Novel Recombinant AAV VP2 Fusion Polypeptide

Abstract

The present disclosure relates to an adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand. The present disclosure further relates to rAAV virions comprising such AAV VP2 fusion polypeptides, and libraries of nucleic acids encoding such AAV VP2 fusion polypeptides, pharmaceutical compositions comprising such rAAV virions, and related methods and uses.

Description

Technical Field

[0001] The present disclosure provides an adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand for improving targeting of AAV in gene therapy approaches. The present disclosure further provides rAAV virions comprising such AAV VP2 fusion polypeptides, and libraries of nucleic acids encoding such AAV VP2 fusion polypeptides, as well as related compositions, methods and uses.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. A copy of the ASCII created on May 5, 2022, is named PAT059281-US-PSP_SL and is 54,417 bytes in size.

Background Art

[0003] Adeno-associated virus (AAV) vectors are one of the most promising gene delivery vectors due to their excellent safety and efficacy profiles. Well-established features of AAV vectors that distinguish them from other vectors include stable long-term expression, broad host range, the ability to transduce both proliferating cells and post-mitotic cells, high titers of AAV vectors produced in tissue culture, derivation from non-pathogenic viruses, and low immunogenicity of both wild-type viruses and recombinant vectors.

[0004] However, due to their broad tropism, AAV vectors have low transduction efficiency into many target organs, and thus, it is necessary to apply high vector doses. It has become increasingly clear that the full potential of this vector system is realized only by modified AAV vectors that exhibit improved cell transduction rates and specificity, which in turn results in an improved safety profile.

[0005] Retargeting attempts are directed at such variable regions for the exposed positions of the loops forming the protrusions and their function in receptor binding. However, these sites tolerate only the insertion of small peptides. U.S. Patent Application Publication No. 20180163229 discloses a variant AAV capsid polypeptide comprising a DARPin fused to the N-terminus of AAV VP2.

[0006] There remains a high unmet need for additional mutant AAV capsid polypeptides that can mediate improved AAV properties for gene therapy, such as increased transduction and / or increased tropism in at least one tissue or cell type, improved cell type selectivity, and / or improved targeting specificity. SUMMARY OF THE INVENTION

[0007] The present disclosure provides an adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, where, for example, the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide directly or via a peptide linker. The peptide linker has a molecular weight of up to 10 kDa. When typically used together with AAV VP1 and / or VP3 capsid polypeptides for rAAV virion assembly, the AAV VP2 fusion polypeptides provided herein can exhibit good modification levels, which means that a satisfactory number of AAV VP2 fusion polypeptides are incorporated into the rAAV virions.

[0008] The AAV VP2 fusion polypeptide described herein can mediate improvement of transduction and / or increase of tropism in at least one tissue or cell type as compared to an AAV VP2 capsid polypeptide that is identical to the VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide except that it is not fused to the polypeptide ligand. The improvement of transduction and / or increase of tropism in at least one tissue or cell type can be mediated by a polypeptide ligand having the ability to bind to cell surface molecules expressed in at least one tissue or cell type. Accordingly, an rAAV virion comprising the AAV VP2 fusion polypeptide provided herein and presenting a polypeptide ligand on its surface confers high cell type selectivity and / or targeting specificity, thereby enabling restriction of biodistribution and safe gene transfer, and thus can be used for cell type-specific gene transfer in therapeutic applications and basic research.

[0009] Accordingly, in one aspect, provided herein is an adeno-associated virus (AAV) VP2 fusion polypeptide comprising, for example, consisting of an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide and the polypeptide ligand has a molecular weight of up to 10 kDa.

[0010] In other aspects, provided herein are nucleic acids encoding such AAV VP2 fusion polypeptides, and cells comprising such AAV VP2 fusion polypeptides or nucleic acids encoding them.

[0011] In other aspects, provided herein are rAAV virions comprising the AAV VP2 fusion polypeptides disclosed herein, and pharmaceutical compositions comprising such rAAV virions.

[0012] In other aspects, provided herein are a library of nucleic acid constructs encoding the AAV VP2 fusion polypeptides disclosed herein, and methods of making AAV VP2 fusion polypeptides having desired characteristics using such libraries.

[0013] In other aspects, provided herein are methods of treatment using rAAV virions comprising the AAV VP2 fusion polypeptides disclosed herein and pharmaceutical compositions comprising such rAAV virions.

[0014] In another aspect, the present specification provides an AAV VP2 capsid polypeptide, wherein a) the AAV VP2 capsid polypeptide is of AAV serotype AAV1 and contains at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 capsid polypeptide contains the amino acid substitutions: D213A, T162R, and / or P191N, or b) the AAV VP2 capsid polypeptide is of an AAV serotype other than AAV1 and contains at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, bi) the AAV VP2 capsid polypeptide is of AAV serotype 8 and contains at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 capsid polypeptide contains the amino acid substitutions: D214A, K163R, and / or P192N, or bii) the AAV VP2 capsid polypeptide is of AAV serotype 9 and contains at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 capsid polypeptide contains the amino acid substitutions: D213A, S162R, and / or P191N.

Embodiments for Carrying out the Invention

[0015] Definitions As used in the specification and the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells including mixtures thereof.

[0016] All numerical notations, such as pH, temperature, time, concentration, and molecular weight, are approximate values that vary in increments of 0.1 (+) or (-), including ranges. Although not necessarily explicitly stated, it should be understood that the term "about" precedes all numerical notations. The term "about" with respect to a numerical value X means, for example, X ± 15% (including all values within that range). Also, although not necessarily explicitly stated, it should be understood that the reagents described herein are merely examples, and that reagents equivalent thereto are known in the art.

[0017] Throughout this specification and the following claims, unless the context requires otherwise, the words "comprise", "comprises", "comprising", and variations thereof are used in an open-ended, non-limiting sense.

[0018] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect, embodiment, and / or claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic novel characteristics of the aspect, embodiment, and / or claim.

[0019] The terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide typically contains at least two amino acids or amino acid variants, and there is no limit to the maximum number of amino acids that can be included in a protein or peptide sequence. A polypeptide encompasses any peptide or protein containing two or more amino acids or variants linked to each other by peptide bonds. This term includes, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0020] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length. These can include one or more of ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, e.g., analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA). Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by creating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al. Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0021] The terms "sequence identity" and "sequence homology" are used interchangeably herein and, when used in reference to a polynucleotide or polypeptide, refer to the percentage of bases or amino acids at the same relative positions when two sequences of the polynucleotide or polypeptide are compared or aligned. Thus, if the subunit positions of both of two molecules are occupied by the same monomeric subunit, for example, if each position of two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer ten subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) match or are homologous, the two sequences are 90% homologous. Sequence identity can be determined in several different ways. For example, the percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, wherein the fragment of the amino acid sequence in the comparison window may include additions or deletions (e.g., gaps or overhangs) as compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue exists in both sequences, obtaining the number of positions that match, dividing the number of positions that match by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence. Sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.). See, for example, Altschul et al., (1990) J. Mol. Bioi., 215:403-10.

[0022] As used herein, the terms "naturally occurring" or "unmodified" when applied to, for example, nucleic acids, polypeptides, cells, or organisms, refer to those that occur in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (such as a virus) is naturally occurring whether it is present in that organism or isolated from one or more components of that organism.

[0023] The term "variant" with respect to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that differs from a parent polynucleotide or polypeptide, also referred to as a non-variant polynucleotide or polypeptide sequence, by at least one residue, i.e., at least one nucleotide in the case of a polynucleotide and at least one amino acid in the case of a polypeptide.

[0024] The term "isolated" with respect to a nucleic acid, polypeptide, or virus discussed herein refers to a nucleic acid, polypeptide, or virus that has been separated from one or more components that are normally associated with it in its natural environment. For example, a nucleic acid or peptide that occurs naturally in a living animal is "not isolated", whereas the same nucleic acid or peptide that has been partially or completely separated from its coexisting substances in its natural state is "isolated". Separation can include removal from a larger nucleic acid (e.g., from a gene or chromosome) or from other proteins or molecules that are normally in contact with the nucleic acid or protein. This term encompasses, but does not require, complete separation. Thus, an isolated nucleic acid or protein may exist in a substantially purified form or, for example, in a non-natural environment such as a host cell.

[0025] As used herein, an isolated nucleic acid comprising a "heterologous nucleic acid sequence" refers to an isolated nucleic acid that contains a portion (i.e., a heterologous nucleic acid portion) that is not operably linked to one or more other components of the isolated nucleic acid in its natural context. For example, a heterologous nucleic acid can include a nucleic acid sequence that is not naturally occurring in a cell, bacterial cell, virus, or organism, where one or more other components of the isolated nucleic acid (e.g., a promoter) are of natural origin, or where one or more other components of the isolated nucleic acid (e.g., a promoter) do not exist in an operably linked state with the heterologous nucleic acid in a cell, bacterial cell, virus, or organism. In some embodiments, the heterologous nucleic acid comprises a transgene. As used herein, a "transgene" is a nucleic acid sequence that encodes a molecule of interest (e.g., a therapeutic protein, a therapeutic RNA molecule, or a reporter protein) that is not naturally bound to one or more components of the nucleic acid molecule. In some embodiments, the heterologous nucleic acid sequence encodes a human protein. In some embodiments, the heterologous nucleic acid sequence encodes an RNA sequence, e.g., shRNA.

[0026] As used herein, the term "reporter sequence" refers to a nucleic acid sequence that encodes a reporter protein, such as a fluorescent protein or an oxidase enzyme, thereby enabling visualization of infection by an rAAV vector comprising such a reporter sequence, i.e., monitoring the success of transduction of a target cell or tissue based on the expression of the reporter protein. A preferred oxidase is firefly luciferase; exemplary fluorescent proteins include GFP and variants thereof, such as eGFP, and sfCherry. The reporter sequence can be packaged into an rAAV virion in addition to, or in place of, a nucleic acid encoding a therapeutic transgene or an AAV VP2 fusion polypeptide disclosed herein.

[0027] As used herein, the term "barcode array" refers to a unique oligonucleotide sequence (e.g., 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 50, 75, 100 nucleotides) having a specific sequence that is used as a means to identify the nucleic acid sequence into which it is incorporated. For example, a barcode can be used as a means to identify or identify individual members (e.g., variants) in a library.

[0028] A DNA sequence or DNA polynucleotide sequence that "encodes" a particular RNA is a DNA sequence that can be transcribed into RNA. A DNA polynucleotide can also encode RNA (mRNA) that is translated into protein, or alternatively, a DNA polynucleotide can encode RNA that is not translated into protein (e.g., tRNA, rRNA, or guide RNA; also referred to as "non-coding" RNA or "ncRNA"). A DNA sequence or DNA polynucleotide sequence can further "encode" a particular polypeptide or protein sequence, in which case, for example, the DNA directly encodes mRNA that can be translated into the polypeptide or protein sequence. A "protein coding sequence" or a sequence encoding a particular protein or polypeptide is a nucleic acid sequence that can be transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence can be determined by the start codon at the 5' end (N-terminus) and the translation stop nonsense codon at the 3' end (C-terminus). The coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. The transcription termination sequence is usually located 3' of the coding sequence.

[0029] As used herein, the term "promoter" or "promoter sequence" refers to a DNA regulatory sequence that can promote the transcription of an operably linked coding or non-coding sequence, such as a downstream (3' direction) coding or non-coding sequence, by binding to, for example, RNA polymerase (e.g., can cause a detectable level of transcription and / or can increase the detectable level of transcription above the level obtained in the absence of the promoter). In some embodiments, the promoter sequence is bound at its 3' end by the transcription start site and extends upstream (5' direction) to include a minimal number of bases or elements for initiating transcription at a detectable level above background. In some embodiments, the promoter sequence can include the transcription start site and a protein binding domain responsible for binding of RNA polymerase. In addition to the sequence sufficient to initiate transcription, the promoter can also include sequences of other regulatory elements involved in the regulation of transcription (e.g., enhancers, Kozak sequences, and introns). A variety of promoters, including inducible promoters and constitutive promoters, can be used to drive expression from the vectors disclosed herein. Examples of promoters known in the art that can be used in some embodiments, such as in the nucleic acid molecules and vectors disclosed herein, include the CMV promoter, 173CMV promoter, HCMV promoter, CBh promoter, CAG promoter, mCCT promoter, CBA promoter, smCBA promoter, and promoters derived from immunoglobulin genes, SV40, or other tissue-specific genes (e.g., RLBP1, RPE, VMD2). Furthermore, standard techniques are also known in the art for creating functional promoters by mixing and matching known regulatory elements. Fragments of promoters, such as fragments that retain at least a minimal number of bases or elements for initiating transcription at a detectable level above background, may also be used.

[0030] In some embodiments, the promoter may be a constitutively active promoter (i.e., a promoter that constitutively drives expression in any cell type and / or under any conditions). In other embodiments, the promoter may be a promoter that is constitutively active in a particular tissue context, such as neurons, heart cells, etc. In other embodiments, the promoter may be an inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, such as the presence of a particular temperature, compound, or protein). In some embodiments, the promoter may be a spatially restricted promoter whose activity can be driven or not depending on the physical situation in which the promoter is found. Non-limiting examples of spatially restricted promoters include tissue-specific promoters, cell-type specific promoters, etc. In some embodiments, the promoter may be a temporally restricted promoter that drives expression depending on the temporal context in which the promoter is found. For example, a temporally restricted promoter may drive expression only at a particular stage of embryonic development or only at a particular stage of a biological process. A non-limiting example of a temporally restricted promoter is the mouse hair follicle cycle promoter.

[0031] In some embodiments, the promoter is tissue-specific in a multicellular organism such that the promoter drives expression only in a particular subset of cells. For example, tissue-specific promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. A neuron-specific promoter refers to a promoter that preferentially drives or controls the expression of a heterologous nucleic acid, such as a nucleic acid encoding a protein or peptide of interest or shRNA, in neurons compared to expression in non-neuronal cells upon delivery to neurons, such as by peripheral administration, direct administration to the central nervous system (CNS), or in vitro, ex vivo, or in vivo.

[0032] The term "operably linked" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this term refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a sequence is continuous with or separated from that sequence by a short spacer sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically continuous with or in close proximity to the coding sequence whose transcription they enhance.

[0033] The terms "DNA regulatory sequence", "control element", and "regulatory element", which are used interchangeably herein, refer to transcriptional and translational control sequences such as promoters, enhancers, silencers, polyadenylation signals, terminators, proteolytic signals, etc., that confer and / or regulate the transcription of non-coding sequences (e.g., short hairpin RNA) or coding sequences (e.g., transgenes) and / or regulate the translation of the encoded polypeptide.

[0034] The terms "polyadenylation (polyA) signal sequence" and "polyadenylation sequence" refer to regulatory elements that confer a signal for transcriptional termination and the addition of an adenosine homopolymer chain to the 3'-end of an RNA transcript. A polyadenylation signal can include a termination signal (e.g., the AAUAAA sequence or other non-canonical sequences), and optionally adjacent auxiliary elements (e.g., GU-rich elements) and / or other elements associated with efficient cleavage and polyadenylation. A polyadenylation sequence can consist of a series of adenosines attached by polyadenylation to the 3'-end of an mRNA. Exemplary polyA signal sequences are the BGH and SV40 polyA signal sequences. In some embodiments, the DNA regulatory sequence or control element is a tissue-specific regulatory sequence.

[0035] The term "post-transcriptional regulatory element" ("PRE") refers to one or more regulatory elements that, when transcribed into mRNA, regulate gene expression at the level of the mRNA transcript. Examples of such post-transcriptional regulatory elements include sequences encoding microRNA binding sites, RNA binding protein binding sites, and the like. Examples of post-transcriptional regulatory elements that can be used with the nucleic acid molecules and vectors disclosed herein include the woodchuck hepatitis post-transcriptional regulatory element (WPRE), and the hepatitis post-transcriptional regulatory element (HPRE).

[0036] The term "intron" refers to a nucleic acid sequence that is non-coding for one or more amino acids of a polypeptide transcript (e.g., a protein of interest) expressed from a nucleic acid, e.g., within an open reading frame. Intron sequences can be transcribed from DNA into RNA (i.e., can be present in pre-mRNA), but can be removed, for example, by splicing, before the protein is expressed from the mature mRNA.

[0037] The term "exon" refers to a nucleic acid sequence that encodes one or more amino acids of a transcript (e.g., a protein of interest) expressed from a nucleic acid, e.g., within an open reading frame (ORF). Exon sequences can be transcribed from DNA into RNA (i.e., can be present in pre-mRNA) and can also be present in the mature mRNA that is translated into a polypeptide (i.e., the processed form of the RNA (e.g., after splicing)).

[0038] In some embodiments, a "vector" is any genetic element (e.g., DNA, RNA, or a mixture thereof) containing a nucleic acid of interest (e.g., a transgene) that can be expressed in a host cell, such as a nucleic acid of interest within a larger nucleic acid sequence or structure suitable for delivery to cells, tissues, and / or organisms, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. For example, a vector can include an insert (e.g., a heterologous nucleic acid including a gene to be expressed or a transgene encoding the open reading frame of that gene) and one or more additional elements suitable for controlling the delivery or expression of the insert. A vector may be capable of replication and / or expression, for example, when associated with appropriate control elements, or may be capable of transmitting genetic information between cells. In some embodiments, the vector may be a vector suitable for expression in a host cell, such as an AAV vector. In some embodiments, the vector may be a plasmid suitable for expression and / or replication, for example, in a cell or bioreactor. In some embodiments, a vector specially designed for the expression of a heterologous nucleic acid sequence in a target cell, such as a transgene encoding a protein of interest, shRNA, etc., may be referred to as an expression vector, which generally has a promoter sequence that drives the expression of the transgene. In other embodiments, a vector, such as a transcription vector, is capable of transcription but not translation, i.e., it can be replicated in a target cell but not expressed. Transcription vectors can be used to amplify their inserts.

[0039] The term "expression vector" refers to a vector containing a polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector may contain sufficient cis-acting elements for expression, either alone, or in combination with other elements for expression supplied by a host cell or in an in vitro expression system. Examples of expression vectors include cosmids, plasmids (e.g., contained in naked or liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0040] The term "plasmid" refers to a non-chromosomal (usually double-stranded) DNA sequence containing an intact "replicon" such that the plasmid is replicated within a host cell. A plasmid may be a circular nucleic acid. When a plasmid is placed within a unicellular organism, the characteristics of that organism are changed or transformed as a result of the plasmid's DNA. For example, a plasmid carrying the gene for tetracycline resistance (TcR) transforms cells that were previously sensitive to tetracycline into cells that are resistant to it.

[0041] As used herein, the term "recombinant virus" is intended to refer to non-wild type and / or artificially produced recombinant viruses (such as parvovirus, adenovirus, lentivirus, or adeno-associated virus, etc.) that contain a transgene or other heterologous nucleic acid. A recombinant virus may include a recombinant viral vector (such as one containing a transgene) packaged within a viral (such as AAV) capsid. A particular type of recombinant virus may be "recombinant adeno-associated virus", or "rAAV". A recombinant viral genome packaged in a viral capsid may be a viral vector. In some embodiments, the recombinant viruses disclosed herein include viral vectors (such as those containing a transgene of interest as described herein). Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, chimeric AAV vectors, adenovirus vectors, retrovirus vectors, lentivirus vectors, DNA virus vectors, herpes simplex virus vectors, baculovirus vectors, or variants or derivatives thereof.

[0042] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. This term encompasses all subtypes, as well as both wild-type and recombinant forms, unless explicitly stated otherwise. The term "rAAV" refers to recombinant adeno-associated virus or recombinant AAV vectors.

[0043] As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, or a vector containing one or more nucleic acid sequences derived therefrom, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5 viral vectors. An AAV vector may contain, for example, one or more AAV wild-type genes that are deleted in part or in whole thereof, such as the rep and / or cap genes, while retaining functional flanking inverted terminal repeat ("ITR") sequences. In some embodiments, the AAV vector may be packaged, for example, in a protein shell containing one or more AAV capsid proteins, i.e., a "capsid", thereby providing a vehicle for delivering the vector nucleic acid to the nucleus of a target cell. In some embodiments, the AAV vector contains one or more AAV ITR sequences (e.g., AAV2 ITR sequences). In some embodiments, the AAV vector contains one or more AAV ITR sequences (e.g., AAV2 ITR sequences) but no additional viral nucleic acid sequences. In some embodiments, the AAV vector components (e.g., ITRs) are derived from a virus of a different serotype than the rAAV capsid (e.g., the AAV vector may contain an ITR derived from AAV2 or the AAV vector may be packaged in an AAV9 capsid). Embodiments of these vector constructs are described, for example, in International Publication No. WO 2019 / 094253 (PCT / US2018 / 058744), which is hereby incorporated by reference in its entirety.

[0044] rAAV vectors include single-stranded AAV vectors and self-complementary AAV vectors (scAAV). scAAV is called "self-complementary" because at least a part of the scAAV vector (for example, at least a part of the coding region) forms an intramolecular double-stranded DNA. In some embodiments, the rAAV is scAAV. In other embodiments, the rAAV is single-stranded AAV. In some embodiments, the viral vector is engineered from naturally occurring adeno-associated virus (AAV) to confer scAAV for use in gene therapy. Embodiments of these vector constructs and methods for preparing and purifying them are provided, for example, in International Publication No. WO 2019 / 094253 (PCT / US2018 / 058744), which is hereby incorporated by reference in its entirety.

[0045] As used herein, "virus" or "virion" refers to a viral particle that includes a viral vector, for example, alone or in combination with one or more additional components such as one or more viral capsids. For example, an AAV virus includes, for example, a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein membrane.

[0046] In some embodiments, terms such as "virus", "virion", "AAV virus", "recombinant AAV virion", "rAAV virion", "AAV vector particle", "full capsid", "full particle" refer to an infectious, replication-deficient virus, e.g., an AAV protein shell encapsulating a heterologous nucleotide sequence of interest within a viral vector flanked on one or both sides by AAV ITRs. An rAAV virion can be produced in a suitable host cell by including sequences that define an AAV vector, e.g., one or more plasmids, alone or in combination with nucleic acids encoding AAV helper functions and accessory functions (such as the rep and cap genes), e.g., on the same plasmid or an additional plasmid. In some embodiments, the host cell is made capable of encoding an AAV polypeptide that achieves packaging of an AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0047] "Packaging" refers to a series of intracellular events that result in the assembly of an AAV virion or AAV particle that encapsulates a nucleic acid sequence. Packaging can mean encapsulation of a nucleic acid sequence into a capsid containing an AAV VP2 fusion polypeptide disclosed herein.

[0048] An "infectious" virion, virus or viral particle contains a polynucleotide component capable of being delivered to a cell tropic for the viral species. This term does not necessarily admit any conclusion regarding the virus's ability to replicate. As used herein, an "infectious" virus or viral particle is one that, when in proximity to a target cell, is capable of infecting the target cell and expressing a heterologous nucleic acid in the target cell. Thus, "infectivity" refers to the ability of a viral particle to approach, infect and express a heterologous nucleic acid in a target cell. Infectivity refers to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are known in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the viral genome copy number. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as "transformation". The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using several techniques, including the evaluation of a marker gene such as the green fluorescent protein (GFP) assay (e.g., here, the virus contains a nucleotide sequence encoding GFP, which is produced in cells infected with the viral particle and detected and / or measured); or, for example, by measuring the produced protein by enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS).

[0049] A "replication-competent" virion or virus (e.g., replication-competent AAV) refers to an infectious virus that can replicate within an infected cell (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, the ability to replicate generally requires the presence of functional AAV packaging genes, namely, the cap gene and the rep gene. In some embodiments, as described herein, the AAV vector lacks one or more AAV packaging genes and is non-replicable in mammalian cells (such as human cells). In some embodiments, the AAV vector lacks any AAV packaging gene sequences, minimizing the possibility of generating replication-competent AAV by recombination between the AAV packaging genes and incoming AAV vectors.

[0050] The term "inverted terminal repeat" or "ITR" refers to a segment of nucleotide sequence that can form a T-shaped palindromic structure, for example, in adeno-associated virus (AAV) and / or recombinant adeno-associated virus vectors (rAAV). Muzyczka et al., (2001) Fields Virology, Chapter 29, Lippincott Williams & Wilkins. In recombinant AAV vectors, these sequences can play a functional role in genome packaging and second-strand synthesis. In some embodiments, the AAV vector contains one or more mutated or shortened ITRs.

[0051] The term "cap gene" or "capsid gene" refers to a nucleic acid sequence that encodes a viral capsid, i.e., the capsid proteins that form or contribute to the formation of the protein shell. In the case of AAV, the capsid proteins are typically VP1, VP2, and VP3. In the case of other parvoviruses, the names and numbers of the capsid proteins may vary. As used herein, the terms "AAV VP1 capsid polypeptide", "AAV VP2 capsid polypeptide", and "AAV VP3 capsid polypeptide" include wild-type AAV capsid polypeptides, as well as variants and fragments thereof, particularly functional variants and fragments. Variants and fragments of functional AAV capsid polypeptides can be used for the assembly of AAV capsids.

[0052] The "rep gene" refers to a nucleic acid sequence that encodes the non-structural proteins (rep78, rep68, rep52, and rep40) required for AAV replication and production.

[0053] The term "AAV helper function" refers to an AAV-derived coding sequence that can be expressed to provide AAV gene products, e.g., those that function in trans for productive AAV replication. For example, the AAV helper function can include both rep and cap, the major AAV open reading frames (ORFs). The rep expression products have been demonstrated to have many functions, among others: recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The Cap expression products provide the necessary packaging functions. The AAV helper function can be used to complement the in trans AAV functions missing in an AAV vector.

[0054] The term "AAV helper construct" generally refers to a nucleic acid molecule that provides or encodes a protein or nucleic acid that confers AAV functions deleted from an AAV vector, e.g., a vector for delivering a nucleotide sequence of interest to a target cell or tissue. AAV helper constructs are commonly used to effect transient expression of the AAV rep and / or cap genes to complement AAV functions that are lacking for AAV replication. Typically, helper constructs lack AAV ITRs and thus cannot replicate or package themselves. AAV helper constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been disclosed, e.g., the commonly used plasmids pAAV / Ad and plM29+45 that encode both Rep and Cap expression products. See, e.g., Samulski et al., (1989) J. Virol., 63:3822-3828; McCarty et al., (1991) J. Virol., 65:2936-2945. Several other vectors encoding Rep and / or Cap expression products have been disclosed. See, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237. Embodiments of these vector constructs, as well as methods for preparing and purifying them, are provided, e.g., in International Publication No. 2019 / 094253 pamphlet (PCT / US2018 / 058744), which is hereby incorporated by reference in its entirety.

[0055] An AAV "helper virus" refers to a virus that enables replication and packaging of AAV in mammalian cells. Such diverse helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass several different subgroups, but adenovirus type 5 of subgroup C is most commonly used as a helper virus.

[0056] The term "helper virus function" refers to a function encoded in a helper virus genome that enables AAV replication and packaging in mammalian cells. Helper virus functions include, for example, adenovirus helper functions. Such helper virus functions can be provided in several ways, such as by providing a helper virus, e.g., by introducing into a producer host cell a nucleic acid sequence encoding a function necessary for AAV production.

