AAV vectors targeting T cells

Abstract

The present disclosure provides variant AAV capsid proteins and AAV capsids, and viral vectors comprising the same.The viral vectors described herein can increase transduction in target cells of interest, such as T cells, compared to native AAV capsid sequences.The present disclosure also provides a method for administering the viral vectors and viral capsids of the present disclosure to cells or patients in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 137,497, filed January 14, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present disclosure relates to variant capsid proteins derived from adeno-associated viruses (AAV), as well as viral capsids and viral vectors comprising the same. In particular, the present disclosure relates to variant AAV capsid proteins and AAV capsids comprising the same that can be incorporated into viral vectors and confer an enhanced cell transduction phenotype of T cells in vivo and / or ex vivo.

[0003] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety: A computer-readable copy of the Sequence Listing (Filename: STRD_022_01WO_Sequence_Listing.txt, Recorded on: January 13, 2022, File size approximately 163.4 kilobytes). [Background technology]

[0004] Adeno-associated viruses (AAV) are small, single-stranded DNA viruses belonging to the genus Dependovirus in the family Parvoviridae. AAV is a promising viral vector for gene therapy due to its ability to infect many cell and tissue types, lack of pathogenicity, low immunogenicity, and ability to effectively transduce non-dividing cells. Each of the known AAV serotypes differs in its ability to infect specific cell types.

[0005] There is growing interest in the use of AAVs to target T cells. For example, AAVs to target T cells can be used in gene therapy to prevent, limit, and / or reverse T cell exhaustion. T cell exhaustion is a state of T cell dysfunction that occurs in many chronic infections and cancers, and has also been shown to reduce the effectiveness of CAR-T therapy. However, AAVs typically do not transduce T cells at high levels.

[0006] Thus, there is a need in the art for improved AAV vectors that can target T cells and enhance transduction efficiency. Summary of the Invention

[0007] The present disclosure relates to adeno-associated virus (AAV) capsid proteins comprising one or more transduction-associated peptides, as well as AAV capsids and viral vectors comprising the same. The disclosed transduction-associated peptides can enhance cellular transduction of AAV vectors into a desired cell type, such as T cells.

[0008] The present disclosure provides a recombinant adeno-associated virus (AAV) vector comprising a capsid protein, the capsid protein comprising a transduction-associated peptide having a sequence of any one of SEQ ID NOs: 17-23. In some embodiments, the capsid protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the transduction-associated peptide replaces amino acids corresponding to amino acids 454-460 of SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0009] The present disclosure provides a recombinant AAV vector comprising a capsid protein, the capsid protein comprising a sequence of SEQ ID NO:1, wherein amino acids 454-460 of SEQ ID NO:1 are replaced by a transduction-associated peptide comprising the sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO:24). In some embodiments, X1 is not G, X2 is not S, X3 is not A, X4 is not Q, X5 is not N, X6 is not K, and / or X7 is not D. In some embodiments, X1 is H, M, A, Q, V, or S. In some embodiments, X2 is A or T. In some embodiments, X3 is P or T. In some embodiments, X4 is R or D. In some embodiments, X5 is V, Q, C, S, or D. In some embodiments, X6 is E, A, or P. In some embodiments, X7 is E, G, N, T, or A. In some embodiments, X1 is H, X2 is A, X3 is P, X4 is R, X5 is V, X6 is E, and X7 is E. In some embodiments, X1 is M, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. In some embodiments, X1 is H, X2 is T, X3 is T, X4 is D, X5 is C, X6 is A, and X7 is N. In some embodiments, X1 is A, X2 is A, X3 is P, X4 is R, X5 is S, X6 is E, and X7 is T. In some embodiments, X1 is Q, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. In some embodiments, X1 is V, X2 is A, X3 is P, X4 is R, X5 is D, X6 is P, and X7 is A. In some embodiments, X1 is S, X2 is A, X3 is P, X4 is R, X5 is S, X46 is E, and X7 is N.

[0010] In some embodiments, the capsid protein comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid sequence having about 99% identity to SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

[0011] The present disclosure provides a recombinant AAV vector comprising a capsid protein, the capsid protein comprising a transduction-associated peptide having the amino acid sequence of SEQ ID NO: 16, the transduction-associated peptide substituting amino acids 454-460 relative to SEQ ID NO: 1. In some embodiments, the transduction-associated peptide has the amino acid sequence of any one of SEQ ID NOs: 17-23.

[0012] The present disclosure provides a nucleic acid encoding a recombinant AAV capsid protein having a sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. In some embodiments, the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid is an RNA sequence. The present disclosure provides an expression vector comprising any one of the nucleic acids disclosed herein. The present disclosure further provides a cell comprising any one of the nucleic acids disclosed herein or any one of the expression vectors disclosed herein.

[0013] In some embodiments, any one of the recombinant AAV vectors disclosed herein further comprises a cargo nucleic acid encapsidated by a capsid protein. In some embodiments, the cargo nucleic acid encodes a therapeutic protein or a therapeutic RNA. In some embodiments, the AAV vector exhibits increased cell transduction compared to an AAV vector that does not include a transduction-associated peptide. In some embodiments, the cell is a T cell. In some embodiments, the AAV vector exhibits increased T cell nucleus transduction compared to an AAV vector that does not include a transduction-associated peptide. In some embodiments, the AAV vector exhibits increased T cell cytosol transduction compared to an AAV vector that does not include a transduction-associated peptide.

[0014] The present disclosure provides a composition comprising any one of the recombinant AAV vectors disclosed herein, any one of the nucleic acids disclosed herein, any one of the expression vectors disclosed herein, or any one of the cells disclosed herein. The present disclosure further provides a pharmaceutical composition comprising any one of the cells disclosed herein, or any one of the recombinant AAV vectors disclosed herein, and a pharma- ceutically acceptable carrier.

[0015] The present disclosure provides a method of delivering an AAV vector to a cell, comprising contacting the cell with any one of the AAV vectors disclosed herein. In some embodiments, the contacting of the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is a T cell. The present disclosure provides a method of treating a subject in need of treatment, comprising administering to the subject an effective amount of any one of the AAV vectors disclosed herein. The present disclosure provides a method of treating a subject in need of treatment, comprising administering to the subject a cell that has been contacted ex vivo with any one of the AAV vectors disclosed herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. The present disclosure provides any one of the AAV vectors disclosed herein for use as a medicament. The present disclosure also provides any one of the AAV vectors disclosed herein for use in a method of treating a subject in need of treatment.

[0016] These and other embodiments are described in further detail in the detailed description that follows. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the volumetric yield of total vector genomes (vg) obtained using the manufacturing process described in Example 2 for various AAV vectors containing variant capsids compared to wild-type AAV6. [Diagram 2] Images from microscopic analysis of T cells transduced with either wild-type AAV6 or AAV vectors containing the indicated AAV6 capsid variants are shown. Each AAV vector packaged a GFP transgene. Images were obtained after transducing cells with AAV vectors using different multiplicities of infection (MOI) as indicated. [Diagram 3]Figure 1 shows the results of flow cytometry analysis of T cells transduced with either wild-type AAV6 packaged with a GFP transgene or the indicated AAVs containing variant capsids packaged with a GFP transgene. A shows the size and granularity (i.e., forward and side scatter) of the cell samples tested from which the cell populations of interest (circled on the figure) were identified. B shows the size and granularity of only the cell populations selected for analysis. C shows the fluorescence (FITC) signal measured for the cell populations of interest. There was an increase in fluorescence in cells transduced with AAV vectors containing STRD-207 capsids compared to cells transduced with wild-type AAV6. [Figure 4] Figure 1 shows plots of the percentage of GFP-positive T cells obtained from flow cytometry experiments performed with wild-type AAV6 or AAV containing the indicated capsid variants. T cells were from two different human donors (donor 11 and donor 12). Different MOIs were used as indicated (10,000, 5,000, and 2,500 for donor 12 T cells, 15,000, 7,500, and 3,750 for donor 11 T cells). [Diagram 5] Figure 1 shows bubble plots representing individual AAV isolates containing variant capsids obtained from nuclear (A) and cytoplasmic (B) fractions of activated T cells after three rounds of evolution and selection for transduction of T cells as described in Example 1. Each bubble represents the amino acid sequence of a distinct capsid protein, and the radius of the bubble is proportional to the number of reads for that variant in each library. The y-axis represents the absolute number of reads. The data are spread along the x-axis for ease of visualization. The predominant isolates were selected for sequencing analysis. [Figure 6] The sequences of transduction-associated peptides identified in AAV vectors that are abundant in the nuclear or cytoplasmic fractions of T cells are shown. These transduction-associated peptides are located at amino acids 464-456 of the capsid protein, with amino acid numbering corresponding to wild-type AAV6 (SEQ ID NO: 1). The sequences shown in FIG. 6 correspond, from top to bottom, to SEQ ID NOs: 17-23. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 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 detailed description of the present invention are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0019] All publications, patent applications, patents, articles, GenBank or other accession numbers, and other references mentioned herein are incorporated by reference in their entirety.

[0020] All amino acid position designations in the AAV capsid protein in this disclosure and the appended claims are with respect to the numbering of the VP1 capsid subunit. Those skilled in the art will understand that the modifications described herein, when inserted into the AAV cap gene, can result in modifications in the VP1, VP2, and / or VP3 capsid subunits. Alternatively, the capsid subunits can be expressed independently, with modifications occurring in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3).

[0021] definition The following terminology is used in the description and appended claims.

[0022] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0023] Furthermore, the term "about," as used herein, when referring to measurable values, such as the amount or length of a polynucleotide or polypeptide sequence, dosage, time, temperature, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0024] Additionally, as used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, including but not limited to combinations when interpreted in the alternative ("or").

[0025] Unless the context indicates otherwise, it is expressly intended that the various features described herein can be used in any combination.

[0026] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. To further illustrate, for example, if the present specification indicates that a particular amino acid may be selected from A, G, I, L, and / or V, this language also indicates that the amino acid may be selected from any subset of these amino acid(s), e.g., A, G, I, or L; A, G, I, or V; A or G; only L; etc., as if each such subcombination were expressly set forth herein. Furthermore, such language also indicates that one or more of the specified amino acids may be excluded. For example, in some embodiments, the amino acid is not A, G, or I; is not A; is not G or V, etc., as if each such possible exclusion were expressly set forth herein.

[0027] As used herein, the terms "reduce", "reduces", "reduction" and similar terms refer to a decrease 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.

[0028] As used herein, the terms "enhance", "enhances", "enhancement" and similar terms refer to an increase 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 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more.

[0029] The term "parvovirus" as used herein encompasses the family Parvoviridae, which includes autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the genera Protoparvovirus, Erythroparvovirus, Bocaparvirus, and the subfamily Densovirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus, and any other autonomous parvovirus now known or later discovered. Other autonomous parvoviruses will be known to those of skill in the art. See, e.g., 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). The terms "subject", "individual", and "patient" are used interchangeably to refer to a vertebrate, e.g., a mammal. A mammal can be, e.g., a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., a cynomolgus monkey, a chimpanzee), or a human. Also included are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject can be an adult, a teenager, a child (2-14 years), an infant (1-24 months), or a neonate (up to 1 month). In some embodiments, an adult is about 65 years of age or older, or about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman planning to become pregnant. In some embodiments, the subject is "in need of" the methods described herein.

[0030] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Many AAV serotypes and clades have been identified (see, e.g., Gao et al, (2004) J. Virology 78:6381-6388; Morris et al, (2004) Virology 33-:375-383; and Table 2).

[0031] As used herein, the term "chimeric AAV" refers to an AAV that comprises a capsid protein having regions, domains, and / or individual amino acids from two or more different serotypes of AAV. In some embodiments, the chimeric AAV comprises a capsid protein composed of a first region from a first AAV serotype and a second region from a second AAV serotype. In some embodiments, the chimeric AAV comprises a capsid protein composed of a first region from a first AAV serotype, a second region from a second AAV serotype, and a third region from a third AAV serotype. In some embodiments, the chimeric AAV may comprise regions, domains, individual amino acids from two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and / or AAV12. For example, a chimeric AAV can include regions, domains and / or individual amino acids from a first and second AAV serotype as shown below (Table 1), where AAVX+Y indicates a chimeric AAV that includes sequences derived from AAVX and AAVY. [Table 1]

[0032] By including individual amino acids or regions from multiple AAV serotypes in one capsid protein, it is possible to obtain capsid proteins with multiple desired properties that are derived separately from multiple AAV serotypes.

