher disease (glucocerebrosidase), Hurler disease (a-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [a-galactosidase] and Pompe disease [lysosomal acid alpha-glucosidase]),and other metabolic disorders, congenital emphysema (alpha-1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyl transferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), frontotemporal dementia, maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina, e.g., inhibitors of PDGF and / or vasohibin or other VEGFs for macular degeneration, or other angiogenesis inhibitors for treating / preventing retinopathy in type I diabetes), diseases of parenchymal organs such as the brain (including Parkinson's disease [GDNF], astrocytoma [endostatin, angiostatin, and / or RNAi against VEGF]), glioblastoma [endostatin, angiostatin, and / or RNAi against VEGF]), liver, kidney, heart (including congestive heart failure or peripheral arterial disease (PAD)) (e.g., by delivering protein phosphatase inhibitor I (I-1) and its fragments (e.g., IlC), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, [32-adrenergic receptor, beta-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylate cyclase type 6, molecules that result in knockdown of G protein-coupled receptor kinase type 2 such as truncated constitutively active bARKct, calbindin, RNAi against phospholamban, phospholamban inhibitors or dominant-negative molecules, e.g., phospholamban S16E, etc.), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivering enos, inos), improvement of heart transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), renal failure (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFa soluble receptor), hepatitis (alpha-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, SCA1, SCA2,Spinal cerebellar ataxia including SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. are included, but not limited thereto. The compositions and methods disclosed herein can be further used after organ transplantation to increase the success rate of organ transplantation and / or reduce the negative side effects of organ transplantation or adjuvant therapy (for example, by administering an immunosuppressant or inhibitory nucleic acid to block cytokine production). As another example, bone morphogenetic proteins (including BNP2, 7, etc., RANKL and / or VEGF) can be administered together with bone allografts, for example, after drug withdrawal or surgical removal in cancer patients.

[0189] In some embodiments, the viral vectors described herein can be used to deliver a heterologous nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent liver diseases or disorders. Liver diseases or disorders include, for example, primary biliary cirrhosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, hepatitis B, hepatitis C, alcoholic liver disease, fibrosis, jaundice, primary sclerosing cholangitis (PSC), Budd-Chiari syndrome, hemochromatosis, Wilson's disease, alcoholic fibrosis, non-alcoholic fibrosis, fatty liver, Gilbert's syndrome, biliary atresia, alpha-1-antitrypsin deficiency, Alagille syndrome, progressive familial intrahepatic cholestasis, hemophilia B, hereditary angioedema (HAE), homozygous familial hypercholesterolemia (HoFH), heterozygous familial hypercholesterolemia (HeFH), von Gierke's disease (GSD I), hemophilia A, methylmalonic acidemia, propionic acidemia, homocystinuria, phenylketonuria (PKU), tyrosinemia type 1, arginase 1 deficiency, argininosuccinate lyase deficiency, carbamoyl-phosphate synthetase 1 deficiency, citrullinemia type 1, citrulline deficiency, Crigler-Najjar syndrome type 1, cystinosis, Fabry disease, glycogenosis 1b, LPL deficiency, N-acetylglutamate synthase deficiency, ornithine transcarbamylase deficiency, ornithine transferase deficiency, primary hyperoxaluria type 1, or ADA SCID.

[0190] Using the compositions and methods described herein, induced pluripotent stem cells (iPS) can also be produced. For example, using the viral vectors described herein, stem cell-related nucleic acid(s) can be delivered to non-pluripotent cells such as adult fibroblasts, skin cells, hepatocytes, kidney cells, adipocytes, cardiomyocytes, neurons, epithelial cells, endothelial cells, etc.

[0191] Nucleic acids encoding factors related to stem cells are known in the art. Non-limiting examples of such factors related to stem cells and pluripotency include Oct-3 / 4, the SOX family (e.g., SOX1, SOX2, SOX3, and / or SOX15), the Klf family (e.g., Klf1, Klf2, Klf4, and / or Klf5), the Myc family (e.g., C-myc, L-myc, and / or N-myc), NANOG, and / or LIN28.

[0192] The methods described herein can also be practiced for the treatment and / or prevention of metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), glycosphingolipid storage disorders such as mucopolysaccharidosis disorders (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [alpha-L-iduronidase], Scheie syndrome [alpha-L-iduronidase], Hurler-Scheie syndrome [alpha-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilippo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:alpha-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [β-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (alpha-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (e.g., Pompe disease, lysosomal acid alpha-glucosidase).

[0193] Gene transfer is substantially useful for understanding disease states and providing therapies. There are several genetic diseases where the defective gene is known and cloned. Generally, the above disease states are generally classified into two categories: a deficiency state of a normal enzyme that generally inherits in a recessive mode, and an imbalance state that may involve a regulatory or structural protein and typically inherits in a dominant mode. For deficiency state diseases, gene transfer can be used to bring a normal gene to the affected tissue for replacement therapy and to create animal models of the disease using antisense mutations. For imbalanced disease states, gene transfer can be used to create the disease state in a model system and then used to counteract the disease state. Thus, the viral vectors described herein enable the treatment and / or prevention of genetic diseases.

[0194] The viral vectors described herein can also be used to deliver functional RNA to cells in vitro or in vivo. The functional RNA can be, for example, non-coding RNA. In some embodiments, the expression of the functional RNA in the cell can reduce the expression of a specific target protein by the cell. Thus, it is possible to administer the functional RNA to a subject in need of a decrease in the expression of a specific protein to effect such a decrease. In some embodiments, the expression of the functional RNA in the cell can increase the expression of a specific target protein by the cell. Thus, it is possible to administer the functional RNA to a subject in need of an increase in the expression of a specific protein to effect such an increase. In some embodiments, the expression of the functional RNA can regulate the splicing of a specific target RNA in the cell. Thus, it is possible to administer the functional RNA to a subject in need of regulation of the splicing of a specific RNA to effect such regulation. In some embodiments, the expression of the functional RNA in the cell can regulate the function of a specific target protein by the cell. Thus, it is possible to administer the functional RNA to a subject in need of regulation of the function of a specific protein to effect such regulation. The functional RNA can also be administered to cells in vitro, for example, to optimize a cell or tissue culture system, or in a screening method, to regulate gene expression and / or cell physiology.

[0195] In addition, the viral vectors described herein are used in diagnostic and screening methods, whereby a nucleic acid of interest is transiently or stably expressed in a cell culture system or, alternatively, in a transgenic animal model.

[0196] Viral vectors can also be used for various non-therapeutic purposes, including but not limited to use in protocols for evaluating gene targeting, clearance, transcription, translation, etc., as will be apparent to those skilled in the art. Viral vectors can also be used for the purpose of evaluating safety (such as spread, toxicity, immunogenicity, etc.). For example, such data are considered by the US Food and Drug Administration as part of the regulatory approval process prior to the evaluation of clinical efficacy.

[0197] In some embodiments, viral vectors can be used to produce an immune response in a subject. According to this embodiment, a viral vector containing a heterologous nucleic acid sequence encoding an immunogenic polypeptide can be administered to the subject, and an active immune response is initiated by the subject against the immunogenic polypeptide. The immunogenic polypeptide is as described above. In some embodiments, a protective immune response is induced.

[0198] Alternatively, the viral vector can be administered to cells ex vivo and the modified cells can be administered to the subject. A viral vector containing a heterologous nucleic acid can be introduced into the cells, the cells can be administered to the subject, the heterologous nucleic acid encoding the immunogen can be expressed, and an immune response against the immunogen can be induced in the subject. In some embodiments, the cells are antigen-presenting cells (e.g., dendritic cells).

[0199] "Active immune response" or "active immunity" is characterized by "the participation of host tissues and cells after encounter with an immunogen". This involves the differentiation and proliferation of immunocompetent cells in lymphoid tissues and leads to the synthesis of antibodies or the development of cell-mediated reactivity, or both. Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). In other words, after exposure to an immunogen by infection or vaccination, an active immune response is initiated by the host. Active immunity can be contrasted with passive immunity, which is acquired by the transfer of preformed substances (antibodies, transfer factors, thymic grafts, interleukin-2) from an actively immunized host to a non-immunized host.

