Adeno-associated viruses with engineered capsids

Abstract

The present disclosure provides recombinant adeno-associated virus (rAAV) virions with engineered capsid proteins identified using high-throughput sequencing screening or rational design. In particular, the present disclosure provides AAV5 virions and capsid proteins with increased transduction efficiency, increased cell type selectivity, and / or other desirable properties in cardiac cells. The present disclosure generally relates to gene therapy using adeno-associated virus vectors.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates generally to gene therapy with adeno-associated virus vectors. In particular, the present disclosure relates to recombinant adeno-associated virus virions having engineered capsid proteins.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 984,197, filed March 2, 2020, and U.S. Provisional Patent Application No. 62 / 827,576, filed April 1, 2019, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0003] Sequence Listing Reference This application has been filed electronically via EFS-Web and includes a sequence listing that has been electronically submitted in .txt format. The text file contains a sequence listing entitled "TENA_012_02WO_SeqList_ST25.txt" that was created on March 31, 2020 and is approximately 2,590 kilobytes in size. The sequence listing contained in this text file is a part of the present specification and is incorporated by reference in its entirety. [Background technology]

[0004] background Adeno-associated viruses (AAVs) are promising for gene therapy and other biomedical applications. In particular, AAVs can be used to deliver gene products to a variety of tissues and cells both in vitro and in vivo. The capsid protein of AAV primarily determines the immunogenicity and tropism of AAV vectors.

[0005] For some tissues, AAV subtype 5 (AAV5) is a preferred AAV vector due to the low incidence of neutralizing antibodies against AAV5 in the human population. However, AAV5 is less efficient at transducing cardiac tissue than other AAV subtypes. In addition, selective transduction of different cell types is desirable but difficult to achieve. For example, in some applications, it may be desirable to transduce cardiac fibroblasts rather than cardiac myocytes, or vice versa. The present disclosure provides variants of AAV5 capsids that form rAAV virions that can transduce cardiac tissues and / or cell types more efficiently and / or selectively than rAAV virions containing wild-type AAV5 capsid proteins. Summary of the Invention [Means for solving the problem]

[0006] Disclosure Summary In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising an AAV5 capsid protein that comprises one or more substitutions selected from S651A, S651G, S651V, S651L, S651I, T578A, T578G, T578V, T578L, T578I, T582A, T582G, T582V, T582L, or T582I compared to the parent AAV5 capsid, and a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0007] In another aspect, the disclosure provides an AAV5 capsid protein that includes one or more substitutions selected from S651A or S651V; T578A or T578V; or T582A or T582V compared to the parent AAV5 capsid.

[0008] In another aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising an AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parental sequence at an insertion site in the GH loop of the parental sequence, and a heterologous nucleic acid com / prising a nucleotide sequence encoding a gene product.

[0009] In another aspect, the disclosure provides an AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence.

[0010] In a further aspect, the disclosure provides a polynucleotide encoding an AAV5 capsid protein, wherein the capsid protein comprises an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence.

[0011] In a further aspect, the disclosure provides a pharmaceutical composition comprising an rAAV virion comprising an AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parental sequence at an insertion site in the GH loop of the parental sequence, and a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0012] In a further aspect, the present disclosure provides a method of transducing a cardiac cell, the method comprising contacting the cardiac cell with a rAAV virion of the present disclosure, wherein the rAAV virion transduces the cardiac cell.

[0013] In a further aspect, the present disclosure provides a method of delivering one or more gene products to a cardiac cell, the method comprising contacting the cardiac cell with an rAAV virion of the present disclosure, wherein the cardiac cell expresses the gene product.

[0014] In a further aspect, the disclosure provides a method of treating a cardiac condition in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a rAAV virion of the disclosure, wherein the rAAV virion transduces cardiac tissue.