[0033] The genomic sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of the natural terminal repeats (TRs), 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. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001 862, AAB95450.1, 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, the disclosures of which are incorporated by reference herein for purposes of teaching parvovirus and AAV nucleic acid and amino acid sequences. 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. 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, the disclosures of which are incorporated by reference herein for the purposes of teaching parvovirus and AAV nucleic acid and amino acid sequences. See also Table 2.The capsid structure of autonomous parvoviruses and AAV is described in further detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). 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. 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. al. (2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al. See also the crystal structure descriptions of BPV (Kailasan et al., (2015) J. Virol. 89:2603-2614), BPV (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]

[0034] Recombinant AAV (rAAV) vectors can be produced in culture using viral producer cell lines. The terms "viral production cell," "viral producer cell line," or "viral producer cell" refer to cells used to produce viral vectors. HEK293 cells and 239T cells are common viral producer cell lines. Table 8 below lists exemplary viral producer cell lines for various viral vectors. Production of rAAV typically requires the presence of three elements in a cell: 1) a transgene flanked by AAV inverted terminal repeat (ITR) sequences, 2) AAV rep and cap genes, and 3) helper virus protein sequences. These three elements can be provided on one or more plasmids and transfected or transduced into the cell. [Table 3]

[0035] As used herein, the term "multiplicity of infection" or "MOI" refers to the number of virions that contact a cell. For example, cultured cells may be infected with approximately 1 x 10 virions per cell. 2 ~Approx. 1×10 5 The cells can be contacted with AAV at an MOI ranging from 10 virions.

[0036] The term "transduction," as used herein, refers to the process by which a nucleic acid (e.g., a transgene) is introduced into a cell by a viral vector. Described herein are modified AAV capsid proteins (e.g., variant capsid proteins) and capsids containing same that can be incorporated into viral vectors and confer an enhanced cell transduction phenotype in vivo or ex vivo. As used herein, "enhanced transduction," "enhanced cell transduction," and similar terms can refer to an increase in transduction of about 1.5-fold to about 100-fold, or more. For example, transduction can be increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or more. Transduction of modified AAV (e.g., AAV comprising a capsid variant) may be enhanced compared to wild-type or native AAV vectors. In some embodiments, transduction of AAV vectors comprising a transduction-associated peptide may be enhanced compared to an otherwise identical AAV vector lacking the transduction-associated peptide.

[0037] The term "transgene" refers to any nucleic acid sequence used to transduce cells, whether maintained ex vivo or in a cell within an organism. A transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or a fragment or portion thereof, a genomic sequence, a regulatory element, etc. A "transgenic" organism, e.g., a transgenic plant or a transgenic animal, is an organism into which a transgene has been delivered or introduced, which can be expressed in the transgenic organism to produce a product, the presence of which can confer an effect (e.g., a therapeutic or beneficial effect) and / or a phenotype (e.g., a desired or altered phenotype) in the organism.

[0038] The term "tropism", as used herein, refers to the preferential invasion of a virus into a particular cell or tissue, and optionally the subsequent expression (e.g., transcription and optionally translation) of a sequence(s) carried in the viral genome within that cell, e.g., in a recombinant virus, expression of a heterologous nucleic acid(s) of interest.

[0039] It will be apparent to one skilled in the art that transcription of a heterologous nucleic acid sequence from a viral genome may not be initiated in the absence of a trans-acting factor, for example, an inducible promoter or an otherwise regulated nucleic acid sequence. In the case of the rAAV genome, gene expression from the viral genome can occur from a stably integrated provirus, a non-integrated episome, and any other form that the virus may assume within a cell.

[0040] As used herein, "systemic tropism" and "systemic transduction" (and equivalent terms) respectively refer to the viral capsid or viral vector of the present disclosure tropizing to or transducing tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney and / or pancreas). In some embodiments, systemic transduction of muscle tissue (e.g., skeletal muscle, diaphragm muscle and cardiac muscle) is observed. In some embodiments, systemic transduction of skeletal muscle tissue occurs. For example, in some embodiments, essentially all skeletal muscle throughout the body is transduced (although the efficiency of transduction may vary depending on muscle type). In some embodiments, systemic transduction of limb muscle, cardiac muscle and diaphragm muscle occurs. 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).

[0041] Alternatively, in some embodiments, the capsid or viral vector is delivered locally (eg, to the footpad, intramuscularly, intradermally, subcutaneously, topically).

[0042] 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 transduction or tropism of the control). In some embodiments, the viral vector efficiently transduces or has efficient tropism for T cells, skeletal muscle, cardiac muscle, diaphragm muscle, pancreas (including islet β cells), spleen, gastrointestinal tract (e.g., epithelial and / or smooth muscle), cells of the central nervous system, lung, joint cells, and / or kidney. A suitable control will be determined by a variety of factors, including the desired tropism profile. In some embodiments, a suitable control is a wild-type or native virus.

[0043] Similarly, whether a virus "does not transduce efficiently" or "does not have efficient tropism" for a target tissue, or similar terms, can be determined by reference to a suitable control. In some embodiments, the viral vector does not efficiently transduce (i.e., does not have efficient tropism) for liver, kidney, gonads, and / or germ cells. In some embodiments, undesired transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 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).

[0044] As used herein, the term "polypeptide" includes both peptides and proteins, unless otherwise indicated.

[0045] A "polynucleotide" is a sequence of nucleotide bases, which may be an RNA, DNA or DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is either a single-stranded or double-stranded DNA sequence.

[0046] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") refers to a polynucleotide that is at least partially separated from at least some other components of a naturally occurring organism or virus, such as cellular or viral structural components, or other polypeptides or nucleic acids that are typically found in association with the polynucleotide. In representative embodiments, the "isolated" nucleotide 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] Similarly, an "isolated" polypeptide refers to a polypeptide that is at least partially separated from at least some other components of a naturally occurring organism or virus, e.g., cellular or viral structural components, or other polypeptides or nucleic acids that are typically found in association with the polypeptide. In some embodiments, an "isolated" polypeptide is enriched at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold, or more, relative to the starting material.

[0048] As used herein, "isolated" or "purified" (or grammatical equivalents) a viral vector means that the viral vector is at least partially separated from at least some other components in the starting material. In some embodiments, an "isolated" or "purified" viral vector is concentrated by at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more compared to the starting material.

[0049] As used herein, the term "transduction-associated peptide" refers to a short amino acid sequence that can be incorporated into an AAV vector and alter the transduction of the AAV vector into any cell. The transduction-associated peptide can have some effect on the transduction of the AAV vector. For example, in some embodiments, the transduction-associated peptide increases the transduction of the AAV vector into a target cell of interest. In some embodiments, the transduction-associated peptide decreases the transduction of the AAV vector into non-targeted cells. The transduction-associated peptide can be inserted into an existing AAV capsid sequence (i.e., causes a net increase in the number of amino acids in the sequence) or can replace an existing portion of the AAV capsid sequence (i.e., causes no net change or a reduction in the number of amino acids in the sequence).

[0050] A "therapeutic polypeptide" or "therapeutic protein" is a polypeptide that can alleviate, reduce, prevent, delay and / or stabilize symptoms resulting from the absence or deficiency of a protein in a cell or subject, and / or that otherwise provides a benefit to the subject, e.g., anti-cancer effect, or improved transplant survival.

[0051] The terms "treat", "treating" or "treatment of" (and grammatical variations thereof) mean reducing the severity, at least partially improving or stabilizing the subject's condition, and / or there is some alleviation, relief, reduction or stabilization of at least one clinical symptom, and / or there is a delay in the progression of a disease or disorder. The term "subject" is used interchangeably with the term "patient."

[0052] "Prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of a disease, disorder and / or clinical symptom(s) in a subject and / or reducing the severity of the onset of the disease, disorder and / or clinical symptom(s) below that which would occur in the absence of the methods of the present disclosure. Prevention can be complete, e.g., the complete absence of the disease, disorder and / or clinical symptom(s). Prevention can also be partial, such that the onset and / or severity of the onset of the disease, disorder and / or clinical symptom(s) in a subject is less than would occur in the absence of the methods of the present disclosure.

[0053] "Therapeutically effective amount" as used herein refers to an amount that, when administered to a subject for treating a disease or at least one of the clinical symptoms of the disease, is sufficient to affect such treatment of the disease or its symptoms. "Therapeutically effective amount" may vary, for example, depending on the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease or disorder, the age, weight and / or health of the patient being treated, and the judgment of the prescribing physician. The appropriate amount in any given case can be ascertained by one skilled in the art or can be determined by routine experimentation.

[0054] As used herein, the term "viral vector," "vector," or "gene delivery vector" refers to a viral (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" may be used to refer only to the vector genome / vDNA.

[0055] An "adeno-associated virus vector" or "AAV vector" typically comprises an AAV capsid and a nucleic acid (e.g., a nucleic acid comprising a transgene) encapsidated by the AAV capsid. An "AAV capsid" is a roughly spherical protein shell comprising approximately 60 "AAV capsid proteins" (interchangeably referred to herein as "AAV capsid protein subunits" or "capsid proteins"), which assemble into a T=1 icosahedral structure. The AAV capsid of the AAV vector described herein comprises a plurality of AAV capsid proteins. When an AAV vector is described as comprising an AAV capsid protein, it will be understood that the AAV vector comprises an AAV capsid, and that the AAV capsid comprises one or more AAV capsid proteins. The term "viral-like particle" or "virus-like particle" refers to a protein capsid that does not contain any vector genome or nucleic acid, including a transfer cassette or transgene. The terms "AAV vector", "AAV capsid" and "AAV capsid protein" may be used interchangeably herein. Based on the context, one of skill in the art will be able to readily infer the meaning of the particular term used.

[0056] In some embodiments, the AAV vector may comprise a nucleic acid that comprises a "transfer cassette," i.e., a nucleic acid that comprises one or more sequences that can be delivered to a cell by an AAV vector. In some embodiments, the nucleic acid is self-complementary (i.e., double-stranded). In some embodiments, the nucleic acid is not self-complementary (i.e., single-stranded).

[0057] "rAAV vector genome" or "rAAV genome" refers to an AAV genome (i.e., vDNA) that contains one or more heterologous nucleic acid sequences. In rAAV vectors, generally, only the terminal repeat(s) (TR(s)) in cis are required for virus production. All other viral sequences are not essential and can be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, rAAV vector genomes will only retain one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Coding sequences for structural and nonstructural proteins can be supplied in trans (e.g., from a vector such as a plasmid or by stably integrating the sequences into the packaging cell). In embodiments, a rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), optionally two TRs (e.g., two AAV TRs), typically located at the 5' and 3' ends of the vector genome, flanking the heterologous nucleic acid, but not necessarily contiguous to the heterologous nucleic acid. The TRs may be the same or different from each other.

[0058] The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function such as replication, viral packaging, integration, and / or proviral rescue). A TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR sequence, such as that of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., SV40 hairpin that functions as an SV40 origin of replication) can be used as a TR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. In addition, a TR can be partially or completely synthetic, such as the "double D sequence" described in U.S. Patent No. 5,478,745 to Samulski et al.

[0059] An "AAV terminal repeat" or "AAV TR" 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). An AAV terminal repeat need not have the native terminal repeat sequence (e.g., the native AAV TR 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.

[0060] The viral vectors of the present disclosure may further be "targeted" viral vectors (e.g., having a targeting tropism) and / or "hybrid" parvoviruses (i.e., in which the viral TR and viral capsid are from different parvoviruses), as described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0061] The viral vectors of the present disclosure can further be double-stranded parvovirus particles, as described in International Patent Publication WO 01 / 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 of the present disclosure.

[0062] In addition, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and / or substitutions.

[0063] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.

[0064] Naturally occurring levorotatory (L-) amino acids are shown in Table 3. [Table 4]

[0065] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are provided in Table 4) and / or an amino acid that has been modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfation). [Table 5-1] [Table 5-2]

[0066] Additionally, 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)). Advantageously, these unnatural amino acids can be used to chemically link a molecule of interest to an AAV capsid protein.

[0067] An "active immune response" or "active immunity" is characterized by "the involvement of host tissues and cells after encounter with an immunogen". It involves the differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, leading 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 1 17 (Joseph A. Bellanti ed., 1985). In other words, an active immune response is initiated by the host after exposure to an immunogen by infection or vaccination. Active immunity can be contrasted with passive immunity, which is obtained by the transfer of preformed substances (antibodies, transfer factors, thymus grafts, interleukin-2) from an actively immunized host to a non-immunized host.

