AAV variants for cardiac delivery

Abstract

Variant adeno-associated virus (AAV) capsids that confer increased transduction in the heart are provided. Also provided are methods of delivering a transgene to the heart and methods of treating cardiac disorders by contacting cardiac cells with a recombinant AAV (rAAV) comprising a variant AAV capsid and a heterologous nucleic acid encoding a transgene.

Description

AAV Variants for Cardiac DeliveryCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 516,031, filed July 27, 2023, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable XML file, entitled “090400-5025 WO Sequence Listing” created on July 17, 2024, with a file size of about 25,300 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Adeno-associated virus (AAV) vectors have been used in clinical trials for cardiac disorders. However, a major impediment is specificity for cardiac tissue, as naturally occurring AAV capsids such as AAV9 exhibit significant transduction of off-target tissues, particularly the liver.SUMMARY OF THE INVENTION

[0004] Described herein are variant AAV capsid sequences that specifically target cardiac tissue, with reduced transduction of liver tissue, with a favorable cardiac to liver distribution of vector genomes to non-human primates relative to parental capsid AAV9. The variant AAV capsids described herein comprise a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) in the GH loop of the capsid.

[0005] In some aspects of the disclosure, recombinant AAV (rAAV) virions are provided, these rAAV virions comprising a variant capsid protein as described herein, wherein the rAAV virions exhibit decreased transduction of liver cells relative to the transduction of liver cells by an AAV virion comprising a native AAV capsid protein of serotype 9. In some embodiments, infectivity of a cardiac cell by an rAAV virion comprising a variant capsid protein as hereindescribed is not significantly reduced relative to infectivity of the cardiac cell by an AAV virion comprising a native AAV capsid protein of serotype 9. Accordingly, in preferred embodiments, the heart-to-liver ratio is higher compared to that of a subject that received an AAV9 vector (native AAV9 vectors exhibit a roughly 1 : 1 heartdiver ratio). Preferably, the heart-to-liver ratio of an rAAV virion comprising a variant capsid protein as described herein is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, or at least 10-fold higher than the heart to liver ratio of AAV9.

[0006] In some embodiments, the rAAV virion comprises a heterologous nucleic acid. In some such embodiments, the heterologous nucleic acid encodes an RNA that encodes a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, e g. the heterologous nucleic acid sequence is an RNA interference agent, a guide RNA for a nuclease, etc.

[0007] Also provided herein are pharmaceutical compositions comprising the subject infectious rAAV virions and a pharmaceutically acceptable carrier.

[0008] Also provided is an isolated nucleic acid comprising a sequence encoding a variant AAV capsid protein as described herein and a host cell comprising the isolated nucleic acid. In yet other embodiments, the isolated nucleic acid and / or isolated host cell comprises the rAAV.

[0009] In some embodiments, a method for delivering a heterologous nucleic acid to a cardiac cell is provided, the method comprising contacting the cardiac cell with a recombinant adeno- associated virus (AAV) comprising a variant AAV capsid protein as described herein. In some aspects, the method is performed in vitro. In other aspects, the method is performed in vivo. In other aspects, the method is performed ex vivo.

[0010] In other embodiments, a method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to a cardiac cell in a mammalian subject is provided, the method comprising administering to the mammal an effective amount of a recombinant AAV (rAAV) comprising a variant AAV capsid protein as herein described or administering a pharmaceutical composition comprising the recombinant AAV and a pharmaceutically acceptable excipient.

[0011] In other embodiments, a method for treating and / or preventing a cardiac disorder in a mammal is provided, the method comprising administering to the mammal a therapeutically effective amount of a recombinant AAV comprising a variant AAV capsid protein as herein described or administering a pharmaceutical composition comprising the recombinant AAV and a pharmaceutically acceptable excipient. In related aspects, a recombinant AAV comprising a variant AAV capsid protein as herein described for use in treating a cardiac disorder is provided. In other related aspects, the use of a recombinant AAV comprising a variant AAV capsid protein as herein described in the manufacture of a medicament for the treatment of cardiac disorder is provided.

[0012] In some aspects, the recombinant AAV comprises (i) a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0013] In some embodiments, the heterologous nucleic acid comprises a nucleotide sequence that encodes a gene product, in which case contacting a cardiac cell with a variant AAV vector as described herein provides a method for delivering a gene product to a cardiac cell. In some aspects, the gene product is an antisense RNA, a microRNA (miRNA), a short hairpin RNA (shRNA) or a small interfering RNA (siRNA) or a precursor or mimic thereof. In some embodiments, the gene product is a long non-coding RNA. In some embodiments, the gene product is a short non-coding RNA. In some embodiments, the gene product is an antisense RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function.

[0014] In some preferred embodiments, the heterologous nucleic acid comprises a nucleotide sequence that encodes a therapeutic gene product capable of ameliorating a cardiac disorder.

[0015] In preferred aspects, the nucleotide sequence encoding the gene is operably linked to at least one transcription control sequence, preferably a transcription control sequence that is heterologous to the nucleic acid. In some aspects, the transcription control sequence is a cell- ortissue-specific promoter that results in cell-specific expression of the nucleic acid e g. in cardiac cells. In other aspects, the transcription control sequence is a constitutive promoter that results in similar expression level of the nucleic acid in many cell types (e.g. a CAG, CBA (chicken beta actin) or CMV promoter). In some preferred embodiments, the transcription control sequence comprises a CAG promoter comprising (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of chicken beta-actin gene and (iii) the splice acceptor of the rabbit beta-globin gene as described in Miyazaki et al., Gene 79(2):269-77 (1989).

[0016] In some preferred aspects, the recombinant AAV (or pharmaceutical composition comprising same) is administered to the mammal by systemic (e.g., intravascular) administration.DESCRIPTION OF THE DRAWINGS

[0017] Figs. 1A-B. Fig. 1A illustrates transduction of human cardiomyocytes at the specified multiplicities of infection (MOIs) by native AAV9 carrying an EGFP reporter gene operably linked to a CAG promoter compared to an rAAV comprising a capsid protein of SEQ ID NO: 11 carrying an EGFP reporter gene operably linked to a CAG promoter. Fig. IB is a graph quantifying the level of transduction by detection of EGFP and cardiac troponin T (cTnT).

[0018] Fig. 2 illustrates transduction of primary hepatocytes at the specified multiplicities of infection (MOIs) by native AAV9 carrying an EGFP reporter gene operably linked to a CAG promoter compared to an rAAV comprising a capsid protein of SEQ ID NO: 11 carrying an EGFP reporter gene operably linked to a CAG promoter.DETAILED DESCRIPTION OF THE INVENTION

[0019] Definitions

[0020] The term "isolated" designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is notisolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered "isolated."

[0021] As used herein, a "coding region" or "coding sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then that a single vector can contain just a single coding region, or comprise two or more coding regions.

[0022] As used herein, the term "regulatory region" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0023] As used herein, the term “nucleic acid” is interchangeable with “polynucleotide” or “nucleic acid molecule” and a polymer of nucleotides is intended.

[0024] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are "operably associated" if induction of promoterfunction results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

[0025] "Transcriptional control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissuespecific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0026] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0027] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene productsdescribed herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0028] “Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissuespecific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0029] The term “interfering RNA” or “RNAi” refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., duplex RNA such as siRNA or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or completeidentity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.

[0030] Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15- 25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21- 22, or 21-23 nucleotides in length, and the double- stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini. Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0031] Preferably, siRNA are chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coll RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Nall. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1 ):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31 :981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).

[0032] With regards to “CRISPR / Cas agents”, the term “CRISPR” encompasses Clustered regularly interspaced short palindromic repeats / CRISPR-associated (Cas) systems that evolved to provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas protein naturally contains DNA endonuclease or RNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA ("sgRNA")). Thus, the Cas protein associates with a DNA-targeting or RNA-targeting RNA (which term encompasses both the two- molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas or Cas-like protein and guides the protein to a target nucleic acid sequence.

[0033] Detailed information regarding CRISPR agents can be found, for example in (a) Jinek et. al., Science. 2012 Aug 17;337(6096):816-21 : "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. 2013 Feb 28; 152(5): 1173-83: "Repurposing CRISPR as an RNA- guided platform for sequence- specific control of gene expression", and (c) US patent application number 13 / 842,859 and PCT application number PCT / US13 / 32589; all of which are hereby incorporated by reference in their entirety. Thus, the term "CRISPR agent" as used herein encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and / or synthetic sequences, that can be used in the Cas- based system (e.g., a Cas9; any component of a DNA-targeting RNA or RNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).