[0057] The terms "tropism" and "transduction" are related but distinct. As used herein, the term "tropism" refers to the ability of an AAV vector or virion to infect one or more specified cell types, and can also include how the vector functions to transduce cells of one or more specified cell types; i.e., tropism refers to the preferential entry of an AAV vector or virion into a particular cell type or tissue type, and / or preferential interaction with a cell surface that facilitates entry into a particular cell type or tissue type, and optionally, and preferably thereto, expression of a sequence carried by the AAV vector or virion in the cell, e.g., in the case of a recombinant virus, expression of a heterologous nucleotide sequence follows. As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more specific cell types; i.e., transduction refers to the entry of an AAV vector or virion into a cell and the transfer of genetic material contained within the AAV vector or virion into the cell to achieve expression of the vector genome. Although not in all cases, in some cases, transduction and tropism can be correlated.

[0058] As used herein, the term "tropism profile" refers to a pattern of transduction of one or more target cells, tissues, and / or organs. Different AAV serotypes exhibit biased tropism profiles and thus tropism can be altered, for example, by manipulation of the capsid.

[0059] The term "host cell" means a cell containing a foreign nucleic acid of interest, e.g., one or more microorganisms, yeast cells, insect cells, mammalian cells. For example, a host cell may contain an AAV helper construct, an AAV vector plasmid, an accessory function vector, and / or other transfer DNA. The term includes the progeny of the original transfected cell. The progeny of a single parental cell may not necessarily be identical in morphology, or genomic or total DNA complement to the original parental cell due to natural, accidental, or intentional mutations.

[0060] As used herein, the term "cell line" refers to a population of cells that can grow and divide continuously or for an extended period in vitro. Under certain circumstances, spontaneous or induced changes in the karyotype can occur during the preservation or introduction of such clonal populations. Thus, cells derived from a cell line may not be strictly identical to the ancestral cells or culture, and such variants are included in the cell line referred to.

[0061] The term "transfection" refers to the uptake of foreign DNA by a cell once exogenous DNA has been introduced into the cell membrane, thereby "transfecting" the cell. See, for example: Graham et al., (1973) Virology, 52:456; Sambrook et al., (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier; Chu et al., (1981) Gene, 13:197. Using such techniques, one or more exogenous DNA sites can be introduced into a suitable host cell. In some embodiments, the term "transduction" is used to refer to the uptake of foreign DNA by a cell, where the foreign DNA is conferred by a virus or viral vector. As a result, when exogenous DNA is introduced into the cell membrane, the cell is said to be "transduced". In some embodiments, the term "transformation" is used to refer to the uptake of foreign DNA by bacterial cells.

[0062] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multiple or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Antibodies can be, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, or chimeric antibodies. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Throughout this document, the term "antibody" or "antibody molecule" also includes, unless the context specifically indicates otherwise, any fragment thereof and any derivative thereof.

[0063] The term "antibody fragment" or "antigen-binding fragment" refers to at least a part of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-bonded Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, bispecific antibodies formed from antibody fragments such as a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody, but are not limited thereto. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a variable region of a light chain and at least one antibody fragment containing a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are continuously linked, for example, via a synthetic linker, such as a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless otherwise specified, an scFv may have VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-linker-VH, or VH-linker-VL.

[0064] The term "complementary determining region" or "CDR" as used herein refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or combinations thereof, and the ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Lefranc et al., (2015) Nucleic Acids Res. 43, D413-422) (the "IMGT" numbering scheme). In some embodiments, in a combined Kabat and Chothia numbering scheme for a given CDR region (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3), the CDR corresponds to the amino acid residues defined as part of the Kabat CDR together with the amino acid residues defined as part of the Chothia CDR. As used herein, a CDR defined according to the "Chothia" numbering scheme is sometimes also referred to as a "hypervariable loop". Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align. Generally, unless otherwise indicated, an antibody molecule can include any combination of one or more Kabat CDRs and / or Chothia CDRs.

[0065] The term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or interact with a molecule. Epitope determinants generally consist of chemically active surface groups of a molecule, such as amino acids, carbohydrates, or sugar side chains, and can have specific three-dimensional structural properties and specific charge properties. Epitopes can be "linear" or "conformational". Conformational and linear epitopes are distinguished, for example, in that binding to the former is lost in the presence of a denaturing solvent while binding to the latter is not.

[0066] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to polypeptides, including antibodies, bispecific antibodies, etc., having substantially the same amino acid sequence or derived from the same genetic source. The term also includes preparations of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0067] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework regions and the CDR regions are derived from sequences of human origin. The constant regions may also be of human sequence, such as human germline sequences, or mutant versions of human germline sequences, or may be derived from antibodies that include consensus framework sequences derived from human framework sequence analysis, as described, for example, in Knappik et al., (2000. J Mol Biol 296, 57-86). The structure and position of immunoglobulin variable domains, such as CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia, and the ImMunoGenTics (IMGT) numbering (see, for example, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Al Lazikani et al., (1997) J. Mol. Bio. 273:927 948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mal. Biol. 273:927-948 and Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Lefranc et al., (2015) Nucleic Acids Res. 43, D413-422).

[0068] Human antibodies can include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or somatic mutations in vivo, or conservative substitutions to promote stability or production). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0069] As used herein, the phrase "recombinant antibody" includes all antibodies prepared, expressed, made, or isolated by recombinant means, including antibodies isolated from transgenic or translchromosomal animals (e.g., mice) for human immunoglobulin genes, or hybridomas prepared therefrom, antibodies isolated from host cells transformed to express human antibodies, e.g., transfectomas, antibodies isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, made, or isolated by any other means that includes splicing of the sequences of all or part of human immunoglobulin genes to other DNA sequences, such as recombinant human antibodies. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if animal transgenics for human Ig sequences are used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are related to, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo, and are derived from human germline VH and VL sequences.

[0070] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. At each antigenic site, the variable regions of the antibody interact with the antigen via weak non-covalent forces at multiple sites; the more interactions, the stronger the affinity. As used herein, the term "high affinity" with respect to an IgG antibody or a fragment thereof (e.g., a Fab fragment) refers to an antibody having an affinity of 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, or 10 -13 M or less for the target antigen. However, high affinity binding can vary for other antibody isotypes. For example, high affinity binding for the IgM isotype refers to an antibody having an affinity of 10 -7 M or less, or 10 -8 M or less.

[0071] As used herein, the terms "binding specificity" or "specific binding" refer to the ability of an individual antibody binding site to react with one antigenic determinant rather than different antigenic determinants. The binding site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. The binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single binding site on the antibody. It is the sum of the attractive and repulsive forces acting between the antigenic determinant and the binding site of the antibody.

[0072] The terms "treating" and "treatment" mean a therapeutic treatment, which is to slow down an undesirable physiological change or disorder in a subject. For the purposes of the present invention, a beneficial or desired clinical result, whether detectable or undetectable, includes, but is not limited to, alleviation of symptoms, reduction of the degree of a disease, stabilization of a medical condition (i.e., not getting worse), delay or slowing of disease progression, alleviation or mitigation of a medical condition, and remission (whether partial or complete). "Treatment" can also mean an extension of survival as compared to the expected survival in the absence of treatment.

[0073] The terms "prevention", "prevent", and "preventing" of any particular disease or disorder refer to prophylactic or preventive measures such as administering a compound of the invention to a subject before any symptoms of the disease or disorder appear.

[0074] The term "subject" refers to an animal, human or non-human to whom treatment by the methods of the invention is provided. Veterinary and non-veterinary applications are contemplated. The term includes, but is not limited to, mammals such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Typical subjects include humans, farm animals, and household pets such as cats and dogs. In some preferred embodiments, the subject is a human.

[0075] The terms "pharmaceutically acceptable" and "physiologically acceptable" are used interchangeably herein and refer to biologically acceptable formulations of a gas, liquid or solid that are suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a substance that is not biologically or otherwise disadvantageous, e.g., such a substance can be administered to a subject without substantially causing undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to administer the rAAV virions disclosed herein to a subject.

[0076] "Effective amount" refers to an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may or may not be the same as a prophylactically effective amount, which is an amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. The "therapeutically effective amount" (i.e., effective dosage) of a therapeutic compound depends on the particular therapeutic compound selected. The composition can be administered one or more times per day to one or more times per week, including every other day. One of ordinary skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments.

[0077] As used herein, a process performed "in vitro" refers to a process performed outside of a normal biological environment, e.g., a test performed in a test tube, flask, Petri dish, or artificial medium. A process performed "in vivo" refers to a process performed within a living organism or cell, e.g., a test performed in cell culture or a mouse. A test performed "ex vivo" refers to a test performed in an external environment, on or within a tissue derived from an organism, that allows manipulation of cells or tissues of the organism under conditions that minimize modification of natural conditions, e.g., under more controlled conditions than are possible in in vivo experiments.

[0078] As used herein, the term "library" refers to a plurality, i.e., at least two, different variant linear nucleic acids, plasmids, virus particles, or viral vectors, etc.

[0079] 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0080] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the detailed description, the drawings, and the claims.

[0081] AAV VP2 Fusion Polypeptide Provided herein is an AAV VP2 fusion polypeptide comprising, from the N-terminus to the C-terminus, a polypeptide ligand, optionally a peptide linker, and an AAV VP2 capsid polypeptide. An rAAV virion comprising the AAV VP2 fusion polypeptide and presenting the polypeptide ligand on its surface results in high cell-type selectivity and / or high targeting specificity, enabling restricted in vivo distribution and safe gene transfer, and thus can be used for cell-type specific gene transfer in therapeutic applications and in basic research applications.

[0082] For rAAV virion assembly, typically when used together with AAV VP1 and / or VP3 capsid polypeptides, the AAV VP2 fusion polypeptide can exhibit a good decoration level, which means that a satisfactory number of AAV VP2 fusion polypeptides are incorporated into the rAAV virion. Surprisingly, it has been found that a good decoration level can be achieved by N-terminally fusing a polypeptide ligand having a molecular weight of up to 10 kDa to the AAV VP2 capsid polypeptide. In the AAV VP2 fusion polypeptide containing a polypeptide ligand located at the N-terminus with a molecular weight exceeding 10 kDa, the decoration level decreases. The AAV VP2 fusion polypeptide containing a polypeptide ligand located at the N-terminus with a molecular weight exceeding 15 kDa, such as a darpin (darpin) with a molecular weight of more than about 18 kDa, shows no decoration or only minimal decoration. Without wishing to be bound by theory, since the AAV VP2 capsid polypeptide is not essential for capsid assembly, providing an AAV VP2 capsid polypeptide with an unfavorable structure, such as an AAV VP2 fusion polypeptide containing a bulky polypeptide ligand, the AAV VP2 capsid polypeptide is not used for capsid assembly, and as a result, rAAV virions may be obtained that have no or very little AAV VP2 fusion polypeptide decoration composed mainly of or only AAV VP1 and VP3 capsid polypeptides.

[0083] The AAV VP2 fusion polypeptides described herein, compared to AAV VP2 capsid polypeptides that are identical to the VP2 capsid polypeptides contained in the AAV VP2 fusion polypeptides except that they are not fused to the polypeptide ligand, can mediate improved transduction and / or increased tropism in at least one tissue or cell type. The improved transduction and / or increased tropism in at least one tissue or cell type is thought to be mediated by the polypeptide ligand, which may have the ability to bind to cell surface molecules expressed in at least one tissue or cell type. Suitable polypeptide ligands having a molecular weight of less than 10 kDa include, but are not limited to, GP2 and Sso7d ligands and affibodies.

[0084] The present invention is based in part on incorporating highly diverse polypeptide ligand libraries, such as highly diverse Sso7d libraries, into rAAV virions by fusing these libraries to the N-terminus of AAV VP2 capsid polypeptides. An AAV library was generated that contains rAAV virions that contain an AAV VP2 fusion polypeptide within the capsid and contain a nucleic acid encoding the AAV VP2 fusion polypeptide encapsulated within the capsid. This AAV library has high diversity and functional titer and can be used for in vivo selection of AAV VP2 fusion polypeptides with desired characteristics. Provided herein are AAV VP2 fusion polypeptides obtained by in vivo selection that enrich for AAV capsids with high transduction of a particular cell type.

[0085] The AAV VP2 fusion polypeptides provided herein can be used in the production of recombinant AAV vectors. Such recombinant AAV vectors are suitable for delivering heterologous nucleic acids, such as therapeutic transgenes, into target cells.

[0086] Accordingly, in one aspect, provided herein is an adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand. The polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, either directly or via a linker. The polypeptide ligand has a molecular weight of up to 10 kDa, such as up to 9 kDa, up to 8 kDa, up to 6 kDa, or up to 5 kDa, for example 3-10 kDa, 4-8 kDa, or 5-7 kDa.

[0087] In one aspect, provided herein is an AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide; and the AAV VP2 capsid polypeptide comprises one or more mutations that abolish or reduce binding to heparan sulfate proteoglycan (HSPG) and / or sialic acid (SIA). In some embodiments, the polypeptide ligand has a molecular weight of up to 10 kDa.

[0088] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or derivatives thereof. This term encompasses all subtypes, as well as both natural and recombinant forms, unless another interpretation is required. The term "AAV" includes, for example, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10, including AAVrh10), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects mammals other than primates, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0089] The genomic sequences of various serotypes of AAV, as well as the sequences of native inverted terminal repeats (ITRs), 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, see the following: GenBank accession numbers: NC-002077 (AAV1), AF063497 (AAV1), NC-001401 (AAV2), AF043303 (AAV2), NC-001729 (AAV3), NC-001829 (AAV4), U89790 (AAV4), NC-006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC-006261 (AAV8); or publications such as WO 2005 / 033321 pamphlet (AAV1-9), the disclosures of which are incorporated herein by reference. Further, for example, also see the following references: Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; WO 00 / 28061 pamphlet, WO 99 / 61601 pamphlet, WO 98 / 11244 pamphlet; and U.S. Patent No. 6,156,303 specification.

[0090] In some embodiments, the polypeptide ligand specifically binds to cell surface molecules expressed in at least one tissue or cell type. In some embodiments, the AAV VP2 fusion polypeptide, which does not contain the polypeptide ligand but is otherwise identical to the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide, mediates an increase in transduction and / or an increase in tropism induction in at least one tissue or cell type.

[0091] In some embodiments, transduction of at least one tissue or cell type increases by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. In some embodiments, the AAV VP2 fusion polypeptide, which does not contain the polypeptide ligand but is otherwise identical to the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide, mediates an increase in transduction in at least one tissue or cell type that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more than 1000-fold compared to the AAV VP2 capsid polypeptide. In some embodiments, tropism in at least one tissue or cell type increases by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.

[0092] In some embodiments, the AAV VP2 fusion polypeptide mediates an increase in transduction and / or an increase in tropic cells in whole blood, brain, liver, spleen, kidney, skeletal muscle, heart, lung, or bone marrow, or in multipotent progenitor cells (MPPs), such as multipotent hematopoietic progenitor cells, or in hematopoietic stem cells (HSCs), such as long-term hematopoietic stem cells (LT-HSCs).

[0093] In one aspect, provided herein is an AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide either directly or via a linker, and which is selected from the group consisting of a GP2 polypeptide, an Sso7d polypeptide, and an affibody.

[0094] As used herein, the term "GP2 polypeptide" refers to a polypeptide scaffold derived from the 45-residue T7 phage gene 2 protein (Gp2). This polypeptide contains an α-helix facing a β-sheet with two adjacent loops suitable for mutagenesis. Mutagenesis of this scaffold can yield high-affinity target-specific binders.

[0095] As used herein, the term "Sso7d polypeptide" refers to a polypeptide derived from the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. This protein is an attractive binding scaffold due to its small size (7 kDa), high thermal stability (Tm of 98 °C), and lack of cysteine and glycosylation sites. In some embodiments, the Sso7d polypeptide is derived from a charge-neutralized variant of the S. solfataricus Sso7d protein, particularly the charge-reduced Sso7d (rcSso7d) variant described in Traxlmayr et al. (DOI 10.1074 / jbc.M116.741314). As used herein, the Sso7d polypeptide also encompasses polypeptides having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over the full length with the wild-type Sso7d or the rcSso7d variant described in Traxlmayr et al. (DOI 10.1074 / jbc.M116.741314).

[0096] In some embodiments, the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity therewith.

[0097] In some embodiments, the polypeptide ligand is optionally selected from the Sso7d polypeptide of SEQ ID NO: 1 having a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.

[0098] In some embodiments, the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1, wherein the amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, and W, and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity therewith.

[0099] In some embodiments, the polypeptide ligand is selected from the Sso7d polypeptide of SEQ ID NO: 1, wherein the amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, W, and amino acid substitutions, and SEQ ID NO: 1 optionally has a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.

[0100] In some embodiments, the AAV VP2 fusion polypeptide further comprises a peptide linker located between the polypeptide ligand and the AAV VP2 capsid polypeptide. In some embodiments, the peptide linker is selected from the group consisting of a glycine-serine (GS) linker and an alanine-proline-serine (APS) linker. The GS linker may be, for example, of the formula [GGGGS]n, where n is an integer in the range of 1 to 10, for example, n is 1, 2, 3, 4, 5 or 6, and in particular, n is 1, 2, 3 or 4. The APS linker may be, for example, of the formula [APS]n, where n is an integer in the range of 1 to 10, for example, n is 1, 2, 3, 4, 5 or 6, and in particular, n is 2, 3, 4 or 5.

[0101] In some embodiments, the AAV VP2 capsid polypeptide comprised in the AAV VP2 fusion polypeptide is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5. In certain embodiments, the AAV VP2 capsid polypeptide comprised in the AAV VP2 fusion polypeptide is of an AAV serotype selected from the group consisting of AAV1, AAV6, AAV8 and AAV9.

[0102] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide comprises at least one mutation in at least one binding site for a natural receptor present on the target cell of an AAV virion containing the AAV VP2 capsid polypeptide. As used herein, the term "natural receptor" refers to, for example, the following: heparan sulfate proteoglycan (HSPG), which has been shown to be the major cellular receptor for AAV serotype 2; N-linked sialic acid-containing glycans, which have been shown to be the major cellular receptors for AAV serotypes 1, 5, and 6; O-linked sialic acid-containing glycans, which have been shown to be the major cellular receptors for AAV serotypes 4 and 9; αVβ5 integrin, α5β1 integrin, CD9, and hepatocyte growth factor receptor, which act as secondary receptors, or rather co-receptors, for AAV serotype 2; basic fibroblast growth factor receptor and 37 / 67 kDa laminin receptor (LamR), which act as secondary receptors, or rather co-receptors, for AAV serotypes 2, 3, 8, and 9; and / or platelet-derived growth factor receptor (PDGFR), which acts as a secondary receptor, or rather co-receptor, for AAV serotype AAV-5. In some embodiments, at least one essential binding site of the AAV VP2 capsid polypeptide for its natural receptor is mutated. In some embodiments, the at least one binding site of the AAV VP2 capsid polypeptide for its natural receptor is located within the VP3 region of the AAV VP2 capsid polypeptide, i.e., within the region shared among the VP1, VP2, and VP3 capsid polypeptides. The regions encoding the VP1 and VP2 proteins represent N-terminal extensions of the region encoding the VP3 protein. Thus, the reading frames of the regions encoding VP1, VP2, and VP3 overlap such that a mutation in the VP3 region is present in all three capsid proteins.In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV6 and within its VP3 region contains at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 3. In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV6 and contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 3: i) K531E and V473D; ii) K531E, K459S, V473D, and N500E; iii) G266A and N269Q; iv) G266A, N269Q, and D590A; v) K531E, V473D, G266A, and N269Q; or vi) K531E, K459S, V473D, N500E, G266A, N269Q, and D590A.

[0103] In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV8 and within its VP3 region contains at least one amino acid substitution selected from the group consisting of: G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4. In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV8 and contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 4: i) G268E and N271Q; ii) S387A; iii) G268E, N271Q, and S387A; iv) A592Q; or v) A592D.

[0104] In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV9 and includes, within its VP3 region, at least one amino acid substitution selected from the group consisting of Q590A, W503A, N562A, and E563A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5. In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV9 and includes the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 5: i) W503A; ii) N562A and E563A; iii) Q590A and W503A; or iv) Q590A, W503A, N562A, and E563A.

[0105] In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV2 and includes the amino acid substitution R585A within its VP3 region relative to the VP1 amino acid sequence of SEQ ID NO: 6.

[0106] In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV1 and includes, within its VP3 region, at least one amino acid substitution selected from the group consisting of V473D, N500E, and R514A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV1 and includes the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 7: i) V473D and N500E; ii) R514A; or iii) V473D, N500E, and R514A.

[0107] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide comprises at least one amino acid substitution relative to each wild-type AAV VP2 capsid polypeptide. The inventors have shown that certain amino acid substitutions in the AAV VP2 capsid polypeptide can result in improved receptor binding, e.g., improved binding of an AAV VP2 fusion polypeptide comprising a mutated AAV VP2 capsid polypeptide to the receptor for the ligand contained in the AAV VP2 fusion polypeptide. Amino acid substitutions in the AAV VP2 capsid polypeptide can also improve transduction. Without wishing to be bound by theory, it is hypothesized that the amino acid substitutions identified by the inventors as improving receptor binding and / or transduction will result in a higher portion of the VP2 N-terminus present in the AAV virion facing the outside of the viral particle. Under natural conditions, most of the VP2 N-terminus present in the AAV virion faces the inside of the AAV capsid, thereby limiting the level of decoration of the AAV virion by the ligand fused to the VP2 N-terminus. By increasing the portion of the VP2 N-terminus present in the AAV virion that faces the outside of the viral particle, i.e., by increasing the VP2 exposure of the AAV virion, the level of decoration by the ligand fused to the VP2 N-terminus can be increased, which can lead to improved receptor binding and transduction levels.

[0108] Accordingly, in some embodiments, the AAV VP2 fusion polypeptide is of AAV serotype AAV1 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In certain such embodiments, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D213A, T162R, and / or P191N, and in particular, the AAV VP2 fusion polypeptide comprises all three amino acid substitutions: D213A, T162R, and P191N.

[0109] In some embodiments, the AAV VP2 fusion polypeptide of AAV serotype AAV1 further comprises the following amino acid substitutions: V473D and N500E with respect to the VP1 amino acid sequence of SEQ ID NO: 7.

[0110] In other embodiments, the AAV VP2 fusion polypeptide is of an AAV serotype other than AAV1 and comprises at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In certain such embodiments, the AAV VP2 fusion polypeptide is of AAV serotype 8 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 fusion polypeptide comprises all three amino acid substitutions: D214A, K163R, and P192N.

[0111] In other such embodiments, the AAV VP2 fusion polypeptide is of AAV serotype 9 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 5. In certain such embodiments, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D213A, S162R, and / or P191N, in particular all three amino acid substitutions: D213A, S162R, and P191N.

[0112] In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide does not contain the native start codon of the AAV VP2 capsid polypeptide. To avoid expression of the native VP2 polypeptide by inhibiting transcription of the VP2 open reading frame, the native start codon of the AAV VP2 capsid polypeptide may be mutated, for example, deleted. Preferably, the mutation does not change the reading frame. In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide lacks its first native amino acid, for example, the first threonine of the corresponding wild-type AAV VP2 capsid polypeptide.

[0113] In some embodiments, the polypeptide ligand contained in the AAV VP2 fusion polypeptide contains a maximum of 3 cysteine residues, a maximum of 2 cysteine residues, a maximum of 1 cysteine residue, or no cysteine residues.

[0114] Nucleic Acids Encoding AAV VP2 Fusion Polypeptides and AAV Cargo Nucleic Acid Sequences In one aspect, provided herein is a nucleic acid encoding the AAV VP2 fusion polypeptide described herein. The nucleic acid encoding the AAV VP2 fusion polypeptide may be RNA, such as mRNA, or DNA, such as cDNA, linear DNA or circular DNA, for example plasmid DNA.

[0115] Also provided herein are nucleic acids that can be encapsulated by an AAV capsid comprising the AAV VP2 fusion polypeptide described herein, and rAAV virions comprising such nucleic acids. These nucleic acids are referred to herein as "AAV genome", "AAV vector genome", "AAV vector", "AAV vector nucleic acid", "AAV cargo", or "cargo plasmid", and these terms are used interchangeably herein. Such AAV cargo or AAV vector genome typically comprises two AAV inverted terminal repeats. The AAV cargo can encode the AAV VP2 fusion polypeptide described herein and / or can include a reporter sequence and / or a barcode sequence, or the AAV cargo can encode a therapeutic RNA or protein, including a therapeutic antibody or a fragment thereof.

[0116] The AAV vector genome can be either single-stranded or self-complementary. “Self-complementary AAV” or “scAAV” refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Without being bound by theory, upon infection, instead of waiting for second-strand cell-mediated synthesis, the two complementary halves of scAAV associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, D M McCarty et al, “Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254 (which is hereby incorporated by reference in its entirety). Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is hereby incorporated by reference in its entirety. For example, the 5’ ITR can be mutated, for example, by deleting the terminal resolution site to allow hairpin formation of the genome.

[0117] In some embodiments, the rAAV vectors disclosed herein lack one or more (e.g., all) AAV rep and / or cap genes. The AAV vector can contain a nucleic acid sequence (e.g., DNA) derived from any suitable AAV serotype (e.g., within its ITR). Suitable AAV serotypes include, but are not limited to, AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5. For example, an AAV vector, e.g., an scAAV vector, can contain a nucleic acid sequence derived from AAV-2, e.g., an ITR sequence derived from AAV-2. An AAV vector, e.g., an scAAV vector, can also contain nucleic acids from two or more serotypes. The nucleotide sequences of the genomes of AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-2 is provided in GenBank accession numbers NC001401 and Srivastava et al., Virol, 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the genome of AAV-5 is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least a portion of the genomes of AAV-7 and AAV-8 are provided in GenBank accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Williams, (2006) Mol. Ther., 13(1):67-76; the AAV-11 genome is provided in Mori et al., (2004) Virology, 330(2):375-383.

[0118] In some embodiments, the functional inverted terminal repeat (ITR) sequences can be used, for example, to support the rescue, replication, and packaging of AAV virions. Thus, the AAV vectors disclosed herein can include sequences (e.g., functional ITRs) that enable viral replication and packaging in cis. The ITRs may be wild-type nucleotide sequences, but they do not have to be, and may be modified, for example, by nucleotide insertions, deletions, or substitutions, as long as the sequences enable functional rescue, replication, and packaging. The ITRs can be from any AAV serotype from which recombinant viruses can be derived, and as such, include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV, or avian AAV.5.

[0119] In some embodiments, the rAAV vectors disclosed herein include one or more ITRs, e.g., two ITRs, one located upstream and the other located downstream of a heterologous nucleic acid (e.g., encoding an AAV VP2 fusion polypeptide and / or a reporter protein or a therapeutic RNA or protein as described herein) and / or other nucleic acid elements described above. In some embodiments, the nucleic acids disclosed herein include, for example, in a scAAV vector, a first ITR located 5’ of the other vector element and a second ITR located 3’, where the ITRs are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 150, 200, 250 nucleotides on the 5’ and / or 3’ side of the other element. The ITR sequence may be wild-type or may include one or more mutations as long as, for example, it retains one or more functions of the wild-type ITR. In some embodiments, the wild-type ITR may be modified to include a deletion of the terminal resolution site. In some embodiments, the scAAV disclosed herein may include two ITR sequences, in which case both are independently wild-type, variant, or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a wild-type, variant, or modified AAV ITR sequence. In some embodiments, both of the two ITR sequences are wild-type, variant, or modified AAV ITR sequences. In some embodiments, the “left” or 5’-ITR is a modified AAV ITR sequence that enables the production of a self-complementary genome, and the “right” or 3’-ITR is a wild-type AAV ITR sequence. In some embodiments, the “right” or 3’-ITR is a modified AAV ITR sequence that enables the production of a self-complementary genome, and the “left” or 5’-ITR is a wild-type AAV ITR sequence. In some embodiments, both ITRs are AAV2 ITRs (optionally, one is wild-type and the other is modified to remove the terminal resolution site or both are wild-type).The AAV ITR embodiments provided in International Publication No. WO 2019 / 094253 (PCT / US2018 / 058744), which is incorporated herein by reference in its entirety, can also be used for any AAV ITRs disclosed herein.