[0068] A "protective" immune response or "protective" immunity, as used herein, indicates that the immune response provides some benefit to the subject in terms of preventing or reducing the onset of disease. Alternatively, a protective immune response or immunity may be useful in the treatment and / or prevention of disease, 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, so long as the benefits of the treatment outweigh any drawbacks thereof.

[0069] As used herein, the term "cancer" includes tumor-forming cancers. Similarly, the term "cancerous tissue" includes tumors. "Cancer cell antigens" include tumor antigens.

[0070] The term "cancer" has its meaning as understood in the art, e.g., the uncontrolled growth of tissue that can spread (i.e., metastasize) to distant sites in the body. Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thymic carcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's 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 malignant condition now known or later identified. In an exemplary embodiment, the present disclosure provides a method for treating and / or preventing tumor-forming cancer.

[0071] The term "tumor" is also understood in the art as, for example, an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0072] The terms "treating cancer", "treatment of cancer" and equivalent terms are intended to reduce or at least partially eliminate the severity of cancer, and / or to slow and / or control the progression of the disease, and / or to stabilize the disease. In some embodiments, these terms refer to preventing, reducing or at least partially eliminating metastasis of cancer, and / or preventing, reducing or at least partially eliminating the growth of metastatic nodules.

[0073] By "cancer prevention" or "preventing cancer" and equivalent terms, it is intended that the method at least partially eliminates or reduces and / or delays the incidence and / or severity of cancer. In other words, the possibility or probability of developing cancer in a subject is reduced and / or the development of cancer may be delayed.

[0074] Modified AAV capsid proteins and capsids containing same The present disclosure provides AAV capsid protein (VP1, VP2 and / or VP3) variants, as well as viral capsids and viral vectors comprising same. Each capsid variant comprises one or more transduction-associated peptides. The transduction-associated peptides are not present in naturally occurring AAV capsid proteins and, in some embodiments, can provide AAV vectors comprising the capsid proteins with enhanced transduction of a target cell of interest (e.g., T cells). The AAV capsid protein variants disclosed herein may be variants to the capsid protein of any AAV serotype currently known or later discovered. In some embodiments, the AAV capsid protein variant is a variant of a capsid protein from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, and avian AAV.

[0075] AAV capsid protein modifications In some embodiments, the transduction-associated peptides described herein can confer one or more desirable properties to a viral vector comprising a modified AAV capsid protein, including, but not limited to, enhanced cell transduction in various cell types (e.g., T cells) in vitro, in vivo, or ex vivo. In some embodiments, the capsid protein of the present disclosure can be incorporated into an AAV vector. In some embodiments, the AAV vector comprising the capsid protein has enhanced cell transduction (e.g., enhanced T cell transduction) compared to wild-type AAV, or an AAV viral particle or AAV viral vector comprising an AAV capsid protein that does not comprise a transduction-associated peptide. In some embodiments, the AAV viral particle or vector of the present disclosure can also evade neutralizing antibodies.

[0076] The transduction-related peptides of the present disclosure can replace the amino acid sequence of a wild-type AAV capsid protein, resulting in no net increase or decrease in the number of amino acids in the AAV capsid protein sequence. In some embodiments, the replacement of the amino acid sequence of a wild-type AAV capsid protein with a transduction-related peptide of the present disclosure can result in a net loss (e.g., deletion) of amino acids compared to the wild-type AAV capsid protein sequence. For example, the transduction-related peptide can replace one or more amino acids of an AAV capsid protein from any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, and avian AAV. In some embodiments, the transduction-associated peptides of the present disclosure may be inserted into the amino acid sequence of a wild-type AAV capsid protein, resulting in an increase in the number of amino acids in the AAV capsid protein sequence.

[0077] In some embodiments, the modification of the AAV capsid protein results in the replacement of one or more amino acid residues of a native AAV capsid protein with an amino acid that is not present in the native capsid sequence. In some embodiments, the modification of the AAV capsid protein results in the replacement of one or more of the following amino acid residues: 454, 455, 456, 457, 458, 459, and 460 with an amino acid that is not present in the native capsid protein sequence, where the amino acid numbering is relative to the corresponding residues in the VP1 sequence of the wild-type AAV6 capsid protein, or the capsid protein of any other AAV serotype. In some embodiments, the modification of the AAV capsid protein results in the deletion of one or more of the following amino acid residues: 454, 455, 456, 457, 458, 459, and 460, where the amino acid numbering is relative to the corresponding residues in the VP1 sequence of the wild-type AAV6 capsid protein, or the capsid protein of any other AAV serotype. In some embodiments, the modification of the AAV capsid protein results in a substitution of one or more of amino acids 454, 455, 456, 457, 458, 459, and / or 460 relative to the amino acid sequence of the native AAV6 capsid protein sequence (SEQ ID NO:1).

[0078] In some embodiments, the AAV capsid protein comprises a transduction-associated peptide of the sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 24). In some embodiments, the AAV capsid protein comprises a transduction-associated peptide of the sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 24), and the capsid protein is of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, or avian AAV. In some embodiments, the AAV capsid protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 or 25-34 comprises a transduction-associated peptide of sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 24). In some embodiments, the AAV capsid protein comprises a sequence of a native AAV6 capsid protein sequence (e.g., SEQ ID NO: 1) and further comprises a transduction-associated peptide of SEQ ID NO: 24. In some embodiments, the AAV capsid protein comprises an amino acid sequence having at least about 80% identity, e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% identity to a wild-type AAV capsid protein sequence, e.g., SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 25-34. In some embodiments, the AAV capsid protein disclosed herein comprises an amino acid sequence having about 99% identity to SEQ ID NO:1.

[0079] The transduction-associated peptide of SEQ ID NO:24 can be used to replace one or more amino acid residues anywhere in the amino acid sequence of the disclosed AAV capsid protein. In some embodiments, the transduction-associated peptide of SEQ ID NO:24 can be used to replace the sequence of a capsid protein, the capsid protein having an amino acid sequence selected from any one of SEQ ID NOs:1 and 25-34. In some embodiments, the transduction-associated peptide of SEQ ID NO:24 can be inserted into the amino acid sequence of an AAV capsid protein disclosed herein. In some embodiments, the replacement of the native sequence of one or more AAV capsid proteins described herein with the transduction-associated peptide of SEQ ID NO:24 can result in one or more amino acids being deleted from the sequence of the AAV capsid protein. In some embodiments, the capsid protein can comprise the sequence of SEQ ID NO:1, except that amino acids 454-460 of SEQ ID NO:1 are replaced with a transduction-associated peptide comprising SEQ ID NO:24. In some embodiments, SEQ ID NO:24 is used to replace the sequence of a wild-type AAV capsid protein such that the resulting sequence contains at least one, two, three, etc. individual amino acids that are not present in the wild-type sequence.

[0080] In some embodiments, SEQ ID NO:24 comprises a sequence where X1 is not G, X2 is not S, X3 is not A, X4 is not Q, X5 is not N, X6 is not K, and / or X7 is not D. In some embodiments, X1 is H, M, A, Q, V, or S. In some embodiments, X2 is A or T. In some embodiments, X3 is P or T. In some embodiments, X4 is R or D. In some embodiments, X5 is V, Q, C, S, or D. In some embodiments, X6 is E, A, or P. In some embodiments, X7 is E, G, N, T, or A. In some embodiments, X1 is H, X2 is A, X3 is P, X4 is R, X5 is V, X6 is E, and X7 is E. In some embodiments, X1 is M, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. In some embodiments, X1 is H, X2 is T, X3 is T, X4 is D, X5 is C, X6 is A, and X7 is N. In some embodiments, X1 is A, X2 is A, X3 is P, X4 is R, X5 is S, X6 is E, and X7 is T. In some embodiments, X1 is Q, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. In some embodiments, X1 is V, X2 is A, X3 is P, X4 is R, X5 is D, X6 is P, and X7 is A. In some embodiments, X1 is S, X2 is A, X3 is P, X4 is R, X5 is S, X46 is E, and X7 is N.

[0081] In some embodiments, the transduction-associated peptide has an amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1=H, M, Q, V, or S; X2=A or T; X3=P or T; X4=R or D; X5=V, Q, C, S, or D; X6=E, A, or P; and X7=E, G, N, T, or A (SEQ ID NO: 16). In some embodiments, the transduction-associated peptide has an amino acid sequence of any one of SEQ ID NOs: 17-23.

[0082] In some embodiments, the AAV capsid protein comprises a transduction-associated peptide having an amino acid sequence of any one of SEQ ID NOs: 17-23. In some embodiments, the transduction-associated peptide having an amino acid sequence of any one of SEQ ID NOs: 17-23 replaces one or more amino acids of the AAV capsid protein. The present disclosure provides a variant of an AAV capsid protein of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, and avian AAV, the variant of the AAV capsid protein comprising an amino acid sequence that comprises a transduction-associated peptide having an amino acid sequence of any one of SEQ ID NOs: 17-23. In some embodiments, the AAV capsid protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 and 25-34, but one or more amino acids are replaced with a transduction-associated peptide having the amino acid sequence of any one of SEQ ID NOs: 17-23.

[0083] In some embodiments, a transduction-associated peptide having an amino acid sequence of any one of SEQ ID NOs: 17-23 replaces one or more amino acids of an AAV capsid protein of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, and avian AAV. In some embodiments, a transduction-associated peptide having an amino acid sequence of any one of SEQ ID NOs: 17-23 replaces one or more amino acids of an AAV capsid protein that comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 and 25-34.

[0084] In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide comprising the sequence of any one of SEQ ID NOs:17-23. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:17. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:18. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:19. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:20. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:21. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:22. In some embodiments, amino acids 454-460 of a native AAV6 capsid protein (e.g., SEQ ID NO:1) are replaced by a transduction-associated peptide of SEQ ID NO:23.

[0085] In some embodiments, the AAV capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or a sequence at least about 80% identical thereto. For example, in some embodiments, the AAV capsid protein comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, at least about 99.5%, or about 100% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14.

[0086] b. Other modifications of AAV capsid proteins The present disclosure contemplates, but is not limited to, that the modified AAV capsid protein 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). 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 modified capsid protein may already be altered 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 now known or later discovered). In some embodiments, the capsid protein may be an engineered AAV, such as AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B. Such AAV capsid proteins are also within the scope of the present disclosure.

[0087] In some embodiments, the AAV capsid protein is chimeric. For example, the chimeric AAV capsid protein may comprise sequences from two or more AAV serotypes, or three or more AAV serotypes. The chimeric AAV capsid protein may comprise sequences from two or more of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV, and avian AAV.

[0088] Thus, in some embodiments, the modified AAV capsid protein may 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 the capsid protein and / or substituted and / or modified by deletion of one or more amino acids. Thus, when a particular AAV capsid protein is referred to herein (e.g., AAV1, 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 disclosed herein. Such modifications include substitutions, insertions, and / or deletions. In some embodiments, the capsid protein includes 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 into the protein (other than the amino acid sequence substitutions of the present disclosure) compared to a native AAV capsid protein sequence. In 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 (other than a transduction-associated peptide according to the present disclosure) compared to a 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, or fewer than 20, fewer than 30, fewer than 40, fewer than 50, fewer than 60, or fewer than 70 amino acid deletions (other than the transduction-associated peptides of the present disclosure) compared to a native AAV capsid protein sequence.

[0089] The modifications to the AAV capsid proteins according to the present disclosure are "selective" modifications. This approach is in contrast to previous studies involving swapping entire subunits or large domains between AAV serotypes (see, for example, International Patent Publication WO 00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774). In some embodiments, the "selective" modifications result in the insertion and / or substitution and / or deletion of no more than about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 consecutive amino acids. The modified capsid proteins and capsids of the present disclosure may further include any other modifications now known or later identified. In the embodiments described herein in which an amino acid residue is replaced by any amino acid residue other than the amino acid residue present in the wild-type or natural amino acid sequence, any other amino acid residue may be any natural or non-natural amino acid residue known in the art (see, for example, Tables 3 and 4). In some embodiments, the substitutions may be conservative substitutions, and in some embodiments, the substitutions may be non-conservative substitutions.

[0090] As described herein, the amino acid and nucleic acid sequences of capsid proteins from many AAVs are known in the art.Therefore, the amino acids that "correspond" to the amino acid positions of the native AAV capsid proteins can be easily determined for any other AAV (e.g., by using sequence alignment).Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity may be determined using standard techniques known in the art, 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), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection.