[0034] By “Zinc-finger nucleases” (ZFNs) it is meant artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on theuse of ZFNs, see, for example: Asuri et al., Mol. Ther. 2012 Feb; 20(2):329-38; Bibikova et al. Science. 2003 May 2;300(5620):764; Wood et al. Science. 2011 Jul 15;333(6040):307; Ochiai et al. Genes Cells. 2010 Aug; 15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 Oct; 40(10):759-65; Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc Natl Acad Sci U S A. 2011 Apr 26; 108(17): 7052-7; Goldberg et al, Cell. 2010 Mar 5;140(5):678-91; Geurts et al, Science. 2009 Jul 24;325(5939):433; Flisikowska et al, PLoS One. 201 l;6(6):e21045. doi: 10.1371 / journal. pone.0021045. Epub 2011 Jun 13; Hauschild et al, Proc Natl Acad Sci U S A. 2011 Jul 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 Nov; 21(1 1): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term "ZFN agent" encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0035] The terminology “Transcription activator-like effector nuclease” or “TALEN” agents refers to Transcription activator-like effector nucleases (TALENs). TALENs are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENs can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul 7;29(8): 731 - 4; Wood et al. Science. 201 1 Jul 15;333(6040):307; Tesson et al. Nat Biotechnol. 201 1 Aug 5;29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug 5;29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term "TALEN agent" encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[0036] The term "plasmid" refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular doublestranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable ofintroducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0037] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. Sequence similarity or sequence homology can be determined in a number of different manners. Commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homologies may be generated by any of a number of computer programs known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0038] % homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0039] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequencecomparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology or identity.

[0040] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible-reflecting higher relatedness between the two compared sequences — may achieve a higher score than one with many gaps. “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0041] Calculation of maximum % homology therefore first requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed. — Chapter 18), FASTA (Altschul et al., 1990 J Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).

[0042] Although the final % homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix — the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0043] Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0044] The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986) “The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.

[0045] Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0046] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or P-alanine residues. A further form of variation, which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

[0047] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment, " as used herein, covers any treatment of a disease in a mammal, particularly in ahuman, and includes: (a) preventing the disease (and / or symptoms caused by the disease) from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., arresting its development; and (c) relieving the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., ameliorating the disease and / or one or more symptoms of the disease. For example, the subject compositions and methods may be directed towards the treatment of muscle disease. Nonlimiting methods for assessing muscle diseases and the treatment thereof include measuring therapeutic protein production (e.g. muscle biopsy followed by immunohistochemistry or serum sampling followed by ELISA or enzyme activity assays), measuring symptoms of heart failure (e.g. the New York Heart Association Functional Classification or the Minnesota Living With Heart Failure Questionnaire), functional cardiac status (e.g. the 6-minute walk test or peak maximum oxygen consumption), bio marker analysis (e.g. N-terminal prohormone brain natriuretic peptide), left ventricular function / remodeling (e.g. left ventricular ejection fraction or left ventricular end-systolic volume), muscle strength (e.g. the Medical Research Council Scales Clinical Investigation of Duchenne Dystrophy, hand-held dynamometry, or maximum weight lift), muscle function (e.g. the Vignos Scale, Timed Function Tests, the Hammersmith Motor Ability Score, timed rise from floor, walk tests, Motor Function Measure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test, or Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders), muscle disease symptoms (e.g. the Neuromuscular Symptoms Score or Clinical Global Impressions), mitochondrial function (e g.31P magnetic resonance spectroscopy), questionnaire-based assessments of quality of life, patient-reported outcomes, or daily activities.

[0048] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, humans; non- human primates, including simians; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0049] The term "effective amount" as used herein is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a compound (e.g., aninfectious rAAV virion) is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of (and / or symptoms associated with) a particular disease state (e.g., a disorder associated with cardiac dysfunction). Accordingly, an effective amount of an infectious rAAV virion is an amount of the infectious rAAV virion that is able to effectively deliver a heterologous nucleic acid to a target cell (or target cells) of the individual. Effective amounts may be determined preclinically by, e.g., detecting in the cell or tissue the gene product (RNA, protein) that is encoded by the heterologous nucleic acid sequence using techniques that are well understood in the art, e.g. RT-PCR, western blotting, ELISA, fluorescence or other reporter readouts, and the like. Effective amounts may be determined clinically by, e.g. detecting a change in the onset or progression of disease using methods known in the art, e.g. 6-minute walk test, left ventricular ejection fraction, hand-held dynamometry, Vignos Scale and the like as described herein and as known in the art.

[0050] As used herein the term “systemic administration” is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation).

[0051] As used herein the term “intravascular administration” refers to the administration of an agent, e.g., a composition comprising a rAAV, into the vasculature of a subject, including the venous and arterial circulatory systems of the subject. Methods for intravascular administration are well known in the art and include for example, use of a hypodermic needle, peripheral cannula, central venous line, etc.

[0052] The term "tropism" as used herein refers to the preferential targeting by a virus (e.g., an AAV) of cells of a particular host species or of particular cell types within a host species. For example, a virus that can infect cells of the heart, lung, liver, and muscle has a broader (i.e., increased) tropism relative to a virus that can infect only lung and muscle cells. Tropism can also include the dependence of a virus on particular types of cell surface molecules of the host. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependencies can be tested using various cellslines deficient in particular classes of molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the virus's relative preferences. For example, a first virus may be able to infect all cell types but is much more successful in infecting those cells with surface glycosaminoglycans. A second virus can be considered to have a similar (or identical) tropism as the first virus if the second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), even if the absolute transduction efficiencies are not similar. For example, the second virus might be more efficient than the first virus at infecting every given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism as the first virus. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein is not altered relative to a naturally occurring virion (e.g., AAV9). In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein is reduced relative to a naturally occurring virion (e.g., AAV9).

[0053] The terminology “directed evolution” refers to a capsid engineering methodology, in vitro and / or in vivo, which emulates natural evolution through iterative rounds of genetic diversification and selection processes, thereby accumulating beneficial mutations that progressively improve the function of a biomolecule. Directed evolution often involves an in vivo method referred to as “biopanning” for selection of AAV variants from a library which variants possess a more efficient level of infectivity of a cell or tissue type of interest

[0054] AAV

[0055] AAV vectors comprising a variant capsid protein as herein described provide for a minimum transduction of off-target tissue, in particular lung. Compared to AAV9, the standard vector presently used for cardiac gene therapy, the observed liver de-targeting of AAV vectors herein described resulted in a ~44-fold lower liver expression, representing a substantial improvement of the off-target profile. This substantial improvement in the off-targeting profile may allow for higher dosing, increased transgene expression and therapeutic efficacy while minimizing adverse off-targeting effects in patients suffering from cardiac disorders such as heart disease.

[0056] Those skilled in the art will appreciate that an AAV vector comprising a transgene and lacking virus proteins needed for viral replication (e.g., cap and rep), cannot replicate since such proteins are necessary for virus replication and packaging. Helper viruses include, typically, adenovirus or herpes simplex virus. Alternatively, as discussed below, the helper functions (El a, Elb, E2a, E4, and VA RNA) can be provided to a packaging cell including by transfecting the cell with one or more nucleic acids encoding the various helper elements and / or the cell can comprise the nucleic acid encoding the helper protein. For instance, HEK 293 were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to El and E3 (see, e.g., Graham et al., 1977, J. Gen. Virol. 36:59-72). Thus, those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them.

[0057] In some aspects, the variant AAV capsid protein comprises an insertion of from about 7 to about 20 amino acids (a “heterologous peptide” or “peptide insertion”) in the GH-loop of a parental AAV capsid protein, wherein the peptide comprises the sequence NLTRVSG (SEQ ID NO:1). Preferably, the variant capsid protein, when present in an AAV virion, exhibits reduced liver tropism relative to that of the corresponding parental capsid protein and / or that of a native AAV virion such as AAV9.

[0058] By the “GH loop,” or loop IV, of the AAV capsid protein it is meant the solvent- accessible portion referred to in the art as the GH loop, or loop IV, of AAV capsid protein. For the GH loop / loop IV of AAV capsid, see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. '19.5QM,' and Shen et al. (2007) Mol. Ther. 15: 1955. Thus, for example, the insertion site can be within about amino acids 570-611 of AAV2 VP1.

[0059] In certain embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any wild-type AAV serotype or AAV variant. For example, the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and580. The insertion site can be between amino acids 590 and 591 , amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600. In some preferred embodiments, the insertion site is located between amino acids 587 and 591 of VP1 of AAV9. For example, the insertion site can be between amino acids587 and 588 of AAV9, between amino acids 588 and 589 of AAV9, between amino acids 589 and 590 of AAV9, between amino acids 590 and 591 of AAV9, or between amino acids 591 and 592 of AAV9. In a particularly preferred embodiment, the insertion site is between amino acids588 and 589 or is between amino acids 589 and 590.