[0120] Polynucleotide sequences can be produced by de novo synthesis (e.g., solid-phase DNA synthesis) or by PCR using an existing sequence as a template. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art such as Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0121] Also provided herein are vectors (e.g., expression vectors) that contain nucleic acids encoding the AAV VP2 fusion polypeptides described herein, and / or contain reporter sequences, and / or contain barcode sequences, or contain nucleic acids encoding therapeutic RNAs or proteins such as therapeutic antibodies or fragments thereof. Using such vectors, an RNA or protein of interest, such as an AAV VP2 fusion polypeptide, can be expressed and / or produced intracellularly, e.g., in vitro, ex vivo, or in vivo, e.g., in one or more tissues of interest in an organism. Various expression vectors can be used to express an RNA or protein of interest, such as the AAV VP2 fusion polypeptides described herein. Viral-based and non-viral expression vectors can be used to produce an RNA or protein of interest in cells, e.g., mammalian cells. Non-viral vectors and systems include plasmids, episomal vectors, typically those containing an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). Such non-viral vectors may be delivered to the target cells using transfection or transduction methods known in the art, e.g., using lipids (e.g., lipofectamine), electroporation, mechanical cell membrane deformation, etc. The term "expression vector" refers to a carrier nucleic acid molecule into which a desired coding sequence can be inserted for introduction into a cell in which it can be expressed. The vector can be a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector, or an artificial chromosome (see, e.g., Harrington et al., Nat Genet 15:345, 1997).For example, non-viral vectors useful for the expression of the RNA and proteins of interest, such as the AAV VP2 fusion polypeptides described herein, in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1 / His, pEBVHis A, B & C, (Invitrogen, San Diego, Calif.), MPSV vectors, and numerous other vectors known in the art for protein expression. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous sarcoma virus, MMTV or MoMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern equine encephalitis virus and flavivirus. Useful viral vectors include retroviruses (e.g., lentiviruses), lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus (HSV)), SV40-based vectors, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus, Sindbis virus, influenza virus, reovirus, Newcastle disease virus (NDV), measles virus, vesicular stomatitis virus (VSV), parvovirus, poliovirus, poxvirus, Seneca Valley virus, coxsackievirus, enterovirus, myxoma virus, Maraba virus or Semliki Forest virus (SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992. In some embodiments, the vector is an adeno-associated virus (AAV) vector, e.g., a recombinant AAV (rAAV) vector.

[0122] In some embodiments, the vector can be a recombinant DNA molecule that contains a nucleic acid encoding an AAV VP2 fusion polypeptide described herein, and / or contains a reporter sequence, and / or contains a barcode sequence, or contains a nucleic acid encoding a therapeutic RNA or protein such as a therapeutic antibody or a fragment thereof. As used herein, "recombinant" means the product of various combinations of cloning, restriction or ligation steps (e.g., related to the polynucleotide or polypeptide contained therein), and / or other procedures that result in a construct different from that found in nature. A recombinant virus or vector is a viral particle containing a recombinant polynucleotide. The term includes, respectively, the replication of the original polynucleotide construct and the progeny of the original viral construct.

[0123] Recombinant vectors typically contain one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). One of ordinary skill in the art will readily recognize that expression of one or more components of the vector in the target cell may require regulatory sequences. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory sequences and / or inducible sequences. Expression vectors may also contain elements designed to optimize the stability and translatability of messenger RNA in the host cell, and / or a drug selection marker for establishing permanent stable cell clones that express an AAV VP2 fusion polypeptide as described herein. The design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. General methods for generating such recombinant expression vectors can be found, inter alia, in Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007 with updated through 2010) Current Protocols in Molecular Biology.

[0124] Certain initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon and adjacent sequences. It may be necessary to provide an exogenous translation control signal that includes the ATG initiation codon. One of ordinary skill in the art will be able to readily determine this and provide the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translation control signal and initiation codon can be either natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements.

[0125] Expression can be achieved using any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. Expression systems for both prokaryotes and eukaryotes are widely available. In some embodiments, the expression system is mammalian cell expression such as the HEK293 expression system. In some embodiments, the nucleic acid can be codon-optimized to facilitate expression in the desired host cell. It will be important to use promoters and / or enhancers that effectively direct the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see, for example, Sambrook et al. (2001)).

[0126] Most eukaryotic RNA molecules can undergo RNA splicing to remove introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see Chandler et al., 1997, Proc. Natl. Acad. Sci. USA, 94(8):3596-601).

[0127] The vectors or constructs of the present disclosure generally include at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal that terminates the production of an RNA transcript is intended. A terminator may be necessary in vivo to achieve a desired message level. In eukaryotic systems, the terminator region may also include specific DNA sequences that allow for site-specific cleavage of the new transcript to expose the polyadenylation site. This signals a special endogenous polymerase to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and are translated more efficiently. Thus, in other embodiments, including eukaryotes, it is preferred that the terminator includes a signal for RNA cleavage, and more preferably that the terminator signal promotes polyadenylation of the mRNA. The terminator and / or polyadenylation site element may serve to enhance the mRNA level and / or minimize readthrough from the cassette into other sequences. Terminators intended for use in the present disclosure include any known terminator of transcription described herein or known to those of skill in the art, including, for example, but not limited to, the termination sequence of a gene such as the bovine growth hormone terminator, or a viral termination sequence such as the SV40 terminator. In certain embodiments, the termination signal may be, for example, the absence of a transcribable or translatable sequence by sequence cleavage.

[0128] In expression, particularly eukaryotic expression, it typically contains a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered important for the successful practice of the present disclosure, and / or any such sequence can be used. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are convenient and / or are known to function well in various target cells. Polyadenylation can increase the stability of the transcript or facilitate cytoplasmic transport.

[0129] To propagate the vector within the host cell, it may contain one or more origins of replication (often referred to as "ori"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0130] In certain embodiments of the present disclosure, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers impart an identifiable change to the cell, enabling easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that enables selection. A positive selectable marker is one whose presence enables its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0131] Typically, the inclusion of a drug selection marker serves to aid in the cloning and identification of transformants. For example, genes conferring resistance to kanamycin, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype enabling the discrimination of transformants based on the implementation of conditions, other types of markers whose basis includes screenable markers such as GFP by colorimetric analysis are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (HSV-tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art also know how to use immunological markers, perhaps in combination with FACS analysis. The marker used is not considered important as long as it can be co-expressed with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are well known to those skilled in the art.

[0132] The selection of an expression vector depends on the cell type for which one or more components of the vector are to be expressed. Typically, the vector includes one or more regulatory sequences such as a promoter and other regulatory sequences (e.g., enhancer) operably linked to the AAV VP2 fusion polypeptide open reading frame.

[0133] A "promoter" is a regulatory sequence that is a region within a nucleic acid sequence where the initiation and rate of transcription are controlled. This may include genetic elements to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can bind. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence to control the initiation and / or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer" (which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence).

[0134] A promoter may be one that is naturally associated with a gene or sequence, as may be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such a promoter can be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence and is located either downstream or upstream of the sequence. Alternatively, certain advantages can be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter (which usually refers to a promoter that is not associated with a nucleic acid sequence in its natural environment). A recombinant or heterologous enhancer also usually refers to an enhancer that is not associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and "non-naturally occurring" promoters or enhancers, i.e., those containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing the nucleic acid sequences of promoters and enhancers, the sequences may also be produced using recombinant cloning and / or nucleic acid amplification techniques related to the compositions disclosed herein, such as PCR (see U.S. Patent No. 4,683,202 and U.S. Patent No. 5,928,906). Further, it is contemplated that control sequences that direct transcription and / or expression of sequences within organelles other than the nucleus, such as mitochondria, chloroplasts, etc., may be similarly utilized.

[0135] The promoter utilized can be constitutive, inducible, synthetic, tissue-specific or cell-specific, and / or under appropriate conditions, useful for directing high-level expression of an introduced DNA segment and advantageous for large-scale production of recombinant proteins and / or peptides. In addition, other regulatory elements, such as enhancers, ribosome binding sites, transcription termination sequences, etc., can also be incorporated to improve the expression of the nucleic acid encoding the AAV VP2 fusion polypeptide described herein.

[0136] In some embodiments, a constitutive promoter is used to achieve a constant expression of the AAV VP2 fusion polypeptide described herein, or a reporter sequence, therapeutic RNA or protein encoded by the nucleic acid contained in the rAAV virion described herein. Examples of constitutive promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, elongation factor-1 promoter, hemoglobin promoter, and creatine kinase promoter.

[0137] In some embodiments, a ubiquitous promoter is used. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives such as CAG, CBh, etc.), EF-1a, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0138] In some embodiments, an inducible promoter is utilized to prevent the inserted sequence from expressing except under inducing conditions. Using an inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, for example, the arabinose promoter, lacZ promoter, tetracycline promoter, metallothionein promoter, glucocorticoid promoter, progesterone promoter, or heat shock promoter.

[0139] In some embodiments, a tissue-specific or cell-specific promoter is used to achieve expression of the AAV VP2 fusion polypeptide described herein, or a reporter protein, therapeutic RNA or protein encoded by the nucleic acid contained in the rAAV virion described herein, in only a specific tissue or cell. The identity of tissue-specific or cell-specific promoters or elements, as well as assays for characterizing their activity, are well known to those skilled in the art. Examples include the human LIMK2 gene (Nomoto et al. 1999, Gene, 236(2):259-271), the somatostatin receptor 2 gene (Kraus et al., 1998, FEES Lett., 428(3):165-170), the mouse testicular retinoic acid binding gene (Lareyre et al., 1999, J. Biol. Chem., 274(12):8282-8290), human CD4 (Zhao-Emonet et al., 1998, Biochim. Biophys. Acta, 1442(2-3):109-119), mouse α2(XI) collagen (Tsumaki, et al., 1998, J. Biol. Chem., 273(36):22861-22864), D1A dopamine receptor gene (Lee, et al., 1997, J. Auton. Nerv. Syst., 74(2-3):86-90), insulin-like growth factor II (Wu et al., 1997, Biochem. Biophys. Res. Commun., 233(1):221-226), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996, J. Immunol., 157(12):5411-5421), muscle creatine kinase (MCK) promoter (Wang et al., Gene Ther. 2008 Nov;15(22):1489-99).

[0140] In some embodiments, a promoter that is not cell-specific is used. In some embodiments, a strong or weak promoter (classified according to its affinity for RNA polymerase and / or sigma factor and the affinity of other promoters) is used.

[0141] In some embodiments, a synthetic promoter is used to achieve expression of the AAV VP2 fusion polypeptides described herein, or a therapeutic RNA or protein encoded by a nucleic acid contained in an rAAV virion described herein. Synthetic promoters can significantly exceed the transcriptional capacity of natural promoters. For example, a synthetic promoter that is not subject to blockade or reduction of activity by endogenous cellular mechanisms or factors can be selected. Other elements, including trans-acting factor binding sites and enhancers, may be inserted into the synthetic promoter to improve transcriptional efficiency. The synthetic promoter can be rationally designed and chemically synthesized to combine the best features of both synthetic and biological promoters. Through several steps, synthetic oligos are annealed and ligated to generate a chemically synthesized full-length promoter. The synthetic promoter may be an inducible or cell-type specific promoter.

[0142] In some embodiments, the promoter operably linked to the AAV VP2 fusion polypeptide ORF, or the nucleic acid sequence encoding a therapeutic nucleic acid or protein, or the reporter sequence, is selected from the 173CMV promoter, the HCMV promoter, the CBh promoter, the CAG promoter, and the mCCT promoter.

[0143] Those skilled in the art will readily recognize that a particular promoter is particularly suitable for expressing the AAV VP2 fusion polypeptides, reporter sequences, or therapeutic RNAs or proteins described herein in target cells, and such promoters include, but are not limited to, species-specific, inducible, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997); the contents of which are hereby incorporated by reference in their entirety).

[0144] In some embodiments, the promoter operably linked to the AAV VP2 fusion polypeptide, reporter sequence, or therapeutic RNA or protein encoded by the nucleic acid contained in the rAAV virion described herein is less than 1 kb. The promoter can have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or more than 800 bp. The promoter can have a length of 200 - 300, 200 - 400, 200 - 500, 200 - 600, 200 - 700, 200 - 800, 300 - 400, 300 - 500, 300 - 600, 300 - 700, 300 - 800, 400 - 500, 400 - 600, 400 - 700, 400 - 800, 500 - 600, 500 - 700, 500 - 800, 600 - 700, 600 - 800, or 700 - 800 bp.

[0145] In one embodiment, the promoter operably linked to the AAV VP2 fusion polypeptide, reporter sequence, or therapeutic RNA or protein encoded by the nucleic acid contained in the rAAV virion described herein may be the same or different promoters, for example, but not limited to, a combination of two or more components, regions or sequences such as CMV and CBA. Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or more than 800 bp. Each component may have a length of 200 - 300, 200 - 400, 200 - 500, 200 - 600, 200 - 700, 200 - 800, 300 - 400, 300 - 500, 300 - 600, 300 - 700, 300 - 800, 400 - 500, 400 - 600, 400 - 700, 400 - 800, 500 - 600, 500 - 700, 500 - 800, 600 - 700, 600 - 800 or 700 - 800 bp.

[0146] In some embodiments, the promoter operably linked to the AAV VP2 fusion polypeptide, reporter sequence, or therapeutic RNA or protein encoded by the nucleic acid contained in the rAAV virion described herein is a combination of a CMV - enhancer sequence, for example, an immediate - early CMV enhancer sequence (e.g., 382 - nucleotide CMV enhancer sequence) and a chicken β - actin (CBA) promoter sequence (e.g., 260 - nucleotide CBA promoter sequence).

[0147] In addition to a promoter, other regulatory elements may be required or desired for efficient expression of the AAV VP2 fusion polypeptides described herein, or a reporter protein, therapeutic RNA or therapeutic protein encoded by a nucleic acid contained in the rAAV virions described herein. Such elements include an ATG start codon and adjacent ribosome binding site or other sequences. Further, the efficiency of expression can be enhanced by including an enhancer appropriate for the cell line used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0148] In some embodiments, the AAV vectors provided herein optionally include an intron disposed between a promoter element and the polynucleotide to be expressed. Without being bound by theory, the inclusion of a 5' intron can increase the level and steady state of the expressed mRNA. Non-limiting examples of introns include introns derived from SV40, introns derived from CBA-MVM, MVM (67-97 bp), FIX short form intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp), hybrid adenovirus splice donor / IgG splice acceptor (230 bps).

[0149] In one embodiment, the intron or intron portion may be 100 to 500 nucleotides in length. The intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. The intron may be 80 to 100, 80 to 120, 80 to 140, 80 to 160, 80 to 180, 80 to 200, 80 to 250, 80 to 300, 80 to 350, 80 to 400, 80 to 450, 80 to 500, 200 to 300, 200 to 400, 200 to 500, 300 to 400, 300 to 500, or 400 to 500 nucleotides in length.

[0150] The expression vector can also be provided with a secretion signal sequence position to form a fusion protein with the protein to be expressed. More generally, the insertion sequence encoding the protein to be expressed is ligated to the signal sequence prior to integration into the vector.

[0151] The generation of expression vectors can utilize vectors containing multiple cloning sites (MCSs), which are nucleic acid regions containing multiple restriction enzyme sites, and any one of them can be used in combination with standard recombinant techniques to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific positions in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, the vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable ligation of an exogenous sequence to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in recombinant techniques.

[0152] In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide described herein further comprises a regulatory sequence. In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide described herein further comprises a promoter. In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide described herein comprises a regulatory sequence, particularly a promoter, that is efficient at driving expression in the target cell.

[0153] In some embodiments, the open reading frame of the AAV VP2 fusion polypeptide is operably linked to the promoter. In some embodiments, the promoter is selected from the HCMV promoter, 173CMV promoter, CAG promoter, CBh promoter, and mCCT promoter. In some embodiments, the nucleic acid further comprises a polyadenylation signal, which can be selected, for example, from the BGH or SV40 polyadenylation signals. In one embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 8.

[0154] In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide further comprises a reporter sequence. In some embodiments, the reporter sequence is operably linked to a promoter, such as a second promoter. In some embodiments, the reporter sequence encodes a fluorescent or luminescent reporter protein. A number of reporter proteins are known in the art and include the following: green fluorescent protein (GFP), variants of green fluorescent protein (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS66T, TagGFP2, mUKGEmerald GFP, Superfolder GFP, GFPuv, destabilized EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, azunte BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, cerulean CFP, SCFP3A, destabilized ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP, mTFPI, mMidonishi-Cyan, aquamanne, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFP I, Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal,mStable, mMaroonl, mGarnet2, tdTomato, mTangerine, mStrawberry, TagRFP, TagRFP667, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, far-red fluorescent protein, yellow fluorescent protein (YFP), eYFP, destabilized EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, sfCherry, sfCherry2, Citrine, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFPI, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange 2, mKusabira Orange, mOrange 2, mKOv, mKO2, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanm, shBFP-N168S / L1731, near-infrared protein, iFP1.4, iRFP713, iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, Brilliant Violet (BV) 421, BV605, BV510, BV711, BV786, PerCP, PerCP / Cy5.5, DsRed, DsRed2, mRFPI, pocilloporin, Renilla GFP, Monster GFP, paGFP, or Phycobihprotein, or a biologically active variant or fragment thereof. The reporter protein can be selected from the group consisting of, for example: EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase, and can be particularly selected from EGFP and sfCherry2.,

[0155] In some embodiments, the vector, such as an expression vector, is an adeno-associated vector (AAV). In some embodiments, the AAV vector comprises an open reading frame encoding the AAV VP2 fusion polypeptide described herein, with inverted terminal repeat (ITR) sequences flanking one or both sides. This nucleic acid may further comprise one or more additional elements, such as, for example, a promoter, an enhancer, one or more intron sequences, a poly(A) sequence, a stuffer sequence, such as a stuffer sequence derived from the HPRT intron, and combinations thereof. In some embodiments, the vector comprises a polynucleotide AAV vector encoding the AAV VP2 fusion polypeptide described herein, encapsulated in an AAV capsid.

[0156] In some embodiments, the vector comprises an open reading frame encoding the AAV VP2 fusion polypeptide described herein, operably linked to at least one target cell-compatible regulatory sequence, such as a promoter. In some embodiments, the promoter is selected from the 173CMV promoter, the HCMV promoter, the CBh promoter, the CAG promoter, and the mCCT promoter.

[0157] In some embodiments, the ITRs in the AAV vector are derived from the same AAV serotype. In some embodiments, the ITRs in the AAV vector are derived from different AAV serotypes. In some embodiments, the ITRs are the same. In some embodiments, the ITRs are different.

[0158] In some embodiments, the ITRs in the AAV vector are derived from the same AAV serotype as the AAV capsid. In some embodiments, the ITRs in the AAV vector are derived from a different serotype than the AAV capsid. In some embodiments, the ITRs are derived from AAV2, and the AAV capsid is derived from a serotype other than AAV2, such as AAV9.

[0159] In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide comprises two AAV inverted terminal repeat (ITR) sequences located upstream and downstream of the AAV VP2 fusion polypeptide open reading frame. In some embodiments, the two ITR sequences are located at the 5' and 3' termini of the nucleic acid molecule. In some of these embodiments, the nucleic acid further comprises a reporter sequence. The reporter sequence can encode, for example, a fluorescent or luminescent reporter protein. For example, the reporter protein can be selected from the group consisting of: EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase, particularly EGFP and sfCherry2. In some embodiments, the reporter sequence is operably linked to a second promoter. In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide further comprises a post-transcriptional regulatory element, such as WPRE or a derivative thereof. In some embodiments, the nucleic acid further comprises a polyadenylation signal selected, for example, from the BGH and SV40 polyadenylation signals. In one embodiment, the nucleic acid encoding the AAV VP2 fusion polypeptide comprises the sequence of SEQ ID NO: 2.

[0160] In one aspect, provided herein is a kit comprising the isolated nucleic acid encoding the AAV VP2 fusion polypeptide.

[0161] Production of Host Cells and AAV Methods for introducing an expression vector containing a polynucleotide sequence of interest vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (see generally, Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistics / gene gun, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22, drug-enhanced uptake of DNA, ex vivo transduction, protoplast fusion, retroviral transduction, viral transfection, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are grown in culture and screened for appropriate activity. For long-term, high-yield production of recombinant proteins, stable expression is often desired. For example, cell lines that stably express a polypeptide can be prepared using an expression vector containing a viral origin of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, the cells may be grown in enriched medium for 1-2 days before switching to selective medium. The purpose of the selectable marker is to confer resistance to selection, the presence of which allows the growth of cells that successfully express the introduced sequence in selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate for the cell type. Methods and conditions for culturing the resulting transfected cells and recovering the antibody produced are known to those of skill in the art and can vary or be optimized based on the specific expression vector and mammalian host cell used, based on this specification.

[0162] In one aspect, provided herein are cells comprising the AAV VP2 fusion polypeptide described herein or a nucleic acid encoding the same. Such cells can be, for example, host cells or therapeutic cells. The terms “host cell” and “recombinant host cell” are used interchangeably herein and refer to not only a particular target cell but also the progeny or potential progeny of such a cell. Because of either mutation or environmental influences, certain modifications may occur in the next generation, and such progeny may not actually be identical to the parental cell, but are still included within the scope of the terms used herein.

[0163] Using a host cell, an AAV VP2 fusion polypeptide can be produced or expressed, and optionally, AAV virions containing the same can be assembled. Accordingly, the present disclosure also features a method of producing an AAV VP2 fusion polypeptide using a host cell and a method of producing AAV virions containing the AAV VP2 fusion polypeptide. In some embodiments, the method includes culturing a host cell (into which a recombinant expression vector encoding the AAV VP2 fusion polypeptide has been introduced) in a suitable medium such that the AAV VP2 fusion polypeptide is produced. In some embodiments, the method further includes isolating the AAV VP2 fusion polypeptide or AAV virions containing the same from the medium or the host cell.

[0164] In one embodiment, the host cell is genetically engineered to contain a nucleic acid encoding the AAV VP2 fusion polypeptide. In one embodiment, the host cell is genetically engineered by using an expression cassette. The term “expression cassette” refers to a nucleotide sequence capable of effecting the expression of a gene in a host compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without an intron, and a termination signal. Additional factors necessary or useful to achieve expression, such as an inducible promoter, may be used.

[0165] The host cell can be a eukaryotic cell, or a prokaryotic cell such as a bacterial cell, an insect cell, or a mammalian cell such as a human cell, but is not limited thereto. Escherichia coli (E. coli) is one prokaryotic host useful for the cloning and expression of polynucleotides. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other enterobacterial species such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, typically, an expression vector containing a control sequence (e.g., origin of replication) compatible with the host cell can also be replicated. Furthermore, there are any number of various well-known promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system from phage λ. These promoters typically control expression, optionally using an operator sequence, and have a ribosome binding site sequence and the like for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express the AAV VP2 fusion polypeptide described herein. Eukaryotic host cell lines capable of secreting intact heterologous proteins have been developed, and examples of such include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. Suitable eukaryotic host cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. The use of mammalian tissue cell cultures for expressing polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information processing sites such as a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator sequence.These expression vectors typically contain a promoter derived from a mammalian gene or a mammalian virus. Suitable promoters can be constitutive, cell-type specific, stage-specific, and / or modifiable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and combinations of promoters-enhancers known in the art.

[0166] In some embodiments, the host cell is suitable for the assembly of AAV virions. Accordingly, methods for producing rAAV virions are also disclosed herein.

[0167] Naturally occurring AAVs contain distinct sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration. Typically, three AAV promoters (named p5, p19, and p40 from their relative map positions) drive the expression of two AAV internal open reading frames that encode the rep and cap genes of the wild-type virus. Two rep promoters (p5 and p19), in combination with alternative splicing of a single AAV intron (at nucleotides 2107 and 2227), are thought to result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties and ultimately are responsible for replication of the viral genome. The cap gene is typically expressed from the p40 promoter. Alternative splicing and non-consensus translation initiation sites are responsible for the production of three related capsid proteins, VP1, VP2, and VP3. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, (1992) Curr. Topics Microbiol. Immunol., 158:97-129.

[0168] In some embodiments, a nucleic acid encoding an AAV VP2 fusion polypeptide, such as a plasmid, is used to produce rAAV virions. In some embodiments, at least one other nucleic acid, such as a plasmid, comprising an AAV rep gene and / or an AAV cap gene is used in the preparation of rAAV virions. In some embodiments, the start codon of VP2 of the cap gene is mutated such that the nucleic acid encoding the AAV VP2 fusion polypeptide is the sole source of the VP2 subunit, and thus all VP2 capsid polypeptides in the produced rAAV virions are fused to the polypeptide ligand. Also disclosed herein is a nucleic acid sequence, such as a plasmid, comprising at least one adenoviral helper function gene. In some embodiments, the nucleic acids encoding AAV rep, AAV cap, and / or the adenoviral helper gene may be present in the same construct, such as a single plasmid, or in separate constructs. In some embodiments, one or more plasmids are co-transfected into competent cells together with the cargo nucleic acid, i.e., the nucleic acid to be encapsulated within the rAAV virion, and then the cells are cultured to produce rAAV virions. Optionally, the cargo plasmid containing the nucleic acid sequence to be encapsulated (“AAV genome”), as well as the plasmid containing the AAV rep and / or cap genes, are introduced into cells permissive to infection by an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpes virus). Techniques for producing rAAV particles in which the AAV genome, rep and cap genes, and helper virus functions are provided to the cells are known in the art and include, for example, electroporation. In some embodiments, the production of rAAV requires the following components present within a single cell (referred to herein as a packaging cell): an rAAV vector, AAV rep and cap genes separate from the rAAV vector (i.e., not within the rAAV vector), and a helper virus function. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is hereby incorporated by reference in its entirety.In various embodiments, the AAV capsid protein may be modified to enhance delivery of the recombinant vector. Modifications to the capsid protein are generally known in the art. See, for example, U.S. Patent Application Publication Nos. 2005 / 0053922 and 2009 / 0202490.

[0169] In various embodiments, the general principles of viral vector production can be utilized to produce the vectors and viruses disclosed herein, such as rAAV. Carter, (1992) Curr. Opinions Biotech., 1533 - 539; Muzyczka, (1992) Curr. Topics Microbial. Immunol., 158:97 - 129. Various methods are disclosed in the following references: Ratschin et al., (1984) Mol. Cell. Biol., 4:2072; Hennonat et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6466; Tratschin et al., (1985) Mol. Cell. Biol., 5:3251; McLaughlin et al. (1988) J. Virol., 62:1963; Lebkowski et al., (1988) Mol. Cell. Biol., 7:349; Samulski et al. (1989) J. Virol., 63:3822 - 3828; U.S. Patent No. 5,173,414; International Publication No. 95 / 13365 Pamphlet and corresponding U.S. Patent No. 5,658,776; International Publication No. 95 / 13392 Pamphlet; No. 96 / 17947 Pamphlet; PCT / US98 / 18600 Specification; International Publication No. 97 / 09441 Pamphlet (PCT / US96 / 14423 Specification); International Publication No. 97 / 08298 Pamphlet (PCT / US96 / 13872 Specification); International Publication No. 97 / 21825 Pamphlet (PCT / US96 / 20777 Specification); International Publication No. 97 / 06243 Pamphlet (PCT / FR96 / 01064 Specification); International Publication No. 99 / 11764 Pamphlet; Perrin et al., (1995) Vaccine, 13:1244 - 1250; Paul et al., (1993) Hum. Gene Ther., 4:609 - 615; Clark et al. (1996) Gene Therapy, 3:1124 - 1132; U.S. Patent No. 5,786,211; No. 5,871,982; and No. 6,258,595.The above literature places particular emphasis on the section of literature related to rAAV production, and the whole of it is incorporated herein by reference.