[0091] Another suitable 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 available from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are optionally set to default values. The parameters are dynamic values, established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched, but the values ​​may be adjusted to increase sensitivity.

[0092] Yet an additional useful algorithm is Gapped BLAST, as reported by Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402.

[0093] Unless otherwise indicated, percent identity calculations are performed in this disclosure using the BLAST algorithm, available on the world wide web at the following address: blast.ncbi.nlm.nih.gov / Blast.cgi.

[0094] C modified viral capsid The present disclosure also provides a viral capsid comprising at least one of the variant capsid proteins disclosed herein. In some embodiments, the viral capsid is a parvovirus capsid, which may further be an autonomous parvovirus capsid or a dependent virus 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 of the present disclosure may be any of the AAV serotypes listed in Table 2, or may be derived from any of the foregoing by one or more insertions, substitutions, and / or deletions. The modified viral capsid may be used as a "capsid carrier" as described, for example, in U.S. Pat. No. 5,863,541. The viral capsid according to the present disclosure may be produced using any method known in the art, for example, by expression from baculovirus (Brown et al., (1994) Virology 198:477-488). In some embodiments, the AAV capsid comprises about 60 variant capsid proteins as described herein.

[0095] In some embodiments, the viral capsid may 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 a cell surface molecule present on a desired target tissue(s) (see, e.g., International Patent Publication WO 00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774; Shi et al., Human Gene Therapy 17:353-361 (2006) [describing 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 insertion of a PI peptide containing the RGD motif following amino acids 447, 534, 573, and 587 of the AAV2 capsid subunit]). Other positions within the AAV capsid subunit that tolerate insertions are known in the art (e.g., positions 449 and 588 as described by Grifman et al., Molecular Therapy 3:964-975 (2001)).

[0096] For example, the viral capsids of the present disclosure may have relatively inefficient tropism to a particular target tissue of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells and / or lungs). Advantageously, targeting sequences can be incorporated into these low-transduction vectors to confer the viral capsids with the desired tropism, and optionally, selective tropism to a particular tissue or cell, such as T cells. AAV capsid proteins, capsids, and vectors containing targeting sequences are described, for example, in International Patent Publication WO00 / 28004. As another example, one or more non-naturally occurring amino acids described in Wang et al., Annu Rev Biophys Biomol Struct.35:225-49 (2006) can be incorporated into the AAV capsid subunits of the present disclosure at orthogonal sites as a means of redirecting low-transduction vectors to the desired target tissue(s). Advantageously, these unnatural amino acids can be used to chemically link molecules of interest to AAV capsid proteins, including, but 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 to target specific cell surface receptors, such as growth factor receptors, integrins, etc. Methods for chemically modifying amino acids are known in the art (see, for example, Greg T. Hermanson, Bioconjugate Techniques, 1st edition, Academic Press, 1996). In some embodiments, the targeting sequence can 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 a specific cell type(s).

[0097] As another non-limiting example, a heparin-binding domain or a heparan sulfate (HS)-binding domain (e.g., a respiratory syncytial virus heparin-binding domain) may be inserted or substituted into a capsid subunit that typically does not bind to HS receptors (e.g., AAV4, AAV5) to confer heparin and / or heparan sulfate binding to the resulting variant. Binding to HS / heparin is known in the art to be mediated by a "basic patch" rich in arginine and / or lysine. In an exemplary embodiment, a sequence following the motif BXXB (SEQ ID NO: 105) (wherein "B" is a basic residue and X is neutral and / or hydrophobic) can be used. As a non-limiting example, BXXB can be RGNR (SEQ ID NO: 106). As another non-limiting example, BXXB replaces amino acids 262-265 of the native AAV2 capsid protein or is substituted at the corresponding position(s) of the capsid protein of another AAV serotype.

[0098] Parvovirus B19 uses globoside as its receptor to infect 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 globoside has been mapped to amino acids 399-406 (Chapman et al, (1993) Virology 194:419), in the 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 substituted into the AAV capsid protein of the present disclosure to target the viral capsid or a viral vector containing same to erythroid cells.

[0099] In some embodiments, the exogenous targeting sequence may be any amino acid sequence that encodes a peptide that modifies the tropism of a viral capsid or viral vector, including a modified AAV capsid protein. In some embodiments, the targeting peptide or protein may be naturally occurring or may be fully or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as ROD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor 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 substance P, keratinocyte growth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2, hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin, β-endorphin, Leu-enkephalin, limorphin, alpha-neoenkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments of the above. As yet a further option, a binding domain from a toxin (e.g., a snake toxin such as tetanus toxin or alpha-bungarotoxin) can be substituted into the capsid protein as a targeting sequence. In yet another exemplary embodiment, the AAV capsid protein can be modified by substituting into the AAV capsid protein a "non-classical" import / export signal peptide (e.g., fibroblast growth factor-1 and -2, interleukin 1, HIV-1 Tat protein, herpes virus VP22 protein, etc.) as described by Cleves (Current Biology 7:R318 (1997)). Also encompassed are peptide motifs that induce uptake by certain cells, such as the FVFLP (SEQ ID NO: 104) peptide motif, which triggers uptake by liver cells.

[0100] Phage display techniques and other techniques known in the art can be used to identify peptides that recognize any cell type of interest. Targeting sequences can code for any peptide that targets a cell surface binding site, including receptors (e.g., proteins, carbohydrates, glycoproteins, or proteoglycans). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties found in mucins, glycoproteins, and carbohydrate moieties found in gangliosides, MHC1 glycoproteins, membrane glycoproteins (including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, etc.). Table 7 provides other non-limiting examples of suitable targeting sequences. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0101] In some embodiments, the targeting sequence can be a peptide (e.g., can contain arginine and / or lysine residues that can be chemically coupled via their R groups) that can be used to chemically couple to another molecule that targets entry into a cell. In some embodiments, the AAV capsid protein or viral capsid of the present disclosure can contain mutations described in WO2006 / 066066. For example, the capsid protein can contain selective amino acid substitutions at amino acid positions 263, 705, 708, and / or 716 of the native AAV2 capsid protein, or the corresponding alteration(s) in a capsid protein from another AAV serotype.

[0102] Additionally or alternatively, in some embodiments, the capsid protein, viral capsid, or vector comprises an insertion of a selective amino acid immediately after amino acid position 264 of the AAV2 capsid protein, or a corresponding modification in a capsid protein from another AAV. By "immediately after amino acid position X" it is intended that the insertion immediately follows the indicated amino acid position (e.g., "after amino acid position 264" indicates a point insertion at position 265, or a more extensive insertion, such as an insertion from positions 265-268). Further, in some embodiments, the capsid protein, viral capsid, or vector of the present disclosure can comprise amino acid modifications, such as those described in PCT Publication No. WO2010 / 093784 (e.g., 2i8) and / or PCT Publication No. WO2014 / 144229 (e.g., double glycans).

[0103] Heterologous molecules are defined as those that are not naturally found in AAV infection, e.g., are not encoded by the wild-type AAV genome. Furthermore, therapeutically useful molecules can be associated with the outside of the chimeric virus capsid to transfer the molecules into the host target cells. Such associated molecules can include DNA, RNA, small organic molecules, metals, sugar chains, lipids, and / or polypeptides. In one embodiment of the present disclosure, therapeutically useful molecules are covalently attached (i.e., conjugated or chemically coupled) to the capsid protein. Methods for covalently attaching molecules are known to those skilled in the art.

[0104] d. Modified viral vectors The present disclosure provides a viral vector comprising the capsid protein variants and capsids of the present disclosure. In some embodiments, the viral vector is a parvovirus vector (e.g., comprising a parvovirus capsid and / or vector genome), such as an AAV vector (e.g., comprising an AAV capsid and / or vector genome). In some embodiments, the viral vector comprises a modified AAV capsid and vector genome comprising the modified capsid protein of the present disclosure.

[0105] For example, in some embodiments, the viral vector comprises (a) a viral capsid (e.g., AAV capsid) comprising the capsid protein variant of the present disclosure, and (b) a nucleic acid comprising a terminal repeat sequence (e.g., AAV TR), wherein the nucleic acid comprising the terminal repeat sequence is encapsidated by the viral capsid. The nucleic acid may optionally comprise two terminal repeats (e.g., two AAV TRs). In a representative embodiment, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide or functional RNA of interest.

[0106] AAV typically does not transduce T cells at high levels. In contrast, in some embodiments, the viral vectors of the present disclosure show enhanced transduction of one or more cell types (e.g., T cells) and / or tissues compared to the level of transduction by wild-type viral vectors, or viral vectors that do not include a capsid protein variant. In some embodiments, the AAV viral vector increases cell transduction compared to wild-type or native AAV viral vectors. In some embodiments, the AAV viral vector increases transduction in one or more cell types (e.g., T cells) compared to wild-type or native AAV viral vectors, or AAV viral vectors that do not include any one of the capsid protein variants disclosed herein. In some embodiments, the AAV viral vector may increase transduction in hematopoietic stem cells. In some embodiments, the AAV viral vector may increase transduction in monocytes, basophils, eosinophils, neutrophils, dendritic cells, macrophages, B cells, T cells, and / or natural killer cells. In some embodiments, the AAV viral vectors can increase transduction in satellite cells, mesenchymal stem cells, and / or basal cells. In some embodiments, the AAV viral vectors can increase transduction in lung epithelial cells, hepatocytes, and / or skeletal muscle cells.

[0107] Known receptors and co-receptors for AAV include heparan sulfate proteoglycans, integrins, O-linked sialic acid, N-linked sialic acid, AAV receptor (AAVR, KIAA0319L), hepatocyte growth factor receptor (c-Met), CD9, FGFR-1, 37 / 67 kDa laminin receptor, and platelet derived growth factor receptor. In embodiments, the AAV viral vector of the present disclosure has increased affinity for one or more of these receptors and / or co-receptors. For example, in some embodiments, the AAV viral vector has increased heparin and / or heparan sulfate binding compared to wild-type or native AAV viral vectors. In some embodiments, the AAV viral vector has increased sialic acid binding compared to wild-type or native AAV viral vectors. In some embodiments, the AAV viral vector has increased integrin binding compared to wild-type or native AAV viral vectors. In some embodiments, the AAV viral vector has increased binding to integrins including alpha and beta subunits compared to wild-type or native AAV viral vectors. The integrin can be, for example, α4β7, α4β1, α1β1, α2β1, αEβ7, αLβ2, α5β1, α5β6, α5β5, α5β8, α5β8, α3β1, α5β1, α11β1, α5β3, α11β3, αVβ3, αVβ5, αVβ6, αVβ8.

[0108] The present disclosure also provides a nucleotide sequence encoding a capsid protein variant (e.g., an AAV capsid protein variant) of the present disclosure or one or more capsids (e.g., AAV capsids) comprising a capsid protein variant, or an expression vector comprising the same. In some embodiments, the nucleic acid encodes a recombinant AAV capsid protein having a sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some embodiments, the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. The nucleotide sequence can be a DNA sequence or an RNA sequence. The expression vector can be, but is not limited to, a viral vector (e.g., adenovirus, AAV, herpesvirus, vaccinia, poxvirus, baculovirus, etc.), or a non-viral vector, such as a plasmid, phage, YAC, BAC, etc. The present disclosure also provides a cell comprising one or more nucleotide sequences or expression vectors of the present disclosure. The cell can be in vitro, ex vivo, or in vivo.

[0109] Methods for Producing Viral Vectors The present disclosure further provides a method for producing a viral vector disclosed herein. Thus, in some embodiments, the present disclosure provides a method for producing an AAV vector that increases cell transduction (e.g., increases T cell transduction), comprising: a) identifying surface-exposed residues on an AAV capsid protein; b) generating a library of AAV capsid proteins that contain amino acid substitutions of the surface-exposed amino acid residues identified in (a); c) producing AAV particles that contain capsid proteins from the library of AAV capsid proteins of (b); d) contacting cells with the AAV particles of (c) under conditions that allow infection and replication; e) selecting AAV particles that can complete at least one infection cycle and replicate to a titer equal to or greater than that of a control AAV particle. In some embodiments, steps (d) and (e) are repeated multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Non-limiting examples of methods for identifying surface-exposed residues include cryo-electron microscopy. 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. 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. al. (2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al. See also the crystal structure descriptions of BPV (Kailasan et al., (2015) J. Virol. 89:2603-2614), BPV (Xie et al., (1996) J. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251:1456-64).