[0015] In a further aspect, the disclosure provides a method for identifying AAV capsid proteins that confer increased transduction efficiency on rAAV virions in a target cell, the method comprising the steps of providing a population of rAAV virions, the rAAV genomes comprising a library of cap polynucleotides encoding variant AAV capsid proteins; contacting a target cell with the population for a period of time sufficient to allow transduction of the cap polynucleotides into the target cell by the rAAV virions; and sequencing the cap polynucleotides from the target cell, thereby identifying the AAV capsid proteins that confer increased transduction efficiency in the target cell. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows an embodiment of a library screening strategy that incorporates an initial negative selection step on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) followed by a positive selection step on human cardiac fibroblasts (hCFs).

[0017] [Figure 2A-2B] 2A-2B show the generation of an AAV5 random peptide display library and its mapping onto the crystal structure of AAV5. FIG. 2A shows a schematic diagram of the AAV genome, including the AAV2 rep gene ("Rep2") and the AAV cap gene ("Cap5") flanked by 3' and 5' inverted repeats (ITRs). In this embodiment, a random 7-mer polypeptide library (AXXXXXXXA; SEQ ID NO: 100) flanked by alanine residues is inserted at position 575 as indicated. FIG. 2B shows the structure of the AAV5 capsid. The approximate insertion site of the polypeptide library (at one of the 60 copies of the capsid protein shown) is indicated by a black arrow.

[0018] [Figure 3A-3D]Figures 3A-3D visualize the library complexity after each screening round, reporting the total number of reads and the percentage of reads that are unique peptides. Each variant is represented by a different shade of grey based on the hydrophobicity of the peptide sequence, and each pixel in the image represents a single read in the library and clusters with other reads of the same sequence. Increased enrichment is evident based on the excess of reads (i.e., the decrease in the percentage of reads that are unique peptides) after round 1 (Figure 3B), after round 2 (Figure 3C), or after round 3 (Figure 3D) compared to the parent library (Figure 3A).

[0019] [Figure 4A-4D] Figures 4A-4D show the transduction efficiency of various rAAV virions identified using the library screening strategy for hCF cells. Figure 4A shows the transduction efficiency of 15 AAV5 variants referenced to unmodified AAV5 (right-most bar) packaged with a green fluorescent protein (GFP) reporter transgene. hCF were infected at a genome multiplicity of infection (MOI) of 100,000 and the percentage of GFP-positive cells was quantified using a Cytation5™ cell imaging multimode reader. Representative fluorescent images are shown for hCF transduced with either unmodified AAV5 (Figure 4B) or AAV5.CR17 (Figure 4C). Figure 4D shows the transduction efficiency of unmodified AAV5 versus AAV.CR17 at MOI 1,000.

[0020] [Figure 5A-5D]Figures 5A-5D show the transduction efficiency of various rAAV virions identified using the library screening strategy on iPS-CM cells. Figure 5A shows the transduction efficiency of 15 AAV5 variants referenced to unmodified AAV5 (right-most bar) packaged with a GFP reporter transgene. hCFs were infected at a genome multiplicity of infection (MOI) of 100,000 and the percentage of GFP-positive cells was quantified using a Cytation 5™ cell imaging multimode reader. Representative fluorescent images are shown for hCFs transduced with either unmodified AAV5 (Figure 5B) or AAV5.CR23 (Figure 5C). Figure 5D shows the transduction efficiency of unmodified AAV5 versus AAV.CR23 at MOI 100,000.