[0170] An exemplary method of creating packaging cells is to create a cell line that stably expresses some or all of the components necessary for AAV particle production. For example, a plasmid (or plasmids) encoding an rAAV vector lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV vector, and a selectable marker such as the neomycin resistance gene is integrated into the cell's genome. The AAV genome is introduced into the cell plasmid by procedures such as GC tailing (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al. (1983) Gene, 23:65-73), or direct blunt-end ligation (Senapathy et al. (1984) J. Biol. Chem., 259:4661-4666). Next, this packaging cell line is infected with a helper virus such as adenovirus and / or a plasmid encoding the helper virus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. In examples of other suitable methods, an adenovirus or baculovirus, rather than a plasmid, is used to introduce the rAAV vector and / or the rep and cap genes into the packaging cells.

[0171] In some embodiments, a method of producing a recombinant virus comprises providing a nucleic acid to be packaged. In some embodiments, the nucleic acid is a plasmid. In other embodiments, the nucleic acid comprises a heterologous nucleic acid sequence intervening between a first AAV terminal repeat and a second AAV terminal repeat. In some embodiments, the heterologous nucleic acid encodes an AAV VP2 fusion polypeptide and / or a reporter protein described herein. In some embodiments, the heterologous nucleic acid encodes a therapeutic protein such as a therapeutic RNA or a therapeutic antibody. In some embodiments, when the heterologous nucleic acid does not encode an AAV VP2 fusion polypeptide, the method of producing a recombinant virus comprises providing an additional nucleic acid encoding an AAV VP2 fusion polypeptide. In some embodiments, the method of producing a recombinant virus comprises providing one or more additional nucleic acids. In some embodiments, the one or more additional nucleic acids comprise an AAV rep gene and / or an AAV cap gene. In some embodiments, the one or more additional nucleic acids comprise an AAV rep gene derived from: AAV serotype 1, AAV serotype 2, AAV serotype 3, AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9. In some embodiments, the one or more additional nucleic acids comprise an AAV cap gene derived from AAV serotype 1, AAV serotype 2, AAV serotype 3, AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, or AAV serotype 9. In some embodiments, the one or more additional nucleic acids comprise one or more adenovirus helper function genes.

[0172] In some embodiments, the nucleic acid is co-transfected into competent cells or packaging cells. Methods of co-transfection are known in the art and include, but are not limited to, transfection with Lipofectamine, electroporation, and polyethyleneimine. The competent cells or packaging cells may be non-adherent cells cultured in suspension or adherent cells. In one embodiment, any suitable packaging cell line can be used, such as HeLa cells, HEK 293 cells, and PerC.6 cells (isogenic 293 strain). In one embodiment, the packaging cells are human cells. In one embodiment, the packaging cells are HEK 293 cells. In one embodiment, the packaging cells are insect cells. In one embodiment, the packaging cells are Sf9 cells. In some embodiments, the method includes culturing the transfected cells to produce recombinant virus. In some embodiments, the method includes recovering the recombinant virus. Methods for recovering recombinant virus include, for example, those disclosed in U.S. Patent Nos. 6,143,548 and 9,408,904. In some embodiments, the recombinant virus is secreted into the cell culture medium and purified from the medium. In some embodiments, the packaging cells are lysed and the contents are purified to recover the recombinant virus. In some embodiments, the virus is recovered from the packaging cells by filtration or centrifugation. In some embodiments, the virus is recovered from the packaging cells by chromatography.

[0173] In some embodiments, mammalian host cells are used to express an AAV VP2 fusion polypeptide and typically incorporate it into AAV virions together with AAV VP1 and VP3 polypeptides. For this purpose, derivatives thereof such as HEK293 or HEK293T / 17 or AAV293 cells can be transfected with a nucleic acid encoding the AAV VP2 fusion polypeptide and nucleic acids encoding the AAV VP1, VP2, and rep polypeptides. In some embodiments, HEK293 cells are further transfected with nucleic acids encoding adenoviral helper functions for AAV replication, such as adenoviral E2, E4, and / or VA gene products. In some embodiments, HEK293 cells are co-transfected with one nucleic acid molecule encoding the AAV VP2 fusion polypeptide, one nucleic acid molecule encoding the AAV VP1, VP2, and rep polypeptides (specifically, the AAV VP1 and VP2 polypeptides are encoded by the AAV cap gene, and more specifically, the AAV VP2 start codon in the AAV cap gene is mutated), at least one, at least two, at least three, or at least one nucleic acid molecule encoding adenoviral helper functions for AAV replication (specifically, the nucleic acid molecule encodes the adenoviral E2, E4, and VA gene products), and optionally, yet another nucleic acid molecule encoding a therapeutic RNA or protein. In some embodiments, the nucleic acid molecule encoding the therapeutic RNA or protein is encapsulated in an AAV capsid composed of the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides. In these embodiments, the nucleic acid molecule encoding the therapeutic RNA or protein typically further comprises flanking AAV ITR sequences. In other embodiments, the nucleic acid molecule encoding the AAV VP2 fusion polypeptide is encapsulated in an AAV capsid composed of the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides. In these embodiments, the nucleic acid molecule encoding the AAV VP2 fusion polypeptide further comprises flanking AAV ITR sequences.In other embodiments, a nucleic acid molecule comprising a barcode array and flanking AAV ITR sequences is encapsulated in an AAV capsid composed of an AAV VP2 fusion polypeptide and AAV VP1 and VP3 polypeptides.

[0174] In some embodiments, a method of producing rAAV virions includes transfecting insect cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the method includes transfecting insect cells with a baculovirus comprising a nucleic acid disclosed herein. In some embodiments, the method includes transfecting insect cells with a baculovirus having a heterologous nucleic acid sequence intervening between a first AAV terminal repeat and a second AAV terminal repeat. In some embodiments, the method includes transfecting insect cells with a baculovirus comprising one or more additional nucleic acids. In some embodiments, the one or more additional nucleic acids include an AAV rep gene and / or an AAV cap gene. In some embodiments, the one or more additional nucleic acids include one or more adenovirus helper function genes. In some embodiments, when the heterologous nucleic acid does not encode the AAV VP2 fusion polypeptide described herein, the method further includes transfecting the insect cells with an additional nucleic acid encoding the AAV VP2 fusion polypeptide described herein. In some embodiments, the insect cells are cultured under conditions suitable for producing recombinant virus. In some embodiments, the virus is recovered from the insect cells. In some embodiments, the virus is recovered from the insect cells by filtration or centrifugation. In some embodiments, the virus is recovered from the insect cells by chromatography.

[0175] In some embodiments, the cells are selected from mammalian cells including, but not limited to, mouse cells, non-human primate cells or human cells. The mammalian cells can be selected from the group consisting of hepatocytes, brain cells, spleen cells, kidney cells, blood cells, lung cells, muscle cells, heart cells, bone marrow cells, multipotent progenitor cells (MPP) such as multipotent hematopoietic progenitor cells, and hematopoietic stem cells (HSC) such as long-term hematopoietic stem cells (LT-HSC).

[0176] rAAV Virions Comprising AAV VP2 Fusion Polypeptides In one aspect, provided herein are recombinant AAV (rAAV) virions comprising an AAV VP2 fusion polypeptide described herein.

[0177] The abbreviation "rAAV" refers to recombinant adeno-associated virus. As used herein, an "rAAV vector" refers to an AAV-derived polynucleotide sequence (i.e., a polynucleotide heterologous to AAV), typically an AAV vector that contains a polynucleotide sequence of interest for cell gene transformation. In some embodiments, the heterologous nucleic acid encodes an AAV VP2 fusion polypeptide described herein. In some embodiments, the heterologous nucleic acid encodes a therapeutic nucleic acid, a therapeutic protein, or a therapeutic antibody or antibody fragment. In some embodiments, the heterologous polynucleotide may be flanked by at least one, and optionally two, AAV inverted terminal repeat (ITR) sequences. The term rAAV vector encompasses both rAAV vector particles and rAAV vector nucleic acids. The rAAV vector can be either single-stranded (ssAAV) or self-complementary (scAAV). An "AAV virus" or "AAV virus particle" or "AAV virion" refers to a virus particle composed of at least one AAV capsid protein (typically, by all of one or more wild-type AAV capsid proteins, or one or more capsid proteins derived from wild-type AAV) and an encapsulated polynucleotide rAAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to mammalian cells), it is typically referred to as an "rAAV vector particle", "rAAV virion" or simply "rAAV vector".

[0178] In some embodiments, the AAV virions disclosed herein comprise one or more AAV capsid proteins. Typically, in AAV, three capsid proteins, VP1, VP2, and VP3, multimerize to form the capsid. The rAAV virions disclosed herein comprise an AAV VP2 fusion polypeptide and optionally further comprise an AAV VP1 and / or VP3 polypeptide. Typically, the capsid of the rAAV virions disclosed herein is composed of VP1, VP3, and VP2 fusion polypeptide subunits. Typically, the capsid of the rAAV virions disclosed herein does not contain an AAV VP2 capsid polypeptide that is not fused to a polypeptide ligand, i.e., typically, all VP2 subunits are the AAV VP2 fusion polypeptides described herein. The AAV capsid polypeptides included in the rAAV virions disclosed herein may be derived from any AAV serotype from which a recombinant virus can be obtained, and as such, include, but are not limited to, the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5, or any variant thereof, or a variant having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the full length of, for example, the VP1, VP2, and / or VP3 capsid polypeptides.

[0179] The polypeptide sequences of the capsid proteins are known in the art and may be derived from the AAV genome. These can be used as exemplary capsids in the AAV virus compositions disclosed herein.

[0180] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide contained in the rAAV virion described herein include, but are not limited to, the following: AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43- 21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-l l / rh.53, AAV4-8 / r 11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC 12, AAV-2-pre-miRNA-lOl, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV10 and / or Japanese AAV10 capsid polypeptides, or, for example, a variant having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length of the VP1, VP2, and / or VP3 capsid polypeptides, and can be a naturally occurring, or mutant / variant capsid polypeptide, independently selected from the group comprising.

[0181] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide contained in the rAAV virion described herein, are AAV serotypes as described in U.S. Patent Application Publication No. 20030138772 (the content of which is hereby incorporated by reference in its entirety), for example, but not limited to, the following: AAV1 (SEQ ID NOs: 6 and 64 in U.S. Patent Application Publication No. 20030138772), AAV2 (SEQ ID NOs: 7 and 70 in U.S. Patent Application Publication No. 20030138772), AAV3 (SEQ ID NOs: 8 and 71 in U.S. Patent Application Publication No. 20030138772), AAV4 (SEQ ID NO: 63 in U.S. Patent Application Publication No. 20030138772), AAV5 (SEQ ID NO: 114 in U.S. Patent Application Publication No. 20030138772), AAV6 (SEQ ID NO: 65 in U.S. Patent Application Publication No. 20030138772), AAV7 (SEQ ID NOs: 1 - 3 in U.S. Patent Application Publication No. 20030138772), AAV8 (SEQ ID NOs: 4 and 95 in U.S. Patent Application Publication No. 20030138772), AAV9 (SEQ ID NOs: 5 and 100 in U.S. Patent Application Publication No. 20030138772), AAV10 (SEQ ID NO: 117 in U.S. Patent Application Publication No. 20030138772), AAV11 (SEQ ID NO: in U.S. Patent Application Publication No. 20030138772), AAV12 (SEQ ID NO: 119 in U.S. Patent Application Publication No. 20030138772), AAVrh10 (amino acids 1 - 738 of SEQ ID NO: 81 in U.S. Patent Application Publication No. 20030138772), AAV16.3 (SEQ ID NO: 10 in U.S. Patent Application Publication No. 20030138772), AAV29.3 / bb.l (SEQ ID NO: 11 in U.S. Patent Application Publication No. 20030138772), AAV29.4 (SEQ ID NO: 12 in U.S. Patent Application Publication No. 20030138772), AAV29.5 / bb.2 (SEQ ID NO: 13 in U.S. Patent Application Publication No. 20030138772), AAV1.3 (SEQ ID NO: 14 in U.S. Patent Application Publication No. 20030138772), AAV13.3 (SEQ ID NO: 15 in U.S. Patent Application Publication No. 20030138772),AAV24.1 (SEQ ID NO: 16 in US Patent Application Publication No. 20030138772), AAV27.3 (SEQ ID NO: 17 in US Patent Application Publication No. 20030138772), AAV7.2 (SEQ ID NO: 18 in US Patent Application Publication No. 20030138772), AAVC1 (SEQ ID NO: 19 in US Patent Application Publication No. 20030138772), AAVC3 (SEQ ID NO: 20 in US Patent Application Publication No. 20030138772), AAVC5 (SEQ ID NO: 21 in US Patent Application Publication No. 20030138772), AAVF1 (SEQ ID NO: 22 in US Patent Application Publication No. 20030138772), AAVF3 (SEQ ID NO: 23 in US Patent Application Publication No. 20030138772), AAVF5 (SEQ ID NO: 24 in US Patent Application Publication No. 20030138772), AAVH6 (SEQ ID NO: 25 in US Patent Application Publication No. 20030138772), AAVH2 (SEQ ID NO: 26 in US Patent Application Publication No. 20030138772), AAV42-8 (SEQ ID NO: 27 in US Patent Application Publication No. 20030138772), AAV42-15 (SEQ ID NO: 28 in US Patent Application Publication No. 20030138772), AAV42-5b (SEQ ID NO: 29 in US Patent Application Publication No. 20030138772), AAV42-lb (SEQ ID NO: 30 in US Patent Application Publication No. 20030138772), AAV42-13 (SEQ ID NO: 31 in US Patent Application Publication No. 20030138772), AAV42-3a (SEQ ID NO: 32 in US Patent Application Publication No. 20030138772), AAV42-4 (SEQ ID NO: 33 in US Patent Application Publication No. 20030138772), AAV42-5a (SEQ ID NO: 34 in US Patent Application Publication No. 20030138772), AAV42-10 (SEQ ID NO: 35 in US Patent Application Publication No. 20030138772), AAV42-3b (SEQ ID NO: 36 in US Patent Application Publication No. 20030138772), AAV42-11 (SEQ ID NO: 37 in US Patent Application Publication No. 20030138772), AAV42-6b (SEQ ID NO: 38 in US Patent Application Publication No. 20030138772), AAV43-1 (SEQ ID NO: 39 in US Patent Application Publication No. 20030138772),AAV43-5 (SEQ ID NO: 40 in US Patent Application Publication No. 20030138772), AAV43-12 (SEQ ID NO: 41 in US Patent Application Publication No. 20030138772), AAV43-20 (SEQ ID NO: 42 in US Patent Application Publication No. 20030138772), AAV43-21 (SEQ ID NO: 43 in US Patent Application Publication No. 20030138772), AAV43-23 (SEQ ID NO: 44 in US Patent Application Publication No. 20030138772), AAV43-25 (SEQ ID NO: 45 in US Patent Application Publication No. 20030138772), AAV44.1 (SEQ ID NO: 46 in US Patent Application Publication No. 20030138772), AAV44.5 (SEQ ID NO: 47 in US Patent Application Publication No. 20030138772), AAV223.1 (SEQ ID NO: 48 in US Patent Application Publication No. 20030138772), AAV223.2 (SEQ ID NO: 49 in US Patent Application Publication No. 20030138772), AAV223.4 (SEQ ID NO: 50 in US Patent Application Publication No. 20030138772), AAV223.5 (SEQ ID NO: 51 in US Patent Application Publication No. 20030138772), AAV223.6 (SEQ ID NO: 52 in US Patent Application Publication No. 20030138772), AAV223.7 (SEQ ID NO: 53 in US Patent Application Publication No. 20030138772), AAV A3.4 (SEQ ID NO: 54 in US Patent Application Publication No. 20030138772), AAV A3.5 (SEQ ID NO: 55 in US Patent Application Publication No. 20030138772), AAV A3.7 (SEQ ID NO: 56 in US Patent Application Publication No. 20030138772), AAV A3.3 (SEQ ID NO: 57 in US Patent Application Publication No. 20030138772), AAV42.12 (SEQ ID NO: 58 in US Patent Application Publication No. 20030138772), AAV44.2 (SEQ ID NO: 59 in US Patent Application Publication No. 20030138772), AAV42-2 (SEQ ID NO: 9 in US Patent Application Publication No. 20030138772), or variants or derivatives thereof may be used.

[0182] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide contained in the rAAV virion described herein, are AAV serotypes described in U.S. Patent Application Publication No. 20150159173, the content of which is hereby incorporated by reference in its entirety, such as, but not limited to, the following: AAV2 (SEQ ID NOs: 7 and 23 of U.S. Patent Application Publication No. 20150159173), rh20 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150159173), rh32 / 33 (SEQ ID NO: 2 of U.S. Patent Application Publication No. 20150159173), rh39 (SEQ ID NOs: 3, 20 and 36 of U.S. Patent Application Publication No. 20150159173), rh46 (SEQ ID NOs: 4 and 22 of U.S. Patent Application Publication No. 20150159173), rh73 (SEQ ID NO: 5 of U.S. Patent Application Publication No. 20150159173), rh74 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 20150159173), AV6.1 (SEQ ID NO: 29 of U.S. Patent Application Publication No. 20150159173), rh.8 (SEQ ID NO: 41 of U.S. Patent Application Publication No. 20150159173), rh.48.1 (SEQ ID NO: 44 of U.S. Patent Application Publication No. 20150159173), hu.44 (SEQ ID NO: 45 of U.S. Patent Application Publication No. 20150159173), hu.29 (SEQ ID NO: 42 of U.S. Patent Application Publication No. 20150159173), hu.48 (SEQ ID NO: 38 of U.S. Patent Application Publication No. 20150159173), rh54 (SEQ ID NO: 49 of U.S. Patent Application Publication No. 20150159173), AAV2 (SEQ ID NO: 7 of U.S. Patent Application Publication No. 20150159173), cy.5 (SEQ ID NOs: 8 and 24 of U.S. Patent Application Publication No. 20150159173), rh.10 (SEQ ID NOs: 9 and 25 of U.S. Patent Application Publication No. 20150159173), rh.13 (SEQ ID NOs: 10 and 26 of US Patent Application Publication No. 20150159173), AAV1 (SEQ ID NOs: 11 and 27 of US Patent Application Publication No. 20150159173), AAV3 (SEQ ID NOs: 12 and 28 of US Patent Application Publication No. 20150159173), AAV6 (SEQ ID NOs: 13 and 29 of US Patent Application Publication No. 20150159173), AAV7 (SEQ ID NOs: 14 and 30 of US Patent Application Publication No. 20150159173), AAV8 (SEQ ID NOs: 15 and 31 of US Patent Application Publication No. 20150159173), hu.13 (SEQ ID NOs: 16 and 32 of US Patent Application Publication No. 20150159173), hu.26 (SEQ ID NOs: 17 and 33 of US Patent Application Publication No. 20150159173), hu.37 (SEQ ID NOs: 18 and 34 of US Patent Application Publication No. 20150159173), hu.53 (SEQ ID NOs: 19 and 35 of US Patent Application Publication No. 20150159173), rh.43 (SEQ ID NOs: 21 and 37 of US Patent Application Publication No. 20150159173), rh2 (SEQ ID NO: 39 of US Patent Application Publication No. 20150159173), rh.37 (SEQ ID NO: 40 of US Patent Application Publication No. 20150159173), rh.64 (SEQ ID NO: 43 of US Patent Application Publication No. 20150159173), rh.48 (SEQ ID NO: 44 of US Patent Application Publication No. 20150159173), ch.5 (SEQ ID NO: 46 of US Patent Application Publication No. 20150159173), rh.67 (SEQ ID NO: 47 of US Patent Application Publication No. 20150159173), rh.58 (SEQ ID NO: 48 of US Patent Application Publication No. 20150159173), or, without limitation, those of their variants including, but not limited to, Cy5Rl, Cy5R2, Cy5R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.48.1, rh.48.2, rh.48.1.2, hu.44Rl, hu.44R2, hu.44R3, hu.29R, ch.5R1, rh64R1, rh64R2, AAV6.2, AAV6.1, AAV6.12, hu.48Rl, hu.48R2, and hu.48R3, or those derived therefrom may be used.

[0183] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide included in the rAAV virion described herein, are of, or are derived from, an AAV serotype described in U.S. Patent No. 7,198,951 (the contents of which are hereby incorporated by reference in their entirety), such as, but not limited to, the following: AAV9 (SEQ ID NOs: 1-3 of U.S. Patent No. 7,198,951), AAV2 (SEQ ID NO: 4 of U.S. Patent No. 7,198,951), AAV1 (SEQ ID NO: 5), AAV3 (SEQ ID NO: 6 of U.S. Patent No. 7,198,951), and AAV8 (SEQ ID NO: 7 of U.S. Patent No. 7,198,951).

[0184] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide included in the rAAV virion described herein may be derived from an AAV serotype, which may be a mutation in the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011), the content of which is hereby incorporated by reference in its entirety), for example, but not limited to, the following: AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, or may have or have them. In some embodiments, the AAV capsid comprises one or more sequences engineered to deliver the vector across the blood-brain barrier (see, for example, B.E. Deverman et al, Nature Biotech, Vol. 34, No. 2, p 204-211 (published online on February 1, 2016) and Caltech press release, A. Wetherston, www.neurology-cenfrd.com / 2016 / 02 / 10 / successfd / brain-barrier; see also International Publication No. 2016 / 0492301 pamphlet and US Patent No. 8,734,809 (the content of each of which is hereby incorporated by reference in its entirety)).

[0185] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide contained in the rAAV virion described herein, are of, or derived from, an AAV serotype described in U.S. Patent No. 6,156,303, the content of which is hereby incorporated by reference in its entirety, for example, but not limited to, the following: AAV3B (SEQ ID NOs: 1 and 10 of U.S. Patent No. 6,156,303), AAV6 (SEQ ID NOs: 2, 7 and 11 of U.S. Patent No. 6,156,303), AAV2 (SEQ ID NOs: 3 and 8 of U.S. Patent No. 6,156,303), AAV3A (SEQ ID NOs: 4 and 9 of U.S. Patent No. 6,156,303), or derivatives thereof.

[0186] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide and / or AAV VP3 polypeptide contained in the rAAV virion described herein, are of, or derived from, an AAV serotype described in U.S. Patent Application Publication No. 2014 / 0359799, the content of which is hereby incorporated by reference in its entirety, for example, but not limited to, the following: AAV8 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 2014 / 0359799), AAVDJ (SEQ ID NOs: 2 and 3 of U.S. Patent Application Publication No. 2014 / 0359799), or variants thereof.

[0187] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, may be of or derived from AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12):5887-5911(2008), the content of which is hereby incorporated by reference in its entirety). The amino acid sequence of AAVDJ8 may contain two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Patent No. 7,588,772 (the content of which is hereby incorporated by reference in its entirety) may contain two mutations: (1) R587Q in which the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T in which the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, it may contain three mutations: (1) K406R in which the lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q in which the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T in which the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0188] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, may be of or derived from an AAV serotype described in International Publication No. WO 98 / 011244 (the content of which is hereby incorporated by reference in its entirety), such as, but not limited to, AAV4 (SEQ ID NOs: 1-20 of International Publication No. WO 98 / 011244).

[0189] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein may be those of an AAV serotype that is a mutation in the AAV2 sequence to produce AAV2G9, as described in WO 2014 / 144229 (the contents of which are incorporated herein by reference in their entirety), or may have or be derived from such.