[0200] As used herein, a "protective" immune response or "protective" immunity indicates that the immune response confers some benefit to the subject in terms of preventing or reducing the occurrence of a disease. Alternatively, a protective immune response or protective immunity may be useful in the treatment and / or prevention of diseases, particularly cancer or tumors (e.g., by preventing cancer or tumor formation, by causing regression of cancer or tumors, and / or by preventing metastasis and / or by preventing the growth of metastatic nodules). The protective effect may be complete or partial as long as the therapeutic benefit outweighs any of its drawbacks.

[0201] In some embodiments, a viral vector or cell comprising a heterologous nucleic acid can be administered in an immunogenically effective amount as described below.

[0202] In some embodiments, the viral vector can be administered for cancer immunotherapy by administration of a viral vector that expresses one or more cancer cell antigens (or immunologically similar molecules), or any other immunogen that elicits an immune response against cancer cells. By way of example, the immune response can be elicited against cancer cell antigens in a subject by administering a viral vector comprising a heterologous nucleic acid encoding a cancer cell antigen, for example, to treat a patient having cancer and / or to prevent the onset of cancer in a subject. The viral vector can be administered to a subject in vivo or by using ex vivo methods, as described herein.

[0203] Alternatively, the cancer antigen can be expressed as part of the viral capsid or can associate with the viral capsid in other ways (e.g., as described above).

[0204] As another alternative, any other therapeutic nucleic acid (e.g., RNAi) or polypeptide (e.g., cytokine) known in the art can be administered to treat and / or prevent cancer.

[0205] As used herein, the term “cancer” includes neoplastic cancers. Similarly, the term “cancer tissue” includes tumors. “Cancer cell antigen” includes tumor antigens.

[0206] The term “cancer” has its understood meaning in the art and is, for example, an uncontrolled tissue growth that has the potential to spread (i.e., metastasize) to distant sites in the body. Exemplary cancers include melanoma, adenocarcinoma, thymic carcinoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignancy now known or later identified, but are not limited thereto. In some embodiments, methods of treating and / or preventing neoplastic cancers are provided.

[0207] The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In some embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0208] The terms "treating cancer", "treatment of cancer", and equivalent terms are intended to mean that the severity of cancer is reduced or at least partially eliminated, and / or the progression of the disease is slowed and / or controlled, and / or the disease is stabilized. In some embodiments, these terms indicate that cancer metastasis is prevented or reduced, or at least partially eliminated, and / or the growth of metastatic nodules is prevented or reduced, or at least partially eliminated.

[0209] The terms "prevention of cancer" or "preventing cancer", and equivalent terms are intended to mean that the method at least partially eliminates or reduces, and / or delays the incidence and / or severity of cancer development. Alternatively, the incidence of cancer in a subject can be reduced or delayed in likelihood or probability.

[0210] In some embodiments, cells can be removed from a subject having cancer and contacted with a viral vector expressing a cancer cell antigen described herein. The modified cells are then administered to the subject, thereby inducing an immune response against the cancer cell antigen. This method can be advantageously used in immunocompromised subjects who cannot initiate a sufficient immune response in vivo (i.e., cannot produce a sufficient amount of enhancing antibodies).

[0211] It is known in the art that immune responses can be enhanced by immunomodulatory cytokines (e.g., alpha-interferon, beta-interferon, gamma-interferon, omega-interferon, tau-interferon, interleukin-1-alpha, interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin 5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-alpha, tumor necrosis factor-beta, monocyte chemoattractant protein-1, granulocyte macrophage colony stimulating factor, and lymphotoxin). Thus, immunomodulatory cytokines (preferably CTL-inducing cytokines) can be administered to a subject together with a viral vector. The cytokines can be administered by any method known in the art. Exogenous cytokines can be administered to the subject or, alternatively, nucleic acids encoding the cytokines can be delivered to the subject using a suitable vector and the cytokines can be produced in vivo.

[0212] Subjects, Pharmaceutical Preparations, and Methods of Administration The viral vectors and capsids described herein are used in both veterinary and medical applications. Suitable subjects include both birds and mammals. As used herein, the term "bird" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots, budgerigars, etc. As used herein, the term "mammal" includes, but is not limited to, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects include neonates, infants, adolescents, adults, and elderly subjects.

[0213] In some embodiments, the subject "is in need of" the methods described herein.

[0214] In some embodiments, there is provided a pharmaceutical composition comprising a viral vector and / or a capsid and / or a capsid protein and / or viral particles in a pharmaceutically acceptable carrier, optionally including other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, and the like. For injection, the carrier is typically liquid. For other administration methods, the carrier can be either solid or liquid. For inhalation administration, the carrier is respirable and can optionally be in solid or liquid particle form.

[0215] "Pharmaceutically acceptable" means a material that is not toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects.

[0216] Also provided herein is a method for transferring nucleic acids into cells in vitro. The viral vector can be introduced into cells by standard transduction methods suitable for specific target cells at an appropriate multiplicity of infection. The titer of the viral vector to be administered may vary depending on the target cell type and number, as well as the specific viral vector, and can be determined by one of ordinary skill in the art without undue experimentation. In some embodiments, at least about 10 3 infectious units, optionally at least about 10 5 infectious units are introduced into the cells.

[0217] The cell(s) into which the viral vector is introduced can be of any type including, but not limited to, nerve cells (cells of the peripheral and central nervous systems, particularly brain cells such as neurons and oligodendrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelial cells, and corneal cells), epithelial cells (e.g., intestinal and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiomyocytes, smooth muscle cells, and / or diaphragm muscle cells), dendritic cells, pancreatic cells (including islet cells), hepatocytes, cardiomyocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, splenocytes, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. In some embodiments, the cell can be any progenitor cell. As a further possibility, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). As yet a further alternative, the cell can be a cancer or tumor cell. Additionally, the cell can be from a species of any origin, as described above.

[0218] The viral vector can be introduced into the cells in vitro for the purpose of administering the modified cells to a subject. In some embodiments, the cells are removed from the subject, the viral vector is introduced therein, and then the cells are readministered to the subject. Methods for removing cells from a subject for ex vivo manipulation and subsequent reintroduction into the subject are known in the art (see, e.g., U.S. Patent No. 5,399,346). Alternatively, the recombinant viral vector can be introduced into cells from a donor subject, cultured cells, or any other suitable source, and the cells are administered to a subject in need thereof (i.e., the "recipient" subject).

[0219] Cells suitable for ex vivo nucleic acid delivery are as described above. The dosage of cells administered to a subject will vary depending on the age, condition, and species of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, etc. Typically, at least about 10 2 ~ about 10 8 cells or at least about 10 3 ~ about 10 6Administer the cells. In some embodiments, cells transduced with a viral vector are administered to a subject in a therapeutically effective amount in combination with a pharmaceutical carrier.

[0220] In some embodiments, a viral vector can be introduced into cells and the cells administered to a subject to induce an immunogenic response to the delivered polypeptide (e.g., expressed as a transgene or in the capsid). Typically, an amount of cells expressing an immunogenic amount of the polypeptide is administered, in combination with a pharmaceutically acceptable carrier. An "immunogenically effective amount" is the amount of the expressed polypeptide that is sufficient to induce an active immune response to the polypeptide in the subject to whom the pharmaceutical formulation is administered. In some embodiments, the dosage is sufficient to effect a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, so long as the advantages of administration of the immunogenic polypeptide outweigh any of its disadvantages.

[0221] Accordingly, in some embodiments, a method of administering a nucleic acid to a cell comprises contacting the cell with a viral vector, viral particle, and / or composition described herein.

[0222] Also provided herein are methods of administering a viral vector, viral particle, and / or viral capsid described herein to a subject. In some embodiments, a method of delivering a nucleic acid to a subject comprises administering to the subject a viral particle, viral vector, and / or composition described herein. Administration of a viral vector, viral particle, and / or capsid to a human or animal subject in need thereof can be effected by any means known in the art. Optionally, the viral vector, viral particle, and / or capsid are delivered in a therapeutically effective amount in a pharmaceutically acceptable carrier. In some embodiments, a therapeutically effective amount of the viral vector, viral particle, and / or capsid is delivered.