[0060] In some embodiments, a peptide insertion disclosed herein has a length of 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids. In some embodiments, the peptide insertion has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and / or carboxyl terminus of the amino acid sequence NLTRVSG (SEQ ID NO: 1). Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, a peptide insertion comprises 1 to 3 spacer amino acids at the N-terminus and 1 to 3 spacer amino acids at the C-terminus. In other embodiments, a peptide insertion comprises 1 spacer amino acid at the N-terminus and 1 spacer amino acid at the C-terminus. In some embodiments, a peptide insertion comprises a serine and / or glycine at the N-terminus and an alanine at the C- terminus. In other embodiments, a peptide insertion comprises an alanine and serine at the N- terminus and an alanine, leucine and serine at the C-terminus. In some preferred embodiments, the peptide insertion comprises or consists of an amino acid sequence selected from NLTRVSG (SEQ ID NO: 1), ASNLTRVSGALS (SEQ ID NO:2) and GNLTRVSGA (SEQ ID NO:3).

[0061] In some aspects, the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and further comprises one or more amino acid substitutions relative to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in an AAV virion, exhibits reduced liver tropism relative to that of the corresponding parental capsid protein and / or that of a native AAV virion such as AAV9.

[0062] In some preferred embodiments, the variant AAV capsid protein comprises a peptide insertion comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1).

[0063] In some preferred embodiments, the variant capsid protein comprises the following amino acid sequence or comprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 98%, or at least 99% identical to an amino acid sequence selected from the following amino acid sequences, wherein the amino acid sequence in each case comprises an insertion peptide comprising SEQ ID NO: 1 (in each case, the core insertion is in bold underline and the spacer amino acids are in italics):MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHOSGNLTRVSG^AOAOAOTGWVONOGILPGMVWQDRDVYLQGPIWAKIPHTDGN FHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:4; Spacer G - A; insertion between amino acids 586 and 587 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSD YQLPYVLGSAHEGCLPPFP AD VFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHOS^ NLTRVSG^Z AOAOAOTGWVQNQGILPGMVWODRDVYLQGPIWAKIPHTD GNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:5; Spacer AS - ALS; insertion between amino acids 586 and 587 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQ S AG'NLTRVSGd Q AQ AQTGW VQNQGILPGM VWQDRD VYLQGPIWAKIPHTDGN FHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID N0:6; Spacer G - A; insertion between amino acids 587 and 588 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAAS'NLTRVSGdLS'QAOAOTGWVONQGILPGWWQDRDVYLOGPIWAKIPHTD GNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:7; Spacer AS - ALS; insertion between amino acids 587 and 588 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAQGNLTRVSG^AQAQTGWVQNQGILPGMVWODRD VYLQGPIWAKIPHTDGN FHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID N0:8; Spacer G - A; insertion between amino acids 588 and 589 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHOSAQ^5NLTRVSG^Z5AOAQTGWVQNQGILPGMVWQDRDVYLOGPIWAKIPHTD GNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID N0:9; Spacer AS - ALS; insertion between amino acids 588 and 589 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQ S AQ AGNLTRVSGd Q AQTGW VQNQGILPGM VWQDRD V YLQGPIWAKIPHTDGN FHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 10;Spacer G - A; insertion between amino acids 589 and 590 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHOSAOA^ANLTRVSG^LAOAOTGWVONOGILPGMVWODRDVYLQGPIWAKIPHTD GNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO:11; Spacer AS - ALS; insertion between amino acids 589 and 590 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHOSAOAOGNLTRVSG^AOTGWVONOGILPGMVWODRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQ KENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 12; Spacer G A; insertion between amino acids 590 and 591 relative to AAV9)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHOSAOAONSNLTRVSG.4 / ..SAOTGWVONOGILPGMVWODRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ IDNO: 13; Spacer AS - ALS; insertion between amino acids 590 and 591 relative to AAV9)

[0064] The amino acid sequence of native AAV9 VP1 capsid is provided below:MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE IQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 14)

[0065] In some embodiments, a variant capsid as herein described comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:4-13 and comprises a NLTRVSG (SEQ ID NO: 1), ASNLTRVSGALS(SEQ ID N0:2) or GNLTRVSGA (SEQ ID NO:3) peptide insertion between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, between amino acids 589 and 590 or between amino acids 590 and 591 relative to AAV9. In preferred embodiments, a variant capsid as herein described comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:8-l 1 and 13 and comprises a NLTRVSG (SEQ ID NO:1), ASNLTRVSGALS (SEQ ID NO:2) or GNLTRVSGA (SEQ ID NO:3) peptide insertion between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, between amino acids 589 and 590 or between amino acids 590 and 591 relative to AAV9 In a particularly preferred embodiment, the variant capsid comprises a peptide insertion of SEQ ID NO:2 and comprises an amino acid sequence at least 85% identical to SEQ ID NO: 11.

[0066] A full complement of AAV Cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins may be provided.

[0067] The invention includes packaging cells, which are encompassed by "host cells," which may be cultured to produce packaged viral vectors of the invention. The packaging cells of the invention generally include cells with heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). Each of these component functions is discussed in the ensuing sections.

[0068] Initially, the vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013 / 063379). A preferred method is described in Grieger, etal. 2015, Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allow for rapid and scalable rAAV production. Using the triple transfection method (e.g., WO 96 / 40240), the suspension HEK293 cell line generates greater than 105vector genome containing particles (vg) / cell or greater than 1014vg / L of cell culture when harvested 48 hours post-transfection.More specifically, triple transfection refers to the fact that the packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as Ela, Elb, E2a, E4, and VA RNA, and another plasmid encodes the transgene and its various control elements (e.g., modified GLA gene and CAG promoter).

[0069] To achieve the desired yields, a number of variables are optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that resulted in high purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids. This user- friendly process can be completed within one week, results in high full to empty particle ratios (>90% full particles), provides post-purification yields (>lxl0A13 vg / L) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. This scalable manufacturing technology has been utilized to manufacture GMP Phase I clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), Giant Axonal Neuropathy (scAAV9) and Retinitis Pigmentosa (AAV2), which have been administered into patients. In addition, a minimum of a 5-fold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.

[0070] The packaging cells include viral vector functions, along with packaging and vector functions. The viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced by the modified GLA sequence and its associated expression control sequences. The viral vector functions include sufficient expression control sequences to result in replication of the viral vector for packaging. Typically, the viral vector includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and replaced by the transgene and its associated expression control sequences. The transgene is typically flanked by two AAV TRs, in place of the deleted viral rep and cap ORFs. Appropriate expression control sequences are included, such as a tissue-specific promoter and other regulatory sequences suitable for use in facilitating tissue-specific expression of thetransgene in the target cell. The transgene is typically a nucleic acid sequence that can be expressed to produce a therapeutic polypeptide or a marker polypeptide.

[0071] The terminal repeats (TR(s)) (resolvable and non-resolvable) selected for use in the viral vectors are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5 and 6 being preferred. Resolvable AAV TRs need not have a wild-type TR sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the TR mediates the desired functions, e.g., virus packaging, integration, and / or provirus rescue, and the like. The TRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double-D sequence" as described in U.S. Pat. No. 5,478,745 to Samulski et al., the entire disclosure of which is incorporated in its entirety herein by reference. Typically, but not necessarily, the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.

[0072] The packaging functions include capsid components. The capsid components are preferably from a parvoviral capsid, such as an AAV capsid or a chimeric AAV capsid function. Examples of suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependovirus. For example, the capsid components may be selected from AAV capsids, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAVrhlO, AAVrh74, RHM4-1, RHM1 5-1 , RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAV Hu 26, AAV1 .1 , AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, AAV2i8, AAV2G9, AAV2i8G9, AAV2-TT, AAV2- TT-S312N, AAV3B-S312N, and AAV-LK03, and other novel capsids as yet unidentified or from non-human primate sources. Capsid components may include components from two or more AAV capsids.

[0073] The packaged viral vector generally includes a gene sequence (e.g., a therapeutic gene to treat a cardiac disorder) and expression control sequences flanked by TR elements, referred to herein as the "transgene" or "transgene expression cassette," sufficient to result in packaging of the vector DNA and subsequent expression of the gene sequence in the transduced cell. The viral vector functions may, for example, be supplied to the cell as a component of a plasmid or an amplicon. The viral vector functions may exist extrachromosomally within the cell line and / or may be integrated into the cell's chromosomal DNA.

[0074] Any method of introducing the nucleotide sequence carrying the viral vector functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments wherein the viral vector functions are provided by transfection using a virus vector; standard methods for producing viral infection may be used.