[0190] In some embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virions described herein and / or the AAV VP1 capsid polypeptide and / or the AAV VP2 capsid polypeptide contained in the AAV VP3 capsid polypeptide are, but are not limited to, AAV3-3 (SEQ ID NO: 217 of WO 2005 / 033321), AAV1 (SEQ ID NO: 219 and 202 of WO 2005 / 033321), AAV106.1 / hu.37 (SEQ ID NO: 10 of WO 2005 / 033321), AAV114.3 / hu.40 (SEQ ID NO: 11 of WO 2005 / 033321), AAV127.2 / hu.41 (SEQ ID NO: 6 and 8 of WO 2005 / 033321), AAV128.3 / hu.44 (SEQ ID NO: 81 of WO 2005 / 033321), AAV130.4 / hu.48 (SEQ ID NO: 78 of WO 2005 / 033321), AAV145.1 / hu.53 (SEQ ID NO: 176 and 177 of WO 2005 / 033321), AAV145.6 / hu.56 (SEQ ID NO: 168 and 192 of WO 2005 / 033321), AAV16.12 / hu.11 (SEQ ID NO: 153 and 57 of WO 2005 / 033321), AAV16.8 / hu.10 (SEQ ID NO: 156 and 56 of WO 2005 / 033321), AAV161.10 / hu.60 (SEQ ID NO: 170 of WO 2005 / 033321), AAV161.6 / hu.61 (SEQ ID NO: 174 of WO 2005 / 033321), AAV1-7 / rh.48 (SEQ ID NO: 32 of WO 2005 / 033321), AAVl-8 / rh.49 (SEQ ID NO: 103 and 25 of WO 2005 / 033321), AAV2 (SEQ ID NO: 211 and 221 of WO 2005 / 033321), AAV2-15 / rh.62 (SEQ ID NO: 33 and 114 of WO 2005 / 033321), AAV2-3 / rh.61 (SEQ ID NO: 21 of WO 2005 / 033321), AAV2-4 / rh.50 (SEQ ID NOs: 23 and 108 of WO 2005 / 033321 Pamphlet), AAV2-5 / rh.51 (SEQ ID NOs: 104 and 22 of WO 2005 / 033321 Pamphlet), AAV3.1 / hu.6 (SEQ ID NOs: 5 and 84 of WO 2005 / 033321 Pamphlet), AAV3.1 / hu.9 (SEQ ID NOs: 155 and 58 of WO 2005 / 033321 Pamphlet), AAV3-11 / rh.53 (SEQ ID NOs: 186 and 176 of WO 2005 / 033321 Pamphlet), AAV3-3 (SEQ ID NO: 200 of WO 2005 / 033321 Pamphlet), AAV33.12 / hu.17 (SEQ ID NO: 4 of WO 2005 / 033321 Pamphlet), AAV33.4 / hu.15 (SEQ ID NO: 50 of WO 2005 / 033321 Pamphlet), AAV33.8 / hu.16 (SEQ ID NO: 51 of WO 2005 / 033321 Pamphlet), AAV3-9 / rh.52 (SEQ ID NOs: 96 and 18 of WO 2005 / 033321 Pamphlet), AAV4-19 / rh.55 (SEQ ID NO: 117 of WO 2005 / 033321 Pamphlet), AAV4-4 (SEQ ID NOs: 201 and 218 of WO 2005 / 033321 Pamphlet), AAV4-9 / rh.54 (SEQ ID NO: 116 of WO 2005 / 033321 Pamphlet), AAV5 (SEQ ID NOs: 199 and 216 of WO 2005 / 033321 Pamphlet), AAV52.1 / hu.20 (SEQ ID NO: 63 of WO 2005 / 033321 Pamphlet), AAV52 / hu.19 (SEQ ID NO: 133 of WO 2005 / 033321 Pamphlet), AAV5-22 / rh.58 (SEQ ID NO: 27 of WO 2005 / 033321 Pamphlet), AAV5-3 / rh.57 (SEQ ID NO: 105 of WO 2005 / 033321 Pamphlet), AAV5-3 / rh.57 (SEQ ID NO: 26 of WO 2005 / 033321 Pamphlet), AAV58.2 / hu.25 (SEQ ID NO: 49 of WO 2005 / 033321 Pamphlet), AAV6 (SEQ ID NOs: 203 and 220 of WO 2005 / 033321 Pamphlet), AAV7 (SEQ ID NOs: 222 and 213 of WO 2005 / 033321 Pamphlet), AAV7.3 / hu.7 (SEQ ID NO: 55 of WO 2005 / 033321 Pamphlet), AAV8 (SEQ ID NOs: 223 and 214 of WO 2005 / 033321 Pamphlet), AAVH-l / hu.1 (SEQ ID NO: 46 of WO 2005 / 033321 Pamphlet), AAVH-5 / hu.3 (SEQ ID NO: 44 of WO 2005 / 033321 Pamphlet), AAVhu.1 (SEQ ID NO: 144 of WO 2005 / 033321 Pamphlet), AAVhu.10 (SEQ ID NO: 156 of WO 2005 / 033321 Pamphlet), AAVhu.11 (SEQ ID NO: 153 of WO 2005 / 033321 Pamphlet), AAVhu.12 (SEQ ID NO: 59 of WO 2005 / 033321 Pamphlet), AAVhu.13 (SEQ ID NO: 129 of WO 2005 / 033321 Pamphlet), AAVhu.14 / AAV9 (SEQ ID NOs: 123 and 3 of WO 2005 / 033321 Pamphlet), AAVhu.15 (SEQ ID NO: 147 of WO 2005 / 033321 Pamphlet), AAVhu.16 (SEQ ID NO: 148 of WO 2005 / 033321 Pamphlet), AAVhu.17 (SEQ ID NO: 83 of WO 2005 / 033321 Pamphlet), AAVhu.18 (SEQ ID NO: 149 of WO 2005 / 033321 Pamphlet), AAVhu.19 (SEQ ID NO: 133 of WO 2005 / 033321 Pamphlet), AAVhu.2 (SEQ ID NO: 143 of WO 2005 / 033321 Pamphlet), AAVhu.20 (SEQ ID NO: 134 of WO 2005 / 033321 Pamphlet), AAVhu.21 (SEQ ID NO: 135 of WO 2005 / 033321 Pamphlet), AAVhu.22 (SEQ ID NO: 138 of WO 2005 / 033321 Pamphlet), AAVhu.23.2 (SEQ ID NO: 137 of WO 2005 / 033321 Pamphlet), AAVhu.24 (SEQ ID NO: 136 of WO 2005 / 033321 Pamphlet), AAVhu.25 (SEQ ID NO: 146 of WO 2005 / 033321 Pamphlet), AAVhu.27 (SEQ ID NO: 140 of WO 2005 / 033321 Pamphlet), AAVhu.29 (SEQ ID NO: 132 of WO 2005 / 033321 Pamphlet), AAVhu.3 (SEQ ID NO: 145 of WO 2005 / 033321 Pamphlet), AAVhu.31 (SEQ ID NO: 121 of WO 2005 / 033321 Pamphlet), AAVhu.32 (SEQ ID NO: 122 of WO 2005 / 033321 Pamphlet), AAVhu.34 (SEQ ID NO: 125 of WO 2005 / 033321 Pamphlet), AAVhu.35 (SEQ ID NO: 164 of WO 2005 / 033321 Pamphlet), AAVhu.37 (SEQ ID NO: 88 of WO 2005 / 033321 Pamphlet), AAVhu.39 (SEQ ID NO: 102 of WO 2005 / 033321 Pamphlet), AAVhu.4 (SEQ ID NO: 141 of WO 2005 / 033321 Pamphlet), AAVhu.40 (SEQ ID NO: 87 of WO 2005 / 033321 Pamphlet), AAVhu.41 (SEQ ID NO: 91 of WO 2005 / 033321 Pamphlet), AAVhu.42 (SEQ ID NO: 85 of WO 2005 / 033321 Pamphlet), AAVhu.43 (SEQ ID NO: 160 of WO 2005 / 033321 Pamphlet), AAVhu.44 (SEQ ID NO: 144 of WO 2005 / 033321 Pamphlet), AAVhu.45 (SEQ ID NO: 127 of WO 2005 / 033321 Pamphlet), AAVhu.46 (SEQ ID NO: 159 of WO 2005 / 033321 Pamphlet), AAVhu.47 (SEQ ID NO: 128 of WO 2005 / 033321 Pamphlet), AAVhu.48 (SEQ ID NO: 157 of WO 2005 / 033321 Pamphlet), AAVhu.49 (SEQ ID NO: 189 of WO 2005 / 033321 Pamphlet), AAVhu.51 (SEQ ID NO: 190 of WO 2005 / 033321 Pamphlet), AAVhu.52 (SEQ ID NO: 191 of WO 2005 / 033321 Pamphlet), AAVhu.53 (SEQ ID NO: 186 of WO 2005 / 033321 Pamphlet), AAVhu.54 (SEQ ID NO: 188 of WO 2005 / 033321 Pamphlet), AAVhu.55 (SEQ ID NO: 187 of WO 2005 / 033321 Pamphlet), AAVhu.56 (SEQ ID NO: 192 of WO 2005 / 033321 Pamphlet), AAVhu.57 (SEQ ID NO: 193 in WO 2005 / 033321 Pamphlet), AAVhu.58 (SEQ ID NO: 194 in WO 2005 / 033321 Pamphlet), AAVhu.6 (SEQ ID NO: 84 in WO 2005 / 033321 Pamphlet), AAVhu.60 (SEQ ID NO: 184 in WO 2005 / 033321 Pamphlet), AAVhu.61 (SEQ ID NO: 185 in WO 2005 / 033321 Pamphlet), AAVhu.63 (SEQ ID NO: 195 in WO 2005 / 033321 Pamphlet), AAVhu.64 (SEQ ID NO: 196 in WO 2005 / 033321 Pamphlet), AAVhu.66 (SEQ ID NO: 197 in WO 2005 / 033321 Pamphlet), AAVhu.67 (SEQ ID NO: 198 in WO 2005 / 033321 Pamphlet), AAVhu.7 (SEQ ID NO: 150 in WO 2005 / 033321 Pamphlet), AAVhu.8 (SEQ ID NO: 12 in WO 2005 / 033321 Pamphlet), AAVhu.9 (SEQ ID NO: 155 in WO 2005 / 033321 Pamphlet), AAVLG-10 / rh.40 (SEQ ID NO: 14 in WO 2005 / 033321 Pamphlet), AAVLG-4 / rh.38 (SEQ ID NO: 86 in WO 2005 / 033321 Pamphlet), AAVLG-4 / rh.38 (SEQ ID NO: 7 in WO 2005 / 033321 Pamphlet), AAVN721-8 / rh.43 (SEQ ID NO: 163 in WO 2005 / 033321 Pamphlet), AAVN721-8 / rh.43 (SEQ ID NO: 43 in WO 2005 / 033321 Pamphlet), AAVpi.1 (SEQ ID NO: 28 in WO 2005 / 033321 Pamphlet), AAVpi.2 (SEQ ID NO: 30 in WO 2005 / 033321 Pamphlet), AAVpi.3 (SEQ ID NO: 29 in WO 2005 / 033321 Pamphlet), AAVrh.38 (SEQ ID NO: 86 in WO 2005 / 033321 Pamphlet), AAVrh.40 (SEQ ID NO: 92 in WO 2005 / 033321 Pamphlet), AAVrh.43 (SEQ ID NO: 163 in WO 2005 / 033321 Pamphlet), AAVrh.44 (SEQ ID NO: 34 in WO 2005 / 033321 Pamphlet), AAVrh.45 (International Publication No. WO 2005 / 033321 Pamphlet Sequence No. 41), AAVrh.47 (International Publication No. WO 2005 / 033321 Pamphlet Sequence No. 38), AAVrh.48 (International Publication No. WO 2005 / 033321 Pamphlet Sequence No. 115), AAVrh.49 (International Publication No. WO 2005 / 033321 Pamphlet Sequence No. 103), AAVrh.50 (International Publication No. WO 2005 / 033321 Pamphlet... SEQ ID NO: 108), AAVrh.51 (SEQ ID NO: 104 in WO 2005 / 033321 pamphlet), AAVrh.52 (SEQ ID NO: 96 in WO 2005 / 033321 pamphlet), AAVrh.53 (SEQ ID NO: 97 in WO 2005 / 033321 pamphlet), AAVrh.55 (SEQ ID NO: 37 in WO 2005 / 033321 pamphlet), AAVrh.56 (SEQ ID NO: 152 in WO 2005 / 033321 pamphlet), AAVrh.57 (SEQ ID NO: 105 in WO 2005 / 033321 pamphlet), AAVrh.58 (SEQ ID NO: 106 in WO 2005 / 033321 pamphlet), AAVrh.59 (SEQ ID NO: 42 in WO 2005 / 033321 pamphlet), AAVrh.60 (SEQ ID NO: 31 in WO 2005 / 033321 pamphlet), AAVrh.61 (SEQ ID NO: 107 in WO 2005 / 033321 pamphlet), AAVrh.62 (SEQ ID NO: 114 in WO 2005 / 033321 pamphlet), AAVrh.64 (SEQ ID NO: 99 in WO 2005 / 033321 pamphlet), AAVrh.65 (SEQ ID NO: 35 in WO 2005 / 033321 pamphlet), AAVrh.68 (SEQ ID NO: 16 in WO 2005 / 033321 pamphlet), AAVrh.69 (SEQ ID NO: 39 in WO 2005 / 033321 pamphlet), AAVrh.70 (SEQ ID NO: 20 in WO 2005 / 033321 pamphlet), AAVrh.72 (SEQ ID NO: 9 in WO 2005 / 033321 pamphlet), or variants thereof, including but not limited to, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.25 / 42 15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37. It can be an AAV serotype as described in WO 2005 / 033321 pamphlet (the content of which is incorporated herein by reference in its entirety), including AAVrhl4, or can be derived therefrom. Non-limiting examples of variants include SEQ ID NOs: 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62, 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219, 224-236 of WO 2005 / 033321 pamphlet (the content of which is incorporated herein by reference in its entirety).

[0191] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, can be of an AAV serotype as described in WO 2015 / 168666 pamphlet (the content of which is incorporated herein by reference in its entirety), such as, but not limited to, AAVrh8R (SEQ ID NO: 9 of WO 2015 / 168666 pamphlet), AAVrh8R A586R mutant (SEQ ID NO: 10 of WO 2015 / 168666 pamphlet), AAVrh8R R533A mutant (SEQ ID NO: 11 of WO 2015 / 168666 pamphlet), or can be derived therefrom.

[0192] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide included in the rAAV virion described herein, are of an AAV serotype described in International Publication No. WO 2018 / 160582 (the content of which is incorporated herein by reference in its entirety), for example, but not limited to, AAVhu68 (e.g., SEQ ID NO: 2 of International Publication No. WO 2018 / 160582), or variants thereof, or may be derived therefrom.

[0193] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, is of an AAV serotype described in U.S. Patent No. 9,233,131, the contents of which are hereby incorporated by reference in their entirety, for example, but not limited to, the following: AAVhE1.1 (SEQ ID NO: 44 of U.S. Patent No. 9,233,131), AAVhErl.5 (SEQ ID NO: 45 of U.S. Patent No. 9,233,131), AAVhER1.14 (SEQ ID NO: 46 of U.S. Patent No. 9,233,131), AAVhErl.8 (SEQ ID NO: 47 of U.S. Patent No. 9,233,131), AAVhEr1.16 (SEQ ID NO: 48 of U.S. Patent No. 9,233,131), AAVhEr1.18 (SEQ ID NO: 49 of U.S. Patent No. 9,233,131), AAVhEr1.35 (SEQ ID NO: 50 of U.S. Patent No. 9,233,131), AAVhEr1.7 (SEQ ID NO: 51 of U.S. Patent No. 9,233,131), AAVhEr1.36 (SEQ ID NO: 52 of U.S. Patent No. 9,233,131), AAVhEr2.29 (SEQ ID NO: 53 of U.S. Patent No. 9,233,131), AAVhEr2.4 (SEQ ID NO: 54 of U.S. Patent No. 9,233,131), AAVhEr2.16 (SEQ ID NO: 55 of U.S. Patent No. 9,233,131), AAVhEr2.30 (SEQ ID NO: 56 of U.S. Patent No. 9,233,131), AAVhEr2.31 (SEQ ID NO: 58 of U.S. Patent No. 9,233,131), AAVhEr2.36 (SEQ ID NO: 57 of U.S. Patent No. 9,233,131), AAVhER1.23 (SEQ ID NO: 53 of U.S. Patent No. 9,233,131), AAVhEr3.1 (SEQ ID NO: 59 of U.S. Patent No. 9,233,131), AAV2.5T (SEQ ID NO: 42 of U.S. Patent No. 9,233,131), or variants thereof, or may be derived therefrom.

[0194] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein are AAV serotypes described in U.S. Patent Application Publication No. 20150376607 (the content of which is incorporated herein by reference in its entirety), for example, but not limited to, the following: AAV-PAEC (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150376607), AAV-LKOl (SEQ ID NO: 2 of U.S. Patent Application Publication No. 20150376607), AAV-LK02 (SEQ ID NO: 3 of U.S. Patent Application Publication No. 20150376607), AAV-LK03 (SEQ ID NO: 4 of U.S. Patent Application Publication No. 20150376607), AAV-LK04 (SEQ ID NO: 5 of U.S. Patent Application Publication No. 20150376607), AAV-LK05 (SEQ ID NO: 6 of U.S. Patent Application Publication No. 20150376607), AAV-LK06 (SEQ ID NO: 7 of U.S. Patent Application Publication No. 20150376607), AAV-LK07 (SEQ ID NO: 8 of U.S. Patent Application Publication No. 20150376607), AAV-LK08 (SEQ ID NO: 9 of U.S. Patent Application Publication No. 20150376607), AAV-LK09 (SEQ ID NO: 10 of U.S. Patent Application Publication No. 20150376607), AAV-LK10 (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150376607), AAV-LK11 (SEQ ID NO: 12 of U.S. Patent Application Publication No. 20150376607), AAV-LK12 (SEQ ID NO: 13 of U.S. Patent Application Publication No. 20150376607), AAV-LK13 (SEQ ID NO: 14 of U.S. Patent Application Publication No. 20150376607), AAV-LK14 (SEQ ID NO: 15 of U.S. Patent Application Publication No. 20150376607), AAV-LK15 (SEQ ID NO: 16 of U.S. Patent Application Publication No. 20150376607), AAV-LK16 (SEQ ID NO: 17 of U.S. Patent Application Publication No. 20150376607), AAV-LK17 (SEQ ID NO: 18 of U.S. Patent Application Publication No. 20150376607), AAV-LK18 (SEQ ID NO: 19 of U.S. Patent Application Publication No. 20150376607),AAV-LK19 (SEQ ID NO: 20 in US Patent Application Publication No. 20150376607), AAV-PAEC2 (SEQ ID NO: 21 in US Patent Application Publication No. 20150376607), AAV-PAEC4 (SEQ ID NO: 22 in US Patent Application Publication No. 20150376607), AAV-PAEC6 (SEQ ID NO: 23 in US Patent Application Publication No. 20150376607), AAV-PAEC (SEQ ID NO: 24 in US Patent Application Publication No. 20150376607), AAV-PAEC 8 (SEQ ID NO: 25 in US Patent Application Publication No. 20150376607), AAV-PAEC11 (SEQ ID NO: 26 in US Patent Application Publication No. 20150376607), AAV-PAEC12 (SEQ ID NO: 27 in US Patent Application Publication No. 20150376607), or variants or derivatives thereof may be used.

[0195] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide included in the rAAV virion described herein, may be of an AAV serotype described in U.S. Patent No. 9,163,261 (the entire content of which is incorporated herein by reference), for example, but not limited to, the following: AAV-2-pre-miRNA-101 (SEQ ID NO: 1 in U.S. Patent No. 9,163,261), or variants or derivatives thereof may be used.

[0196] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, are of the AAV serotypes described in U.S. Patent Application Publication No. 20150376240, the contents of which are hereby incorporated by reference in their entirety, for example, but not limited to, the following: AAV-8h (SEQ ID NO: 6 of U.S. Patent Application Publication No. 20150376240), AAV-8b (SEQ ID NO: 5 of U.S. Patent Application Publication No. 20150376240), AAV-h (SEQ ID NO: 2 of U.S. Patent Application Publication No. 20150376240), AAV-b (SEQ ID NO: 1 of U.S. Patent Application Publication No. 20150376240), or variants thereof, or may be derived therefrom.

[0197] In some embodiments, the AAV particles of the present invention include, but are not limited to, AAV SM 10-2 (SEQ ID NO: 22 in US Patent Application Publication No. 20160017295), AAV Shuffle 100-1 (SEQ ID NO: 23 in US Patent Application Publication No. 20160017295), AAV Shuffle 100-3 (SEQ ID NO: 24 in US Patent Application Publication No. 20160017295), AAV Shuffle 100-7 (SEQ ID NO: 25 in US Patent Application Publication No. 20160017295), AAV Shuffle 10-2 (SEQ ID NO: 34 in US Patent Application Publication No. 20160017295), AAV Shuffle 10-6 (SEQ ID NO: 35 in US Patent Application Publication No. 20160017295), AAV Shuffle 10-8 (SEQ ID NO: 36 in US Patent Application Publication No. 20160017295), AAV Shuffle 100-2 (SEQ ID NO: 37 in US Patent Application Publication No. 20160017295), AAV SM 10-1 (SEQ ID NO: 38 in US Patent Application Publication No. 20160017295), AAV SM 10-8 (SEQ ID NO: 39 in US Patent Application Publication No. 20160017295), AAV SM 100-3 (SEQ ID NO: 40 in US Patent Application Publication No. 20160017295), AAV SM 100-10 (SEQ ID NO: 41 in US Patent Application Publication No. 20160017295), or variants thereof, and may include or be derived from an AAV serotype having a sequence as described in US Patent Application Publication No. 20160017295 (the contents of which are hereby incorporated by reference in their entirety).

[0198] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, are of an AAV serotype described in US Patent Application Publication No. 20150238550, the content of which is hereby incorporated by reference in its entirety, such as, but not limited to, the following: BNP61 AAV (SEQ ID NO: 1 in US Patent Application Publication No. 20150238550), BNP62 AAV (SEQ ID NO: 3 in US Patent Application Publication No. 20150238550), BNP63 AAV (SEQ ID NO: 4 in US Patent Application Publication No. 20150238550), or variants thereof, or may be derived therefrom.

[0199] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide included in the rAAV virion described herein are AAV serotypes described in U.S. Patent Application Publication No. 20150315612, the content of which is hereby incorporated by reference in its entirety, for example, but not limited to, the following: AAVrh.50 (SEQ ID NO: 108 of U.S. Patent Application Publication No. 20150315612), AAVrh.43 (SEQ ID NO: 163 of U.S. Patent Application Publication No. 20150315612), AAVrh.62 (SEQ ID NO: 114 of U.S. Patent Application Publication No. 20150315612), AAVrh.48 (SEQ ID NO: 115 of U.S. Patent Application Publication No. 20150315612), AAVhu.19 (SEQ ID NO: 133 of U.S. Patent Application Publication No. 20150315612), AAVhu.ll (SEQ ID NO: 153 of U.S. Patent Application Publication No. 20150315612), AAVhu.53 (SEQ ID NO: 186 of U.S. Patent Application Publication No. 20150315612), AAV4-8 / rh.64 (SEQ ID NO: 15 of U.S. Patent Application Publication No. 20150315612), AAVLG-9 / hu.39 (SEQ ID NO: 24 of U.S. Patent Application Publication No. 20150315612), AAV54.5 / hu.23 (SEQ ID NO: 60 of U.S. Patent Application Publication No. 20150315612), AAV54.2 / hu.22 (SEQ ID NO: 67 of U.S. Patent Application Publication No. 20150315612), AAV54.7 / hu.24 (SEQ ID NO: 66 of U.S. Patent Application Publication No. 20150315612), AAV54.1 / hu.21 (SEQ ID NO: 65 of U.S. Patent Application Publication No. 20150315612), AAV54.4R / hu.27 (SEQ ID NO: 64 of U.S. Patent Application Publication No. 20150315612), AAV46.2 / hu.28 (SEQ ID NO: 68 of U.S. Patent Application Publication No. 20150315612), AAV46.6 / hu.29 (SEQ ID NO: 69 of U.S. Patent Application Publication No. 20150315612), AAV128.1 / hu.It may be of or derived from SEQ ID NO: 43 (SEQ ID NO: 80 in US Patent Application Publication No. 2015 / 0315612), or a variant thereof.

[0200] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, may be of an AAV serotype described in WO 2015 / 121501 (the content of which is incorporated herein by reference in its entirety), such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 in WO 2015 / 121501), "UPenn AAV10" (SEQ ID NO: 8 in WO 2015 / 121501), "Japanese AAV10" (SEQ ID NO: 9 in WO 2015 / 121501), or a variant thereof, or derived from any of them.

[0201] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 polypeptide capsid polypeptide and / or AAV VP3 capsid polypeptide contained in the rAAV virion described herein, may be selected from the capsid polypeptides disclosed in WO 2015 / 191508 (the content of which is incorporated herein by reference in its entirety).

[0202] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein, may be of a hybrid serotype that provides enhanced transduction or tropism, extended transgene expression, and / or an improved safety profile in a particular cell type of interest. Hybrid serotypes can be generated by transcapsidation, adsorption of bispecific antibodies to the capsid surface, mosaic capsids, and chimeric capsids, and / or other capsid protein modifications.

[0203] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein, may be further modified for specific therapeutic applications by rational mutagenesis of the capsid protein (see, e.g., Pulicherla et al, Mol Ther, 2011, 19:1070 - 1078), incorporation of peptide ligands into the capsid, and directed evolution to generate new AAV variants.

[0204] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein, may be of an AAV serotype selected from a variety of species including, but not limited to, human, non - human primate, avian, and bovine. In one embodiment, the AAV may be avian AAV (AAAV). The AAAV serotype may be the sequences described in U.S. Patent No. 9,238,800 (the contents of which are hereby incorporated by reference in their entirety), e.g., AAAV (SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Patent No. 9,238,800), or variants thereof, or may include them.

[0205] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein may be of an AAV serotype that is or is derived from bovine AAV (BAAV). The BAAV serotype may be a sequence as described in U.S. Patent No. 9,193,769, the contents of which are hereby incorporated by reference in their entirety, for example, but not limited to, BAAV (SEQ ID NOs: 1 and 6 of U.S. Patent No. 9,193,769), or a variant thereof, or may have it. The BAAV serotype may be a sequence as described in U.S. Patent No. 7,427,396, the contents of which are hereby incorporated by reference in their entirety, for example, but not limited to, BAAV (SEQ ID NOs: 5 and 6 of U.S. Patent No. 7,427,396), or variants thereof, or may contain them.

[0206] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein may be of an AAV serotype that is or is derived from caprine AAV. The caprine AAV serotype may be a sequence as described in U.S. Patent No. 7,427,396, the contents of which are hereby incorporated by reference in their entirety, for example, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Patent No. 7,427,396), or variants thereof, or may have it.

[0207] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein, may be of an AAV serotype that can be engineered as a hybrid AAV from two or more parental serotypes. In some embodiments, the serotype may be AAV2G9, which contains sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be, or may have, a sequence as described in U.S. Patent Application Publication No. 20160017005, the entire contents of which are incorporated herein by reference.

[0208] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein, may be of an AAV serotype produced by an AAV9 capsid library having mutations at amino acids 390-627 (VP1 numbering) as described in Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011); the content of which is hereby incorporated by reference in its entirety). Serotypes and corresponding nucleotide and amino acid substitutions can be, but are not limited to, the following: AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80 (G1441A,; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

[0209] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein, may include the capsid sequences of SEQ ID NOs: 1 and 3 of WO 2014 / 160092 (the content of which is incorporated herein by reference in its entirety).

[0210] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein, may include the AAV capsid sequences of SEQ ID NO: 1 or SEQ ID NOs: 2-4 of WO 2014 / 052789 (the content of which is incorporated herein by reference in its entirety).

[0211] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein, may have an enhanced ability to cross the blood-brain barrier as disclosed in U.S. Patent No. 8,927,514 (the content of which is incorporated herein by reference in its entirety). Examples of such amino acid sequences and nucleic acid sequences of the capsid protein include, but are not limited to, SEQ ID NOs: 2-17 and SEQ ID NOs: 25-33 of U.S. Patent No. 8,927,514.

[0212] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein, may be an AAV5 capsid protein or a variant thereof, such as those described in U.S. Patent No. 7,056,502 (the content of which is incorporated herein by reference in its entirety).

[0213] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein may be an AAV6 capsid protein or a variant thereof.

[0214] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein may be an AAV8 capsid protein or a variant thereof, such as that described in SEQ ID NO: 2 of the amino acid sequence of U.S. Patent No. 8,318,480, the entire content of which is incorporated herein by reference.

[0215] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptide contained in the rAAV virion described herein may be an AAV9 capsid protein or a variant thereof, which includes, for example, Amino Acid Sequence 2 of U.S. Patent No. 7,198,951, the entire content of which is incorporated herein by reference, or the sequences of SEQ ID NO: 2, 4, or 6 disclosed in U.S. Patent Application Publication No. 20130224836, the entire content of which is incorporated herein by reference, wherein at least one of the surface-exposed tyrosine residues of the above amino acid sequences is substituted with another amino acid residue.

[0216] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein may be variant capsid proteins engineered to acquire certain properties. Such methods for obtaining engineered capsids are described, for example, in WO 2011 / 038187 pamphlet (the content of which is incorporated herein by reference in its entirety). Such methods and vectors are also described, for example, in WO 2012 / 112832 pamphlet and WO 2015 / 054653 pamphlet (the content of which is incorporated herein by reference in its entirety). Such variant capsids include, for example, SEQ ID NO: 23 of WO 2015 / 054653 pamphlet, or variants thereof. Further variant capsids include, for example, the capsid sequences described in WO 2017 / 019994 pamphlet (the content of which is incorporated by reference in its entirety). In embodiments, the AAV capsid polypeptide may include the capsid of the Anc80 AAV capsid sequence (e.g., SEQ ID NO: 1 of WO 2017019994 pamphlet), for example, Anc80L65 (e.g., SEQ ID NO: 23 of WO 2017019994 pamphlet), or, for example, Anc110 (e.g., SEQ ID NO: 42 of WO 2017019994 pamphlet).

[0217] In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides contained in the rAAV virions described herein may be the AAV10 capsid protein or a variant thereof, which includes, for example, the amino acid sequence of SEQ ID NO: 81 of EP 2341068 B1.

[0218] In some embodiments, the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide described herein, and / or the AAV VP1 and / or VP3 polypeptides included in the rAAV virions described herein, may be an AAVDJ capsid protein, an AAVDJ / 8 capsid protein, or a variant thereof. In some embodiments, the AAVDJ capsid protein and / or the AAVDJ / 8 capsid protein may comprise an amino acid sequence including a first region derived from a first AAV serotype (e.g., AAV2), a second region derived from a second AAV serotype (e.g., AAV8), and a third region derived from a third AAV serotype (e.g., AAV9), wherein the first, second, and third regions may comprise any amino acid sequences disclosed herein.