[0223] The viral vectors and / or capsids described herein can be further administered to induce an immunogenic response (e.g., as a vaccine). Typically, the immunogenic composition comprises an immunogenically effective amount of the viral vector and / or capsid in combination with a pharmaceutically acceptable carrier. Optionally, the dosage is sufficient to effect a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent as long as the advantages of administration of the immunogenic polypeptide outweigh any of its disadvantages. The subject and the immunogen are as described above.

[0224] The dosage of the viral vector and / or capsid administered to a subject depends on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular viral vector or capsid, and the nucleic acid to be delivered, among other factors, and can be determined in a routine manner. Exemplary dosages for achieving a therapeutic effect are at least about 10 5 about 10 6 about 10 7 about 10 8 about 10 9 about 10 10 about 10 11 about 10 12 about 10 13 about 10 14 or about 10 15 transducing units, optionally at a titer of about 10 8 ~10 13 transducing units. In some embodiments, the dosage of AAV is from about 2.0×10 13 vg / kg body weight of the subject to about 4.0×10 13 vg / kg body weight of the subject, e.g., about 2.0×10 13 vg / kg, about 2.1×10 13 vg / kg, about 2.2×10 13 vg / kg, about 2.3×10 13 vg / kg, about 2.4×10 13 vg / kg, about 2.5×10 13 vg / kg, about 2.6×10 13 vg / kg, about 2.7×10 13 vg / kg, about 2.8×1013 vg / kg, about 2.9×10 13 vg / kg, about 3.0×10 13 vg / kg, about 3.1×10 13 vg / kg, about 3.2×10 13 vg / kg, about 3.3×10 13 vg / kg, about 3.4×10 13 vg / kg, about 3.5×10 13 vg / kg, about 3.6×10 13 vg / kg, about 3.7×10 13 vg / kg, about 3.8×10 13 vg / kg, about 3.9×10 13 vg / kg, or about 4.0×10 13 vg / kg could be. In some embodiments, the dose of AAV is about 2×10 13 vg to about 4.0×10 13 vg, for example, about 2.0×10 13 vg, about 2.1×10 13 vg, about 2.2×10 13 vg, about 2.3×10 13 vg, about 2.4×10 13 vg, about 2.5×10 13 vg, about 2.6×10 13 vg, about 2.7×10 13 vg, about 2.8×10 13 vg, about 2.9×10 13 vg, about 3.0×10 13 vg, about 3.1×10 13 vg, about 3.2×10 13 vg, about 3.3×10 13 vg, about 3.4×10 13 vg, about 3.5×10 13 vg, about 3.6×10 13 vg, about 3.7×10 13 vg, about 3.8×10 13 vg, about 3.9×10 13 vg, or about 4.0×10 13 vg could be.

[0225] In some embodiments, two or more administrations (e.g., 2, 3, 4 or more administrations) may be used to achieve the desired level of gene expression over various intervals, e.g., periods of daily, weekly, monthly, yearly, etc.

[0226] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovarian), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm, and / or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both the skin and mucosa including the airway surface, and transdermal administration), intralymphatic, etc., as well as direct injection into a tissue or organ (e.g., to the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration can also be to a tumor (e.g., within or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated and / or prevented, as well as the nature of the particular vector being used.

[0227] Administration to skeletal muscle includes, but is not limited to, administration to the skeletal muscle of the extremities (e.g., upper arm, forearm, thigh, and / or calf), back, neck, head (e.g., tongue), chest, abdomen, pelvis / perineum, and / or fingers.Suitable skeletal muscles include, but are not limited to, the abductor digiti minimi (hand), abductor digiti minimi (foot), abductor pollicis, abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor pollicis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articularis genus, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, digastric, dorsal interossei (hand), dorsal interossei (foot), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor digitorum brevis, extensor hallucis longus, extensor pollicis brevis, extensor pollicis longus, extensor indicis, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (hand), flexor digiti minimi brevis (foot), flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, genioglossus, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis, iliocostalis lumborum, iliocostalis thoracis, iliopsoas, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, interspinales, intertransversi, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator labii superioris alaeque nasi, levator palpebrae superioris, levator scapulae, long rotators, longissimus capitis, longissimus cervicis, longissimus thoracis, longus capitis, longus colli, lumbricals (hand), lumbricals (foot), masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, obliquus capitis inferior, obliquus capitis superior, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interossei, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis, plantar interossei, plantar fascia, platysma, popliteus, posterior scalene, quadratus femoris, quadratus plantae, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, sartorius, scalenus minimus, semimembranosus, semispinalis capitis, semispinalis cervicis, semispinalis thoracis, semitendinosus, serratus anterior, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fasciae latae, teres major, teres minor, thorax, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor, and other suitable skeletal muscles known in the art.

[0228] The viral vector and / or capsid can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion, (optionally, isolated limb perfusion of the leg and / or arm, see, for example, Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In some embodiments, the viral vector and / or capsid is administered to the limbs (arms and / or legs) of a subject (e.g., a subject having muscular dystrophy such as Duchenne muscular dystrophy (DMD) or limb-girdle muscular dystrophy (LGMD)) by limb perfusion, optionally isolated limb perfusion (e.g., intravenous or intra-articular administration). In some embodiments, the viral vector and / or capsid can be advantageously administered without using "hydrodynamic" techniques. Conventional vector tissue delivery (e.g., to muscle) is often enhanced by hydrodynamic techniques (e.g., large-volume intravenous / intravenous administration), which increase pressure in the vasculature and facilitate the ability of the vector to cross the endothelial cell barrier. In some embodiments, the viral vector and / or capsid can be administered in the absence of hydrodynamic techniques such as bolus injection and / or elevation of intravascular pressure (e.g., greater than normal systolic pressure, e.g., 5%, 10%, 15%, 20%, 25% or less of intravascular pressure relative to normal systolic pressure). Such methods can reduce or avoid side effects associated with hydrodynamic techniques such as edema, nerve damage, and / or compartment syndrome. Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The viral vector and / or capsid can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion.

[0229] Administration to the diaphragm muscle can be performed by any suitable method including intravenous administration, intra-arterial administration, and / or intraperitoneal administration.

[0230] Delivery to the target tissue can also be achieved by delivering a depot comprising a viral vector and / or a capsid. In some embodiments, the depot comprising a viral vector and / or a capsid is implanted into skeletal muscle, cardiac muscle, and / or diaphragm muscle tissue, or the tissue can be contacted with a film or other matrix comprising a viral vector and / or a capsid. Such implantable matrices or substrates are described in U.S. Patent No. 7,201,898.

[0231] In some embodiments, the viral vector and / or viral capsid is appropriately administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat and / or prevent muscular dystrophy, heart disease [e.g., PAD or congestive heart failure]).

[0232] In some embodiments, the compositions and methods described herein are used to treat and / or prevent diseases or disorders of skeletal muscle, cardiac muscle, and / or diaphragm muscle. Muscle diseases or disorders can be, for example, muscular dystrophy, myopathy, motor neuron disease, and cardiomyopathy. Muscle diseases or disorders can be, for example, dystrophinopathy, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy (e.g., myotonic dystrophy 1 and 2), facioscapulohumeral muscular dystrophy (FDHD), Emery - Dreifuss muscular dystrophy, limb - girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy, juvenile macular dystrophy, central core myopathy, central nuclear myopathy, and inclusion body myositis.

[0233] In some embodiments, a method of doing so in a subject in need of treatment and / or prevention of muscular dystrophy is provided, the method comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector, the viral vector comprising dystrophin, minidystrophin, microdystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, an anti-inflammatory polypeptide such as an Ikappa B dominant mutant, sarcospan, utrophin, microdystrophin, laminin-a2, alpha-sarcoglycan, beta-sarcoglycan, gamma-sarcoglycan, delta-sarcoglycan, IGF-1, an antibody or antibody fragment against myostatin or myostatin propeptide, and / or a heterologous nucleic acid encoding an RNAi against myostatin. In some embodiments, the viral vector can be administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle, as described elsewhere herein.