[0075] The packaging functions include genes for viral vector replication and packaging. Thus, for example, the packaging functions may include, as needed, functions necessary for viral gene expression, viral vector replication, rescue of the viral vector from the integrated state, viral gene expression, and packaging of the viral vector into a viral particle. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, a Baculovirus, or HSV helper construct. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA. Examples include genes encoding AAV Rep and Cap proteins.

[0076] The helper functions include helper virus elements needed for establishing active infection of the packaging cell, which is required to initiate packaging of the viral vector. Examples include functions derived from adenovirus, baculovirus and / or herpes virus sufficient to result in packaging of the viral vector. For example, adenovirus helper functions will typically include adenovirus components Ela, Elb, E2a, E4, and VA RNA. The packaging functions may be supplied by infection of the packaging cell with the required virus. The packaging functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon. See, e.g., pXR helper plasmids as described in Rabinowitz et al., 2002, J. Virol. 76:791, and pDG plasmids described in Grimm et al., 1998, Human Gene Therapy 9:2745-2760. The packaging functions may exist extrachromosomally within the packaging cell, but are preferably integrated into the cell's chromosomal DNA (e.g., El or E3 in HEK 293 cells).

[0077] Any suitable helper virus functions may be employed. For example, where the packaging cells are insect cells, baculovirus may serve as a helper virus. Herpes virus may also be used as a helper virus in AAV packaging methods. Hybrid herpes viruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes.

[0078] Any method of introducing the nucleotide sequence carrying the helper functions into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal. In embodiments wherein the helper functions are provided by transfection using a virus vector or infection using a helper virus; standard methods for producing viral infection may be used.

[0079] Any suitable permissive or packaging cell known in the art may be employed in the production of the packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of packaging cells in the practice of the invention include, for example, human cell lines, such as VERO, WI38, MRC5, A549, HEK 293 cells (which express functional adenoviral El under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, and HT1080 cell lines. In one aspect, the packaging cell is capable of growing in suspension culture, more preferably, the cell is capable of growing in serum-free culture. In one embodiment, the packaging cell is a HEK293 that grows in suspension in serum free medium. In another embodiment, the packaging cell is the HEK293 cell described in U.S. Pat. No. 9,441,206 and deposited as ATCC No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002 / 46359. In another aspect, the packaging cell is cultured in the form of a cell stack (e.g. 10-layer cell stack seeded with HEK293 cells).

[0080] Cell lines for use as packaging cells include insect cell lines. Any insect cell which allows for replication of AAV and which can be maintained in culture can be used in accordance with the present invention. Examples include Spodoptera frugiperda, such as the Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references are incorporated herein for their teachings concerning use of insect cells for expression of heterologous polypeptides, methods of introducing nucleic acids into such cells, and methods of maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, N J (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., 1989, J. Virol. 63:3822-3828; Kajigaya et al., 1991, Proc. Nat'l. Acad. Sci. USA 88: 4646-4650; Ruffing et al., 1992, J. Virol. 66:6922-6930; Kimbauer et al.,1996, Virol. 219:37-44; Zhao et al., 2000, Virol. 272:382-393; and Samulski et al., U.S. Pat. No. 6,204,059.

[0081] Virus capsids according to the invention can be produced using any method known in the art, e.g., by expression from a baculovirus (Brown et al., (1994) Virology 198:477-488). As a further alternative, the virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and rAAV template as described, for example, by Urabe et al., 2002, Human Gene Therapy 13: 1935-1943.

[0082] In another aspect, the present invention provides for a method of rAAV production in insect cells wherein a baculovirus packaging system or vectors may be constructed to carry the AAV Rep and Cap coding region by engineering these genes into the polyhedrin coding region of a baculovirus vector and producing viral recombinants by transfection into a host cell. Notably when using Baculovirus production for AAV, preferably the AAV DNA vector product is a self- complementary AAV like molecule without using mutation to the AAV ITR. This appears to be a by-product of inefficient AAV rep nicking in insect cells which results in a self-complementary DNA molecule by virtue of lack of functional Rep enzyme activity. The host cell is a baculovirus-infected cell or has introduced therein additional nucleic acid encoding baculovirus helper functions or includes these baculovirus helper functions therein. These baculovirus viruses can express the AAV components and subsequently facilitate the production of the capsids.

[0083] During production, the packaging cells generally include one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together or separately to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line or integrated into the cell's chromosomes.

[0084] The cells may be supplied with any one or more of the stated functions already incorporated, e.g., a cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, a cell line with one or more packaging functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA, or a cell line with helper functions incorporated extrachromosomally or integrated into the cell's chromosomal DNA.

[0085] The rAAV vector may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods described in Clark et al., 1999, Human Gene Therapy 10(6): 1031 - 1039; Schenpp and Clark, 2002, Methods Mol. Med. 69:427-443; U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0086] The AAV variants disclosed herein were generated through the use of in vivo directed evolution involving the use of primate cardiac screens following intravenous administration. In some embodiments, the variant capsid proteins disclosed herein, when present in an AAV virion, confer decreased transduction of a liver cell compared to the transduction of the liver cell by a native AAV9 virion, when administered systemically. In some such embodiments, the AAV variant virion or variant rAAV exhibits at least 1.5-fold, 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 50-fold, at least 100-fold, at least 500-fold or more than 500-fold, decreased transduction of a liver cell, compared to the transduction of the liver cell by a wild-type AAV virion of serotype 9. In some embodiments, the AAV variant virion preferentially transduces a cardiac cell, e.g., a subject rAAV virion infects a cardiac cell with 2-fold, 5- fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-muscle cell (e.g., a liver cell). In some embodiments, the transduced cardiac cell is cardiomyocyte, cardiac fibroblast, smooth muscle cell or a cardiac progenitor cell. Transduction of a cardiac cell, e.g. increased efficiency of transduction, broader transduction, more preferential transduction, etc. may be readily assessed in vitro or in vivo by any number of methods in the art for measuring gene expression. For example, the AAV may be packaged with a genome comprising an expression cassette comprising a reporter gene, e.g. a fluorescent protein, under the control of a ubiquitous or tissue specific promoter, and the extent of transduction assessed by detecting the fluorescent protein by, e.g., fluorescence microscopy. As another example, the AAV may be packaged with a genome comprising a barcoded nucleic acid sequence, and the extent of transduction assessed by detecting the nucleic acid sequence by, e.g., PCR. As another example, the AAV may be packaged with a genome comprising an expression cassette comprising a therapeutic gene for the treatment of a cardiac disease, and the extent of transduction assessed by detecting the treatment of the cardiac disease in an afflicted patient that was administered the AAV.

[0087] Heterologous nucleic acid

[0088] In preferred embodiments, an AAV comprising a variant capsid protein comprising a peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) in the GH-loop of the capsid further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0089] In some embodiments, the gene product is a reporter gene, non-limiting examples of which include fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme that produces a detectable product, such as luciferase (e.g., from Gaussia, Renilla, or Photinus), P-galactosidase, P-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene, or proteins that can be directly detected. Virtually any protein can be directly detected by using, for example, specific antibodies to the protein.

[0090] In other embodiments, the gene product is a therapeutic protein suitable for the treatment of one or more cardiac disorders. In some aspects, the gene product is a protein selected from the group of cardiac repair factors, calcium regulators, and pro-angiogenic factors. Nonlimiting examples of such proteins include a cardiac repair factor, such as the human myeloid derived growth factor (huMydgf), a calcium regulator selected from the group consisting of the calcium regulator proteins sarcoplasmic / endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), small ubiquitin-related modifier 1 (SUMO1), S100 calcium-binding protein Al (S100A1), and the pro-angiogenic vascular endothelial growth factor (VEGF). In some aspects, the protein supplements a corresponding defective gene in the subject to be treated, including, without limitation, a protein selected from beta-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3), troponin I type 3 (TNNI3), cardiac troponin T (TNNT2), tropomyosin alpha-1 chain (TPM1), myosin light chain (MYL3), plakophilin-2 (PKP2), 5 ’-AMP-activated protein kinase subunit gamma-2 (PRKAG2), titin (TTN), myosin, light chain 2 (MYL2), actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT- like subfamily, member 1 (KCNQ1), myocyte enhancer factor 2c (MEF2C), cardiac LIM protein (CSRP3), a cardiac sarcomeric protein, beta-myosin heavy chain, myosin ventricular essential light chain 1, cardiac troponin I, caveolin 3 (CAV3), galactosidase alpha (GLA), lysosomal-associated membrane protein 2 (LAMP2), mitochondrial transfer RNA glycine (MTTG), mitochondrialtransfer RNA isoleucine (MTTI), mitochondrial transfer RNA lysine (MTTK), mitochondrial transfer RNA glutamine (MTTQ), myosin light chain 3 (MYL3), troponin C (TNNC1), transthyretin (TTR), stromal-derived factor- 1 (SDF-1), adenylate cyclase-6 (AC6), beta-ARKct (beta-adrenergic receptor kinase C terminus), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor, hypoxia inducible growth factor, thymosin beta 4 (TMSB4X), nitric oxide synthase-3 (NOS3), unocartin 3 (UCN3), melusin, apoplipoprotein-E (ApoE), and superoxide dismutase (SOD).