[0219] In some embodiments, the serotype of the AAV capsid polypeptide may depend on the desired distribution, transduction efficiency, and required cell targeting. As described by Sorrentino et al. (comprehensive map of CNS transduction by eight adeno-associated virus serotypes upon cerebrospinal fluid administration in pigs, Molecular Therapy accepted article preview online 07 December 2015; doi:10.1038 / mt.2015.212; the content of which is incorporated herein by reference in its entirety), AAV serotypes have resulted in different distributions, transduction efficiencies, and cell targeting. To achieve the desired efficacy, it is necessary to select an AAV serotype that best matches not only the target cells but also the desired transduction efficiency and distribution.

[0220] In some embodiments, the rAAV virion exhibits an increase in transduction and / or an increase in tropism in at least one tissue or cell type as compared to an rAAV virion comprising the same AAV VP2 capsid polypeptide except that it is not fused to the polypeptide ligand.

[0221] In some embodiments, transduction of at least one tissue or cell type is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. In some embodiments, the rAAV virion exhibits transduction that is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100, at least 1000-fold, or more than 1000-fold in at least one tissue or cell type as compared to an rAAV virion comprising the same AAV VP2 capsid polypeptide except that it is not fused to the polypeptide ligand. In some embodiments, tropism in at least one tissue or cell type is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.

[0222] In some embodiments, all AAV VP2 capsid polypeptides contained in the rAAV virion are fused to the polypeptide ligand, either directly or via a linker.

[0223] In some embodiments, the rAAV virion further comprises a nucleic acid sequence selected from the group consisting of non-coding nucleic acids, protein or RNA coding sequences, expression cassettes, multiple expression cassettes, sequences for homologous recombination, and genomic gene targeting cassettes.

[0224] In some embodiments, the rAAV virion further comprises a nucleic acid sequence encoding an AAV VP2 fusion polypeptide as described herein. In some of these embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virion is the same as the AAV VP2 fusion polypeptide encoded by the nucleic acid sequence contained in the rAAV virion.

[0225] In some embodiments, the rAAV virion comprises a nucleic acid sequence encoding a therapeutic nucleic acid, a therapeutic protein, or a therapeutic antibody or antibody fragment.

[0226] The therapeutic nucleic acid can be selected from the group consisting of mRNA, siRNA, miRNA, shRNA, and antisense oligonucleotides.

[0227] The antibody fragment can be selected from the group consisting of, for example, Fab, Fab’, F(ab’)2, Fv, single domain antibody (dAb), and single chain variable fragment (scFv).

[0228] In some embodiments, the rAAV virion comprising the AAV VP2 fusion polypeptide further comprises AAV VP1 and VP3 polypeptides. In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of an AAV serotype independently selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV, or avian AAV. In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of an AAV serotype selected from the group consisting of AAV1, AAV6, AAV8, and AAV9. In some of these embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of the same AAV serotype.

[0229] In some embodiments, the AAV VP1, VP2, and VP3 polypeptides contained in the rAAV virion comprise at least one mutation in at least one binding site for a natural receptor present on the target cell of the AAV virion composed of the AAV VP1, VP2, and VP3 polypeptides. In certain embodiments, at least one essential binding site for the natural receptor is mutated in the AAV VP1, VP2, and VP3 polypeptides. In some embodiments, the AAV VP1, VP2, and VP3 polypeptides comprise the same at least one mutation in the shared VP3 region of each AAV capsid polypeptide. In some of these embodiments, the AAV VP2 fusion polypeptide, and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV6, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A with respect to the VP1 amino acid sequence of SEQ ID NO: 3, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise an amino acid substitution with respect to the VP1 amino acid sequence of SEQ ID NO: 3: i) K531E and V473D; ii) K531E, K459S, V473D, and N500E; iii) G266A and N269Q; iv) G266A, N269Q, and D590A; v) K531E, V473D, G266A, and N269Q; or vi) K531E, K459S, V473D, N500E, G266A, N269Q, and D590A.

[0230] In some embodiments, the AAV VP2 fusion polypeptide, and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV8, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: G268E, N271Q, S387A, A592Q and A592D, relative to the VP1 amino acid sequence of SEQ ID NO: 4, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise an amino acid substitution relative to the VP1 amino acid sequence of SEQ ID NO: 4: i) G268E and N271Q; ii) S387A; iii) G268E, N271Q and S387A; iv) A592Q; or v) A592D.

[0231] In some embodiments, the AAV VP2 fusion polypeptide, and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV9, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: Q590A, W503A, N562A and E563A, relative to the VP1 amino acid sequence of SEQ ID NO: 5, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide, and the AAV VP1 and VP3 polypeptides each comprise an amino acid substitution relative to the VP1 amino acid sequence of SEQ ID NO: 5: i) W503A; ii) N562A and E563A; iii) Q590A and W503A; or iv) Q590A, W503A, N562A and E563A.

[0232] In some embodiments, the AAV VP2 fusion polypeptide, and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV2, each comprising, within the shared VP3 region, the amino acid substitution R585A relative to the VP1 amino acid sequence of SEQ ID NO: 6.

[0233] In some embodiments, the AAV VP2 fusion polypeptide, and said AAV VP1 and VP3 polypeptides, are of AAV serotype AAV1, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: V473D, N500E, and R514A relative to the VP1 amino acid sequence of SEQ ID NO: 7, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide, and said AAV VP1 and VP3 polypeptides each comprise an amino acid substitution relative to the VP1 amino acid sequence of SEQ ID NO: 7: i) V473D and N500E; ii) R514A; or iii) V473D, N500E, and R514A.

[0234] In one aspect, provided herein are cells comprising the rAAV virions described herein.

[0235] In some embodiments, the cell is a host cell. In some embodiments, the rAAV virion is assembled in said host cell. The host cell can be selected from the group consisting of insect cells (e.g., Sf9 cells) and HEK293 cells or derivatives thereof, e.g., HEK293T / 17 cells or AAV293 cells.

[0236] In some embodiments, the cell is selected from mammalian cells such as, but not limited to, mouse cells, non-human primate cells, or human cells. In some embodiments, the mammalian cell is selected from the group consisting of hepatocytes, brain cells, spleen cells, kidney cells, blood cells, lung cells, muscle cells, heart cells, bone marrow cells, multipotent progenitor cells (MPP) such as multipotent hematopoietic progenitor cells, and hematopoietic stem cells (HSC) such as long-term hematopoietic stem cells (LT-HSC).

[0237] Pharmaceutical Compositions and Methods of Treatment In one aspect, provided herein is a pharmaceutical composition comprising the rAAV virions described herein and a pharmaceutically acceptable excipient.

[0238] The rAAV virions described in this specification can be formulated to prepare a composition useful as a formulation. Exemplary formulations include, for example, those disclosed in U.S. Patent Nos. 9,051,542 and 6,703,237, which are incorporated herein by reference in their entirety. The compositions of the present disclosure can be formulated for administration to mammalian subjects, such as humans. In some embodiments, the delivery system can be formulated for intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, injectable depot-type devices, or topical administration.

[0239] In some embodiments, when the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. can be used. These compositions may be sterilized and / or sterile filtered. The resulting aqueous solution may be packaged for use as is or lyophilized. In some embodiments, the lyophilized formulation is combined with a sterile solution prior to administration.

[0240] In some embodiments, the composition, such as a pharmaceutical composition, may contain pharmaceutically acceptable auxiliary substances for approximating physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the pharmaceutical composition contains a preservative. In some other embodiments, the pharmaceutical composition does not contain a preservative.

[0241] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Non-limiting examples of routes of administration include parenteral administration, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion. Methods of formulating suitable pharmaceutical compositions are known in the art, see, for example: Remington: The Science and Practice of Pharmacy, 21st ed, 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0242] Also provided herein are rAAV virions or pharmaceutical compositions containing the same for use as a medicament. For example, the rAAV virions disclosed herein can be administered to deliver a heterologous nucleic acid encoding a therapeutic RNA or protein to target cells. Accordingly, as used herein, the terms “therapeutic use” or “treatment” include gene therapy for the treatment or prevention of diseases, and the term “gene therapy” can be broadly defined as the concept of the directed introduction of foreign genetic material into cells, tissues or organs for the purpose of correcting defective genes aimed at improving the clinical condition of a patient. For example, the heterologous nucleic acid can replace a gene in a target cell that has a mutation or deletion causing a genetic disease. Administration of the rAAV virions disclosed herein can help treat, prevent, delay, slow down or improve such genetic diseases. In some embodiments, “gene therapy” refers only to “somatic cell therapy” and does not include “germline therapy” which can induce heritable changes passed from generation to generation, where somatic cell therapy limits the therapeutic effect to the individual being treated. The rAAV virions described herein can be administered in vivo or ex vivo. As used herein, the terms “treat” and “treatment” include the step of administering a composition containing the rAAV virions disclosed herein in an effective dose or effective multiple doses to an animal (including humans) in need thereof. When the above dose is administered before the onset of a disorder / disease, this administration is prophylactic. When the dose is administered after the onset of a disorder / disease, this administration is therapeutic. In some embodiments, an effective amount is an amount that detectably alleviates (either eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, an amount that delays or prevents progression to a disorder / disease state, an amount that delays or prevents progression of a disorder / disease state, an amount that reduces the severity of a disease, an amount that results in remission (partial or complete) of a disease, and / or an amount that extends survival. The term encompasses complete treatment (i.e., cure) and / or prevention, but does not necessarily require it.

[0243] In one aspect, provided herein is a method of delivering a transgene to a cell, the method comprising contacting the rAAV virions described herein or a pharmaceutical composition comprising the same with the cell.

[0244] In one aspect, provided herein is a method of delivering a transgene to a cell, wherein the cell expresses a cell surface molecule to which the polypeptide ligand specifically binds.

[0245] In some embodiments, the cell is in a living subject. In some embodiments, the subject is a mammalian subject, such as a human.

[0246] In one aspect, provided herein is a method of using the rAAV virions described herein or a pharmaceutical composition comprising the same in a therapeutic treatment regimen or as a vaccine.

[0247] In one aspect, provided herein is a method of using the rAAV virions described herein or a pharmaceutical composition comprising the same to reduce the amount of total rAAV virions administered to a subject in a treatment method, the method comprising administering to the subject a lesser amount of total rAAV virions compared to the dosage of rAAV comprising an AAV VP2 capsid polypeptide that is identical except that it is not fused to the polypeptide ligand, whereby a similar therapeutic effect can be achieved.

[0248] In one aspect, provided herein is a method of using the rAAV virions described herein or a pharmaceutical composition comprising the same to increase the transduction efficiency of rAAV virions administered to a subject in a treatment method, the method comprising administering to the subject a predetermined amount of rAAV virions, wherein the rAAV virions comprising an AAV VP2 fusion polypeptide can transduce a cell type or tissue at a faster rate compared to rAAV virions comprising an AAV VP2 capsid polypeptide that is identical except that it is not fused to the polypeptide ligand.

[0249] AAV VP2 Fusion Polypeptide Library In one aspect, provided herein is a library construct comprising a nucleic acid sequence encoding an AAV VP2 fusion polypeptide comprising, for example consisting of, an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the polypeptide ligand has a molecular weight of up to 10 kDa.

[0250] In some embodiments, the polypeptide ligand encoded by the library construct comprises randomized amino acids at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions.

[0251] In one aspect, provided herein is a library construct comprising a nucleic acid sequence encoding an AAV VP2 fusion polypeptide comprising, for example consisting of, an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the polypeptide ligand is selected from the group consisting of a GP2 polypeptide, an Sso7d polypeptide, and an affibody.

[0252] In some embodiments, the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 and an Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0253] In some embodiments, the polypeptide ligand is optionally selected from the Sso7d polypeptide of SEQ ID NO: 1 having a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.

[0254] In some embodiments, the polypeptide ligand has amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 that are independently selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 in which X is D, R, H, N, A, I, Y, and W, and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0255] In some embodiments, the polypeptide ligand is selected from the Sso7d polypeptide of SEQ ID NO: 1 in which the amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, W, and amino acid substitutions, where SEQ ID NO: 1 optionally has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.

[0256] In some embodiments, the encoded AAV VP2 fusion further comprises a peptide linker between the polypeptide ligand and the AAV VP2 capsid polypeptide. In some of these embodiments, the peptide linker is selected from the group consisting of a glycine-serine (GS) linker and an alanine-proline-serine (APS) linker. In some of these embodiments, the GS linker has the formula [GGGGS]n, where n is an integer in the range of 1 to 10, for example, n is 1, 2, 3, 4, 5, or 6, and in particular, n is 1, 2, 3, or 4. In some embodiments, the APS linker has the formula [APS]n, where n is an integer in the range of 1 to 10, for example, n is 1, 2, 3, 4, 5, or 6, and in particular, n is 2, 3, 4, or 5.

[0257] In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide does not contain the native start codon of the AAV VP2 capsid polypeptide. The native start codon of the AAV VP2 capsid polypeptide may be mutated, for example, it may be deleted. In some embodiments, the AAV VP2 capsid polypeptide contained in the AAV VP2 fusion polypeptide lacks its first native amino acid, for example, the first threonine of the corresponding wild-type AAV VP2 capsid polypeptide.

[0258] In one aspect, provided herein is a library comprising a plurality of library constructs described herein. In some embodiments, the library comprises at least two different library constructs described herein. In some embodiments, at least two different library constructs contained in the library differ in the nucleic acid sequences encoding the polypeptide ligand.

[0259] In some embodiments, the library comprises at least 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、or 10 9 unique library constructs. In some embodiments, the library comprises 10 2 ~10 3 、10 3 ~10 4 、10 4 ~10 5 、10 5 ~10 6 、10 6 ~10 7 、10 7 ~10 8 、or 10 8 ~10 9 unique library constructs.

[0260] In some embodiments, the library comprises at least 10 2 、103 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 and includes members of. In some embodiments, the library is 10 2 to 10 3 , 10 3 to 10 4 , 10 4 to 10 5 , 10 5 to 10 6 , 10 6 to 10 7 , 10 7 to 10 8 , 10 8 to 10 9 , or 10 9 to 10 10 and includes members of.

[0261] In some embodiments, the library includes a library construct that encodes all possible variants of the polypeptide ligand.

[0262] In some embodiments, the library construct is an RNA molecule, e.g., an mRNA molecule, or a DNA molecule, e.g., a cDNA molecule, a linear DNA molecule or a circular DNA molecule, e.g., a plasmid DNA molecule. In some embodiments, the library construct further includes a promoter. In some embodiments, the open reading frame of the AAV VP2 fusion polypeptide is operably linked to the promoter.

[0263] In some embodiments, the library construct is a plasmid DNA molecule comprising the nucleic acid sequence encoding the AAV VP2 fusion polypeptide operably linked to a promoter. In some embodiments, the library construct further comprises a reporter sequence. In some embodiments, the reporter sequence encodes a fluorescent or luminescent reporter protein. In some embodiments, the reporter protein is selected from the group consisting of EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase.

[0264] In some embodiments, the library construct further comprises a barcode sequence. In some of these embodiments, a specific barcode sequence is assigned to each of the specific AAV VP2 fusion polypeptide sequences, which means that each of the specific barcode sequences corresponds to one specific AAV VP2 fusion polypeptide sequence.

[0265] In some embodiments, the library is an AAV library, i.e., each library construct is present within an rAAV virion.

[0266] In some embodiments, the rAAV virion contains, for example, within its capsid, the AAV VP2 fusion polypeptide described herein. In some embodiments, the AAV VP2 fusion polypeptide contained within a given rAAV virion, for example, within its capsid, is the same as the AAV VP2 fusion polypeptide encoded by the library construct present in said given rAAV virion. In some embodiments, most of the rAAV virions contain a library construct encoding an AAV VP2 fusion polypeptide that is the same as the AAV VP2 fusion polypeptide contained within a given rAAV virion. This is referred to as the genotype-phenotype linkage or capsid-genome correlation of the AAV library.

[0267] In some embodiments, the AAV library comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 unique AAV variants. In some embodiments, the AAV library comprises 10 2 to 10 3 , 10 3 to 10 4 , 10 4 to 10 5 , 10 5 to 10 6 , 10 6 to 10 7 , 10 7 to 10 8 , or 10 8 to 10 9 unique AAV variants.

[0268] In some embodiments, the AAV library comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 AAV members. In some embodiments, the library comprises 10 2 to 10 3 , 10 3 to 10 4 , 10 4 to 10 5 , 10 5 to 10 6 , 10 6 to 10 7 , 10 7 to 10 8 , 10 8 to 10 9 , or 10 9 to 10 10 AAV members.

[0269] The AAV libraries described herein can be used for phenotypic selection of capsid polypeptides having desired characteristics, such as AAV VP2 fusion polypeptides. The AAV VP2 fusion polypeptides are encoded in cis from a replicating AAV genome. This enables the recovery of capsid DNA after phenotypic selection. Thus, during the production of the viral library, it is desirable to minimize random mixing of capsomers and mismatched viral genome envelopes. This can be achieved, for example, by selecting appropriate conditions for rAAV virion production, such as using an appropriate ratio of a nucleic acid molecule encoding an AAV VP2 fusion polypeptide and nucleic acid molecules encoding AAV VP1 and VP3 capsid polypeptides for transfection of host cells for rAAV virion production. Typically, in a transfection reaction, the nucleic acids encoding VP1 and VP3 are used in excess compared to the nucleic acid encoding the AAV VP2 fusion polypeptide. The nucleic acid encoding the AAV VP2 fusion polypeptide is used in an amount such that, on average, each host cell is transfected with only a single nucleic acid encoding the AAV VP2 fusion polypeptide.

[0270] In some embodiments, all AAV VP2 capsid polypeptides included in the rAAV virions of the AAV library are fused to a polypeptide ligand, either directly or via a linker.

[0271] In some embodiments, each library construct of the AAV library comprises two AAV inverted terminal repeat (ITR) sequences located upstream and downstream of the AAV VP2 fusion polypeptide open reading frame, e.g., at the 5' and 3' ends of the library construct. In some embodiments, the AAV VP2 fusion polypeptide open reading frame is operably linked to a promoter. In some embodiments, the library construct further comprises a reporter sequence. In some embodiments, the reporter sequence encodes a fluorescent or luminescent reporter protein. In some embodiments, the reporter protein is selected from the group consisting of: EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase. In some embodiments, the reporter sequence is operably linked to a promoter, e.g., a second promoter comprised by the library construct.

[0272] In some embodiments, the rAAV virions of the AAV library comprise the AAV VP2 fusion polypeptide described herein and further comprise AAV VP1 and VP3 polypeptides. In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides contained in the rAAV virions are independently selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5. In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides contained in the rAAV virions are of an AAV serotype independently selected from the group consisting of: AAV1, AAV6, AAV8 and AAV9. In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides contained in the rAAV virions are of the same AAV serotype.

[0273] In some embodiments, the AAV VP1, VP2 and VP3 polypeptides contained in the rAAV virions of the AAV library comprise at least one mutation in at least one binding site for a natural receptor present on the target cell of the AAV virion consisting of the AAV VP1, VP2 and VP3 polypeptides. In certain embodiments, at least one essential binding site for the natural receptor is mutated in the AAV VP1, VP2 and VP3 polypeptides. In some embodiments, the AAV VP1, VP2 and VP3 polypeptides comprise the same at least one mutation in the shared VP3 region of each AAV capsid polypeptide.

[0274] In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV6, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of K531E, V473D, K459S, N500E, G266A, N269Q, and D590A with respect to the VP1 amino acid sequence of SEQ ID NO: 3, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise an amino acid substitution with respect to the VP1 amino acid sequence of SEQ ID NO: 3: i) K531E and V473D; ii) K531E, K459S, V473D, and N500E; iii) G266A and N269Q; iv) G266A, N269Q, and D590A; v) K531E, V473D, G266A, and N269Q; or vi) K531E, K459S, V473D, N500E, G266A, N269Q, and D590A.

[0275] In some embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV8, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of G268E, N271Q, S387A, A592Q, and A592D with respect to the VP1 amino acid sequence of SEQ ID NO: 4, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise an amino acid substitution with respect to the VP1 amino acid sequence of SEQ ID NO: 4: i) G268E and N271Q; ii) S387A; iii) G268E, N271Q, and S387A; iv) A592Q; or v) A592D.

[0276] In some embodiments, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV9, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: Q590A, W503A, N562A, and E563A, relative to the VP1 amino acid sequence of SEQ ID NO: 5, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each comprise an amino acid substitution relative to the VP1 amino acid sequence of SEQ ID NO: 5: i) W503A; ii) N562A and E563A; iii) Q590A and W503A; or iv) Q590A, W503A, N562A, and E563A.

[0277] In some embodiments, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV2, each comprising, within the shared VP3 region, the amino acid substitution R585A relative to the VP1 amino acid sequence of SEQ ID NO: 6.

[0278] In some embodiments, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV1, each comprising, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: V473D, N500E, and R514A, relative to the VP1 amino acid sequence of SEQ ID NO: 7, or any combination thereof. In some of these embodiments, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each comprise an amino acid substitution relative to the VP1 amino acid sequence of SEQ ID NO: 7: i) V473D and N500E; ii) R514A; or iii) V473D, N500E, and R514A.

[0279] In some embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virions of the AAV library is of AAV serotype AAV1 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D213A, T162R, and / or P191N, and particularly all three amino acid substitutions: D213A, T162R, and P191N.

[0280] In other embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virions of the AAV library is of an AAV serotype other than AAV1 and comprises at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, relative to the VP1 amino acid sequence of SEQ ID NO: 7.

[0281] In certain such embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virions is of AAV serotype 8 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D214A, K163R and / or P192N, and particularly all three substitutions: D214A, K163R and P192N.

[0282] In other such embodiments, the AAV VP2 fusion polypeptide contained in the rAAV virion is of AAV serotype 9 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D213A, S162R and / or P191N, especially all three amino acid substitutions D213A, S162R and P191N.

[0283] In one aspect, provided herein is a library comprising a plurality of rAAV virions each containing a variant AAV VP2 fusion polypeptide described herein.

[0284] In some embodiments, the library comprises at least two different rAAV virions each containing a variant AAV VP2 fusion polypeptide described herein. In some embodiments, the at least two different rAAV virions differ in the polypeptide ligand contained in the AAV VP2 fusion polypeptide.

[0285] In some embodiments, the AAV library is at least 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、or 10 9 of unique rAAV virions. In some embodiments, the AAV library is 10 2 ~10 3 、10 3 ~10 4 、10 4 ~10 5 、10 5 ~10 6 、10 6 ~10 7 、10 7 ~10 8 、or 10 8 ~109 comprises unique rAAV virions.

[0286] In some embodiments, the AAV library comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 rAAV virion members. In some embodiments, the AAV library comprises 10 2 ~10 3 , 10 3 ~10 4 , 10 4 ~10 5 , 10 5 ~10 6 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , or 10 9 ~10 10 rAAV virion members.

[0287] In some embodiments, the rAAV virions included in the library comprise nucleic acids containing barcode sequences. In some embodiments, specific barcode sequences are assigned to specific AAV VP2 fusion polypeptides, which means that rAAV virions containing an AAV VP2 fusion polypeptide of a given amino acid sequence comprise nucleic acids having the corresponding barcode sequences. Barcode sequences comprise, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides.

[0288] Methods of Identifying AAV VP2 Fusion Polypeptide Variants In one aspect, provided herein is a method of making an AAV VP2 fusion polypeptide comprising at least one desired feature, the method comprising screening a library described herein for an encoded AAV VP2 fusion polypeptide comprising the at least one desired feature.

[0289] In some embodiments, the at least one desired feature is an increase in transduction and / or tropism in at least one tissue or cell type mediated by the AAV VP2 fusion polypeptide as compared to an AAV VP2 capsid polypeptide that is identical to the AAV VP2 capsid polypeptide included in the AAV VP2 fusion polypeptide except that it does not include the polypeptide ligand.

[0290] In one aspect, provided herein is a method of making an AAV VP2 fusion polypeptide comprising at least one desired feature, the method comprising: b) contacting a library described herein with a plurality of cells; c) isolating nucleic acid molecules from at least a portion of the cells; and d) determining at least a portion of a sequence encoding a polypeptide ligand or a fragment thereof of at least one nucleic acid molecule isolated in step b). In some embodiments, the plurality of cells in step b) are present within a non-human model animal, such as a mouse model or a non-human primate model.

[0291] In some embodiments, the method further comprises, prior to step b), step a) of providing an AAV library as described herein. In some embodiments, the library is packaged into HEK293 cells or HEK293 derivative cells in which helper functions (e.g., E2A, E4, VA, E1A, and E1B) are supplied in trans. In some embodiments, the AAV rep function comprises the rep78, rep68, rep52, and rep40 gene products. In some embodiments, the rep gene is supplied in trans. In some embodiments, the start codons of the rep78 and / or rep68 open reading frames are modified from ACG to ATG to increase the replication of the capsid library construct containing the inverted terminal repeats (ITRs), thereby improving the AAV library production yield. In some embodiments, the cap gene is supplied in trans. In some embodiments, the cap gene is controlled by the p40 promoter such that it is expressed only during production in HEK293 cells in the presence of helper virus functions. In some embodiments, the start codon of the VP2 open reading frame of the cap gene provided in trans is mutated. In some embodiments, the nucleic acid encoding the AAV VP2 fusion polypeptide is supplied as a payload to the production cell line upon AAV production.

[0292] In some embodiments, step a) of providing an AAV library comprises: ai) transfecting a cell with a library of nucleic acid sequences encoding AAV rep, a nucleic acid sequence encoding an AAV VP1 capsid polypeptide, a nucleic acid sequence encoding an AAV VP3 capsid polypeptide, and a nucleic acid encoding the AAV VP2 fusion polypeptide as described herein, wherein each library construct further comprises two AAV inverted terminal repeat (ITR) sequences at its 5' and 3' ends; and aii) incubating the cell under conditions suitable for AAV virion formation, optionally wherein the cell is a mammalian cell, particularly a HEK293 cell, or an insect cell, particularly an Sf9 cell.

[0293] In some embodiments, the AAV VP1 capsid polypeptide and the AAV VP3 capsid polypeptide are encoded by the same nucleic acid sequence containing the AAVcap gene, and in particular here, the start codon of VP2 in the cap gene is mutated.

[0294] In some embodiments, the plurality of cells in step b) are present in a non-human model animal. In some embodiments, the animal is treated with the AAV library by intravenous, intracranial, or intrathecal injection, or injection by some other route (e.g., intranasal, intrahepatic, intraventricular, intravitreous, intravestibular, etc.). After a sufficient time (e.g., 7, 10, 14, 18, 21, 24, 28, 30 days or more) has elapsed for the AAV to migrate to the tissue or organ of interest, the animal is sacrificed and the tissue / organ of interest is harvested. In some embodiments, the tissue / organ of interest to be harvested is selected from the central nervous system, brain, heart, lung, trachea, esophagus, muscle, bone, cartilage, bone marrow, stomach, pancreas, intestine, liver, spleen, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testis, prostate, eye, blood, lymph, and oral mucosa. In some embodiments, the tissue / organ of interest to be harvested is selected from whole blood, brain, liver, spleen, kidney, skeletal muscle, heart, lung, and bone marrow.