[0234] Alternatively, the methods described herein can be carried out to deliver a nucleic acid to skeletal muscle, cardiac muscle, or diaphragm muscle for use as a platform for systemic delivery to other tissues for the production of a polypeptide (e.g., an enzyme) or a functional RNA (e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood, or for the treatment and / or prevention of a disorder (e.g., a metabolic disorder such as diabetes [e.g., insulin], hemophilia [e.g., factor IX or factor VIII], a mucopolysaccharidosis [e.g., Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteaux-Lamy syndrome, etc.], or a lysosomal storage disorder such as Gaucher disease [glucocerebrosidase] or Fabry disease [alpha-galactosidase A], or a glycogen storage disorder such as Pompe disease [lysosomal acid alpha-glucosidase]). Other suitable proteins for the treatment and / or prevention of metabolic disorders are described herein. The use of muscle as a platform for expressing a nucleic acid of interest is described in U.S. Patent Publication No. US2002 / 0192189.

[0235] In some embodiments, a method of doing so in a subject in need of treatment and / or prevention of a metabolic disorder comprises administering to the skeletal muscle of the subject a therapeutically or prophylactically effective amount of a viral vector, the viral vector comprising a heterologous nucleic acid encoding a polypeptide, the metabolic disorder being the result of a deficiency and / or defect of the polypeptide. Exemplary metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein. Optionally, a polypeptide (e.g., a polypeptide secreted in its native state or engineered to be secreted, e.g., by operable association with a secretion signal sequence known in the art) is secreted. Without being bound to any particular theory, according to this embodiment, administration to skeletal muscle can result in secretion of the polypeptide into the systemic circulation and delivery to the target tissue(s). Methods of delivering the viral vector to skeletal muscle are described in more detail herein.

[0236] The methods described herein can also be practiced to produce non-coding RNAs such as antisense RNAs, RNAi, or other functional RNAs (e.g., ribozymes) for systemic delivery.

[0237] In some embodiments, a method of doing so in a subject in need of treatment and / or prevention of congenital heart failure or PAD comprises administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector, the viral vector comprising, for example, sarcoendoplasmic reticulum Ca 2+-ATPase (SERCA2a), angiogenic factors, phosphatase inhibitor I (I-1) and its fragments (e.g., I1C), RNAi against phospholamban, phospholamban inhibitors or dominant negative molecules, e.g., phospholamban S16E, zinc finger proteins that regulate the phospholamban gene, beta-2-adrenergic receptor, beta-2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, beta-adrenergic receptor kinase inhibitor (PARKct), inhibitor 1 of protein phosphatase 1 and its fragments (e.g., I1C), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that result in knockdown of G protein-coupled receptor kinase type 2 such as truncated constitutively active bARKct, Pim-1, PGC-I alpha, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-p4, mir-1, mir-133, mir-206, mir-208, and / or mir-26a, including heterologous nucleic acids encoding the same.

[0238] The injectable can be prepared in conventional forms, either as a liquid solution or suspension, a solid form suitable for solution or suspension in a liquid prior to injection, or an emulsion. Alternatively, the viral vector and / or viral capsid may be administered in a localized manner rather than a systemic manner, e.g., in a depot or sustained release formulation. Further, the viral vector and / or viral capsid can be delivered by attachment to a surgically implantable matrix (e.g., as described in U.S. Patent Publication No. US-2004-0013645-A1).

[0239] The viral vectors and / or viral capsids disclosed herein can be administered to the lungs of a subject by any suitable means, optionally, by administering an aerosol suspension of respirable particles consisting of the viral vectors and / or viral capsids that the subject inhales. The respirable particles can be liquid or solid. An aerosol of liquid particles containing the viral vectors and / or viral capsids can be produced by any suitable means known to those of skill in the art, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, for example, U.S. Patent No. 4,501,729. An aerosol of solid particles containing the viral vectors and / or capsids can likewise be produced with any solid particle drug aerosol generator by techniques known in the pharmaceutical arts.

[0240] The viral vectors and viral capsids can be administered to tissues of the CNS (e.g., the brain, the eye) and can advantageously result in a broader distribution of the viral vector or capsid than is observed in the absence of the compositions and methods described herein.

[0241] In some embodiments, the delivery vectors described herein can be administered to treat CNS diseases including genetic disorders, neurodegenerative disorders, mental disorders, and tumors. Exemplary diseases of the CNS include adrenomyeloneuropathy (AMN), Alzheimer's disease, Angelman syndrome, frontotemporal dementia, Parkinson's disease, Huntington's disease, fragile X syndrome, Canavan disease, Leigh disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease (GM2 gangliosidosis), Lesch-Nyhan disease, MC4R obesity, metachromatic leukodystrophy (MLD), MPS I (Hurler / Scheie), MPS IIIA (Sanfilippo A), Niemann-Pick C1, Rett syndrome, spinal muscular atrophy (SMA), AADC deficiency, sporadic amyotrophic lateral sclerosis (ALS), alpha-mannosidosis, aspartylglucosaminuria, Dravet syndrome, giant axonal neuropathy, globoid cell leukodystrophy (Krabbe), Glut 1 deficiency, GM1 gangliosidosis, infantile neuronal ceroid lipofuscinosis (INCL, Batten), juvenile neuronal ceroid lipofuscinosis (JNCL, Batten), late infantile neuronal ceroid lipofuscinosis (LINCL, Batten), MPS II (Hunter), MPS IIIB (Sanfilippo B), MPS IIIC (Sanfilippo C), MPS IVA (Morquio syndrome), MPS VI (Maroteaux-Lamy), peroxisome biogenesis disorders (Zellweger syndrome spectrum), Sandhoff disease (GM2 gangliosidosis), epilepsy, cerebral infarction, mental disorders including mood disorders (e.g., depression, bipolar disorder, persistent mood disorder, secondary mood disorder), schizophrenia, substance use disorders (e.g., alcohol use disorder and other substance use disorders), neuroses (anxiety, obsessive-compulsive disorder, somatic symptom disorder, dissociative disorder, grief, postpartum depression), psychoses (e.g., hallucinations and delusions), dementia, delusional disorder, attention deficit disorder, psychological disorders, sleep disorders, pain disorders, eating or weight disorders (e.g., obesity, cachexia, anorexia nervosa, and bulimia nervosa), as well as CNS cancers and tumors (e.g., pituitary tumors), but are not limited thereto.

[0242] Examples of CNS disorders include ophthalmic diseases involving the retina, posterior segment, and optic nerve (such as retinitis pigmentosa, diabetic retinopathy, and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

[0243] Although not all, most ophthalmic diseases and disorders are associated with one or more of three types of symptoms: (1) angiogenesis, (2) inflammation, and (3) degeneration. The viral vectors described herein can be used to deliver anti-angiogenic factors, anti-inflammatory factors, factors that delay cell degeneration, factors that promote cell survival, or factors that promote cell growth, and combinations thereof.

[0244] For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intravitreally (e.g., into the vitreous humor) or peribulbar (e.g., into the sub-Tenon's space). One or more neurotrophic factors can also be co-delivered either intravitreally (e.g., into the vitreous humor) or peribulbar.

[0245] Uveitis is associated with inflammation. One or more anti-inflammatory factors can be administered by intravitreal (e.g., into the vitreous humor or anterior chamber) administration of the delivery vector.

[0246] In contrast, retinitis pigmentosa is characterized by retinal degeneration. In some embodiments, retinitis pigmentosa can be treated by intravitreal (e.g., vitreous) administration of a delivery vector encoding one or more neurotrophic factors.

[0247] Age-related macular degeneration is associated with both angiogenesis and retinal degeneration. This disorder can be treated by administering a delivery vector of the present invention encoding one or more neurotrophic factors intravitreally (e.g., into the vitreous humor) and / or one or more anti-angiogenic factors either intravitreally or peribulbar (e.g., into the sub-Tenon's space).