[0091] Alternatively, the heterologous nucleic acid may encode a functional RNA, e.g., an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. Nos. 6,013,487; 6,083,702), interfering RNAs (RNAi) including small interfering RNAs (siRNA) that mediate gene silencing (see, Sharp et al., (2000) Science 287:2431), microRNA, or other non-translated “functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), and the like. Exemplary untranslated RNAs include RNAi or antisense RNA against the multiple drug resistance (MDR) gene product (e.g., to treat tumors and / or for administration to the heart to prevent damage by chemotherapy), RNAi or antisense RNA against myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against VEGF or a tumor immunogen including but not limited to those tumor immunogens specifically described herein (to treat tumors), RNAi or antisense oligonucleotides targeting mutated dystrophins (Duchenne or Becker muscular dystrophy), RNAi or antisense RNA against the hepatitis B surface antigen gene (to prevent and / or treat hepatitis B infection), RNAi or antisense RNA against the HIV tat and / or rev genes (to prevent and / or treat HIV) and / or RNAi or antisense RNA against any other immunogen from a pathogen (to protect a subject from the pathogen) or a defective gene product (to prevent or treat disease). RNAi or antisense RNA against the targets described above or any other target can also be employed as a research reagent. In some aspects, the interfering RNA knocks-down expression of an mRNA encoding a protein associated with a cardiac disorder. In some preferred embodiments, the gene product is an interfering RNA, more preferably an miRNA.

[0092] In some preferred aspects, the gene product encodes a microRNA (miRNA). The microRNA preferably is one that is involved in the regulation of the Mitogen-activated proteinkinase (MAPK) pathway, the MYOD pathway, the FOXO3 pathway, or the ERK-MAPK pathway. In a particularly preferred embodiment, the microRNA encoded by the transgene of the viral vector for use in the method of the invention is selected from the group consisting of miR- 378, miR669a, miR-21 miR212, and miR132.

[0093] In other aspects, a gene product is a site-specific endonuclease that provides for sitespecific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a muscle disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a muscle structural protein and / or provides for normal muscle function, a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.

[0094] In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., a subject rAAV virion can be used to deliver both a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional muscle protein (e.g., functional lamin A / C, functional fibrillin, functional collagen type VI, etc.). In some embodiments, a rAAV virion disclosed herein comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional muscle protein. Functional muscle proteins include, e.g., lamin A / C, fibrillin 1, COL6A1, COL6A2, COL6A3, and the like.

[0095] Site-specific endonucleases that are suitable for use include, e.g., meganucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and Clustered regularly interspaced short palindromic repeats / CRISPR-associated (Cas), where such sitespecific endonucleases are non-naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as “INDELs”) then throw theprotein out of frame and effectively knock out the gene. See, e.g., U.S. Patent Publication No. 2011 / 0301073, the contents of which are incorporated herein by reference.

[0096] In preferred embodiments, the nucleotide sequence encoding the gene product is operably linked to a promoter. Suitable promoters may be selectively or constitutively active in heart tissue, and in particular in cardiomyocytes.

[0097] In some preferred embodiments, the promoter is a ubiquitous promoter such as a CMV promoter, a CAG promoter, a PGK-1 promoter, or an EF-la promoter. In one aspect, the ubiquitous promoter is a CAG promoter comprising (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of chicken beta-actin gene and (iii) the splice acceptor of the rabbit beta-globin gene as described in Miyazaki et al., Gene 79(2):269-77 (1989). In a particularly preferred embodiment, the promoter functionally linked to the transgene is the CAG promoter. In another preferred embodiment, the promoter functionally linked to the transgene is the CMV promoter.

[0098] In yet another preferred embodiment, the promoter operably linked to the transgene is a tissue- or cell-specific promoter. In some preferred embodiments, the promoter is a cardiomyocyte-specific promoter. Examples of cardiac-specific or preferred promoters include, without limitation, cardiac muscle a-actin promoter (Minty and Kedes (1986) Mol. Cell. Biol. 6:2125— 2136); cardiac troponin T (TNNT2) promoter (Farza et al. (1998) J. Mol. Cell Cardiol. 30(6): 1247-53); cardiac troponin C (TNNC1) promoter (Scheier et al. (1990) Journal of Biological Chemistry 34(5 ):21247-21253 ); cardiac myosin-binding protein C promoter (Lin et al. (2013) PLoS ONE 8(7): e69671); cardiac troponin I (TNNI3) promoter (Bhavsar et al. (1996) Genomics 35(1 ): 1 1-23), myosin light chain 2A promoter; myosin light chain 2v (MLC-2V) promoter; muscle creatine kinase (MCK) promoter; cytomegalovirus enhancer + myosin light chain 2 promoter (CMV-MLC2, or CMV-MLC1.5, CMV-MLC260); and phosphoglycerate kinase (PGK) promoter,

[0099] In some aspects, the gene product is also under the control of one or more enhancers, preferably one or more cardiac-specific enhancers such as human calsequestrin 1 (CASQ1) enhancer.

[0100] Methods of Delivering Nucleic Acids to Cardiac Tissue

[0101] In several embodiments, methods for delivering heterologous nucleotide sequences to cardiac tissue or the heart while minimizing delivery to peripheral organs such as the liver are provided utilizing an rAAV comprising a variant capsid protein as herein described. The rAAV may be employed to deliver a nucleotide sequence of interest to a cardiac cell in vitro, e.g., to produce a polypeptide or nucleic acid in vitro for ex vivo gene therapy. The rAAV are additionally useful in a method of delivering a nucleotide sequence to a subject in need thereof, e g., to express a therapeutic or immunogenic polypeptide or nucleic acid. In this manner, the polypeptide or nucleic acid may thus be produced in vivo in the subject. The subject may be in need of the polypeptide or nucleic acid because the subject has a deficiency of the polypeptide, or because the production of the polypeptide or nucleic acid in the subject may impart some therapeutic effect, as a method of treatment or otherwise.

[0102] In particular embodiments, the rAAV are useful to express a polypeptide or nucleic acid that provides a beneficial effect to the heart. The ability to target rAAV to cardiac cell while minimizing delivery to the liver may be particularly useful to treat diseases or disorders involving cardiac dysfunction.

[0103] Thus, in one aspect, a method of delivering a nucleic acid of interest to a cardiac cell is provided, the method comprising contacting the cardiac cell with an AAV particle as herein described.

[0104] In another aspect, a method of delivering a nucleic acid of interest to a cardiac cell in a mammalian subject is provided, the method comprising administering an effective amount of the AAV particle as herein described or a pharmaceutical formulation comprising same to a mammalian subject. In related aspects, delivery of the nucleic acid to the liver is minimized.

[0105] Representative cardiac cells to which the nucleic acid can be delivered include, cardiomyocytes, cardiomyoblasts, cardiac fibroblasts, smooth muscle cells and cardiac progenitor cells. In some preferred embodiments, the nucleic acid is delivered to cardiomyocytes.

[0106] The rAAV virion can be delivered to cardiac tissue or the heart by intravascular (intravenous or intra-arterial) administration, direct cardiac injection (into the left atrium, right atrium, right ventricle and / or septum), antegrade or retrograde infusion into the coronary artery (via the left anterior descending or left circumflex coronary arteries), recirculation, intraperi cardial injection, trans endocardial injection, or by any other convenient mode or route of administration that will result in the delivery of the rAAV virion to cardiac tissue or the heart. In other embodiments, the rAAV virion is administered by intramyocardial injection. In a preferred embodiment, a subject rAAV virion is delivered to cardiac tissue or the heart by systemic intravenous administration.

[0107] Treatment methods

[0108] In certain embodiments, a method is provided for the treatment of a cardiac disorder in a subject in need of such treatment by administering to the subject, the method comprising administering to the subject a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO:1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter, or administering to the mammal a pharmaceutical composition comprising said rAAV and a pharmaceutically acceptable carrier. In some aspects, the variant AAV capsid protein is at least 90%, at least 95%, at least 98%, at least 99% or is 100% identical to the amino acid sequence set forth as any one of SEQ ID NOs:4-13. In preferred embodiments, the variant AAV capsid protein is at least 90%, at least 95%, at least 98%, at least 99% or is 100% identical to the amino acid sequence set forth as any one of SEQ ID NOs:8-l 1 and 13.