[0021] [Figure 6A] Figures 6A-6E demonstrate that AAV5.CR17 has improved transduction efficiency in primary mouse cardiac fibroblasts (mCFs). mCFs were infected with either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP at a high (100,000), medium (10,000), or low (1,000) MOI. 72 h post-infection, transduction was assessed using flow cytometry. Figure 6A shows histograms and gating of the "total GFP" and "high GFP" populations. Increased percent GFP+ (Figure 6B) and GFP intensity (Figure 6C) were observed in the "total GFP" population when cells were transduced with AAV5.CR17 at an MOI of 103, 104, or 105 compared to unmodified AAV5. Increased percent GFP+ (Figure 6D) and GFP intensity (Figure 6E) were observed in the "high GFP" population when cells were transduced with AAV5.CF17 at an MOI of 103, 104, or 105 compared to unmodified AAV5. [Figure 6B-6E]Figures 6A-6E demonstrate that AAV5.CR17 has improved transduction efficiency in primary mouse cardiac fibroblasts (mCFs). mCFs were infected with either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP at a high (100,000), medium (10,000), or low (1,000) MOI. 72 h post-infection, transduction was assessed using flow cytometry. Figure 6A shows histograms and gating of the "total GFP" and "high GFP" populations. Increased percent GFP+ (Figure 6B) and GFP intensity (Figure 6C) were observed in the "total GFP" population when cells were transduced with AAV5.CR17 at an MOI of 103, 104, or 105 compared to unmodified AAV5. Increased percent GFP+ (Figure 6D) and GFP intensity (Figure 6E) were observed in the "high GFP" population when cells were transduced with AAV5.CF17 at an MOI of 103, 104, or 105 compared to unmodified AAV5.

[0022] [Figure 7A-7D] Figures 7A-7H show the transduction profiles of AAV5 and AAV5.CR17 in CD1 mice with and without myocardial infarction (MI). Unmodified AAV5 showed limited transduction of cardiac tissue in control mice (GFP-Figure 7A; DAPI-Figure 7B) or induced MI mice (GFP-Figure 7C; DAPI-Figure 7D). AAV5.CR17 more effectively transduced cardiac tissue in both control mice (GFP-Figure 7E; DAPI-Figure 7F) and induced MI mice (GFP-Figure 7G; DAPI-Figure 7H). 1.2 x 10vg of either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP was injected into 6-week-old CD1 mice via intramyocardial injection. GFP intensity and distribution were assessed 7 days after injection. Increased transduction of cardiomyocytes and fibroblasts is observed by AAV5.CR17 in both non-MI and MI animals. All cardiac tissue sections were imaged using a 20x objective with equal gain and exposure time. Anti-GFP immunostaining is shown in green and nuclear counterstaining with DAPI blue is shown in blue. [Fig. 7E-7H]Figures 7A-7H show the transduction profiles of AAV5 and AAV5.CR17 in CD1 mice with and without myocardial infarction (MI). Unmodified AAV5 showed limited transduction of cardiac tissue in control mice (GFP-Figure 7A; DAPI-Figure 7B) or induced MI mice (GFP-Figure 7C; DAPI-Figure 7D). AAV5.CR17 more effectively transduced cardiac tissue in both control mice (GFP-Figure 7E; DAPI-Figure 7F) and induced MI mice (GFP-Figure 7G; DAPI-Figure 7H). 1.2 x 10vg of either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP was injected into 6-week-old CD1 mice via intramyocardial injection. GFP intensity and distribution were assessed 7 days after injection. Increased transduction of cardiomyocytes and fibroblasts is observed by AAV5.CR17 in both non-MI and MI animals. All cardiac tissue sections were imaged using a 20x objective with equal gain and exposure time. Anti-GFP immunostaining is shown in green and nuclear counterstaining with DAPI blue is shown in blue.

[0023] [Figure 8A-8D] Figures 8A-8E show sequence motifs based on the top 100 (Figure 8A), top 50 (Figure 8B), top 20 (Figure 8C) sequences in Table 2 (SEQ ID NOs: 101-600), or sequences with data reported in Table 1 (Figure 8D) (SEQ ID NOs: 101-115). Figure 8E shows sequence motifs for all sequence reads in the library, weighted by the number of sequence reads in the library after round 3 (SEQ ID NOs: 101-600). [Figure 8E] Figures 8A-8E show sequence motifs based on the top 100 (Figure 8A), top 50 (Figure 8B), top 20 (Figure 8C) sequences in Table 2 (SEQ ID NOs: 101-600), or sequences with data reported in Table 1 (Figure 8D) (SEQ ID NOs: 101-115). Figure 8E shows sequence motifs for all sequence reads in the library, weighted by the number of sequence reads in the library after round 3 (SEQ ID NOs: 101-600).