[0248] Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment of glaucoma involves administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the delivery vectors of the present invention. Such agents include N-methyl-D-aspartic acid (NMDA) antagonists, cytokines, and neurotrophic factors, which are delivered intravitreally, optionally intravitreally.

[0249] In some embodiments, the compositions and methods described herein can be used to treat seizures, for example, to reduce the onset, incidence, or severity of seizures. The effectiveness of a therapeutic treatment for seizures can be evaluated by behavior (e.g., shaking, eye or mouth twitching) and / or electrographic means (most seizures have characteristic electrographic abnormalities). Thus, epilepsy, which is characterized by multiple seizures over time, can also be treated.

[0250] In some embodiments, a method of treating a subject in need of treatment comprises administering to the subject an AAV vector comprising a capsid protein, the capsid protein comprising any one of the amino acid sequences of SEQ ID NOs: 165-187. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 175, or a sequence at least 95% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 175, or a sequence at least 95% identical thereto. In some embodiments, the subject has Dravet syndrome. In some embodiments, the subject has Rett syndrome. In some embodiments, the subject has Angelman syndrome. In some embodiments, the subject has Niemann-Pick disease. In some embodiments, the subject has fragile X syndrome. In some embodiments, the subject has Alzheimer's disease. In some embodiments, the subject has Gaucher disease. In some embodiments, the subject has Huntington's disease. In some embodiments, the subject has Parkinson's disease. In some embodiments, the subject has Friedreich's ataxia. In some embodiments, the AAV vector is administered to the subject by intracerebroventricular (ICV) injection. In some embodiments, the AAV vector is administered to the subject by intrathecal (IT) injection. In some embodiments, the AAV vector is administered to the subject by intravenous (IV) injection.

[0251] In some embodiments, a method of treating a subject in need of treatment comprises administering to the subject an AAV vector comprising a capsid protein, the capsid protein comprising the amino acid sequence of SEQ ID NO: 175 or 180, the subject having Dravet syndrome, Rett syndrome, Angelman syndrome, Niemann-Pick disease, or fragile X syndrome, and the AAV vector being administered to the subject by ICV or IT injection.

[0252] In some embodiments, a method of treating a subject in need of treatment comprises administering to the subject an AAV vector comprising a capsid protein, the capsid protein comprising the amino acid sequence of SEQ ID NO: 175 or 180, the subject having Gaucher disease, Huntington's disease, Parkinson's disease, or Friedreich's ataxia, and the AAV vector being administered to the subject by ICV or IT injection.

[0253] In some embodiments, somatostatin (or an active fragment thereof) is administered to the brain using a delivery vector to treat a pituitary tumor. According to this embodiment, the delivery vector encoding somatostatin (or an active fragment thereof) is administered by microinjection into the pituitary gland. Similarly, such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary gland). The nucleic acid (e.g., GenBank accession number J00306) and amino acid (e.g., GenBank accession number P01166, containing the processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.

[0254] In some embodiments, the vector can include a secretion signal described in U.S. Patent No. 7,071,172.

[0255] In some embodiments, the viral vector and / or viral capsid is administered to the CNS (e.g., to the brain or eye). The viral vector and / or capsid can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (including the cerebrum, cortex, basal ganglia, hippocampus, and amygdala, including the striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. The viral vector and / or capsid can also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve.

[0256] The viral vector and / or capsid can be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more distributed administration of the delivery vector. The viral vector and / or capsid can also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0257] The viral vector and / or capsid can be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of sugars such as mannitol), intranasal, intra-aural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and peribulbar (e.g., sub-Tenon's space) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons. In some embodiments, the viral vector and / or capsid are administered as a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment in the CNS. In some embodiments, the viral vector and / or capsid can be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye can be by topical application of droplets. As a further alternative, the viral vector and / or capsid can be administered as a solid sustained-release formulation (see, e.g., U.S. Patent No. 7,201,898).

[0258] In some embodiments, the viral vector can be used for retrograde transport for treating and / or preventing diseases and disorders associated with motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, the viral vector can be delivered to muscle tissue that can migrate to neurons.

[0259] Numbered embodiments Notwithstanding the appended claims, the present disclosure describes the following numbered embodiments.

[0260] 1. An adeno-associated virus (AAV) vector comprising (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises a peptide having any one of the sequences of SEQ ID NOs: 12 to 20.

[0261] 2. The AAV vector according to embodiment 1, wherein the cargo nucleic acid comprises 5' and 3' AAV inverted terminal repeats.

[0262] 3. The AAV vector according to embodiment 1 or 2, wherein the cargo nucleic acid comprises a transgene.

[0263] 4. The AAV vector according to embodiment 3, wherein the transgene encodes a therapeutic protein or RNA.

[0264] 5. The AAV vector according to any one of embodiments 1 to 4, wherein the recombinant capsid protein has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the natural sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV capsid.

[0265] 6. The AAV vector according to embodiment 5, wherein the recombinant capsid protein has at least 90% sequence identity to the natural sequence of the AAV9 capsid.

[0266] 7. The AAV vector according to any one of Embodiments 1 to 6, wherein the peptide is located at amino acid positions corresponding to amino acids 451 to 458 of the native AAV9 capsid, or equivalent amino acid residues of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV, and the peptide is selected from any one of SEQ ID NOs: 12 to 18.

[0267] 8. The AAV vector according to any one of Embodiments 1 to 6, wherein the peptide is located at amino acid positions corresponding to amino acids 587 to 594 of the native AAV9 capsid, or equivalent amino acid residues in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV, and the peptide is selected from SEQ ID NO: 19 or 20.

[0268] 9. The AAV vector according to Embodiment 1, wherein the recombinant capsid protein comprises: a) a first peptide having any one of the sequences of SEQ ID NOs: 12 to 18; and b) a second peptide having any one of the sequences of SEQ ID NOs: 19 to 20.

[0269] 10. The AAV vector according to Embodiment 9, wherein the first peptide is at amino acid positions 451 to 458, the second peptide is at amino acid positions 587 to 594, and the amino acid numbering is based on equivalent amino acid residues in the native AAV9 capsid, or AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV.

[0270] 11. The AAV vector according to any one of Embodiments 1 to 10, wherein the peptide inhibits the binding of at least one antibody to the capsid protein.

[0271] 12. The AAV vector according to Embodiment 11, wherein the peptide inhibits the neutralization of the infectivity of the AAV vector by the antibody.

[0272] 13. The AAV vector according to any one of Embodiments 1 to 12, wherein the peptide selectively binds to a receptor expressed on the cell surface of the central nervous system (CNS).

[0273] 14. The AAV vector according to Embodiment 13, wherein the cell is in the prefrontal cortex, thalamus, cerebellar cortex, dentate nucleus, spinal cord, or dorsal root ganglion.

[0274] 15. The AAV vector according to any one of Embodiments 1 to 14, wherein the peptide selectively binds to a receptor expressed on the cell surface of the heart.

[0275] 16. The AAV vector according to any one of Embodiments 1 to 15, wherein the capsid protein further comprises a peptide that modifies the HI loop of the capsid.

[0276] 17. An adeno-associated virus (AAV) vector comprising (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein contains a peptide having the sequence of X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 at amino acids 451 to 458 of the natural AAV9 capsid protein sequence (SEQ ID NO: 158), and the peptide is not present in the natural AAV9 capsid protein sequence.

[0277] 18.X 1 is not I, but X 2 is not N, but X 3 is not G, but X 4 is not S, but X 5 is not G, but X 6 is not Q, but X 7 is not N and / or is X 8 The AAV vector according to Embodiment 17, which is not Q

[0278] 19.X 1 The AAV vector according to Embodiment 18, which is S, F, Q, G, K, or R

[0279] 20.X 2 The AAV vector according to Embodiment 18 or 19, which is C, G, R, D, T, or Q

[0280] 21.X 3 The AAV vector according to any one of Embodiments 18 to 20, which is Q, V, G, Y, R, F, or D

[0281] 22.X 4 The AAV vector according to any one of Embodiments 18 to 21, which is P, Q, A, or R

[0282] 23.X 5 The AAV vector according to any one of Embodiments 18 to 22, which is T, N, A, P, or I

[0283] 24.X 6 The AAV vector according to any one of Embodiments 18 to 23, which is V, Q, A, or I

[0284] 25.X 7 The AAV vector according to any one of Embodiments 18 to 24, which is M, P, R, Q, or N

[0285] 26.X 8The AAV vector according to any one of Embodiments 18 to 25, which is N, L, F, E, H, or A.