[0110] Resolution and identification of surface exposed residues allows for subsequent modification by random, rational, and / or degenerate mutagenesis to generate AAV capsids that can be identified through further selection and / or screening. Thus, in a further embodiment, the disclosure provides a method for producing an AAV vector that increases cell transduction (e.g., increases T cell transduction), comprising: a) identifying surface exposed amino acid residues on an AAV capsid protein; b) generating an AAV capsid protein that includes amino acid substitutions of the surface exposed amino acid residues identified in (a) by random, rational, and / or degenerate mutagenesis; c) producing AAV particles that include the capsid protein from the AAV capsid protein of (b); d) contacting a cell with the AAV particles of (c) under conditions that allow infection and replication to occur; and e) selecting AAV particles that are able to complete at least one infection cycle and replicate to titers equal to or greater than control AAV particles.

[0111] Methods for generating AAV capsid proteins containing amino acid substitutions of surface-exposed amino acid residues by random mutagenesis, rational mutagenesis and / or degenerate mutagenesis are known in the art. This comprehensive approach provides a platform technology that can be applied to the modification of any AAV capsid. The application of this platform technology can result in AAV variants that are derived from the original AAV capsid template and have enhanced transduction efficiency. One advantage and benefit is that the application of this technology can expand the cohort of patients that can be targeted for gene therapy by AAV vectors.

[0112] In some embodiments, the disclosure provides a method of producing a viral vector, comprising providing a cell with (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence) and (b) an AAV sequence sufficient for replication and encapsidation of the nucleic acid template into an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding the AAV capsid of the disclosure). 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, which are located 5' and 3' to the heterologous nucleic acid sequence (if present), but need not be directly contiguous to the heterologous nucleic acid sequence.

[0113] The nucleic acid template and 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 in the cell. The method may further comprise a step of collecting the viral vector from the cell. The viral vector may be collected from the medium and / or by lysing the cell. The cell may be a cell permissive for AAV viral replication. Any suitable cell known in the art may be used. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell may be a trans-complementing packaging cell line that provides a function deleted from a replication-deficient helper virus, such as 293 cells or other E1a trans-complementing cells.

[0114] The AAV replication and capsid sequences may 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 do not need to be provided together, but it may be convenient to do so. The AAV rep and / or cap sequences may be provided by any viral or non-viral vector. For example, the rep / cap sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). An EBV vector may be used to express the AAV cap and rep genes. One advantage of this method is that the EBV vector is episomal but maintains high copy number throughout successive cell divisions (i.e., stably integrated into the cell as an extrachromosomal element designated as an "EBV-based nuclear episome," see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67). As a further alternative, the rep / cap sequences may be stably integrated into the cell. Typically, AAV rep / cap sequences are not flanked by TRs to prevent rescue and / or packaging of these sequences.

[0115] The nucleic acid template can be provided to the cell using any method known in the art. For example, the template can be provided by a non-viral vector (e.g., a plasmid) or a viral vector. In some embodiments, the nucleic acid template is provided by a herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). As another example, Palombo et al., (1998) J. Virology 72:5025, describes a baculovirus vector carrying a reporter gene flanked by AAV TR. The EBV vector can be used to deliver the template, as described above for the rep / cap gene.

[0116] 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. To enhance viral titer, the cell can be provided with helper virus functions (e.g., adenovirus or herpesvirus) that facilitate productive AAV infection. The helper virus sequences required for AAV replication are known in the art. Typically, these sequences are provided by a helper adenovirus or herpesvirus vector. Alternatively, the adenovirus or herpesvirus sequences can be provided as another non-viral or viral vector, such as a non-infectious adenovirus mini-plasmid that carries all of the helper genes that facilitate efficient AAV production described in Ferrari et al., (1997) Nature Med.3:1295, and U.S. Patent Nos. 6,040,183 and 6,093,570.

[0117] In addition, helper virus functions can be provided by packaging cells in which the helper sequences are embedded in the chromosome or maintained as stable extrachromosomal elements. Generally, the helper virus sequences cannot be packaged into AAV virions, e.g., they are not adjacent to the TR. Those skilled in the art will recognize that the helper virus sequences are not packaged into AAV virions, e.g., they are not adjacent to the TR.

[0118] It will be appreciated that it may be advantageous to provide the AAV replication and capsid sequences, as well as the helper virus sequences (e.g., adenovirus sequences) on a single helper construct. This helper construct may be a non-viral or viral construct. As one non-limiting example, the helper construct may be a hybrid adenovirus or hybrid herpesvirus that contains the AAV rep / cap genes. In some embodiments, the AAV rep / cap sequences and the adenovirus helper sequences are provided by a single adenovirus helper vector. This vector may further comprise a nucleic acid template. The AAV rep / cap sequences and / or the rAAV template may be inserted into a deleted region of the adenovirus (e.g., E1a or E3 region).

[0119] In some embodiments, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper vector. The rAAV template can be provided, for example, as a plasmid template. In some embodiments, the AAV rep / cap sequence and the adenovirus helper sequence 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 in the cell as an extrachromosomal element (e.g., as an EBV-based nuclear episome).

[0120] In some embodiments, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper. The rAAV template can be provided as a separate replicating viral vector. For example, the rAAV template can be provided by a rAAV particle or a second recombinant adenovirus particle. According to the above method, the hybrid adenovirus vector typically contains sufficient adenovirus 5' and 3' cis sequences (i.e., adenovirus terminal repeat and PAC sequences) for adenovirus replication and packaging. The AAV rep / cap sequence and, if present, the rAAV template are embedded in the adenovirus backbone and adjacent to the 5' and 3' cis sequences, so that these sequences can be packaged into the adenovirus capsid. As mentioned above, the adenovirus helper sequence and the AAV rep / cap sequence 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 an adenovirus and chimeric helper that contains both the AAV rep and cap genes.

[0121] Herpes viruses may also be used as helper viruses in AAV packaging methods. Hybrid herpes viruses encoding AAV Rep protein(s) can advantageously facilitate scalable AAV vector production schemes. Hybrid herpes simplex virus type I (HSV-1) vectors expressing AAV-2 rep and cap genes have been described (Conway et al., (1999) Gene Therapy 6:986 and WO00 / 17377). In some embodiments, the viral vectors of the present disclosure can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template, for example, as described by Urabe et al., (2002) Human Gene Therapy 13:1935-43.

[0122] AAV vector stocks free of contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be separated from helper viruses based on affinity for heparan substrates (Zolotukhin et al. (1999) Gene Therapy 6:973). Replication-defective helper viruses with deletions can be used to render any contaminating helper viruses replication-incompetent. In some embodiments, adenovirus helpers lacking late gene expression can be used, since only adenovirus early gene expression is required to mediate AAV viral packaging. Adenovirus mutants defective in late gene expression are known in the art (e.g., adenovirus mutants ts100K and ts149).

[0123] Recombinant viral vectors The present disclosure provides a recombinant viral vector (e.g., a recombinant AAV vector) comprising at least one of the capsid proteins (e.g., AAV capsid proteins) or at least one of the capsids (e.g., AAV capsids) disclosed herein, wherein the capsid protein comprises one or more transduction-associated peptides disclosed herein. In some embodiments, the AAV vector exhibits increased transduction into cells, such as T cells, compared to wild-type AAV vectors or AAV vectors that do not comprise a transduction-associated peptide. In some embodiments, the AAV vector exhibits increased transduction into the nucleus of T cells, compared to wild-type AAV vectors or AAV vectors that do not comprise a transduction-associated peptide. In some embodiments, the AAV vector exhibits increased transduction into the cytosol of T cells, compared to wild-type AAV vectors or AAV vectors that do not comprise a transduction-associated peptide.

[0124] The recombinant viral vectors of the present disclosure are useful for delivering nucleic acids to cells in vitro, ex vivo, and in vivo. Molecules that can be packaged and transferred into cells by modified viral capsids include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof. In particular, viral vectors can be advantageously used to deliver or transfer nucleic acids to animal cells, including mammalian cells. Thus, in some embodiments, nucleic acids ("cargo nucleic acids") can be encapsidated by the capsid proteins of the present disclosure. The cargo nucleic acid sequence delivered by the viral vectors of the present disclosure can be any heterologous nucleic acid sequence(s) of interest.

[0125] In some embodiments, expression of a heterologous nucleic acid delivered by an AAV vector disclosed herein is increased compared to expression of a heterologous nucleic acid delivered by a wild-type AAV vector (such as an AAV6 vector) or an AAV vector that does not include a transduction-associated peptide disclosed herein. In some embodiments, expression of a heterologous nucleic acid delivered by an AAV vector disclosed herein is increased by at least about 1.5-fold, such as about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold (including all values ​​and subranges therebetween) compared to expression of a heterologous nucleic acid delivered by a wild-type AAV vector (such as an AAV6 vector) or an AAV vector that does not include a transduction-associated peptide disclosed herein. In some embodiments, expression of a heterologous nucleic acid delivered by an AAV vector disclosed herein is increased by at least about 10%, e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% (including all values ​​and subranges therebetween) compared to expression of a heterologous nucleic acid delivered by a wild-type AAV vector (such as an AAV6 vector) or an AAV vector that does not contain a transduction-associated peptide disclosed herein.

[0126] Nucleic acids of interest include nucleic acids that encode 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 encodes a therapeutic protein or therapeutic RNA.

[0127] Therapeutic polypeptides include, but are not limited to, chimeric antigen receptors (CARs), ABCD1, beta globin (HBB), hemoglobin A, hemoglobin F, cystic fibrosis transmembrane conductance regulator protein (CFTR), dystrophin (mini-dystrophin and micro-dystrophin).See, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003 / 017131; International Publication No. WO / 2008 / 088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:1 3714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, activin type 11 soluble receptor, IGF-1, anti-inflammatory polypeptides such as IκB dominant mutants, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, a-globin, spectrin, α-1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, γ-interferon, etc. , interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, lymphotoxins, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial-derived growth factor, transforming growth factor-α and -β, etc.), lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis growth factor soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, molecules regulating calcium handling (e.g., SERCA). 2A, inhibitors of PP1 and fragments thereof [e.g., WO 2006 / 029319 and WO 2007 / 100465]), molecules that act to knockdown G protein-coupled receptor kinase type 2, such as truncated constitutively active bARKct, anti-inflammatory factors, such as IRAP, anti-myostatin protein, aspartoacylase, monoclonal antibodies (including single chain monoclonal antibodies; an exemplary Mab is the Herceptin® Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see U.S. Pat. No. 7,071,172), angiogenesis inhibitors, such as vasohibin and other VEGF inhibitors (e.g., vasohibin 2 [WO JP2006 / 073052]). 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-inactive Cas9 linked to a transcription enhancer or inhibitor element, zinc finger proteins linked to a transcription enhancer or inhibitor element, transcription activator-like (TAL) effectors linked to a transcription 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 that has a therapeutic effect in a subject in need of such a therapeutic effect. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, such as antibodies or antibody fragments directed against myostatin (e.g., Fang et al., Nature Biotechnology 23:584-590 (2005). Heterologous nucleic acid sequences encoding a polypeptide include sequences 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 chloramphenicol acetyltransferase genes.Optionally, the heterologous nucleic acid encodes a secreted polypeptide (e.g., a polypeptide that is a secreted polypeptide in its natural state or that has been engineered to be secreted, e.g., by operable association with a secretory signal sequence known in the art).

[0128] Alternatively, in some embodiments of the present disclosure, the heterologous nucleic acid may encode antisense nucleic acids, ribozymes (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediated / ram splicing (see Puttaraju et al, (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi) including siRNAs, shRNAs, or miRNAs that mediate 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; U.S. Pat. No. 5,869,248 to Yuan et al.). Exemplary non-coding RNAs include RNAi against multiple drug resistance (MDR) gene products (e.g., for administration to the heart to treat and / or prevent tumors and / or to prevent chemotherapy damage), 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 (e.g., for treating cardiovascular disease; see, e.g., Hoshijima et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)), and RNAi against phospholamban inhibitory or dominant negative molecules, such as phospholamban S16E (e.g., for treating cardiovascular disease; see, e.g., Hoshijima et al., J. al. Nat. Med. 8:864-871 (2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi directed against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).