[0024] [Figure 9] Figure 9 shows that AAV5 surface-exposed serine / threonine / lysine / tyrosine mutant vectors demonstrate increased transduction efficiency of hCF in vitro. hCF cells were infected with either AAV5-wild type or AAV5-mutant vectors packaging a ubiquitously expressed GFP reporter. Substitutions S651A, T578A, or T582A in the AAV5 capsid resulted in improved human cardiac fibroblast transduction 72 hours post-infection at a multiplicity of infection of 100,000 vg / cell.

[0025] [Figure 10A-10C] Figures 10A-10C show transduction of human cardiac fibroblasts (hCF) with either AAVx1 vector (upper line) or AAV5 vector (lower line). Figure 10A shows the percentage of transduced cells. Figure 10B shows the expression of the GFP transgene. Figure 10C shows the relative transduction (relative transduction = transduced cells (%) x cell intensity).

[0026] [Figure 11] FIG. 11 shows the relative transduction of cardiac fibroblast (CF) cells in vivo.

[0027] [Figure 12] FIG. 12 shows that AAVx1 vector carrying a MyA expression cassette is effective against myocardial infarction by kinetic echocardiography data of % ejection fraction.

[0028] [Figure 13] FIG. 13 shows a graph of the change in percent ejection fraction up to 9 weeks after injection in treated and untreated pigs with ischemic injury. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description The present disclosure generally relates to recombinant adeno-associated virus (rAAV) virions for use as viral vectors having capsid proteins with (1) substitutions at selected surface-exposed amino acid residues; (2) insertions in the GH loop; or (3) both. The rAAV virions can transduce cardiac tissue, e.g., cardiac fibroblasts, at least as well as or better than rAAV virions having parent capsid proteins that do not have these substitutions and / or insertions.

[0030] The present disclosure provides recombinant adeno-associated virus (rAAV) virions. In particular, the present disclosure provides novel capsid proteins, methods for identifying them, and methods for using them. The methods for identifying novel capsid proteins disclosed herein have broad applicability for any serotype of AAV, including hybrid capsid proteins. In addition, they can be applied to iteratively improve capsid proteins with mutations from this and other methods. In general, the methods of the present disclosure involve the preparation of a randomized or semi-randomized library of AAV capsids in the form of cap gene polynucleotides, the preparation of AAV virions containing such capsids (either by incorporating the cap gene library into the AAV genome or by providing it in trans, such as on a plasmid transfected into a packaging system), positively or negatively selecting the AAV virions, and recovering the cap genes for sequencing. In some embodiments, the recovery and sequencing includes nanopore sequencing. Other high-throughput or next-generation sequencing (NGS) methods can be used.

[0031] In some embodiments, the rAAV virion disclosed herein comprises an AAV5 capsid protein that comprises an insertion of 5-11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence. The rAAV virion generally comprises a heterologous nucleic acid that comprises a nucleotide sequence that encodes a gene product. A suitable insertion site for insertion can be determined by a variety of methods, including, but not limited to, modeling the GH loop based on the crystal structure of the AAV5 capsid; selecting flexible residues; or screening various insertion sites. The insertion can be between two adjacent amino acid residues, or non-adjacent residues may be selected, in which case the insertion causes the deletion of one or more intervening residues.

[0032] In some embodiments, AAV5 capsid protein comprises the substitution of residues that are rationally designed; introduced by mutagenesis; or randomized by generating a library of sequences with random codon usage at one or more sites.Exemplary non-limiting substitutions that regulate transfection efficiency include, but are not limited to, S651A, T578A, or T582A.Substituted residues can be incorporated into the AAV5 capsid protein that further comprises one or more insertion sites, such as insertions at GH loop or other loops.