[0286] 27.X 1 is S, and X 2 is C, and X 3 is Q, and X 4 is P, and X 5 is T, and X 6 is V, and X 7 is M, and X 8 The AAV vector according to Embodiment 17, which is N.

[0287] 28.X 1 is F, and X 2 is G, and X 3 is V, and X 4 is P, and X 5 is N, and X 6 is Q, and X 7 is P, and X 8 The AAV vector according to Embodiment 17, which is L.

[0288] 29.X 1 is Q, and X 2 is R, and X 3 is G, and X 4 is Q, and X 5 is A, and X 6 is A, and X 7 is P, and X 8 The AAV vector according to Embodiment 17, which is F.

[0289] 30.X 1 is G, and X 2 is D, and X 3 is Y, and X 4 is A, and X 5 is P, and X 6 is I, and X 7 is R, and X 8 The AAV vector according to Embodiment 17, which is E.

[0290] 31.X 1 is K, and X2 is T, and X 3 is R, and X 4 is R, and X 5 is I, and X 6 is V, and X 7 is Q, and X 8 is H, the AAV vector according to Embodiment 17.

[0291] 32.X 1 is F, and X 2 is G, and X 3 is F, and X 4 is P, and X 5 is N, and X 6 is Q, and X 7 is P, and X 8 is L, the AAV vector according to Embodiment 17.

[0292] 33.X 1 is R, and X 2 is Q, and X 3 is D, and X 4 is Q, and X 5 is P, and X 6 is I, and X 7 is N, and X 8 is A, the AAV vector according to Embodiment 17.

[0293] 34. An adeno-associated virus (AAV) vector comprising (i) a mutant AAV9 capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein has X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 a peptide having the sequence (SEQ ID NO: 158) at amino acids 587-594 of the native AAV9 capsid protein sequence, and the peptide is not present in the native AAV9 capsid protein sequence, the adeno-associated virus (AAV) vector.

[0294] 35.X 1 is not A, but X 2 is not Q, but X 3 is not A, but X 4 is not Q, but X 5 is not A, but X 6 is not Q, but X 7 is not T, and / or X 8 An AAV vector according to Embodiment 34, wherein it is not G

[0295] 36.X 1 An AAV vector according to Embodiment 35, wherein it is S

[0296] 37.X 2 An AAV vector according to Embodiment 35 or 36, wherein it is K or T

[0297] 38.X 3 An AAV vector according to any one of Embodiments 35 to 37, wherein it is V

[0298] 39.X 4 An AAV vector according to any one of Embodiments 35 to 38, wherein it is E or D

[0299] 40.X 5 An AAV vector according to any one of Embodiments 35 to 39, wherein it is S

[0300] 41.X 6 An AAV vector according to any one of Embodiments 35 to 40, wherein it is W or I

[0301] 42.X 7 An AAV vector according to any one of Embodiments 35 to 41, wherein it is T or A

[0302] 43.X 8 An AAV vector according to any one of Embodiments 35 to 42, wherein it is E or I

[0303] 44.X 1 is S, and X2 is K, and X 3 is V, and X 4 is E, and X 5 is S, and X 6 is W, and X 7 is T, and X 8 is E, the AAV vector according to Embodiment 34.

[0304] 45.X 1 is S, and X 2 is T, and X 3 is V, and X 4 is D, and X 5 is S, and X 6 is I, and X 7 is A, and X 8 is I, the AAV vector according to Embodiment 34.

[0305] 46. An adeno-associated virus (AAV) vector comprising (i) a recombinant capsid protein and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the capsid protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 165 to 187.

[0306] 47. The AAV vector according to Embodiment 46, wherein the capsid protein comprises an amino acid sequence of any one of SEQ ID NOs: 165 to 187.

[0307] 48. The AAV vector according to Embodiment 47, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 175.

[0308] 49. The AAV vector according to Embodiment 47, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 180.

[0309] 50. The AAV vector according to any one of embodiments 46 to 49, wherein the AAV vector selectively delivers the cargo nucleic acid to cells or tissues of the central nervous system.

[0310] 51. The AAV vector according to embodiment 50, wherein the tissue of the central nervous system is the premotor area, thalamus, cerebellar cortex, dentate nucleus, spinal cord, or dorsal root ganglion.

[0311] 52. The AAV vector according to any one of embodiments 46 to 49, wherein the AAV vector delivers the cargo nucleic acid to the brain but does not deliver the AAV vector to the heart.

[0312] 53. The AAV vector according to any one of embodiments 46 to 49, wherein the AAV vector delivers the cargo nucleic acid to the brain and the heart.

[0313] 54. The AAV vector according to embodiment 53, wherein the delivery of the cargo nucleic acid is more to the brain than to the heart.

[0314] 55. The AAV vector according to embodiment 53, wherein the delivery of the cargo nucleic acid is substantially equal in the heart and the brain.

[0315] 56. A nucleic acid sequence encoding a recombinant capsid protein of the AAV vector according to any one of embodiments 1 to 55.

[0316] 57. The nucleic acid sequence according to embodiment 56, wherein the nucleic acid sequence is a DNA sequence.

[0317] 58. The nucleic acid sequence according to embodiment 56, wherein the nucleic acid sequence is an RNA sequence.

[0318] 59. An expression vector comprising the nucleic acid sequence according to any one of embodiments 56 to 58.

[0319] 60. A cell comprising the nucleic acid sequence according to any one of embodiments 56 to 58.

[0320] 61. A cell comprising the expression vector according to Embodiment 59.

[0321] 62. A pharmaceutical composition comprising the AAV vector according to any one of Embodiments 1 to 55.

[0322] 63. The pharmaceutical composition according to Embodiment 62, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0323] 64. A pharmaceutical composition comprising the cell according to Embodiment 60 or 61.

[0324] 65. The pharmaceutical composition according to Embodiment 64, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0325] 66. A method for treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the AAV vector according to any one of Embodiments 1 to 55.

[0326] 67. The method according to Embodiment 66, wherein the subject has a disease or disorder of the central nervous system.

[0327] 68. The method according to Embodiment 67, wherein the disease or disorder of the central nervous system is Dravet syndrome.

[0328] 69. The method according to Embodiment 67, wherein the disease or disorder of the central nervous system is Rett syndrome.

[0329] 70. The method according to Embodiment 67, wherein the disease or disorder of the central nervous system is Angelman syndrome.

[0330] 71. The method according to Embodiment 67, wherein the disease or disorder of the central nervous system is Niemann-Pick disease.

[0331] 72. The method according to Embodiment 67, wherein the disease or disorder of the central nervous system is fragile X syndrome.

[0332] 73. The method according to embodiment 67, wherein the disease or disorder of the central nervous system is Gaucher disease.

[0333] 74. The method according to embodiment 67, wherein the disease or disorder of the central nervous system is Huntington's disease.

[0334] 75. The method according to embodiment 67, wherein the disease or disorder of the central nervous system is Parkinson's disease.

[0335] 76. The method according to embodiment 67, wherein the disease or disorder of the central nervous system is Friedreich's ataxia.

[0336] 77. The method according to any one of embodiments 66 to 76, wherein the AAV vector comprises a capsid protein, and the capsid protein comprises any one amino acid sequence of SEQ ID NOs: 165 to 187.

[0337] 78. The method according to any one of embodiments 66 to 76, wherein the AAV vector comprises a capsid protein, and the capsid protein comprises the amino acid sequence of SEQ ID NO: 175.