[0109] In some embodiments, the cardiac disorder is heart disease. In some aspects, the method of treating or preventing a heart disease comprises transduction of primate (e.g., human) cardiomyocytes.

[0110] In some aspects, the heart disease is a cardiomyopathy, preferably selected from hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic rightventricular cardiomyopathy (ARVC), restrictive cardiomyopathy (RCM) and left ventricular non-compaction cardiomyopathy (LVNC). The cardiomyopathy may be primary cardiomyopathy, preferably inherited cardiomyopathy, cardiomyopathy caused by spontaneous mutations, and acquired cardiomyopathy, preferably ischemic cardiomyopathy caused by atherosclerotic or other coronary artery diseases, cardiomyopathy caused by infection or intoxication of the myocardium.

[0111] In some aspects, the heart disease is heart failure or chronic heart failure, optionally selected from heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mid-range ejection fraction (HFmrEF). In other aspects, the heart disease is congestive heart failure, coronary heart disease.

[0112] In some aspects, the heart disease is selected from angina pectoris, cardiac fibrosis, myocarditis, endocarditis, congenital cardiovascular defects, a valvular heart disease, myocardial infarction, atrial arrhythmia, ventricular arrhythmia, diastolic heart failure, systolic heart failure, left ventricular non-compaction, ventricular septal defect, ischemia and cardiac hypertrophy.

[0113] In other aspects, the cardiac disorder is selected from Barth syndrome, Frei dri ch’s Ataxia, Catecholaminergic polymorphic ventricular tachycardia (CPVT), Marfan syndrome, Noonan syndrome, Brugada syndrome, Long QT Syndrome, Myotonic Dystrophy 1 , Limb Girdle Dystrophy Type IB, Naxos disease, Loeys-Dietz syndrome, X-Linked Emery-Dreifuss muscular dystrophy (EDMD) and SV Aortic Stenosis.

[0114] In a preferred embodiment, the use of an rAAV in the treatment of a cardiac disorder or for the manufacture of a medicament for the treatment of cardiac disorder is provided, wherein the rAAV comprises (i) a capsid comprising a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO:1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter. In particularly preferred embodiments, the rAAV comprises a capsid comprising a capsid protein having the amino acid sequence of SEQID NO: 1 1 . In some aspects, the rAAV is administered by intravascular (e g. intravenous) injection.

[0115] In some preferred embodiments, a pharmaceutical composition for use in treating a cardiac disorder is provided, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an infectious rAAV comprising (i) a capsid comprising a variant capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, preferably wherein the peptide insertion is 7 to 20 amino acids long comprising the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter. In related aspects, the infectious rAAV comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs:4-13 or an amino acid sequence at least 85% identical thereto and (ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat (b) a CAG or CMV promoter (c) a nucleic acid encoding a gene product (d) a polyadenylation sequence and (e) an AAV2 terminal repeat. In preferred embodiments, the infectious rAAV comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs:8-l 1 and 13 or an amino acid sequence at least 85% identical thereto and (ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat (b) a CAG or CMV promoter (c) a nucleic acid encoding a gene product (d) a polyadenylation sequence and (e) an AAV2 terminal repeat

[0116] The subject to be treated is a human or non-human primate. Non-human primates include, but not limited to, monkeys, squirrel monkeys, owl monkeys, baboons, chimpanzees, marmosets, gorillas, apes, lemurs, macaques and gibbons. Further, as used herein a human primate comprises a human.

[0117] In some embodiments, the pharmaceutical composition comprises 1 x 1012to 1 x 1015vector particles / kg or vector genomes / kg, 1 x 1012to 1 x 1015vector particles or vector genomes, or about 1 x 1012, about 2 x 1012, 3x 1012, about 4 x 1012, about 5 x 1012, about 6 x 1012, about 7 x 1012, about 8 x 1012, about 9 x 1012, about 1 x 1013, about 2 x 1013, about 3 x 1013, about 4 x 1013, about 5 x 1013, about 6 x 1013, about 7 x 1013, about 8 x 1013, about 9 x 1013, about 1 x 1014,about 2 x T O14, about 3 x IO14, about 4 x 1014, about 5 x TO14, about 6 x 1014, about 7 x TO14, about 8 x 1014, about 9 x 1014or about 1 x 1015vector particles / kg or vector genomes / kg. In some aspects, the pharmaceutical composition comprises about 3 x 1012, about 1 x 1013or about 5 x 1013vector particles / kg or vector genomes / kg. In some preferred embodiments, the pharmaceutical composition is administered to a human with a disorder associated with cardiac dysfunction via intravenous injection.EXAMPLES

[0118] The following examples illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention in any way. While this invention has been described in relation to its preferred embodiments, various modifications thereof will be apparent to one skilled in the art from reading this application.Example 1

[0119] Materials and Methods

[0120] AAV Library Preparation

[0121] From a diverse Round 1 IV (intravenous) primate selection, the top 125 AAV peptide insertions detected in heart but minimally in liver were identified from a Round 1 non-human primate (NHP) selection. The peptide insertion variants identified were re-synthesized by Twist Bioscience (South San Francisco, CA) and re-diversified to include positional insertion refinement and two linker options. Synthesized Cap fragment libraries were cloned into replication-competent backbone plasmids.

[0122] The composition balance and identity of the resulting plasmid library was confirmed by full-length sequencing on a PacBio Sequel lie NGS instrument. For AAV manufacturing, the plasmid library was transfected into a 293T-derived cell line using FuGene. Following transfection, cells and supernatant were collected and lysed, endonuclease treated, clarified via sterile filtration, and frozen. Thawed harvest material was clarified and loaded onto relevant affinity resins, eluted at low pH and immediately neutralized. The manufactured AAV library was sequenced on a PacBio Sequel lie NGS and tittered using digital droplet PCR. In addition,aggregation by dynamic light scattering and endotoxin composition by the limbus amebocyte lysate (LAL) method was determined.

[0123] AAV Library Screening in Non-Human Primates[00124J Two male cynomolgus macaque (Macaca fascicularis) were dosed intravenously with the AAV library using a ~3.5 mL injection volume of the library at 4.3xl012vector genomes (vg) / mL. Three weeks after dosing, the heart, liver, skeletal muscle, brain, kidney, dorsal root ganglia, spinal cord, spleen and select lymph nodes were harvested from each animal. The heart was sectioned into right and left atrium, right and left ventricles, and septum, while the liver was divided into four sagittal lobes and frozen on dry ice.

[0125] Frozen tissue was thawed, homogenized and subject to genomic DNA isolation followed by full length AAV variant Cap gene PCR amplification using serotype agnostic primers. The resulting capsid PCR product was sequenced on a PacBio Sequel lie NGS instrument and output sequences were aligned to WT AAV reference sequences to identify the specific unnatural variations in each sequence. Data was normalized to total read count per sample.

[0126] Cell Culture

[0127] iPSC-CM

[0128] iPSC-CMs were obtained from FujiFilm and cultured according to manufacturer’s instructions. Briefly, cells were seeded into 24-well plate at a density of 156,000 cells / cm2in plating medium and incubated for 48 hours. Subsequently, the cells were fed with maintenance medium, with medium changes performed every 48 hours.

[0129] Hepatocytes

[0130] Primary hepatocytes were obtained from LifeNet Health and cultured according to manufacturer’s instructions. Briefly, cells were thawed from liquid nitrogen into thawing medium and then diluted to 700,000 cells / ml in plating medium and seeded into a collagen type 1 -coated 48-well plate at 0.2 ml per well. Cells were incubated with periodic gentle shaking for 3 hours, at which point the medium was exchanged for maintenance medium. Medium changeswere subsequently performed daily and cells were maintained at 37 °C in a 5% CO2 environment.

[0131] Transduction[00132J Human iPSC-cardiomyocytes were transduced with Round 2 library pool at an MOI of 1,000 and 10,000. Cells were harvested post infection for sequencing analysis.

[0133] Human iPSC-cardiomyocytes were transduced 6 days after seeding with a multiplicity of infection (MOI, vg / cell) of 5,000, 10,000 or 20,000. Samples were collected post infection for analysis.

[0134] Human primary hepatocytes were transduced one day after seeding with a multiplicity of infection (MOI, vg / cell) of 10,000, 20,000 or 50,000. Samples were collected post infection for analysis.