[0129] Additionally, nucleic acid sequences that induce alternative splicing can be delivered. For example, antisense sequences (or other inhibitory sequences) that are complementary to the 5' and / or 3' splice sites of dystrophin exon 51 can be delivered together with U1 or U7 small nuclear (sn)RNA promoters to induce skipping of this exon. For example, DNA sequences that include U1 or U7 snRNA promoters located 5' to the antisense / inhibitory sequence(s) can be packaged and delivered in the modified capsids of the present disclosure.

[0130] In some embodiments, the nucleic acid sequence that induces gene editing can be delivered.For example, the nucleic acid can code gene editing molecule, such as guide RNA or nuclease.In some embodiments, the nucleic acid can code zinc finger nuclease, homing endonuclease, TALEN (transcription activator-like effector nuclease), NgAgo (argonaute endonuclease), SGN (structure-guided endonuclease), or RGN (RNA-guided nuclease), such as Cas9 nuclease or Cpf1 nuclease.

[0131] Viral vectors can also contain heterologous nucleic acid that shares common homology with and recombines with a locus on a host chromosome. This approach can be used, for example, to correct a genetic defect in a host cell.

[0132] The present disclosure also provides viral vectors expressing immunogenic polypeptides, e.g., for vaccination. The nucleic acid may encode any immunogen of interest 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 proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.

[0133] The use of parvoviruses as vaccine vectors is known in the art (see, e.g., 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 to Samulski et al.; U.S. Patent No. 5,863,541 to Samulski et al.). Antigens may be presented in the parvovirus capsid. Alternatively, antigens may be expressed from heterologous nucleic acids introduced into the recombinant vector genome. Any immunogen of interest described herein and / or known in the art may be provided by the viral vectors of the present disclosure.

[0134] An immunogenic polypeptide can be any polypeptide suitable for eliciting an immune response and / or protecting a subject against infection and / or disease, including, but not limited to, microbial, bacterial, protozoan, parasitic, fungal, and / or viral infection and disease. For example, an immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an 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 an HIV or SIV envelope GP 160 protein, an HIV or SIV matrix / capsid protein, and an HIV or SIV gag, pol, and env gene product). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a Lassa virus immunogen, such as a Lassa virus nucleocapsid protein, and a Lassa virus envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia LI gene product or an 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 a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide may further be a polio immunogen, a herpes immunogen (e.g., an immunogen for CMV, EBV, HSV), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or any other vaccine immunogen now known in the art or later identified as an immunogen.

[0135] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell.

[0136] Exemplary cancer and tumor cell antigens are described in SA Rosenberg (Immunity 10:281 (1991)). Other exemplary cancer 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, KIAA0205, 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. Pat. No. 4,968,603), CA125, LK26, FB5 (endosialin), TAG72, AFP, CA19-9, 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 WO90 / 05142), telomerase, nuclear matrix The antigens include, but are not limited to, antigens now known or later discovered to be associated with cancer, such as melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's 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 malignant condition now known or later identified, or metastases thereof (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

[0137] One skilled 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.

[0138] Furthermore, regulated expression of the heterologous nucleic acid(s) of interest can be achieved at the post-transcriptional level, for example by modulating 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).

[0139] Those skilled 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 natural or foreign, and can be natural or synthetic sequences. By foreign, it is meant that the transcription initiation region is not found in the wild-type host into which it is introduced.

[0140] In some embodiments, the promoter / enhancer element may be native to the target cell or subject to be treated. In representative 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. Furthermore, in some embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0141] Inducible expression control elements are typically advantageous in those applications where it is desirable to provide regulation over the expression of a heterologous nucleic acid sequence(s). Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferred promoter / enhancer elements, including muscle-specific or preferred (including cardiac, skeletal, and / or smooth muscle specific or preferred), neural tissue specific or preferred (including brain specific or preferred), eye-specific or preferred (including retina specific and cornea specific), liver-specific or preferred, bone marrow specific or preferred, pancreas specific or preferred, spleen specific or preferred, and lung specific or preferred promoter / enhancer elements. In some embodiments, inducible expression control elements such as promoters and / or enhancers promote selective expression in T cells. Other inducible promoter / enhancer elements include hormone-inducible elements and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and metallothionein promoter.

[0142] In embodiments in which the heterologous nucleic acid sequence(s) are transcribed and then translated in the target cell, specific initiation signals are generally included for efficient translation of the inserted protein-coding sequence. These exogenous translational control sequences may include the ATG initiation codon and adjacent sequences, and can be of a variety of origins, both natural and synthetic.

[0143] Pharmaceutical Compositions and Methods of Use The present disclosure also provides a composition comprising at least one of the AAV capsid protein, AAV capsid, viral vector, nucleic acid, expression vector, and / or cell disclosed herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided comprising the viral vector and / or capsid and / or capsid protein and / or viral particle of the present disclosure in a pharmaceutically acceptable carrier, and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other modes of administration, the carrier can be either a solid or liquid. For inhalation administration, the carrier is inhalable, and can optionally be in solid or liquid particulate form. "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.

[0144] The viral vectors according to the present disclosure provide a means for delivering heterologous nucleic acids into a wide range of cells, including dividing and non-dividing cells. In some embodiments, the cells are T cells. The viral vectors can be used to deliver a nucleic acid of interest to a cell in vitro, for example, to produce a polypeptide in vitro or to perform ex vivo gene therapy. The viral vectors are further useful in methods for delivering a nucleic acid to a subject in need of delivery of the nucleic acid to express, for example, an immunogenic polypeptide, or a therapeutic polypeptide, or a functional RNA. In this way, the polypeptide or functional RNA can be produced in vivo in the subject. The subject may need the polypeptide because it is deficient in the polypeptide. Furthermore, the method can be performed because producing the polypeptide or functional RNA in the subject may have some beneficial effect. In some embodiments, the method includes expressing the polypeptide or RNA in a cell in vitro, ex vivo, or in vivo, and optionally isolating the polypeptide or RNA from the cell. Viral vectors can also be used to produce a polypeptide or functional RNA of interest in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effect of the functional RNA on the subject, e.g., in connection with a screening method).

[0145] The present disclosure provides a method of administering any one of the viral vectors, viral particles, and / or compositions of the present disclosure to a subject. Accordingly, the present disclosure provides a method of treating a subject in need of treatment, comprising administering to the subject an effective amount of any one of the viral vectors (e.g., AAV vectors), any one of the viral particles (e.g., AAV particles), and / or any one of the compositions disclosed herein. Accordingly, the present disclosure provides any one of the viral vectors (e.g., AAV vectors), any one of the viral particles (e.g., AAV particles), and / or any one of the compositions disclosed herein for use as a medicament and / or in a method of treating a subject in need of treatment.

[0146] In some embodiments, the viral vectors of the present disclosure can be used to deliver heterologous nucleic acids encoding a polypeptide or functional RNA to treat and / or prevent any disease condition in which it is beneficial to deliver a therapeutic polypeptide or functional RNA, in some embodiments the disease condition is associated with, correlated with, or caused by T cell dysfunction or expansion. In some embodiments, disease states include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane conductance regulator protein) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin), and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (cytokines including endostatin, angiostatin, TRAIL, FAS-ligand, interferons;RNAi, including RNAi against VEGF or multidrug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma]), diabetes mellitus (insulin), muscular dystrophies, e.g., Duchenne (RNAi against dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., a, β, γ], myotrophin pro-peptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides, e.g., IκB dominant mutant, sarcospan, utrophin, mini-utrophin, antisense or RNAi against splice junctions in the dystrophin gene to induce exon skipping [see, e.g., WO / 2003 / 095647], U7 to induce exon skipping antisense to snRNA [see, e.g., WO / 2006 / 021724], and antibodies or antibody fragments to myostatin or myostatin propeptide) and Becker's, Gaucher's (glucocerebrosidase), Hurler's (aL-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry's [a-galactosidase] and Pompe's [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α-1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (as well as other diseases of the eye and retina;For example, inhibitors of PDGF and / or vasohibin or other VEGF for macular degeneration or other angiogenesis inhibitors to treat / prevent retinal disorders, for example in type I diabetes), diseases of solid organs, for example the brain (for example Parkinson's disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin and / or VEGF], glioblastoma [RNAi against endostatin, angiostatin and / or VEGF]), liver, kidney, heart, for example congestive heart failure or peripheral arterial disease (P AD) (e.g., protein phosphatase inhibitor I (I-1) and its fragments (e.g., IlC), serca2a, zinc finger proteins regulating the phospholamban gene, Barkct, [32-adrenergic receptor, 2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that act on G protein-coupled receptor kinase type 2 knockdown, e.g., truncated constitutively active bARKct;calsarcin, RNAi against phospholamban, delivery of phospholamban inhibitory or dominant negative molecules such as phospholamban S16E), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., delivery of enos, inos), improved survival of cardiac transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), renal failure (erythropoietin), These include inflammatory diseases such as osteoarthritis, osteoporosis ...

[0147] In some embodiments, the viral vectors of the present disclosure can be used to deliver heterologous nucleic acids encoding polypeptides or functional RNAs for treating and / or preventing liver diseases or disorders, such as primary biliary cirrhosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic 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, nonalcoholic 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), and others. I), hemophilia A, methylmalonic acidemia, propionic acidemia, homocystinuria, phenylketonuria (PKU), tyrosinemia type 1, arginase 1 deficiency, argininosuccinate lyase deficiency, carbamoyl phosphate synthase 1 deficiency, citrullinemia type 1, citrin deficiency, Crigler-Najjar syndrome type 1, cystinosis, Fabry disease, glycogen storage disease 1b, LPL deficiency, N-acetylglutamate synthase deficiency, ornithine transcarbamylase deficiency, ornithine translocase deficiency, primary hyperoxaluria type 1, or ADA SCID.

[0148] The viral vectors described herein can also be used to generate induced pluripotent stem cells (iPS).For example, the viral vectors of the present disclosure can be used to deliver stem cell-related nucleic acid(s) to non-pluripotent cells, such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, cardiac cells, neural cells, epithelial cells, endothelial cells, etc.

[0149] Nucleic acids encoding factors associated with stem cells are known in the art. Non-limiting examples of such factors associated with stem cells and pluripotency include Oct-3 / 4, SOX family (e.g., SOX1, SOX2, SOX3 and / or SOX15), Klf family (e.g., Klf1, KHZ Klf4 and / or Klf5), Myc family (e.g., C-myc, L-myc and / or N-myc), NANOG and / or LIN28.

[0150] In some embodiments, the modified vectors disclosed herein are used to treat lysosomal storage diseases, such as mucopolysaccharidoses, as described herein (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], [Ag-galactosidase], C [acetyl-CoA:alpha-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfatase], B [beta-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage diseases (e.g., Pompe disease; lysosomal acid alpha-glucosidase). In some embodiments, the disclosure can be practiced to treat metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), lysosomal storage diseases such as mucopolysaccharidoses (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-acetylglucosamine sulfatase], The enzymes may also be used to treat and / or prevent diseases such as Fabry disease (alpha-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage diseases (e.g., Pompe disease; lysosomal acid alpha-glucosidase), including but not limited to: C [acetyl-CoA:alpha-glucosaminidase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-sulfate sulfatase], B [beta-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.

[0151] Gene transfer is of great help in understanding disease states and providing therapy. There are many genetic diseases for which the defective genes are known and have been cloned. Generally, the disease states are divided into two categories: deficiency states of normal enzymes, which are generally inherited in a recessive manner, and imbalanced states, which may involve regulatory or structural proteins and are typically inherited in a dominant manner. For deficiency state diseases, gene transfer can be used to bring normal genes to the affected tissue for replacement therapy, and antisense mutations can be used to create animal models of the disease. For imbalanced disease states, gene transfer can be used to create disease states in model systems, which can then be used in attempts to combat the disease state. Thus, the viral vectors according to the present disclosure allow for the treatment and / or prevention of genetic diseases.

[0152] Viral vectors according to the present disclosure may be used to provide functional RNA to cells in vitro or in vivo. The functional RNA may be, for example, a non-coding RNA. In some embodiments, expression of a functional RNA in a cell may reduce expression of a specific target protein by the cell. Thus, functional RNA may be administered to reduce expression of a specific protein in a subject in need of reducing expression of the protein. In some embodiments, expression of a functional RNA in a cell may increase expression of a specific target protein by the cell. Thus, functional RNA may be administered to increase expression of a specific protein in a subject in need of increasing expression of the protein. In some embodiments, expression of a functional RNA may regulate splicing of a specific target RNA in a cell. Thus, functional RNA may be administered to regulate splicing of a specific RNA in a subject in need of regulating splicing of the specific RNA. In some embodiments, expression of a functional RNA in a cell may regulate the function of a specific target protein by the cell. Thus, functional RNA may be administered to regulate the function of a specific protein in a subject in need of regulating the function of the specific protein. Functional RNAs can also be administered to cells in vitro to modulate gene expression and / or cell physiology, for example, to optimize cell or tissue culture systems or in screening methods.