[0338] 79. The method according to any one of embodiments 66 to 76, wherein the AAV vector comprises a capsid protein, and the capsid protein comprises the amino acid sequence of SEQ ID NO: 180.

[0339] 80. The method according to any one of embodiments 66 to 79, wherein the AAV vector is administered to the subject by intracerebroventricular (ICV) injection.

[0340] 81. The method according to any one of embodiments 66 to 79, wherein the AAV vector is administered to the subject by intrathecal (IT) injection.

[0341] 82. The method according to any one of embodiments 66 to 79, wherein the AAV vector is administered to the subject by intravenous (IV) injection.

[0342] 83. The method according to any one of embodiments 66 to 82, wherein the subject is a mammal.

[0343] 84. The method according to embodiment 83, wherein the subject is a human.

[0344] 85. An in vitro method for introducing a nucleic acid molecule into a cell, the method comprising contacting the cell with an AAV vector according to any one of embodiments 1 to 55.

[0345] 86. An AAV vector according to any one of embodiments 1 to 55 for use as a medicament.

[0346] 87. An AAV vector according to any one of embodiments 1 to 55 for use in a method of treating a subject in need of treatment.

[0347] 88. An AAV vector according to any one of embodiments 1 to 55 for use in a method of treating or preventing a CNS disease or disorder in a subject in need of treatment.

Examples

[0348] The following examples included in this specification are for illustrative purposes only and are not intended to be limiting. As used herein, the terms STRD.101 and STRD.102 are used to describe capsid protein sequences, and AAV-STRD.101 and AAV-STRD.102 are used to describe AAV vectors containing capsid proteins. However, the terms STRD.101 and STRD.102 can be used to describe AAV vectors containing the named capsids in some contexts, as will be apparent to those skilled in the art.

[0349] Example 1. Combinatorial engineering and selection of AAV vectors that avoid antibodies The method for generating AAV variants that avoid antibodies is as follows. The first step involves identifying the three-dimensional antigen epitopes on the AAV capsid surface, for example, using cryo-electron microscopy. Next, the selected residues within the antigen motif are subjected to mutagenesis using degenerate primers that have each codon replaced by nucleotide NNK and gene fragments combined together by Gibson assembly and / or multi-step PCR. The capsid-encoding gene containing the degenerate library of the mutant antigen motif is cloned into the wild-type AAV genome to replace the original Cap encoding the DNA sequence, yielding a plasmid library. Next, the plasmid library is transfected into 293 producer cell lines with an adenovirus helper plasmid to generate an AAV capsid library, which can be subjected to selection. The success of AAV library generation is confirmed via DNA sequencing.

[0350] To select for new AAV strains that can avoid neutralizing antibodies (NAb) and / or target the central nervous system (CNS), the AAV library is subjected to multiple rounds of infection in non-human primates. At each stage, the tissue of interest is isolated from the animal subject. Cell lysates taken from the tissue of interest are sequenced to identify AAV isolates that avoid antibody neutralization. After multiple rounds of infection in non-human primates, the isolated sequences from each mutagenized region are combined in all permutations and combinations.

[0351] As a specific example, the common antigen motif on the AAV capsid protein (VP1) was subjected to mutagenesis as described above. The degenerate library (Figure 1A) was then subjected to the first round of infection in non-human primates (intravenous injection). Tissues were harvested on day 7 post-infection and sequenced to identify a single AAV isolate.

[0352] Various recombinant AAV isolates were identified in tissue samples including the spinal cord, dorsal root ganglia, frontal lobe, temporal lobe, occipital lobe, putamen, globus pallidus, thalamus, tonsil, hippocampus, substantia nigra, pons, cerebellum, and medulla oblongata. The results of this first round of evolution are shown in Figure 1B.

[0353] Subsequently, the recombinant AAV isolated during the first round of evolution (Figure 1B) was reintroduced into a second non-human primate. Tissues were harvested on day 7 post-infection and sequenced to identify a single AAV isolate. The results of this second round of evolution are shown in Figure 1C.

[0354] The most frequently occurring recombinant AAVs were sequenced. The substitutions present in these AAVs are shown in Tables 6.1 and 6.2. These data indicate that recombinant AAV virions having capsid proteins containing the substitutions listed in Tables 6.1 and 6.2 evade neutralizing antibodies in vivo in non-human primates and have tropism for the desired target tissues.

[0355] Example 2: Manufacturability of Recombinant AAV Vectors To determine whether the various recombinant AAVs identified in Example 1 could be produced in large-scale systems, AAV was produced according to standard methods and the yields were compared to the yields of wild-type AAV vectors.

[0356] AAV was produced in HEK293 cells according to a standard triple transfection protocol. Briefly, cells were transfected with (i) a plasmid containing either the wild-type AAV9 capsid sequence, the STRD.101 capsid variant sequence (SEQ ID NO: 180), or the STRD.102 capsid variant sequence (SEQ ID NO: 175), (ii) a plasmid containing the 5’ ITR, transgene, and 3’ ITR sequences, and (ii) a plasmid containing the helper genes necessary for AAV production. Two different transgenes were used with each capsid in a self-complementary construct. Cells were then lysed and the virions were purified using affinity columns, CsCl density ultracentrifugation, and dialysis. Subsequently, a PCR-based quantification approach was used to measure the yield of each AAV.

[0357] As shown in Fig. 2, the recombinant AAV vectors containing the STRD.101 and STRD.102 capsids had a yield similar to that of wild-type AAV9. This data confirms that recombinant AAVs containing recombinant capsid proteins are suitable for commercial production.

[0358] Example 3: In Vitro Transduction Using Recombinant AAV Viral Vectors To confirm whether the recombinant AAV vectors of Example 1 are generally infectious and can transduce cells in culture, various AAV vectors were prepared according to standard protocols.

[0359] The infectivity of the recombinant AAVs was tested using a standard TCID50 assay. Briefly, HeLa RC32 cells were infected with recombinant AAV particles at doses ranging from 5 digits in the presence of adenovirus (Ad5). After 72 hours, DNA was extracted and vector genome replication was quantified by qPCR.

[0360] The particle-to-infectivity ratio was calculated to determine infectivity. As shown in Fig. 3, the infectivity ratio of the AAV-STRD.101 vector was lower compared to that of wild-type AAV9. Since a lower infectivity ratio leads to higher potency, AAV-STRD.101 is more infectious than wild-type AAV9.

[0361] Individually, infectivity was also determined in various cell lines. Recombinant AAVs packaging the luciferase transgene were generated and contacted with cells in culture at a dose of 10,000 vector genomes (vg) per cell. Forty-eight hours after infection, the cells were lysed. The lysates were contacted with a bioluminescence substrate and relative fluorescence units (RFUs) were measured. As shown in Figs. 4A - 4D, the AAV-STRD.101 vector infected U87 cells (human glioblastoma cell line, Fig. 4A), N2A cells (mouse neural crest-derived cell line, Fig. 4B), SY5Y cells (human neuroblastoma cell line, Fig. 4C), and U2OS cells (human osteosarcoma cell line, Fig. 4D) at levels comparable to wild-type AAV9.

[0362] Therefore, this data shows that the recombinant AAV vector of Example 1 can effectively transduce cells in culture.

[0363] Example 4: In Vivo Characterization of Recombinant AAV Targeting the Central Nervous System Two recombinant capsid proteins, STRD.101 and STRD.102, were selected for in vivo characterization. Recombinant AAVs containing these capsid proteins and packaging the native tdTomato fluorescent transgene were generated. The recombinant AAVs were administered to neonatal mice by intracerebroventricular injection on day 0. Three weeks after injection, brain tissues were harvested, fixed, and expression was evaluated by visual assessment of tdTomato fluorescence. Figure 5 provides representative images showing tdTomato expression in coronal vibratome sections 24 hours after fixation with 4% PFA. These same sections were also visualized using immunohistochemistry (Figure 6). As shown in the images of Figure 5 and Figure 6, the AAV9, AAV-STRD.102, and AAV-STRD.101 vectors each had different distributions in the brain tissue, and the highest transgene expression was localized near the injection site. Collectively, this data shows that the tested recombinant AAVs were successful in delivering the transgene to target cells in vivo after intracerebroventricular injection.