[0135] Flow Cytometry

[0136] iPSC-CMs were dissociated into single-cell suspensions using TrypLE solution. Subsequently, cells were fixed and permeabilized using Cytofix / Cytoperm solution obtained from BD Bioscience. Cells were then stained using a cardiac Troponin T (cTnT) conjugated antibody at 4 °C for 30 min in dark following with multiple wash steps. Finally, the samples were loaded onto a BD Celesta flow cytometer for analyzing the cTnT and GFP positive populations.

[0137] Immunocytochemistry (ICC)

[0138] Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.01% Triton X-100. Primary antibody was added for 18 hours at 4°C, followed by a secondary antibody and nuclear counterstain for 1 hour at room temperature. Cells were imaged on a Zeiss Axiovert fluorescent microscope in blue, green and red channels.

[0139] ResultsTable 1 depicts the sequence frequencies of top capsid variants as determined by PacBio full- length capsid sequencing compared between in vitro iPSC-cardiomyocytes and NHP selections. NHP, non-human primate; AAV, adeno-associated virus; MOI, multiplicity of infection; iPSC, induced pluripotent stem cell; * indicates SEQ ID NO.l; Linker 1, glycine (G) - alanine (A); Linker 2, alanine serine (AS) - alanine leucine serine (ALS), (dash represents the peptide insertion location); Shift 0, insertion between amino acid 588 and 589 of AAV; Shift -1, insertion between 587 and 588; Shift +1, insertion between 589 and 590; Shift -2 insertion between 586 and 587; Shift +2, insertion between 590 and 591. Sequencing results identified a capsid comprising capsid protein of SEQ ID NO: 11 and other variants having a core insertion of SEQ ID NO: 1 with high frequency in NHP heart and iPSC-cardiomyocytes following transduction with Round 2 library (Table 1). The same variants also showed limited transduction in NHP liver tissue.Table 1

[0140] Human iPSC-cardiomyocytes were transduced with rAAV comprising a capsid of SEQ ID NO: 11 or with wildtype AAV9 virions, each carrying an EGFP transgene operably linked to a CAG promoter, to monitor transduction efficiency. Seven days post transduction, cells were analyzed by immunocytochemistry (ICC) and flow cytometry. Capsid comprising a capsid protein of SEQ ID NO: 11 transduced human iPSC-cardiomyocytes better than AAV9 at all MOIs examined by ICC and quantified by flow cytometry using GFP positive cells within the cTnT positive population (Figures 1A and IB; Transduction efficiency of SEQ ID NO: 1 compared to AAV9 at three MOI in iPSC-cardiomyocytes; Figures 1 A Immunocytochemistry, Figures IB Flow cytometry quantification; EGFP, enhance green fluorescent protein (green); cTnT, cardiac troponin T (red); DAPI, nuclei (blue); MOI, multiplicity of infection; AAV, adeno-associated virus, iPSC, induced pluripotent stem cell. Error bars ± standard deviation; n=3).

[0141] Primary human hepatocytes were transduced with capsid comprising a capsid protein of SEQ ID NO: 11 or wildtype capsid AAV9 carrying CAG-EGFP transgene to monitor transduction efficiency. Six days post transduction, cells were analyzed by immunocytochemistry (ICC). Capsid comprising a capsid protein of SEQ ID NO: 11 transduced hepatocytes better than AAV9 by ICC images (Figure 2; immunocytochemistry of transduction efficiency of SEQ ID NO: 1 compared to AAV9 and positive control at three MOI in primary hepatocytes; EGFP, enhance green fluorescent protein (green); HNF4A, hepatocyte nuclear factor 4 alpha (red); DAPI, nuclei (blue); MOI, multiplicity of infection; AAV, adeno-associated virus).

[0142] While the materials and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Claims

CLAIMS1. A recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

2. The rAAV according to claim 1, wherein the insertion peptide has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and / or carboxyl terminus of amino acid sequence of NLTRVSG (SEQ ID NO:1).

3. The rAAV according to claim 1 or 2, wherein the insertion peptide is GNLTRVSGA(SEQ ID NO:3).

4. The rAAV according to claim 1 or 2, wherein the insertion peptide is ASNLTRVSGALS(SEQ ID NO:2).

5. The rAAV according to any one of claims 1-4, wherein the insertion site is located between amino acids corresponding to amino acids 570 and 627 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

6. The rAAV according to any one of claims 1-5, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

7. The rAAV according to any one of claims 1-6, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

8. The rAAV according to any one of claims 1-6, wherein the insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

9. The rAAV according to any one of claims 1-6, wherein the insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

10. The rAAV according to any one of claims 1-6, wherein the insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

11. The rAAV according to any one of claims 1-3 and 5, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

12. The rAAV according to any one of claims 1-3 and 5, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

13. The rAAV according to any one of claims 1-3 and 5, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

14. The rAAV according to any one of claims 1, 2, 4 and 5, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

15. The rAAV according to any one of claims 1, 2, 4 and 5, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

16. The rAAV according to any one of claims 1, 2, 4 and 5, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

17. The rAAV according to any one of claims 1-10, wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as any one of SEQ ID NOs:4-13, preferably wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as any one of SEQ ID NOs:8-l 1 and 13.

18. The rAAV according to any one of claims 1-17, wherein the gene product is a peptide, a protein, a small interfering RNA, an antisense RNA, a microRNA, a short hairpin RNA or a gene editing system.

19. The rAAV according to any one of claims 1-17, wherein the rAAV preferentially targets cardiac cells over liver cells when administered systemically to a subject.

20. A method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to a cardiac cell, the method comprising contacting the cardiac cell with a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.21 . The method according to claim 20, wherein the insertion peptide has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and / or carboxyl terminus of amino acid sequence of NLTRVSG (SEQ ID NO: 1).

22. The method according to claim 20 or 21, wherein the insertion peptide is GNLTRVSGA(SEQ ID NO:3).

23. The method according to claim 20 or 21, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2).

24. The method according to any one of claims 20-23, wherein the insertion site is located between amino acids corresponding to amino acids 570 and 627 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

25. The method according to any one of claims 20-24, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

26. The method according to any one of claims 20-25, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

27. The method according to any one of claims 20-25, wherein the insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

28. The method according to any one of claims 20-25, wherein the insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

29. The method according to any one of claims 20-25, wherein the insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9(SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

30. The method according to any one of claims 20-22 and 24, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

31. The method according to any one of claims 20-22 and 24, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

32. The method according to any one of claims 20-22 and 24, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

33. The method according to any one of claims 20, 21, 23 and 24, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

34. The method according to any one of claims 20, 21, 23 and 24, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

35. The method according to any one of claims 20, 21, 23 and 24, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site islocated between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

36. The method according to any one of claims 20-35, wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:4-13, preferably wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:8-l 1 and 13.

37. The method according to any one of claims 20-36, wherein the gene product is a peptide, a protein, a small interfering RNA, an antisense RNA, a microRNA, a short hairpin RNA or a gene editing system.

38. The method according to any one of claims 20-37, wherein the rAAV preferentially targets cardiac cells over liver cells when administered systemically to a subject.

39. The method according to any one of claims 20-38, wherein the cardiac cell is a fibroblast, a smooth muscle cell or a cardiomyocyte.

40. The method according to any one of claims 20-39, wherein the nucleotide sequence encoding the gene product is operably linked to a constitutive promoter.

41. The method according to any one of claims 20-39, wherein the nucleotide sequence encoding the gene product is operably linked to a cell-specific promoter, preferably a cardiac cell-specific promoter.

42. A method for delivering a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product to the heart of a mammal, the method comprising administering to the mammal a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion covalently inserted in the GH-loop of the AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

43. The method according to claim 42, wherein the insertion peptide has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and / or carboxyl terminus of amino acid sequence of NLTRVSG (SEQ ID NO: 1).

44. The method according to claim 42 or 43, wherein the insertion peptide is GNLTRVSGA(SEQ ID NO:3).

45. The method according to claim 42 or 43, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2).

46. The method according to any one of claims 42-45, wherein the insertion site is located between amino acids corresponding to amino acids 570 and 627 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

47. The method according to any one of claims 42-46, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

48. The method according to any one of claims 42-47, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

49. The method according to any one of claims 42-47, wherein the insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

50. The method according to any one of claims 42-47, wherein the insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

51. The method according to any one of claims 42-47, wherein the insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9(SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

52. The method according to any one of claims 42-44 and 46, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

53. The method according to any one of claims 42-44 and 46, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

54. The method according to any one of claims 42-44 and 46, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

55. The method according to any one of claims 42, 43, 45 and 46, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

56. The method according to any one of claims 42, 43, 45 and 46, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NOG) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

57. The method according to any one of claims 42, 43, 45 and 46, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site islocated between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

58. The method according to any one of claims 42-57, wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:4-13, preferably wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:8-l 1 and 13.