[0153] In some embodiments, the viral vectors disclosed herein may be contacted with cells ex vivo. In some embodiments, the cells are T cells, e.g., activated T cells. In some embodiments, the cells (e.g., activated T cells) are obtained from a subject, such as a human patient. In some embodiments, the cells contacted with the viral vector are administered to a subject in need thereof.

[0154] In some embodiments, the viral vector comprises a heterologous nucleic acid encoding a chimeric antigen receptor (CAR). Thus, in some embodiments, contacting the viral vector with a T cell results in expression of the chimeric antigen receptor (CAR) and generation of a CAR T cell. Thus, in some embodiments, the present disclosure provides a method of preparing a CAR T cell, comprising contacting any one of the viral vectors disclosed herein with a T cell ex vivo. The present disclosure further provides a CAR T cell produced using any one of the methods disclosed herein, and a method of treating a subject in need of treatment, comprising administering to the subject a CAR T cell disclosed herein. In some embodiments, the CAR T cell is produced using T cells obtained from the same subject (autologous T cells), while in other embodiments, the CAR T cell is produced using T cells obtained from a healthy donor subject (allogeneic T cells). A subject in need of administration of CAR T cells can be identified by a physician or skilled medical practitioner and can have any disease, e.g., cancer, such as acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, acute myeloid leukemia (AML), or multiple myeloma.

[0155] T cell exhaustion is a state of T cell dysfunction that occurs in many chronic infections and cancers, and has also been shown to reduce the effectiveness of CAR-T therapy. In some embodiments, the recombinant viral vectors disclosed herein are used in gene therapy (e.g., CAR-T therapy) to prevent, limit, and / or reverse T cell exhaustion. Thus, the present disclosure provides a method of alleviating, preventing, limiting, and / or reversing T cell exhaustion in a subject, comprising administering to the subject an effective amount of any one of the viral vectors (e.g., AAV vectors), any one of the viral particles (e.g., AAV particles), and / or any one of the compositions disclosed herein.

[0156] In some embodiments, the viral vector comprises a heterologous nucleic acid encoding an immunogen, such as an immunogenic polypeptide. Thus, in some embodiments, contact of the viral vector with the cell results in expression of the immunogen. In some embodiments, the cell can be administered to a subject, thereby inducing an immune response to the immunogen in the subject. In some embodiments, a protective immune response is elicited. In some embodiments, the cell is an antigen-presenting cell (e.g., a dendritic cell). In some embodiments, the cell is removed from the subject, the viral vector is introduced therein, and the cell is then returned to the subject. Methods for removing cells from a subject for ex vivo manipulation and then returning them to the subject are known in the art (see, for example, U.S. Patent No. 5,399,346). Alternatively, the recombinant viral vector can be introduced into cells from a donor subject, cultured cells, or cells from any other suitable source, and the cells are administered to a subject in need thereof (i.e., a "recipient" subject).

[0157] In some embodiments, cells can be removed from a subject with cancer and contacted with a viral vector expressing a cancer cell antigen according to the present disclosure. The modified cells are then administered to a subject to elicit an immune response against the cancer cell antigen. This method can be advantageously used in immunosuppressed subjects who are unable to mount a sufficient immune response in vivo (i.e., unable to produce sufficient amounts of enhanced antibodies). Alternatively, the cancer antigen can be expressed as part of or otherwise associated with the viral capsid (e.g., as described above). Alternatively, 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.

[0158] 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-1 beta, 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 chemotactic protein-1, granulocyte-macrophage colony-stimulating factor, and lymphotoxin). Thus, an immunomodulatory cytokine (preferably a CTL-inducing cytokine) can be administered to a subject together with the viral vector. The cytokine can be administered by any method known in the art. Exogenous cytokines may be administered to a subject, or alternatively, nucleic acids encoding the cytokines may be delivered to a subject using a suitable vector to allow production of the cytokine in vivo.

[0159] Additionally, viral vectors according to the present disclosure find use in diagnostic and screening methods whereby a nucleic acid of interest is expressed transiently or stably in cell culture systems or transgenic animal models.

[0160] The viral vectors of the present disclosure can also be used for a variety of non-therapeutic purposes, including, but not limited to, use in protocols to assess gene targeting, clearance, transcription, translation, etc., as will be apparent to one of skill in the art. The viral vectors can also be used to assess safety (distribution, toxicity, immunogenicity, etc.). Such data are reviewed, for example, by the U.S. Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.

[0161] In some embodiments, the modified viral capsids of the present disclosure are used to generate antibodies against the novel capsid structures, hi some embodiments, exogenous amino acid sequences can be inserted into the modified viral capsids for antigen presentation to cells, e.g., for administration to a subject to generate an immune response against the exogenous amino acid sequence.

[0162] In some embodiments, a viral capsid can be administered prior to and / or simultaneously with (e.g., within minutes or hours of each) administration of a viral vector that delivers a nucleic acid encoding a polypeptide or functional RNA of interest to block specific cellular sites. For example, a capsid of the invention can be delivered to block cellular receptors on liver cells, followed by or simultaneously with administration of a delivery vector, thereby reducing transduction of liver cells and enhancing transduction of other targets (e.g., skeletal muscle, cardiac muscle, and / or diaphragm muscle).

[0163] Dosage and Mode of Administration The viral vector can be introduced into cells at an appropriate multiplicity of infection according to standard transduction methods suitable for the particular target cells. The titer of the viral vector administered can vary depending on the target cell type and number and the particular viral vector, and can be determined by one of skill in the art without undue experimentation. In a representative embodiment, the titer is at least about 10 3 Infectious units, optionally at least about 10 5 The infectious units are introduced into the cells.

[0164] The cell(s) into which the viral vector is introduced can be of any type, including, but not limited to, T cells, neural cells (including cells of the peripheral and central nervous system, particularly brain cells, e.g., neurons and oligodendrocytes), lung cells, cells of the eye (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, cardiac muscle cells, 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, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like. In representative embodiments, the cells can be any progenitor cells. As a further possibility, the cells can be stem cells (e.g., neural stem cells, hepatic stem cells). As yet another option, the cells can be cancer cells or tumor cells. Furthermore, the cells can be from any species of origin, as indicated above.

[0165] Cells suitable for ex vivo nucleic acid delivery are as described above. The dosage of cells administered to a subject varies depending on the age, condition and species of the subject, the type of cells, the nucleic acid expressed by the cells, the mode of administration, etc. Typically, at least about 10 cells per dose are administered in a pharma- ceutically acceptable carrier. 2 ~about 10 8 cells or at least about 10 3 ~about 10 6 In some embodiments, the viral vector-transduced cells are administered to the subject in a therapeutically effective amount in combination with a pharmaceutical carrier.

[0166] In some embodiments, a viral vector can be introduced into a cell and the cell can be administered to a subject to induce an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or in a capsid). Typically, an amount of cells expressing an immunogenically effective amount of the polypeptide is administered in combination with a pharma- ceutically acceptable carrier. An "immunogenically effective amount" is an amount of expressed polypeptide that is sufficient to induce an active immune response against the polypeptide in the subject to whom the pharmaceutical preparation is administered. In some embodiments, the amount administered is sufficient to provide a protective immune response. The degree of protection conferred need not be complete or permanent, as long as the benefits of administering an immunogenic polypeptide outweigh any drawbacks thereof. Thus, the present disclosure provides a method of administering a nucleic acid to a cell, comprising contacting the cell with a viral vector, viral particle, and / or composition of the present disclosure.

[0167] The dosage of 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, and can be determined by routine methods. Exemplary dosages to achieve 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 , about 10 15 transducing units, optionally about 10 8 ~10 13 In some embodiments, two or more administrations (e.g., two, three, four or more administrations) can be used to achieve the desired level of gene expression over various intervals, e.g., daily, weekly, monthly, yearly, etc.

[0168] The administration of the viral vectors, viral particles and / or capsids according to the present disclosure to a human subject or an animal in need thereof can be performed by any means known in the art. Optionally, the viral vectors, viral particles and / or compositions are delivered in a therapeutically effective amount in a pharma- ceutically acceptable carrier. In some embodiments, a therapeutically effective amount of the viral vectors, viral particles and / or capsids is delivered.

[0169] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [e.g., administration to skeletal muscle, diaphragm muscle, and / or cardiac muscle], intradermal, intrathoracic, intracerebral, and intraarticular), topical (e.g., to both cutaneous and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct injection into a tissue or organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration can also be to a tumor (e.g., in 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. The present disclosure can also be implemented to generate non-coding RNA, such as antisense RNA, RNAi, or other functional RNA (eg, ribozymes) for systemic delivery.

[0170] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the viral vectors and / or viral capsids of the present disclosure can be administered in a localized manner rather than a systemic manner, for example, in a depot or sustained release formulation. Additionally, the viral vectors and / or viral capsids can be delivered attached to a surgically implantable matrix (e.g., as described in U.S. Patent Publication No. US-2004-0013645-A1). EXAMPLES

[0171] The following examples are included herein for illustrative purposes only and are not intended to be limiting. As used herein, the terms STRD.201, STRD.202, STRD.203, STRD.204, STRD.205, STRD.206, and STRD.207 are used to represent capsid protein sequences, and the terms AAV-STRD.201, AAV-STRD.202, AAV-STRD.203, AAV-STRD.204, AAV-STRD.205, AAV-STRD.206, and AAV-STRD.207 are used to represent AAV vectors comprising the capsid proteins. However, the terms STRD.201, STRD.202, STRD.203, STRD.204, STRD.205, STRD.206, and STRD.207 may be used to represent AAV vectors comprising the designated capsids, depending on the context, as will be apparent to one of skill in the art.

[0172] Example 1: Evolution of AAV capsid protein variants containing transduction-associated peptides An in vitro evolution process was used to prepare AAV capsid protein variants that, when incorporated into AAV vectors, enhance vector transduction into T cells. The first step of this process involved identifying surface-exposed regions of the AAV capsid surface using cryo-electron microscopy. Selected residues within the surface-exposed regions of the AAV capsid were then subjected to mutagenesis using degenerate primers in which each codon was replaced by the nucleotide NNK, and the gene fragments were combined together by Gibson assembly and / or multi-step PCR. Herein, amino acid residues 454-460 of SEQ ID NO:1 were subjected to random mutagenesis to generate a library of recombinant capsid gene sequences. Each gene of this degenerate library was cloned into the wild-type AAV genome to replace the original Cap-encoding DNA sequence, resulting in a plasmid library. The plasmid library was then transfected into a 293 producer cell line along with an adenovirus helper plasmid to generate an AAV capsid library. DNA sequencing confirmed successful generation of the AAV library.

[0173] To identify AAV vectors that can target and effectively transduce T cells, the AAV library described above was subjected to multiple rounds of in vitro selection. Specifically, an initial transduction into a mixed cell population was performed, followed by two rounds of transduction into activated donor T cells. At each stage, viral DNA was purified, PCR amplified, backcloned into AAV vectors, and used for the next round of selection. Further details of the general methods used for combinatorial manipulation and AAV vector selection are provided in WO2019 / 195449, WO2019 / 195423, and WO2019 / 195444, the contents of each of which are incorporated herein by reference in their entirety. After three rounds of infection, AAV particles were isolated from cultured T cells. Specifically, cells were lysed, viral DNA was purified from the nuclear and cytoplasmic fractions of T cells, PCR amplified, and backcloned into AAV vectors as described above.

[0174] After three rounds of selection and evolution as described in Example 1, AAV variants abundant in the nuclear and cytoplasmic fractions of T cells were sequenced to identify a single AAV isolate. In the bubble plot shown in Figure 5, the size of the bubble is proportional to the number of reads. The most abundant AAV variants in the nuclear fraction (AAV.STRD-203, 205), the cytoplasmic fraction (AAV.STRD-206, 207), or the nuclear and cytoplasmic fractions (AAV.STRD-201, 202, and 204) were sequenced to identify the amino acid residues present at amino acid positions 454-460. See Figure 6 and Table 5. These results demonstrated that recombinant AAV virions containing variant capsid proteins containing the transduction-associated peptides in Table 5 can effectively transduce T cells. [Table 7]

[0175] Example 2: Manufacturability of AAV vectors containing transduction-associated peptides To determine whether the various AAV vectors identified in Example 1 can be manufactured in large-scale systems, AAV was produced according to standard methods and yields were compared to those of wild-type AAV6 vector.