[0364] The AAV-STRD.101 and AAV-STRD.102 vectors packaging tdTomato were also administered to four adult mice by intravenous injection at a dose of 5.5×10 13 vg / kg. Three weeks after injection, the liver and heart were harvested, fixed, and the expression profile was evaluated by visual assessment of tdTomato fluorescence.

[0365] A representative image from one mouse showing TdTomato expression in a vibratome liver section 24 hours after fixation in 4% PFA is provided in Figure 7. Notably, the AAV-STRD.102 and AAV-STRD.101 vectors were non-targeted to the liver compared to wild-type AAV9. This desirable property was unexpected because counter-screening in the liver did not occur during evolution.

[0366] A representative image from one mouse showing TdTomato expression in a vibratome heart section 24 hours after fixation in 4% PFA is provided in Figure 8. Specifically, the vectors tested had different tropisms for the heart. Specifically, the AAV-STRD.102 vector had lower infectivity in the heart compared to AAV-STRD.101. This differential transduction was completely unexpected because screening of the heart did not occur during evolution.

[0367] In summary, this data indicates that the AAV-STRD.102 and AAV-STRD.101 vectors may be successful in targeting CNS tissue in vivo and avoiding clearance by the liver, and are powerful tools for gene therapy. Considering their different tropisms (i.e., AAV-STRD.101 was more infectious in the heart than AAV-STRD.102), these vectors are powerful tools for targeting gene therapy treatment to specific desired tissues.

[0368] Example 5: In Vivo Distribution of Recombinant AAV in Non-Human Primates To determine the in vivo distribution, recombinant AAV was administered to non-human primates. Recombinant AAV was administered by intravenous (IV) and intracerebroventricular (ICV) injection (Figure 9). AAV-STRD.101 was administered at a dose of 2.9×10 13 vg / kg by IV injection and 2.1×10 13 vg (black dots) by ICV injection. AAV-STRD.102 was administered at a dose of 2.8×10 13vg / kg, and by ICV injection at a dose of 3.0×10 13 vg (white dots). After 30 days, the animals were sacrificed and the viral load in various CNS tissues was measured by qPCR.

[0369] As shown in Figure 9, both AAV-STRD.102 and AAV-STRD.101 infected various CNS tissues. In addition, since AAV showed high levels of transduction, this data suggests that these AAVs are likely to avoid neutralizing AAV in vivo.

[0370] Example 6: A cell therapy method for treating a subject in need of treatment Cells are transduced ex vivo using an AAV vector. For some purposes, the cells can be autologous (i.e., derived from the subject being treated) or allogeneic (i.e., derived from a different subject / donor). After transduction of the cells with AAV and verification of the expression of the transgene, the cells are administered to the subject using standard clinical methods.

[0371] The cells can be administered to the subject once, or the administration can be repeated multiple times at therapeutically effective intervals. The number of cells administered varies, for example, depending on the disease or condition being treated, the severity of the subject's disease / condition, and the height and weight of the subject.

[0372] Example 7: A gene therapy method for treating a subject in need of treatment The AAV vectors described herein (e.g., AAV vectors comprising a capsid having the sequence of SEQ ID NO: 175 or 180) are administered to a subject in need thereof, and the subject has a disease or disorder of the CNS. The AAV vector is administered to the subject once, or the administration can be repeated multiple times at therapeutically effective intervals. The administration is performed by one or more therapeutically effective routes such as intravenous (IV), intracerebroventricular (ICV), or intrathecal (IT) injection. The dosage of the AAV vector varies depending on, for example, the disease or condition being treated, the severity of the subject's disease / condition, and the height and weight of the subject. For example, the dosage of AAV administered to the subject can be 2.8×10 13 vg / kg or 2.9×10 13 vg / kg when the AAV vector is administered by IV injection. When the AAV vector is administered by ICV injection, the dosage can be 2.1×10 13 vg or 3.0×10 13 vg. In some protocols, the AAV vector can be administered to the subject by both IV and ICV injection.

[0373] The foregoing are illustrative of the invention and should not be construed as limiting thereof. The invention is defined by the following claims and equivalents of the claims are included therein.

Claims

1. An adeno-associated virus (AAV) vector comprising: (i) a recombinant capsid protein; and (ii) a cargo nucleic acid encapsulated by the capsid protein, wherein the recombinant capsid protein comprises a peptide having the amino acid sequence of SEQ ID NO: 19 at positions 587 to 594 numbered by SEQ ID NO: 9, and the recombinant capsid protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

9. The adeno-associated virus (AAV) vector.

2. The AAV vector according to claim 1, wherein the cargo nucleic acid comprises 5' and 3' AAV inverted terminal repeats.

3. The AAV vector according to claim 2, wherein the cargo nucleic acid comprises a transgene.

4. The AAV vector according to claim 3, wherein the transgene encodes a therapeutic protein or RNA.

5. The AAV vector according to claim 1, wherein the recombinant capsid protein has at least 95% sequence identity to SEQ ID NO:

9.

6. The AAV vector according to claim 5, wherein the recombinant capsid protein has at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 172 and 174-180.

7. The AAV vector according to claim 6, wherein the recombinant capsid protein comprises any one of the amino acid sequences of SEQ ID NOs: 172 and 174-180.

8. The AAV vector according to claim 7, wherein the recombinant capsid protein comprises the amino acid sequence of SEQ ID NO:

175.

9. The AAV vector according to claim 7, wherein the recombinant capsid protein comprises the amino acid sequence of SEQ ID NO:

172.

10. The AAV vector according to claim 1, wherein the recombinant capsid protein comprises a peptide having the amino acid sequence of SEQ ID NO: 18 at positions 451 to 458 of the amino acid sequence of SEQ ID NO:

9.

11. The AAV vector according to claim 10, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:

180.

12. The AAV vector according to any one of claims 1 to 11, wherein the peptide inhibits the binding of at least one antibody to the capsid protein.

13. The AAV vector according to claim 12, wherein the peptide inhibits neutralization of the infectivity of the AAV vector by the antibody.

14. The AAV vector according to any one of claims 1 to 11, wherein the peptide selectively binds to a receptor expressed on the surface of cells of the central nervous system (CNS).

15. The AAV vector according to claim 14, wherein the cells are in the motor cortex, thalamus, cerebellar cortex, dentate nucleus, spinal cord, or dorsal root ganglion.

16. The AAV vector according to any one of claims 1 to 11, wherein the peptide selectively binds to a receptor expressed on the surface of cells of the heart.

17. The AAV vector according to any one of claims 1 to 11, wherein the capsid protein further comprises a peptide that modifies the HI loop of the capsid.

18. A polynucleotide encoding a recombinant capsid protein of the AAV vector according to any one of claims 1 to 11.

19. The polynucleotide according to claim 18, wherein the polynucleotide is a DNA sequence.

20. The polynucleotide according to claim 18, wherein the polynucleotide is an RNA sequence.

21. An expression vector comprising the polynucleotide according to claim 18.

22. A cell comprising the polynucleotide according to claim 18.

23. A cell comprising the expression vector according to claim 21.

24. A pharmaceutical composition comprising the AAV vector according to any one of claims 1 to 11.

25. The pharmaceutical composition according to claim 24, wherein the composition further comprises a pharmaceutically acceptable carrier.

26. A pharmaceutical composition comprising the cell according to claim 22 or 23.

27. The pharmaceutical composition according to claim 26, wherein the composition further comprises a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising the AAV vector according to any one of claims 1 to 11 for use in the treatment of a subject in need of treatment.

29. The pharmaceutical composition according to claim 28, wherein the subject has a disease or disorder of the central nervous system.

30. The pharmaceutical composition according to claim 28 or 29, wherein the subject is a mammal.

31. The pharmaceutical composition according to claim 30, wherein the subject is a human.

32. An in vitro method for introducing a nucleic acid molecule into a cell, the method comprising contacting the cell with an AAV vector according to any one of claims 1 to 11.

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