59. The method according to any one of claims 42-58, wherein the gene product is a peptide, a protein, a small interfering RNA, an antisense RNA, a microRNA, a short hairpin RNA or a gene editing system.

60. The method according to any one of claims 42-59, wherein the rAAV preferentially targets cardiac cells over liver cells when administered systemically to a subject.

61. The method according to claim 60, wherein the cardiac cell is a fibroblast, a smooth muscle cell or a cardiomyocyte.

62. The method according to any one of claims 42-61, wherein the nucleotide sequence encoding the gene product is operably linked to a constitutive promoter.

63. The method according to any one of claims 42-62, wherein the nucleotide sequence encoding the gene product is operably linked to a cell-specific promoter, preferably a cardiac cell-specific promoter.

64. The method according to any one of claims 42-63, wherein the rAAV is administered to the mammal systemically.

65. The method according to claim 64, wherein the rAAV is administered to the mammal intravenously.

66. The method according to any one of claims 42-63, wherein the rAAV is administered directly to the heart of the mammal.

67. The method according to claim 66, wherein the rAAV is administered to the mammal via an injection into the heart.

68. The method according to any one of claims 19-35, wherein the gene product is a cardiac- associated gene selected from cardiac troponin T (TNNT2); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5 ’-AMP- activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT- like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); myocyte enhancer factor 2c (MEF2C); cardiac LIM protein (CSRP3); cardiac troponin T; a cardiac sarcomeric protein; beta-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac a-actin; a-tropomyosin; cardiac troponin I; cardiac myosin binding protein C; four-and-a-half LIM protein 1; 5 ’-AMP-activated protein kinase subunit gamma-2; troponin I type 3, myosin light chain 2, actin alpha cardiac muscle 1; cardiac LIM protein; caveolin 3 (CAV3); galactosidase alpha (GLA); lysosomal-associated membrane protein 2 (LAMP2); mitochondrial transfer RNA glycine (MTTG); mitochondrial transfer RNA isoleucine (MTTI); mitochondrial transfer RNA lysine (MTTK); mitochondrial transfer RNA glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoendoplasmic reticulum calcium- ATPase 2a (SERCA2a); stromal-derived factor- 1 (SDF-1); adenylate cyclase-6 (AC6); beta-ARKct (b-adrenergic receptor kinase C terminus); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia inducible growth factor; thymosin beta 4 (TMSB4X); nitric oxide synthase-3 (NOS3); unocartin 3 (UCN3); melusin; apoplipoprotein-E (ApoE); superoxide dismutase (SOD); and S100A1.

69. A method for treating a cardiac disorder in a mammal, the method comprising administering to the mammal a recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion with covalently inserted in the GH-loop of the AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NLTRVSG (SEQ ID NO: 1) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a therapeutic gene product capable of ameliorating the cardiac disorder, said nucleotide sequence operably linked to a promoter, or administering to the mammal apharmaceutical composition comprising said rAAV and a pharmaceutically acceptable carrier.

70. The method according to claim 69, wherein the insertion peptide has from 1 to 5 spacer amino acids (Y1-Y5) at the amino and / or carboxyl terminus of amino acid sequence of NLTRVSG (SEQ ID NO:1).

71. The method according to claim 69 or 70, wherein the insertion peptide is GNLTRVSGA(SEQ ID NO:3).

72. The method according to claim 69 or 70, wherein the insertion peptide isASNLTRVSGALS (SEQ ID NO:2).

73. The method according to any one of claims 69-72, wherein the insertion site is located between amino acids corresponding to amino acids 570 and 627 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

74. The method according to any one of claims 69-73, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

75. The method according to any one of claims 69-74, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

76. The method according to any one of claims 69-74, wherein the insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

77. The method according to any one of claims 69-74, wherein the insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

78. The method according to any one of claims 69-74, wherein the insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

79. The method according to any one of claims 69-71 and 73, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

80. The method according to any one of claims 69-71 and 73, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

81. The method according to any one of claims 69-71 and 73, wherein the insertion peptide isGNLTRVSGA (SEQ ID NO:3) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

82. The method according to any one of claims 69, 70, 72 and 73, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

83. The method according to any one of claims 69, 70, 72 and 73, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 589 and 590 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

84. The method according to any one of claims 69, 70, 72 and 73, wherein the insertion peptide is ASNLTRVSGALS (SEQ ID NO:2) and the insertion site is insertion site is located between amino acids corresponding to amino acids 590 and 591 of VP1 of AAV9 (SEQ NO: 14) or the corresponding position in the capsid protein of another AAV serotype.

85. The method according to any one of claims 69-84, wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:4-13, preferably wherein the variant capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NOs:8-l 1 and 13.

86. The method according to any one of claims 69-85, wherein the therapeutic gene product is a peptide, a protein, a small interfering RNA, an antisense RNA, a microRNA, a short hairpin RNA or a gene editing system.

87. The method according to any one of claims 69-86, wherein the rAAV preferentially targets cardiac cells over liver cells when administered systemically to a subject.

88. The method according to claim 87, wherein the cardiac cell is a fibroblast, a smooth muscle cell or a cardiomyocyte.

89. The method according to any one of claims 69-88, wherein the nucleotide sequence encoding the therapeutic gene product is operably linked to a constitutive promoter.

90. The method according to any one of claims 69-89, wherein the nucleotide sequence encoding the therapeutic gene product is operably linked to a cell-specific promoter, preferably a cardiac cell-specific promoter.

91. The method according to any one of claims 69-90, wherein the rAAV or the pharmaceutical composition is administered to the mammal systemically.

92. The method according to claim 91, wherein the rAAV or the pharmaceutical composition is administered to the mammal intravenously.

93. The method according to any one of claims 69-90, wherein the rAAV or the pharmaceutical composition is administered directly to the heart of the mammal.

94. The method according to claim 93, wherein the rAAV or the pharmaceutical composition is administered to the mammal via an injection into the heart.

95. The method according to any one of claims 69-93, wherein the therapeutic gene product is a cardiac-associated gene selected from cardiac troponin T (TNNT2); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5 ’-AMP-activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNN13); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT- like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); myocyte enhancer factor 2c (MEF2C); cardiac LIM protein (CSRP3); cardiac troponin T; a cardiac sarcomeric protein; b-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac a-actin; a-tropomyosin; cardiac troponin I; cardiac myosin binding protein C; four-and-a-half LIM protein 1; titin; 5 ’-AMP-activated protein kinase subunit gamma-2; troponin I type 3, myosin light chain 2, actin alpha cardiac muscle 1; cardiac LIM protein; caveolin 3 (CAV3); galactosidase alpha (GLA); lysosomal- associated membrane protein 2 (LAMP2); mitochondrial transfer RNA glycine (MTTG); mitochondrial transfer RNA isoleucine (MTTI); mitochondrial transfer RNA lysine (MTTK); mitochondrial transfer RNA glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); beta-ARKct (b-adrenergic receptor kinase C terminus); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia inducible growth factor; thymosin beta 4 (TMSB4X); nitric oxide synthase-3 (NOS3); unocartin 3 (UCN3); melusin; apoplipoprotein-E (ApoE); superoxide dismutase (SOD); and S100A1.

96. The method according to any one of claims 69-95, wherein the cardiac disorder is selected from the group consisting of: Barth syndrome; Freidrich’s Ataxia; CPVT; Marfan; Noonan; Brugada; Long QT Syndrome; Myotonic Dystrophy 1; Limb Girdle Dystrophy Type IB; Naxos; Loeys-Dietz; X-Linked EDMD; SV Aortic Stenosis; coronary heart disease; cardiomyopathy; endocarditis; congenital cardiovasculardefects; congestive heart failure; hypertrophic cardiomyopathy; a valvular heart disease; myocardial infarction; congestive heart failure; long QT syndrome; atrial arrhythmia; ventricular arrhythmia; diastolic heart failure; systolic heart failure; cardiac valve disease; cardiac valve calcification; left ventricular non-compaction; ventricular septal defect; and ischemia.

97. A pharmaceutical composition comprising the rAAV according to any one of claims 1 to19 and a and a pharmaceutically acceptable carrier.

98. The rAAV of claim 19 or the methods of claims 38, 60 or 87 or the pharmaceutical composition of claim 97, wherein the rAAV preferentially targets cardiac cells over liver cells by preferentially transducing cardiac cells to liver cells by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold higher ratios as compared to AAV9 or the corresponding capsid protein of another AAV serotype .

99. A host cell comprising the rAAV of any one of claims 1 to 19 or 98.

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