[0295] In some embodiments, the cells in the tissue are monitored for the expression of the reporter sequence in the cytoplasm of the cells or in the organelle of interest. In some embodiments, the organelle of interest is the nucleus. In some embodiments, the organelle containing the AAV payload demonstrated by the expression of the reporter sequence is isolated from the tissue. In some embodiments, the organelle is the nucleus. In some embodiments, the method further comprises the step of selecting the cells in which the reporter sequence is expressed.

[0296] In some embodiments, in step c), nucleic acids from cells of a predetermined tissue or cell type are isolated. In some embodiments, the cells of the predetermined tissue are selected from cells of the central nervous system, brain, heart, lung, trachea, esophagus, muscle, bone, cartilage, bone marrow, stomach, pancreas, intestine, liver, spleen, bladder, kidney, ureter, urethra, uterus, fallopian tube, ovary, testis, prostate, eye, blood, lymph, and oral mucosa. In some embodiments, the cells of the predetermined tissue are selected from whole blood, brain, liver, spleen, kidney, skeletal muscle, heart, lung, and bone marrow. In some embodiments, the cells of a predetermined cell type are selected from multipotent hematopoietic progenitor cells (MPP), e.g., multipotent hematopoietic progenitor cells, and hematopoietic stem cells (HSC), e.g., long-term hematopoietic stem cells (LT-HSC). In some embodiments, the cells of a predetermined tissue or cell type are isolated 1-21 days, 1-14 days, 7-21 days, 2-10 days, or 2-5 days after contacting the plurality of cells with the library.

[0297] In some embodiments, nucleic acid is extracted from a cellular organelle (e.g., nucleus). In some embodiments, the extracted nucleic acid is RNA, and in some embodiments, the extracted nucleic acid is DNA. In some embodiments, the extracted RNA is subjected to reverse transcription to produce cDNA, which is then amplified. In some embodiments, primers specific to the barcoded region are used for amplification. In some embodiments, at least a portion of the sequence encoding the variable polypeptide ligand is amplified. In some embodiments, the amplified cDNA is sequenced. In some embodiments, enrichment of specific AAV variants in specific tissues / organs is observed after in vivo selection.

[0298] In some embodiments, step c) comprises isolating RNA from at least a portion of said cells, and step d) comprises the sub-steps di) reverse transcribing said isolated RNA, dii) PCR amplifying at least a portion of the sequence encoding the polypeptide ligand or a fragment thereof from the cDNA generated in step di), and diiii) sequencing at least a portion of the polypeptide ligand sequence, for example by next generation sequencing.

[0299] In some embodiments, the method further comprises step e) of determining the relative occurrence of a particular polypeptide ligand sequence in one tissue or cell type relative to the total number of polypeptide ligand sequences in said tissue or cell type.

[0300] In some embodiments, in vivo selection of AAV VP2 fusion polypeptide variants can be performed in 1, 2, 3, 4, 5 or more rounds, and in any case, pooling the AAV variants obtained from the previous round or synthesizing a subset of the enriched variants for re-selection in vitro or in vivo.

[0301] In some embodiments, the method further comprises step f) of generating a second library of nucleic acid sequences encoding an AAV VP2 fusion polypeptide from the nucleic acid molecules isolated in step c), and repeating steps b), c), d), and optionally a) and e) using said second library.

[0302] In some embodiments, after a desired number of selection rounds, the resulting variants are analyzed. In some embodiments, individual variants are used to deliver a transgene, such as a reporter gene, to a desired cell or organ in vivo. After analyzing the delivery ability of the variants, the best candidates are selected for future use.

[0303] In some embodiments, the selected candidates are subjected to in vivo or in vitro selection of variants with improved characteristics such as functional maturation by random mutagenesis and improved transduction of cells of a particular cell type or tissue.

[0304] In one aspect, provided herein are AAV VP2 fusion polypeptides made by the methods described herein.

[0305] Also provided herein are rAAV virions comprising an AAV VP2 fusion polypeptide produced by the method described herein, and pharmaceutical compositions comprising the same. The rAAV virions and pharmaceutical compositions can be used as pharmaceuticals.

[0306] AAV VP2 Capsid Polypeptides Having Improved Receptor Binding and / or Transduction Levels In one aspect, an AAV VP2 capsid polypeptide of AAV serotype AAV1 is provided herein, which comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In some embodiments, the AAV VP2 capsid polypeptide comprises the amino acid substitutions: D213A, T162R, and / or P191N, particularly all three amino acid substitutions: D213A, T162R, and P191N.

[0307] In another aspect, an AAV VP2 capsid polypeptide of an AAV serotype other than AAV1 is provided herein, which comprises at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, relative to the VP1 amino acid sequence of SEQ ID NO: 7.

[0308] In some embodiments, the AAV VP2 capsid polypeptide is of AAV serotype 8, which comprises at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 capsid polypeptide comprises the amino acid substitutions: D214A, K163R and / or P192N, especially all three amino acid substitutions D214A, K163R and P192N.

[0309] In other embodiments, the AAV VP2 capsid polypeptide is of AAV serotype 9 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 capsid polypeptide comprises the amino acid substitutions: D213A, S162R, and / or P191N, especially all three amino acid substitutions D213A, S162R, and / or P191N.

[0310] Also provided herein is an AAV VP2 fusion polypeptide comprising one of the AAV VP2 capsid polypeptides listed above and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide.

[0311] Embodiments 1. An adeno-associated virus (AAV) VP2 fusion polypeptide comprising, for example, consisting of, an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide and the polypeptide ligand has a molecular weight of up to 10 kDa.

[0312] 2. The AAV VP2 fusion polypeptide of embodiment 1, wherein the polypeptide ligand specifically binds to a cell surface molecule expressed in at least one tissue or cell type and / or wherein the AAV VP2 fusion polypeptide mediates an increase in transduction and / or an increase in tropism in at least one tissue or cell type as compared to an AAV VP2 capsid polypeptide that does not comprise the polypeptide ligand.

[0313] 3. The AAV VP2 fusion polypeptide of embodiment 1 or 2, wherein the polypeptide ligand is selected from the group consisting of a GP2 polypeptide, an Sso7d polypeptide, and an affibody.

[0314] 4. The AAV VP2 fusion polypeptide of embodiment 3, wherein the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0315] 5. The AAV VP2 fusion polypeptide of embodiment 4, wherein the polypeptide ligand is independently selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 (wherein the amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, and W), and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0316] 6. The AAV VP2 fusion polypeptide of any one of embodiments 1-5, further comprising a peptide linker between the polypeptide ligand and the AAV VP2 capsid polypeptide, in particular, the peptide linker is selected from the group consisting of a glycine-serine (GS) linker and an alanine-proline-serine (APS) linker, more specifically, the GS linker is of the formula [GGGGS]n, and the APS linker is of the formula [APS]n, wherein n is an integer in the range of 1 to 10, in particular, n is 1, 2, 3, 4, 5, or 6, more specifically, in the case of the GS linker, n is 1, 2, 3, or 4, and in the case of the APS linker, n is 2, 3, 4, or 5.

[0317] 7. The AAV VP2 fusion polypeptide of any one of embodiments 1-6, wherein the AAV VP2 capsid polypeptide is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV, or avian AAV.5, in particular, the group consisting of AAV1, AAV6, AAV8, and AAV9.

[0318] 8. The AAV VP2 capsid polypeptide comprises at least one mutation at at least one binding site for its natural receptor, in particular here, a) The AAV VP2 fusion polypeptide is of AAV serotype AAV6 and within its VP3 region comprises at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 3. In particular, the AAV VP2 fusion polypeptide comprises the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 3: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide is of AAV serotype AAV8 and within its VP3 region comprises at least one amino acid substitution selected from the group consisting of: G268E, N271Q, S387A, A592Q and A592D, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 fusion polypeptide comprises the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 4: bi) G268E and N271Q; bii) S387A; biii) G268E, N271Q and S387A; biv) A592Q; or bv) A592D; c) The AAV VP2 fusion polypeptide is of AAV serotype AAV9 and contains, within its VP3 region, at least one amino acid substitution selected from the group consisting of Q590A, W503A, N562A, and E563A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 5: ci) W503A; cii) N562A and E563A; ciii) Q590A and W503A; or civ) Q590A, W503A, N562A, and E563A; d) The AAV VP2 fusion polypeptide is of AAV serotype AAV2 and contains, within its VP3 region, the amino acid substitution R585A relative to the VP1 amino acid sequence of SEQ ID NO: 6; or e) The AAV VP2 fusion polypeptide is of AAV serotype AAV1 and contains, within its VP3 region, at least one amino acid substitution selected from the group consisting of V473D, N500E, and R514A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 7: ei) V473D and N500E; eii) R514A; or eiii) V473D, N500E, and R514A, the AAV VP2 fusion polypeptide of embodiment 7.

[0319] 9. a) The AAV VP2 fusion polypeptide is of AAV serotype AAV1 and includes at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 fusion polypeptide includes the amino acid substitutions: D214A, K163R, and / or P192N; or b) The AAV VP2 fusion polypeptide is of an AAV serotype other than AAV1 and includes at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. Specifically, bi) The AAV VP2 fusion polypeptide is of AAV serotype 8 and includes at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 fusion polypeptide includes the amino acid substitutions: D214A, K163R, and / or P192N; or bii) The AAV VP2 fusion polypeptide is of AAV serotype 9 and includes at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 fusion polypeptide includes the amino acid substitutions: D213A, S162R, and / or P191N. The AAV VP2 fusion polypeptide according to any one of Embodiments 1 to 8.

[0320] 10. The AAV VP2 fusion polypeptide according to Embodiment 9a), further including the amino acid substitutions V473D and N500E with respect to the VP1 amino acid sequence of SEQ ID NO: 7.

[0321] 11. The AAV VP2 fusion polypeptide according to any one of embodiments 1 to 10, wherein the AAV VP2 capsid polypeptide lacks the first threonine of the corresponding wild-type AAV VP2 capsid polypeptide.

[0322] 12. The AAV VP2 fusion polypeptide according to any one of embodiments 1 to 11, wherein the polypeptide ligand contains a maximum of 3 cysteine residues, particularly a maximum of 2 cysteine residues, especially a maximum of 1 cysteine residue, or most specifically does not contain a cysteine residue.

[0323] 13. An isolated nucleic acid encoding the AAV VP2 fusion polypeptide according to any one of embodiments 1 to 12.

[0324] 14. The isolated nucleic acid according to embodiment 13, further comprising a promoter.

[0325] 15. The isolated nucleic acid according to embodiment 13 or 14, further comprising two AAV inverted terminal repeat (ITR) sequences located upstream and downstream of the open reading frame (ORF) of the AAV VP2 fusion polypeptide, and optionally further comprising a reporter sequence, particularly wherein the reporter sequence encodes a fluorescent or luminescent reporter protein, and more specifically, the reporter protein is selected from the group consisting of EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase.

[0326] 16. A cell comprising the AAV VP2 fusion polypeptide according to any one of embodiments 1 to 12 or the nucleic acid according to any one of embodiments 13 to 15.

[0327] 17. The cell according to embodiment 16, wherein the cell is selected from the group consisting of insect cells (e.g., Sf9 cells) and HEK293 cells.

[0328] 18. The cell of embodiment 16, which is selected from mammalian cells, including mouse cells, non-human primate cells, or human cells, the cells being selected from the group consisting of, but not limited to, hepatocytes, brain cells, spleen cells, kidney cells, blood cells, lung cells, muscle cells, heart cells, bone marrow cells, multipotent progenitor cells (MPP) such as pluripotent hematopoietic progenitor cells, and hematopoietic stem cells (HSC) such as long-term hematopoietic stem cells (LT-HSC).

[0329] 19. A recombinant adeno-associated virus (rAAV) virion comprising any one of the AAV VP2 fusion polypeptides of embodiments 1-12.

[0330] 20. The rAAV virion of embodiment 19, wherein all AAV VP2 capsid polypeptides are fused to the polypeptide ligand.

[0331] 21. The rAAV virion of embodiment 19 or 20, further comprising a nucleic acid sequence selected from the group consisting of non-coding nucleic acids, proteins or RNA coding sequences, expression cassettes, multiple expression cassettes, sequences for homologous recombination, and genomic gene targeting cassettes.

[0332] 22. The rAAV virion of embodiment 21, wherein the expression cassette encodes any one of the AAV VP2 fusion polypeptides of embodiments 1-12, and in particular, the AAV VP2 fusion polypeptide contained in the rAAV virion is identical to the AAV VP2 fusion polypeptide encoded by the expression cassette.

[0333] 23. The rAAV virion of embodiment 21, wherein the expression cassette encodes a therapeutic nucleic acid, including, but not limited to, therapeutic RNA, mRNA, siRNA, miRNA, shRNA, and antisense oligonucleotides, therapeutic proteins, or therapeutic antibodies or antibody fragments.

[0334] 24. The virion further comprises AAV VP1 and VP3 polypeptides, in particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of the same AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV, especially from the group consisting of AAV1, AAV6, AAV8 and AAV9, the rAAV virion of any one of embodiments 19 to 23.

[0335] 25. The AAV VP1, VP2 and VP3 polypeptides comprise at least one mutation at at least one binding site for the natural receptor of the AAV VP1, VP2 and VP3 capsid polypeptides. In particular, the AAV VP1, VP2 and VP3 polypeptides comprise the same at least one mutation in the shared VP3 region of each AAV capsid polypeptide. More specifically, a) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV6, and each comprises at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 3 in the shared VP3 region. In particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise the following amino acid substitutions with respect to the VP1 amino acid sequence of SEQ ID NO: 3: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV8, and each contains, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of the following with respect to the VP1 amino acid sequence of SEQ ID NO: 4: G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each contain the following amino acid substitutions with respect to the VP1 amino acid sequence of SEQ ID NO: 4: bi) G268E and N271Q; bii) S387A; biii) G268E, N271Q, and S387A; biv) A592Q; or bv) A592D. c) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV9, and each contains, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of the following with respect to the VP1 amino acid sequence of SEQ ID NO: 5: Q590A, W503A, N562A, and E563A, or any combination thereof. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each contain the following amino acid substitutions with respect to the VP1 amino acid sequence of SEQ ID NO: 5: ci) W503A; cii) N562A and E563A; ciii) Q590A and W503A; or civ) Q590A, W503A, N562A, and E563A. d) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV2, and each contains the amino acid substitution R585A with respect to the VP1 amino acid sequence of SEQ ID NO: 6 within the shared VP3 region; or e) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV1, and each contains, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: V473D, N500E, and R514A with respect to the VP1 amino acid sequence of SEQ ID NO: 7, or any combination thereof. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides contain the following amino acid substitutions with respect to the VP1 amino acid sequence of SEQ ID NO: 7: ei) V473D and N500E; eii) R514A; or eiii) V473D, N500E, and R514A. The rAAV virion of embodiment 24.

[0336] 26. A pharmaceutical composition comprising any one of the rAAV virions of embodiments 19-21 and 23-25 and a pharmaceutically acceptable excipient.

[0337] 27. Any one of the rAAV virions of embodiments 19-21 and 23-25 or the pharmaceutical composition of embodiment 26 for use as a medicament.

[0338] 28. A method of delivering a transgene to a cell, the method comprising contacting a cell with any one of the rAAV virions of embodiments 19-21 and 23-25 or the pharmaceutical composition of embodiment 26.

[0339] 29. The method of embodiment 28, wherein the cell is present in a living subject, in particular, the subject is a mammalian subject, more specifically, a human.

[0340] 30. A method of using any one of the rAAV virions of embodiments 19-21 and 23-25 or the pharmaceutical composition of embodiment 26 in a therapeutic treatment regimen or as a vaccine.

[0341] 31. A library construct comprising a nucleic acid sequence encoding an AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, for example, consisting of them, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the polypeptide linker has a molecular weight of at most 10 kDa.

[0342] 32. The library construct of embodiment 31, wherein the polypeptide ligand comprises randomized amino acids at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions.

[0343] 33. The library construct of embodiment 31 or 32, wherein the polypeptide ligand is selected from the group consisting of a GP2 polypeptide, an Sso7d polypeptide, and an affibody.

[0344] 34. The library construct of embodiment 33, wherein the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 and an Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0345] 35. The library construct of embodiment 34, wherein the amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 of the polypeptide ligand are independently selected from D, R, H, N, A, I, Y, and W, and the Sso7d polypeptide of SEQ ID NO: 1, and an Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0346] 36. A library comprising a plurality of the library constructs of any one of embodiments 31 to 35.

[0347] 37. The library is at least 10 2 、10 3 、10 4 、10 5 、10 6 、107 , 10 8 or 10 9 A library according to embodiment 36, comprising a unique library construct of

[0348] 38. The library construct is a plasmid DNA molecule comprising the nucleic acid sequence encoding the AAV VP2 fusion polypeptide operably linked to a promoter, optionally further comprising a reporter sequence, in particular, the reporter sequence encodes a fluorescent or luminescent reporter protein, more specifically, the reporter protein is selected from the group consisting of EGFP, mCherry, sfCherry, sfCherry2, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase, a library of embodiment 36 or 37.

[0349] 39. Each library construct is present within an rAAV virion, in particular, the rAAV virion comprises the AAV VP2 fusion polypeptide of embodiment 4 or 5, and the AAV VP2 fusion polypeptide contained in a given rAAV virion is identical to the AAV VP2 fusion polypeptide encoded by the library construct present within the rAAV virion, a library of embodiment 36 or 37.

[0350] 40. Each library construct further comprises two AAV inverted terminal repeat (ITR) sequences located upstream of the promoter and downstream of the nucleic acid encoding the AAV VP2 fusion polypeptide, optionally further comprising a reporter sequence, in particular, the reporter sequence encodes a fluorescent or luminescent reporter protein, more specifically, the reporter protein is selected from the group consisting of EGFP, mCherry, sfCherry, mClover3, mRuby3, mApple, iRFP, tdTomato, mVenus, YFP, RFP, firefly luciferase, and nanoluciferase, a library of embodiment 39.

[0351] 41. The rAAV virion comprises the AAV VP2 fusion polypeptide of Embodiment 4 or 5, and further comprises AAV VP1 and VP3 polypeptides. In particular, the AAV VP2 fusion polypeptide contained in the rAAV virion and the AAV VP1 and VP3 polypeptides are of the same AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.32.33, bovine AAV or avian AAV.5, and in particular, from the group consisting of AAV1, AAV6, AAV8 and AAV9, of Library of Embodiment 39 or 40.

[0352] 42. The AAV VP1, VP2 and VP3 polypeptides contained in the rAAV virion comprise at least one mutation at at least one binding site for the natural receptor of the AAV VP1, VP2 and VP3 capsid polypeptides. In particular, the AAV VP1, VP2 and VP3 polypeptides comprise the same at least one mutation in the shared VP3 region of each AAV capsid polypeptide. More specifically, a) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV6, and each comprises at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 3 in the shared VP3 region. In particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each comprise the following amino acid substitutions with respect to the VP1 amino acid sequence of SEQ ID NO: 3: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV8, each containing, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each contain, with respect to the VP1 amino acid sequence of SEQ ID NO: 4, the following amino acid substitutions: bi) G268E and N271Q; bii) S387A; biii) G268E, N271Q, and S387A; biv) A592Q; or bv) A592D; c) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV9, each containing, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: Q590A, W503A, N562A, and E563A, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each contain, with respect to the VP1 amino acid sequence of SEQ ID NO: 5, the following: ci) W503A; cii) N562A and E563A; ciii) Q590A and W503A; or civ) Q590A, W503A, N562A, and E563A; d) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV2, each containing the amino acid substitution R585A within the shared VP3 region with respect to the VP1 amino acid sequence of SEQ ID NO: 6; or e) The AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides are of AAV serotype AAV1, each containing, within the shared VP3 region, at least one amino acid substitution selected from the group consisting of: V473D, N500E and R514A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 fusion polypeptide and said AAV VP1 and VP3 polypeptides each contain, relative to the VP1 amino acid sequence of SEQ ID NO: 7, the following amino acid substitutions: ei) V473D and N500E; eii) R514A; or eiii) V473D, N500E and R514A, a library of embodiment 41.

[0353] 43. a) The AAV VP2 fusion polypeptide contained in the rAAV virion is of AAV serotype AAV1 and contains at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N with respect to the VP1 amino acid sequence of SEQ ID NO: 7, or any combination thereof. In particular, the AAV VP2 fusion polypeptide contains the following amino acid substitutions: D213A, T162R, and / or P191N; or b) The AAV VP2 fusion polypeptide contained in the rAAV virion is of an AAV serotype other than AAV1 and contains at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, bi) The AAV VP2 fusion polypeptide contained in the rAAV virion is of AAV serotype 8 and contains at least one amino acid substitution selected from the group consisting of E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N with respect to the VP1 amino acid sequence of SEQ ID NO: 4, or any combination thereof. Among others, the AAV VP2 fusion polypeptide contains the following amino acid substitutions: D214A, K163R, and / or P192N; or bii) The AAV VP2 fusion polypeptide contained in the rAAV virion is of AAV serotype 9 and contains at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, G199R, D213A, S162R, and P191N with respect to the VP1 amino acid sequence of SEQ ID NO: 5, or any combination thereof. In particular, the AAV VP2 fusion polypeptide contains the following amino acid substitutions: D213A, S162R, and / or P191N, the library of embodiment 41.

[0354] A method for producing an AAV VP2 fusion polypeptide comprising at least one desired feature, the method comprising screening any one of the libraries of embodiments 36-43 for the encoded AAV VP2 fusion polypeptide comprising at least one desired feature.

[0355] 45. The method of embodiment 44, wherein the at least one desired feature is an increase in transduction and / or an increase in tropism in at least one tissue or cell type mediated by the AAV VP2 fusion polypeptide as compared to an AAV VP2 capsid polypeptide that does not contain the polypeptide ligand.

[0356] 46. The method of embodiment 44 or 45, comprising: b) contacting any one of the libraries of embodiments 36-43 with a plurality of cells; c) isolating nucleic acid molecules from at least a portion of the cells; and d) determining at least a portion of the sequence encoding the polypeptide ligand or a fragment thereof of at least one nucleic acid molecule isolated in step b).

[0357] 47. Prior to step b), the method of embodiment 46 further comprises step a) of providing the library of embodiment 39 by: ai) transfecting cells with a nucleic acid sequence comprising AAV rep, a nucleic acid sequence encoding an AAV VP1 capsid polypeptide, a nucleic acid sequence encoding an AAV VP1 capsid polypeptide, a nucleic acid sequence encoding an AAV VP3 capsid polypeptide, and the library of embodiment 36 (wherein each library construct further comprises two AAV inverted terminal repeat (ITR) sequences upstream and downstream of the nucleic acid sequence encoding the AAV VP2 fusion polypeptide); and aii) incubating the cells under conditions suitable for AAV virion formation (optionally, the cells are mammalian cells, particularly HEK293 cells, or insect cells, particularly Sf9 cells).

[0358] 48. The method of embodiment 47, wherein the AAV VP1 capsid polypeptide and the AAV VP3 capsid polypeptide are encoded by the same nucleic acid sequence comprising the AAV cap gene, particularly wherein the start codon of VP2 in the cap gene is mutated.

[0359] 49. The method of any one of embodiments 46 to 48, wherein the plurality of cells of step b) is present in a non-human model animal.

[0360] 50. The method of embodiment 49, wherein in step c) nucleic acids from cells of a predetermined tissue or cell type are isolated, in particular the cells of a predetermined tissue or cell type are isolated 1 to 21 days, 1 to 14 days, 7 to 21 days, 2 to 10 days, 2 to 5 days after contacting the plurality of cells with the library.

[0361] 51. The method of any one of embodiments 44 to 50, further comprising the step of selecting cells in which the reporter sequence is expressed.

[0362] 52. The method of any one of embodiments 46 to 51, wherein step c) comprises isolating RNA from at least a portion of the cells, and step d) comprises the following substeps: di) reverse transcription of the isolated RNA, dii) PCR amplification of at least a portion of the sequence encoding the polypeptide ligand or a fragment thereof from the cDNA generated in step di), and diiii) sequencing at least a portion of the polypeptide ligand sequence, in particular by next-generation sequencing.

[0363] 53. The method of any one of embodiments 46 to 52, comprising a step e) of determining the relative occurrence of a particular polypeptide ligand sequence in a tissue or cell type relative to the total number of polypeptide ligand sequences in said tissue or cell type.

[0364] Step f) of preparing a second library of nucleic acid sequences encoding an AAV VP2 fusion polypeptide from the nucleic acid molecule isolated in step c), and repeating steps b), c), d), and optionally a) and e) using the second library, the method according to any one of embodiments 46 to 53.

[0365] 55. An AAV VP2 fusion polypeptide produced by the method according to any one of embodiments 44 to 54.

[0366] 56. A method for producing an AAV virion comprising an AAV VP2 fusion polypeptide, the method comprising transducing a packaging cell with the nucleic acid of embodiment 14 or 15, wherein by said method, a packaging cell producing an AAV virion is obtained.

[0367] 57. A method for producing an AAV virion comprising an AAV VP2 fusion polypeptide, the method comprising culturing the cell of embodiment 16 or 17, wherein by said method, a packaging cell producing an AAV virion is obtained.

[0368] 58. An AAV virion produced by the method of embodiment 56 or 57.

[0369] 59. A method for manufacturing a pharmaceutical composition comprising the AAV virion of embodiment 53 or 58, the method comprising formulating the AAV virion with a pharmaceutically acceptable carrier.

[0370] 60. A kit comprising the isolated nucleic acid according to any one of embodiments 13 to 15.

[0371] 61. An AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, the polypeptide ligand being fused to the N-terminus of the AAV VP2 capsid polypeptide, the polypeptide ligand having a molecular weight of at most 10 kDa.

[0372] 62. An AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the AAV VP2 capsid polypeptide comprises one or more mutations that abolish or reduce binding to heparan sulfate proteoglycan (HSPG) and / or sialic acid (SIA).

[0373] 63. The AAV VP2 fusion polypeptide of embodiment 62, wherein the polypeptide ligand has a molecular weight of up to 10 kDa.

[0374] An AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, wherein: a) the AAV VP2 capsid polypeptide is of AAV serotype AAV1 and has at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 capsid polypeptide comprises the following amino acid substitutions: D213A, T162R, and / or P191N, or b) the AAV VP2 capsid polypeptide is of an AAV serotype other than AAV1 and has at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, relative to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, bi) the AAV VP2 capsid polypeptide is of AAV serotype 8 and has at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 capsid polypeptide comprises the following amino acid substitutions: D214A, K163R and / or P192N, or bii) the AAV VP2 capsid polypeptide is of AAV serotype 9 and has at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 capsid polypeptide comprises the following amino acid substitutions: D213A, S162R and / or P191N, the AAV VP2 fusion polypeptide.

[0375] 65. The AAV VP2 capsid polypeptide of embodiment 64a), further comprising the amino acid substitutions: V473D and N500E with respect to the VP1 amino acid sequence of SEQ ID NO: 7.

[0376] 66. a) The AAV VP2 capsid polypeptide is of AAV serotype AAV1 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, the AAV VP2 capsid polypeptide comprises the amino acid substitutions: D213A, T162R, and / or P191N, or b) The AAV VP2 capsid polypeptide is of an AAV serotype other than AAV1 and comprises at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, with respect to the VP1 amino acid sequence of SEQ ID NO: 7. In particular, bi) The AAV VP2 capsid polypeptide is of AAV serotype 8 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 4. In particular, the AAV VP2 capsid polypeptide comprises the following amino acid substitutions: D214A, K163R and / or P192N, or bii) The AAV VP2 capsid polypeptide is of AAV serotype 9 and comprises at least one amino acid substitution selected from the group consisting of: E147S, P185G, P166R, G199R, D213A, S162R and P191N, or any combination thereof, with respect to the VP1 amino acid sequence of SEQ ID NO: 5. In particular, the AAV VP2 capsid polypeptide comprises the following amino acid substitutions: D213A, S162R and / or P191N, The AAV VP2 capsid polypeptide.