[0176] AAV was produced in HEK293 cells following a standard triple transfection protocol. Briefly, cells were transfected with (i) a plasmid containing either the wild-type AAV9 capsid sequence or a variant capsid sequence listed in Table 5, (ii) a plasmid containing the 5'ITR, transgene, and 3'ITR sequences, and (iii) a plasmid containing the helper genes required for AAV production. AAV was purified from the cell culture supernatant. The yield of each AAV was then measured using a PCR-based quantification approach.

[0177] As shown in Figure 1 and Table 6, a recombinant AAV vector containing the STRD-201 capsid sequence (referred to herein as "AAV.STRD-201") exhibited higher yields than that of wild-type AAV6. Furthermore, the yields of AAV.STRD-204, AAV.STRD-205, AAV.STRD-206, and AAV.STRD-207 were comparable to that of wild-type AAV6.

[0178] These data support that AAV vectors containing capsid variant proteins are suitable for commercial production. [Table 8]

[0179] Example 3: Characterization of GFP transgene expression by AAV variants in T cells Activated T cells were transduced with recombinant AAV variants, AAV.STRD-201, AAV.STRD-202, AAV.STRD-204, AAV.STRD-205, AAV.STRD-206, and AAV.STRD-207, carrying the GFP transgene sequence, or with wild-type AAV6 vector. Because T cells aggregate during proliferation, the cells were pipetted up and down or mixed before imaging. GFP expression was observed by microscopy, and images from the experiment are shown in Figure 2. Higher GFP expression indicates more efficient transduction of viral vectors into T cells. As can be seen from the images in Figure 2, all AAV variants showed brighter green fluorescent signals compared to wild-type AAV6 viral vectors, thus indicating higher expression of GFP in activated T cells. Among the recombinant AAV variants, AAV.STRD-201 and AAV.STRD-207 showed particularly enhanced GFP expression, suggesting more enhanced transduction of T cells. To further analyze the level of GFP expression from AAV variants compared to wild-type AAV6 viral vector, T cells transduced with either AAV6 vector or AAV.STRD-207 variant were subjected to flow cytometry, with T cells alone used as a negative control. As shown in Figure 3C, an increased percentage of cells transduced with AAV.STRD-207 variants showed higher GFP signal (indicated by FITC signal above the blue line) compared to the population transduced with AAV6 parent vector. GFP expression in cells transduced with AAV variants (AAV.STRD-201, AAV.STRD-202, AAV.STRD-204, AAV.STRD-205, AAV.STRD-206 and AAV.STRD-207) is further quantified in Figure 4, which shows the percentage of GFP positive cells in a given population (shown in bar graphs) and the average intensity of GFP in that population (shown in line graphs).The results show that the increase in the number of GFP-positive cells corresponds well with an increase in the average intensity of the GFP signal in cells transduced by the AAV variants compared to wild-type AAV6, indicating that enhanced transduction of AAV variants into T cells resulted in increased GFP expression in T cells.

[0180] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

[0181] Numbered Embodiments The following list of embodiments is included herein for illustrative purposes only and is not intended to be exhaustive or limiting. Claimed subject matter is expressly not limited to the following embodiments. Embodiment 1. A recombinant adeno-associated virus (AAV) vector comprising a capsid protein, wherein the capsid protein comprises a transduction-associated peptide having any one of the sequences of SEQ ID NOs: 17-23. Embodiment 2. The recombinant AAV vector of embodiment 1, wherein the capsid protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. Embodiment 3. The recombinant AAV vector of embodiment 1 or embodiment 2, wherein the transduction-associated peptide replaces amino acids corresponding to amino acids 454 to 460 of SEQ ID NO:1. Embodiment 4. The recombinant AAV vector of embodiment 1, wherein the capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Embodiment 5. A recombinant AAV vector comprising a capsid protein, wherein said capsid protein comprises the sequence of SEQ ID NO:1, and wherein amino acids 454 to 460 of SEQ ID NO:1 are replaced by a transduction-associated peptide comprising the sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO:24). Embodiment 6. The recombinant AAV vector of embodiment 5, wherein X1 is not G, X2 is not S, X3 is not A, X4 is not Q, X5 is not N, X6 is not K, and / or X7 is not D. Embodiment 7. The recombinant AAV vector of any one of embodiments 5 to 6, wherein X1 is H, M, A, Q, V, or S. Embodiment 8. A recombinant AAV vector according to any one of embodiments 5 to 7, wherein X2 is A or T. Embodiment 9. The recombinant AAV vector of any one of embodiments 5 to 8, wherein X3 is P or T. Embodiment 10. A recombinant AAV vector according to any one of embodiments 5 to 9, wherein X4 is R or D. Embodiment 11. A recombinant AAV vector according to any one of embodiments 5 to 10, wherein X5 is V, Q, C, S or D. Embodiment 12. A recombinant AAV vector according to any one of embodiments 5 to 11, wherein X6 is E, A or P. Embodiment 13. A recombinant AAV vector according to any one of embodiments 5 to 12, wherein X7 is E, G, N, T, or A. Embodiment 14. The recombinant AAV vector of embodiment 5, wherein X1 is H, X2 is A, X3 is P, X4 is R, X5 is V, X6 is E, and X7 is E. Embodiment 15. The recombinant AAV vector of embodiment 5, wherein X1 is M, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. Embodiment 16. The recombinant AAV vector of embodiment 5, wherein X1 is H, X2 is T, X3 is T, X4 is D, X5 is C, X6 is A, and X7 is N. Embodiment 17. The recombinant AAV vector of embodiment 5, wherein X1 is A, X2 is A, X3 is P, X4 is R, X5 is S, X6 is E, and X7 is T. Embodiment 18. The recombinant AAV vector of embodiment 5, wherein X1 is Q, X2 is A, X3 is P, X4 is R, X5 is Q, X6 is E, and X7 is G. Embodiment 19. The recombinant AAV vector of embodiment 5, wherein X1 is V, X2 is A, X3 is P, X4 is R, X5 is D, X6 is P, and X7 is A. Embodiment 20. The recombinant AAV vector of embodiment 5, wherein X1 is S, X2 is A, X3 is P, X4 is R, X5 is S, X46 is E, and X7 is N. Embodiment 21. The recombinant AAV vector of embodiment 5, wherein the capsid protein comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:1. Embodiment 22. The recombinant AAV vector of embodiment 21, wherein the capsid protein comprises an amino acid sequence having about 99% identity to SEQ ID NO:1. Embodiment 23. The recombinant AAV vector of embodiment 5, wherein the capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. Embodiment 24. A recombinant AAV vector comprising a capsid protein, wherein the capsid protein comprises a transduction-associated peptide having the amino acid sequence of SEQ ID NO:16, and wherein the transduction-associated peptide substitutes amino acids 454-460 relative to SEQ ID NO:1. Embodiment 25. The recombinant AAV vector described in embodiment 24, wherein the transduction-associated peptide has an amino acid sequence of any one of SEQ ID NOs: 17 to 23. Embodiment 26. A nucleic acid encoding a recombinant AAV capsid protein having the sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. Embodiment 27. The nucleic acid of embodiment 26, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15. Embodiment 28. The nucleic acid of embodiment 26 or embodiment 27, wherein the nucleic acid is a DNA sequence. Embodiment 29. The nucleic acid of embodiment 26 or embodiment 27, wherein the nucleic acid is an RNA sequence. Embodiment 30. An expression vector comprising the nucleic acid according to any one of embodiments 26 to 29. Embodiment 31. A cell comprising a nucleic acid according to any one of embodiments 26 to 29, or an expression vector according to embodiment 30. Embodiment 32. A recombinant AAV vector described in any one of embodiments 1 to 25, further comprising a cargo nucleic acid encapsidated by the capsid protein. Embodiment 33 The recombinant AAV vector of embodiment 32, wherein the cargo nucleic acid encodes a therapeutic protein or RNA. Embodiment 34. A recombinant AAV vector described in any one of embodiments 32 to 33, wherein the AAV vector exhibits increased transduction of cells compared to an AAV vector that does not contain the transduction-associated peptide. Embodiment 35 The AAV vector of embodiment 34, wherein the cell is a T cell. Embodiment 36. The AAV vector of embodiment 35, wherein the AAV vector exhibits increased transduction of the nucleus of the T cell compared to an AAV vector that does not contain the transduction-associated peptide. Embodiment 37. The AAV vector of embodiment 35, wherein the AAV vector exhibits increased transduction of the T cell cytosol compared to an AAV vector not comprising the transduction-associated peptide. Embodiment 38. A composition comprising a recombinant AAV vector according to any one of embodiments 1 to 25 or 32 to 37, a nucleic acid according to any one of embodiments 26 to 29, an expression vector according to embodiment 30, or a cell according to embodiment 31. Embodiment 39. A pharmaceutical composition comprising a cell according to embodiment 31 or a recombinant AAV vector according to any one of embodiments 1 to 25 or 32 to 37, and a pharma- ceutical acceptable carrier. Embodiment 40. A method for delivering an AAV vector to a cell, comprising contacting the cell with an AAV vector described in any one of embodiments 1 to 25 or 32 to 37. Embodiment 41 The method of embodiment 40, wherein the contacting of the cells is performed in vitro, ex vivo, or in vivo. Embodiment 42 The method of embodiment 40 or embodiment 41, wherein the cell is a T cell. Embodiment 43. A method for treating a subject in need of treatment, comprising administering to the subject an effective amount of an AAV vector described in any one of embodiments 1 to 25 or 32 to 37. Embodiment 44. A method for treating a subject in need of treatment, comprising administering to the subject cells that have been contacted ex vivo with an AAV vector described in any one of embodiments 1 to 25 or 32 to 37. Embodiment 45 The method of embodiment 43 or embodiment 44, wherein the subject is a mammal. Embodiment 46 The method of embodiment 45, wherein the subject is a human. Embodiment 47. An AAV vector according to any one of embodiments 1 to 25 or 32 to 37 for use as a medicament. Embodiment 48. An AAV vector described in any one of embodiments 1 to 25 or 32 to 37 for use in a method for treating a subject in need of treatment.

Claims

1. A recombinant adeno-associated virus (AAV) vector comprising a capsid protein, the capsid protein comprising a transduction-associated peptide having a sequence of any one of SEQ ID NOs: 17-23.

2. 2. The recombinant AAV vector of claim 1, wherein the capsid protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:

1.

3. 3. The recombinant AAV vector of claim 1 or 2, wherein the transduction-associated peptide replaces amino acids corresponding to amino acids 454-460 of SEQ ID NO:

1.

4. 2. The recombinant AAV vector of claim 1, wherein the capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

5. 1. A recombinant AAV vector comprising a capsid protein, said capsid protein comprising a transduction-associated peptide having the amino acid sequence of SEQ ID NO:16, said transduction-associated peptide substituting amino acids 454-460 relative to SEQ ID NO:

1.

6. The recombinant AAV vector of claim 5, wherein the transduction-associated peptide has the amino acid sequence of any one of SEQ ID NOs: 17 to 23.

7. A nucleic acid encoding a recombinant AAV capsid protein having the sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

8. The nucleic acid of claim 7, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15.

9. 9. The nucleic acid of claim 7 or 8, wherein the nucleic acid is a DNA sequence or an RNA sequence.

10. An expression vector comprising the nucleic acid according to any one of claims 7 to 9.

11. A cell comprising the nucleic acid according to any one of claims 7 to 9, or the expression vector according to claim 10.

12. A recombinant AAV vector according to any one of claims 1 to 6, comprising a cargo nucleic acid encapsidated by said capsid protein.

13. A pharmaceutical composition comprising the cell of claim 11 or the recombinant AAV vector of any one of claims 1 to 6, and a pharma- ceutically acceptable carrier.

14. A method for delivering an AAV vector to a cell, the method comprising contacting the cell with the AAV vector of any one of claims 1 to 6.

15. 13. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of an AAV vector of any one of claims 1 to 6, or a cell that has been contacted ex vivo with an AAV vector of any one of claims 1 to 6.

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