[0377] An rAAV virion comprising the AAV VP2 capsid polypeptide of Embodiment 66.

[0378] The patent or application file contains at least one color drawing. A copy of this patent or patent application publication (with color drawings) will be provided by the Patent Office upon payment of the claims and the necessary fees.

Brief Description of the Drawings

[0379]

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Example

[0380] Example 1: Initial Assessment of AAV VP2 Fusion Polypeptide Decoration Levels in AAV8 Capsids Using VHH, DARPIN, and Mini-Protein Scaffolds Genetically Fused to the VP2 N-Terminus As described in Munch et al., Nat Commun 6, 6246 (2015), to evaluate the feasibility of generating decorated AAVs with VP2 N-terminal gene fusions, various scaffolds were cloned into the AAV8 VP2 expression plasmid AgC1159 at the N-terminus of the VP2 open reading frame lacking the start codon encoding threonine (VP2ΔT1). The resulting AAV VP2 fusion polypeptide expression plasmids have the AAV VP2 fusion polypeptide open reading frame operably linked to the HCMV promoter, which is depicted schematically in Figure 1.

[0381] Cloning of Scaffolds into VP2 Expression Plasmids: The scaffold sequences shown in Table 1 were ordered from GeneArt as string DNAs with 5' and 3' extensions suitable for Gibson-based cloning. In the case of VHH, the sequences were amplified by PCR using the internal expression plasmid as a template. This fragment was cloned into the HindIII-AgeI digested VP2 expression plasmid AgC1159, and correct assembly was confirmed by Sanger sequencing.

[0382]

Table 1

[0383] Production of Decorated AAVs: The generated VP2 fusion plasmids were transfected into suspension HEK293T / 17F cells together with the rep / cap VP2 KO plasmid (containing the AAV2 rep gene and AAV8 cap gene with a mutation in the VP2 open reading frame start codon, thus encoding AAV8 VP1 and VP3 but not VP2), pHelper (encoding adenoviral proteins E2A, E4, and VA RNA), and HCMVprom-EGFP-SV40pA scAAV as cargo (pNN001; encoding eGFP under the control of the HCMV promoter and flanked by AAV2 ITRs, the schematic of which is shown in Figure 2).

[0384] Briefly, 100 ml (350 ml in the case of AAV8 w.t. production) of HEK293T / 17F suspension cells were diluted to 1.5E+06 cells / ml and then transfected with PEI MAX (PEI:DNA ratio = 3:1) containing 0.8 μg of DNA mix per 1E+06 cells. The plasmid mix was composed of equimolar amounts of three or four plasmids as described in Table 2. Six days after transfection, the samples were centrifuged at 4000 rpm for 20 minutes, and the supernatant was loaded onto a POROS GoPure AAVX 1 ml 0.5cml×5cml (ThermoFisher #A36652) attached to an AKTA Pure instrument at a rate of 4 ml / min. The bound AAV particles were washed with 10 column volumes (CV) of wash buffer, pH 7.3 (50 mM Tris-Cl, 0.5 M NaCl, 0.01% Pluronic F-68) and then eluted with 5 CV of glycine elution buffer, pH 2.7 (0.1 M glycine, 0.2 M NaCl, 0.25 M L-arginine). The eluted fractions (0.25 ml) were immediately neutralized with 25 μl of 1 M Tris-Cl, pH 10. The fractions containing AAV (evaluated by UV280 signal) were pooled and dialyzed overnight against PBS, pH 7.4 + 0.001% Pluronic F-68 using a Float-A-Lyzer Dialysis Device 100 kDa (Spectrum™ #G235071).

[0385] The full particle concentration (vg / ml) was evaluated using qPCR. qPCR was performed on samples pretreated with DNAse using a KAPA Probe Fast qPCR kit and the SV40pA oligo set shown in Table 3 on a Qiagen RotorGene instrument. Plasmid pNN001 was used for standard curve generation. The qPCR results are summarized in Table 4.

[0386]

Table 2

[0387]

Table 3

[0388]

Table 4

[0389] The absence of VP2 (sample AAV8 VP2 KO) and the supply of VP2 in trans (sample AAV8 trans-VP2) had only a slight effect on the AAV titer. Genetically fusing various scaffolds to the VP2 N-terminus resulted in a 2- to 7-fold decrease in the AAV titer.

[0390] Assessment of Decoration Levels 20 μl of affinity-purified AAV preparations were analyzed by SDS-PAGE under denaturing conditions to evaluate the presence of VP2 fusions. The SDS page is shown in Figure 3. The molecular weight shift of VP2 could only be observed for the mini-protein decoration samples, while no detectable level of VP2 fusions was found for any of the other samples (VHH and Darpin fusions).

[0391] Furthermore, CE-SDS analysis (Agilent 2200 Tapestation system, P200 protein kit using denaturing conditions) was performed to semi-quantitatively evaluate the relative amounts of different VP proteins in the purified samples. As a result, a molecular weight shift of VP2 was confirmed in the AAV8-anti-HA mini-protein sample, while no detectable presence of VP2 fusions was found in the other samples. The AAV8-anti-HA mini-protein sample showed relative amounts of various VP proteins similar to those of the AAV8 w.t. sample. In the AAV8 trans-VP2 sample, a slightly increased amount of VP2 was observed. The results are summarized in Table 5.

[0392] Table 5: Molecular weights and estimated relative VP amounts of Darpin-, VHH-, and mini-protein decorated AAV8 preparations (Tapestation)

[0393]

Table 5

[0394]

Table 6

[0395] Mass Spectrometry Since the VP1 band may co-migrate with the VHH and Darpin VP2 fusion, all preparations were analyzed using LC-MS. 5 μl of untreated AAV sample was injected into a UPLC-I class connected to a Synapt G2S (WATERS) quadrupole time-of-flight mass spectrometer. The sample was denatured on the column and separated by chromatography using a Waters Bioresolve RP 1×50 mm column. The column was held at 80 °C using a gradient consisting of an organic mobile phase from 30 to 38% over 40 minutes at a flow rate of 0.1 ml / min. The aqueous mobile phase consisted of 0.1% formic acid in water, and the organic mobile phase consisted of 0.1% formic acid in acetonitrile.

[0396] The predicted masses of VP1 and VP3 could be confirmed in all test samples, and the presence of VP2 w.t. or the VP2 fusion was confirmed only in the AV8 w.t., AAV8 trans-VP2, and AAV8-anti-HA miniprotein samples, which was consistent with the results of SDS and CE-SDS.

[0397] AAV8 Sandwich ELISA: To further evaluate the decoration efficiency and correct exposure of the VP2 fusion scaffold, a more sensitive sandwich ELISA assay was performed. 96-well plates were coated with hEGFR and hTIGIT extracellular domain (ECD) recombinant proteins at 5 μg / ml. After overnight incubation and blocking with 3% BSA / TBST buffer, various amounts of AAV vg were added to the wells and incubated at RT for 2 hours. After the washing step (3×), the wells were incubated with anti-AAV8 monoclonal mIgG2 (Origene AM32478SU-N) at room temperature (RT) for 1 hour. The wells were washed again (3×), incubated with anti-mouse IgG-AP conjugate (Sigma-Aldrich A5153), washed further (3×), and signals were generated using Attophos substrate (Roche 00000011681982001). The results are shown in Figure 4.

[0398] In contrast to MS and CE-SDS, Darpin and VHH were detected on the surface of assembled AAV by AAV8 sandwich Elisa. A small fraction of the generated AAV was suspected to contain VP2 fused to either of these two scaffolds, which were detectable only by sandwich Elisa and not by MS and CE-SDS.

[0399] Example 2: Assessment of AAV8 Decoration Levels Using Small Scaffolds Genetically Fused to the VP2 N-Terminus As observed in Example 1, AAV8 - anti-HA miniprotein seems to be the only AAV decoration preparation with a VP2 decoration level close to wild type. To evaluate whether this phenomenon is due to the small size of the scaffold, various anti-EGFR binding domains listed in Table 6 were cloned into the AAV8 VP2 expression plasmid AgC1159.

[0400]

Table 7

[0401] Production of Decorated AAVs Cloning, expression, purification, and evaluation of virus concentration of various decorated AAV8 particle preparations were carried out as described in Example 1, except for the following exceptions: · The amount of transfection was 300 ml for each sample · Cells and supernatant were harvested 4 days after transfection · Final concentration: 0.5% Triton, 2 mM MgCl 2 and 12.5 U / ml of benzonase was added to the bulk harvest, lysed at 37 °C for 2 hours, then 0.5 M NaCl was added, and the samples were centrifuged at 3500 g for 30 minutes. The lysate was filtered and clarified, and then loaded onto POROS AAVx 1 ml resin.

[0402] As a control, new preparations of AAV8 - anti - EGFR Darpin and AAV8 - anti - HA mini - protein were made in parallel in the same manner. qPCR described in Example 1 was used to evaluate the full - particle concentration (vg / ml). The results are summarized in Table 7c.

[0403] Assessment of Decoration Levels and Total AAV Yields: To evaluate the VP distribution and total VP yield in the purified samples, CE - SDS analysis (using the Agilent 2100 Bioanalyzer system and Protein 230 kit under denaturing conditions) was performed.

[0404] Calculation of total AAV particles / ml (vp / ml) and VP ratio · Divide the ng / μl (= μg / ml) value by the measured MW and multiply by 1E+06 (conversion from μg to g) to calculate the mol / ml value · Multiply the mol / ml value by 6.022E+23 (Avogadro's number) to calculate the number of molecules / ml · VP distribution: Calculate based on the value of the number of molecules / ml assuming that one AAV consists of a total of 60 VPs · AAV particles / ml (vp / ml): Divide the sum of VP1 + VP2 + VP3 molecules / ml by 60

[0405] Summarize the results in Table 7 and Figure 5.

[0406] Table 7: Bioanalyzer Results of AAV8 Decorated Samples

[0407]

Table 8

[0408]

Table 9

[0409]

Table 10

[0410] The successful incorporation of the VP2 fusion polypeptide into the AAV capsid seems to correlate with the molecular weight of the scaffold fused to the VP2 polypeptide. When using scaffolds less than 10 kDa, a satisfactory decoration level (>60% of the w.t. VP2 level) was obtained. Beyond this threshold, a decrease in the incorporation of the VP2 fusion was observed, particularly prominent in the case of the 18 kDa darpin. For all the test anti-EGFR scaffolds used in this experiment, a slight decrease in yield was observed (<2-fold compared to w.t.).

[0411] In Vitro Infectivity of Decorated AAV8 Against EGFR Overexpressing Cell Lines: To evaluate the effect of anti-EGFR modified AAV8 preps at various modification levels, an infectivity assay was performed in HKB11 cells that stably overexpress hEGFR (HKB11_EGFR). On the day of infection, cells were resuspended in DMEM to 5E+05 cells / ml and seeded at 50 μl / well in a 96-well plate (2.5E+04 cells / well). AAV8 decorated preparations were diluted in DMEM medium and applied to the seeded cells at various concentrations ranging from 2E+06 to 1.56E+04 moi based on total vp (final volume 100 μl). Four days after introduction, cells were detached and FACS analysis was performed to calculate the percentage of EGFP-positive cells. Full particles (vg) were back-calculated. Figure 6 represents the number of full particles versus the percentage of infected (EGFP+) cells.

[0412] High levels of decoration of the anti-EGFR VP2 fusion polypeptide showed an increase in uptake into HKB11_EGFR cells (Sso7d > Affibody > GP2). In contrast, the anti-EGFR DARPin and fibronectin scaffold fusions did not show an increase in infectivity compared to the non-decorated AAV8 w.t. control, probably due to the low levels of decoration obtained with these two scaffolds.

[0413] In Vitro Infectivity of Decorated AAV8 Against Glioblastoma Multiforme (GBM) Neurospheres: GBM neurosphere cultures were obtained from the Henri Ford Health System and suspended and grown in DMEM / HamF12 (Gibco #11039-021) supplemented with Gibco N2 supplement (100×; #17202-048), BSA (Sigma #A4919; final concentration 0.5 mg / ml), Gentamicin (Gibco #15710-064, final concentration 0.025 mg / ml), Antibiotic-Antimycotic solution (100×) (Invitrogen #15240-062), EGF (Peprotech #100-15, final concentration 20 ng / ml), and FGF (Peprotech #100-18B, final concentration 20 ng / ml) at 37°C and 5% CO2. To verify the infectivity results obtained with the EGFR overexpressing HKB11 cell line, the GBM neurosphere cell line (HF-2927) with enhanced EGFR expression was infected with anti-EGFR Sso7d (E.18.4.5)-AAV8. Without disrupting the neurospheres, GBM cells were seeded into 96-well plates at approximately 40,000 cells / well (the cell number was determined by collecting 200 μl of the cell suspension, disrupting the spheres, and estimating the cell concentration). The seeded neurospheres were infected with anti-GFR Sso7d decorated AAV8 at 1E+06 and 1E+05 moi and compared to non-decorated AAV8 and w.t. preparations of AAV9 containing the same EGFP cargo. Three days after transduction, the EGFP signal was monitored using a ZOE Fluorescent Cell Imager (Bio-Rad). Representative photos of the neurospheres are shown in Figure 7.

[0414] AAV particles presenting the anti-EGFR Sso7d VP2 fusion polypeptide showed significantly increased cell uptake in the GBM neurosphere cultures of the EGFR overexpressing cell line HF-2927 compared to non-decorated AAV8 and AAV9 preparations.

[0415] Example 3: Assessment of AAV8 Decoration Levels Using Additional GP2 and Affibody Bindings Genetically Fused to the VP2 N-Terminus Summary: Additional exemplary GP2 and affibody arrays were obtained from the literature and cloned into the AAV8 VP2 expression plasmid as described in Example 1.

[0416]

Table 11

[0417] Production of Decorated AAV8: A DNA mix containing the aforementioned pHelper, (DL144)CAG-EGFP-NOX single-stranded cargo, (AgC1157)pAAV rep2_cap8 VP2 KO, and AAV8 VP2 fusion expression plasmid in equimolar ratios was prepared and transfected into 200 ml of HEK293 suspension cells at a concentration of 2E+06 vc / ml using the FectoVIR-AAV transfection reagent (Polyplus #101000022); DNA:FectoVIR = 1 μg:1 μl (1.1 μg of total DNA / 1E+06 cells). After transfection, glucose with a final concentration of 2 g / l was added to the transfection mix, and the mixture was incubated at 37 °C, 110 rpm, and 6% CO2. Two additional transfections were also performed with the combination of non-decorated AAV1Sil1 and its corresponding barcoded cargo plasmid (A02). Three days after transfection, benzonase with a final concentration of 0.1 U / μl and 20 ml of lysis buffer (500 mM HEPES, 10% Tween 20, 20 mM MgCl 2) was added to the transfection reactants, and the mixture was incubated with stirring at 37 °C for 3 hours. After this incubation step, 24 ml of sucrose salt solution (5 M NaCl, 7% sucrose) was added to the lysate and incubated for an additional 20 minutes. The sample was centrifuged at 3500 g for 15 minutes, the supernatant was filtered through a 0.22 μM filter, and after adding EDTA to a final concentration of 5 mM, it was loaded onto an AAVX prepacked column (Pre-packed Column), 0.5 × 5 cm, 1 ml (ThermoFisher #A36652) attached to an AKTA Pure instrument at 1 ml / min. The bound AAV particles were washed with 10 CV of wash buffer (pH 7.3, 50 mM Tris-Cl, 0.5 M NaCl, 0.01% Pluronic F-68) and eluted with 5 CV of glycine elution buffer, pH 2.7 (0.1 M glycine, 0.2 M NaCl, 0.25 M L-arginine). The elution fractions (0.25 ml) were immediately neutralized with 25 μl of 1 M Tris-Cl, pH 10. The fractions containing AAV (evaluated by UV280 signal) were pooled and dialyzed overnight against PBS, pH 7.4 + 0.001% Pluronic F-68 using a Float-A-Lyzer Dialysis Device 100KD (Spectrum™ #G235071), and then passed through a 0.22 μm filter unit.

[0418] Determination of Titers and Decoration Levels: The titer of the AAV vector was measured using the QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad) with the SV40 pA-specific oligo set described in Table 3. The purified preparation was pretreated as follows: 5 μl of AAV was incubated with 1 unit of DNase and 1×DNase buffer (Merck #04716728001) in a total volume of 20 μl at 37 °C for 30 minutes. 1 μl of 20 mg / ml proteinase K (Life technologies #EO0491) was added to the mixture, incubated at 55 °C for 1 hour, and then subjected to an inactivation step at 95 °C for 15 minutes. Serial 1 / 10 dilutions of the treated preparation were prepared with 1×PCR buffer II (Life technologies # N8080010), 2.5 mM MgCl 2 , 0.05% Pluronic F-68 (Sigma Aldrich #K4894), 2 ng / μl sheared salmon sperm DNA (Invitrogen #15632011) and used as templates for PCR amplification.

[0419] Droplets were generated as follows: 5.5 μl of the pretreated AAV dilution, 900 nM each of the forward and reverse SV40 pA primers, 125 nM of the SV40 pA probe, and 11 μl of the 2×ddPCR Supermix for probes (BioRad #1863024) were mixed to a final volume of 22 μl. Technical replicates were performed for each sample. Each 20 μl of the ddPCR assay mixture was loaded into a disposable droplet generation cartridge (Bio-Rad). Next, 70 μL of the droplet generation oil for probes (BioRad #1863005) was loaded into each of the eight oil wells. Subsequently, the cartridge was placed inside the QX200 droplet generation device (Bio-Rad). When droplet generation was complete, 40 μl of volume was transferred to a 96-well PCR plate using a multi-channel pipette.

[0420] The plate was heat-sealed with foil and then placed in a C1000 Touch Thermal Cycler (Bio-Rad). The thermal cycling conditions were as follows: 10 minutes at 95°C, then 40 cycles of 30 seconds at 95°C and 1 minute at 60°C, followed by incubation at 98°C for 10 minutes and then holding at 4°C for an indefinite period. The FAM fluorescence signal that labels the AAV genomic DNA sequence in each droplet was counted using a QX200 digital droplet reader and analyzed with QuantaSoft analysis software (Bio-Rad). To calculate the titer of AAV, the number of droplets was converted by multiplying by the respective total dilution factor and further multiplying by a factor of 2 to account for the possibility of re-annealing of single-stranded genomes.

[0421] To evaluate the VP di...

Claims

1. An adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, An AAV VP2 fusion polypeptide, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the polypeptide is the Sso7d polypeptide of SEQ ID NO: 1 (wherein amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, and W), and the Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto.

2. The AAV VP2 fusion polypeptide according to claim 1, further comprising a peptide linker between the polypeptide ligand and the AAV VP2 capsid polypeptide, wherein the peptide linker is selected from the group consisting of glycine-serine (GS) linkers and alanine-proline-serine (APS) linkers, more specifically, the GS linker is of the formula [GGGGGS]n, and the APS linker is of the formula [APS]n, where n is an integer in the range of 1 to 10, more specifically, n is 1, 2, 3, 4, 5 or 6, more specifically, in the case of the GS linker, n is 1, 2, 3 or 4, and in the case of the APS linker, n is 2, 3, 4 or 5.

3. The AAV VP2 fusion polypeptide according to claim 1, wherein the AAV VP2 capsid polypeptide is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh. 8, AAVrh. 10, AAVrh. 32, 33, bovine AAV, or avian AAV. 5, particularly from the group consisting of AAV1, AAV6, AAV8, and AAV9.

4. The AAV VP2 capsid polypeptide comprises at least one mutation in at least one binding site to its native receptor, and in particular, here, a) The AAV VP2 fusion polypeptide is of AAV serotype AAV6, and its VP3 region contains at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 3, and in particular, the AAV The VP2 fusion polypeptide comprises the following amino acid substitutions to the VP1 amino acid sequence of SEQ ID NO: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide is of AAV serotype AAV8 and contains within its VP3 region at least one amino acid substitution selected from the group consisting of G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4, in particular the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 4: bi) G268E and N271Q; bii) S387A; biiii) G268E, N271Q, and S387A; biv) A592Q; or bv) A592D; c) The AAV VP2 fusion polypeptide is of AAV serotype AAV9 and contains within its VP3 region at least one amino acid substitution selected from the group consisting of Q590A, W503A, N562A and E563A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5, in particular the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: ci) W503A; cii) N562A and E563A; ciii) Q590A and W503A; or civ) Q590A, W503A, N562A and E563A; d) The AAV VP2 fusion polypeptide is of AAV serotype AAV2 and contains the amino acid substitution R585A for the VP1 amino acid sequence of SEQ ID NO: 6 within its VP3 region; or e) The AAV VP2 fusion polypeptide is of AAV serotype AAV1, and its VP3 region contains at least one amino acid substitution selected from the group consisting of V473D, N500E, and R514A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7, and in particular, the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: ei) V473D and N500E; eii) R514A; or eiii) V473D, N500E, and R514A. The AAV VP2 fusion polypeptide according to claim 3.

5. a) The AAV VP2 fusion polypeptide is of AAV serotype AAV1 and comprises at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7, in particular, the AAV VP2 fusion polypeptide comprises the amino acid substitutions: D213A, T162R and / or P191N; or b) The AAV VP2 fusion polypeptide is of an AAV serotype other than AAV1, and includes at least one amino acid substitution corresponding to at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, relative to the VP1 amino acid sequence of SEQ ID NO:

7. The AAV VP2 fusion polypeptide according to claim 1.

6. The AAV VP2 fusion polypeptide according to claim 5, further comprising amino acid substitutions V473D and N500E for the VP1 amino acid sequence of SEQ ID NO:

7.

7. An isolated nucleic acid encoding an AAV VP2 fusion polypeptide according to any one of claims 1 to 6.

8. A cell comprising the AAV VP2 fusion polypeptide described in any one of claims 1 to 6, wherein the cell is selected from the group consisting of insect cells (e.g., Sf9 cells) and HEK293 cells.

9. A cell comprising nucleic acid as described in Claim 7, wherein the cell is selected from the group consisting of insect cells (e.g., Sf9 cells) and HEK293 cells.

10. A recombinant AAV (rAAV) virion comprising the AAV VP2 fusion polypeptide described in any one of claims 1 to 6.

11. A recombinant AAV (rAAV) virion comprising a nucleic acid sequence encoding the AAV VP2 fusion polypeptide according to any one of claims 1 to 6, wherein the AAV VP2 fusion polypeptide contained in the AAV virion is the same as the AAV VP2 fusion polypeptide encoded by the nucleic acid sequence, wherein the AAV VP2 fusion polypeptide is the same as the AAV VP2 fusion polypeptide according to any one of claims 1 to 6.

12. The rAAV virion according to claim 10, further comprising a nucleic acid sequence encoding a therapeutic nucleic acid, a therapeutic protein, or a therapeutic antibody or antibody fragment.

13. The rAAV virion according to claim 10, wherein the virion further comprises AAV VP1 and VP3 polypeptides, and in particular the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of the same AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh. 8, AAVrh. 10, AAVrh. 32, 33, bovine AAV, or avian AAV. 5, and in particular the group consisting of AAV1, AAV6, AAV8, and AAV9.

14. The AAV VP1, VP2, and VP3 polypeptides each contain at least one mutation in at least one binding site to the native receptor of the AAV VP1, VP2, and VP3 capsid polypeptide, and more specifically, the AAV VP1, VP2, and VP3 polypeptides each contain the same at least one mutation in the shared VP3 region of the AAV capsid polypeptide, and more specifically, a) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV6, and each contains within the shared VP3 region at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 3, in particular the AAV VP2 fusion polypeptide and the AAV Each of the VP1 and VP3 polypeptides comprises the following amino acid substitutions to the VP1 amino acid sequence of SEQ ID NO: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV8, and each contains within the shared VP3 region at least one amino acid substitution selected from the group consisting of: G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4, in particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each contain the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 4: bi) G268E and N271Q; bii) S387A; biiii) G268E, N271Q, and S387A; biv) A592Q; or bv) A592D. c) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV9, and each contains within the shared VP3 region at least one amino acid substitution selected from the group consisting of: Q590A, W503A, N562A and E563A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 5, in particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides each contain the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: ci) W503A; cii) N562A and E563A; ciii) Q590A and W503A; or civ) Q590A, W503A, N562A and E563A. d) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV2, and each contains the amino acid substitution R585A for the VP1 amino acid sequence of SEQ ID NO: 6 within the shared VP3 region; or e) The AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides are of AAV serotype AAV1, and each contains within the shared VP3 region at least one amino acid substitution selected from the group consisting of V473D, N500E, and R514A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7, in particular, the AAV VP2 fusion polypeptide and the AAV VP1 and VP3 polypeptides contain the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: ei) V473D and N500E; eii) R514A; or eiii) V473D, N500E, and R514A. The rAAV billion according to claim 13.

15. A pharmaceutical composition comprising the rAAV virion described in claim 10 and a pharmaceutically acceptable excipient.

16. The rAAV virion according to claim 10, for use as a pharmaceutical product.

17. The pharmaceutical composition according to claim 15 for use as a pharmaceutical product.

18. A library construct comprising a nucleic acid sequence encoding an AAV VP2 fusion polypeptide, The AAV VP2 fusion polypeptide, for example, consists of an AAV VP2 capsid polypeptide and a polypeptide ligand. A library construct wherein the polypeptide ligand is fused to the N-terminus of an AAV VP2 capsid polypeptide, and the polypeptide ligand is the Sso7d polypeptide of SEQ ID NO: 1 (wherein amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, and W), and the Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto.

19. A library comprising a plurality of library constructs according to claim 18, wherein the library comprises at least 10 ２ , 10 ３ , 10 ４ , 10 ５ , 10 ６ , 10 ７ , 10 ８ , or 10 ９ A library containing unique library constructs.

20. The library according to claim 19, wherein each library construct resides within an rAAV vilion, and the AAV VP2 fusion polypeptide contained in a given rAAV vilion is identical to the AAV VP2 fusion polypeptide encoded by the library construct residing within the given rAAV vilion.

21. A method for producing an AAV VP2 fusion polypeptide having at least one desired feature, comprising: b) contacting a library according to claim 19 or 20 with a plurality of cells; c) isolating nucleic acid molecules from at least a portion of the cells; and d) determining at least a portion of the sequence encoding a polypeptide ligand or a fragment thereof of the at least one nucleic acid molecule isolated in step c), wherein the plurality of cells in step b) are present in a non-human model animal.

22. An AAV VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, The polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide, and the polypeptide ligand has a maximum molecular weight of 10 kDa. AAV VP2 fusion polypeptide wherein the AAV VP2 capsid polypeptide contains one or more mutations that eliminate or reduce binding to heparan sulfate proteoglycan (HSPG) and / or sialic acid (SIA).

23. a) The AAV VP2 capsid polypeptide is of AAV serotype AAV1 and contains at least one amino acid substitution selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 7, in particular, the AAV VP2 capsid polypeptide contains the following amino acid substitutions: D213A, T162R, and / or P191N, or b) The AAV VP2 capsid polypeptide is of an AAV serotype other than AAV1, and contains at least one amino acid substitution corresponding to at least one of the amino acid substitutions selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, relative to the VP1 amino acid sequence of SEQ ID NO:

7. AAV VP2 fusion polypeptide.

24. An rAAV virion comprising the AAV VP2 capsid polypeptide as described in claim 23.

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