[0033] The capsid proteins of the present disclosure include any insertions identified as enriched by directed evolution methods, followed by sequences including, but not limited to, those shown in the Examples. While not limited to any particular insertion site, in some embodiments, the insertion is between about residues 574 and 575 in the AAV5 capsid protein (SEQ ID NO:1). In some embodiments, the insertion site is between any two, optionally adjacent amino acids, in the parent sequence corresponding to positions 560-594 of SEQ ID NO:1. In some embodiments, the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1. The N-terminal residues of VP1, VP2, and VP3 are shown above the sequence of full-length VP1. [ka]

[0034] Insertion at this insertion site splits the VP3 capsid protein into an N-terminal segment (SEQ ID NO:2) and a C-terminal segment (SEQ ID NO:3), disclosed herein for clarity as separate sequences, which may be linked by any of the insertions of this disclosure. In some embodiments, the capsid protein comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:1 (with a gap in the alignment at the insertion site for clarity). In some embodiments, the capsid protein comprises a sequence sharing at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:2, and a sequence sharing at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:3, wherein SEQ ID NO:2 and SEQ ID NO:3 are linked by an insertion of 5-11 amino acids. In some embodiments, the insertion is selected from SEQ ID NOs:101-600. In some embodiments, adjacent alanine (A) residues in SEQ ID NOs:101-600 are replaced by other residues, such as flexible residues serine (S) or glycine (G), and the adjacent A residues are absent. MGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTG NNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQ (SEQ ID NO:2) SSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO:3)

[0035] In some embodiments, the capsid protein comprises a sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4, where X represents any amino acid. In some embodiments, the capsid protein comprises a sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5, where X represents any amino acid. [ka] [ka]

[0036] In some embodiments, the insertion is of the formula: -X0-(X)n -X 10 - (In the formula, n is 5 to 9, and X0 and X 10 are each independently A, S, G, or absent (SEQ ID NO: 1101). It is a peptide.

[0037] In some embodiments, the insertion is of the formula: -X0-X1-X2-X3-X4-X5-X6-X7-X8- (wherein X0 and X8 are each independently A, S, G, or absent) (SEQ ID NO: 1102). It is a peptide.

[0038] In some embodiments, the insertion is of the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (wherein X1 is P, R, or G; X2 is K, L, or R; X3 is any amino acid; X4 is N, H, K, or Q; X5 is any amino acid or optionally X5 is G, K, or S; X6 is any amino acid or optionally X6 is T or V; X7 is any amino acid or optionally X7 is K or V) (SEQ ID NO: 1103). It is a peptide.

[0039] In some embodiments, the insertion is of the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (wherein X1 is P, R, or G; X2 is K, L, or R; X3 is any amino acid; X4 is N, H, K, or Q; X5 is any amino acid, or optionally X5 is G, K, or S; X6 is any amino acid; and X7 is any amino acid) (SEQ ID NO: 1104). It is a peptide.

[0040] In some embodiments, the insertion is of the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (wherein X1 is P, R, or G; X2 is K, L, or R; X3 is any amino acid; X4 is N, H, K, or Q; X5 is any amino acid, or optionally X5 is G, K, or S; X6 is T or V; and X7 is any amino acid) (SEQ ID NO: 1105). It is a peptide.

[0041] In some embodiments, the insertion is of the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (wherein X1 is P, R, or G; X2 is K, L, or R; X3 is any amino acid; X4 is N, H, K, or Q; X5 is any amino acid or optionally X5 is G, K, or S; X6 is T or V; and X7 is K or V) (SEQ ID NO: 1106). It is a peptide.

[0042] In some embodiments, X1 through X7 are any amino acids and the insertions are defined by one or more of the following criteria: X1 is P, R, or G; X2 is K, L, or R; X3 is any amino acid; X4 is N, H, K, or Q; X5 is G, K, or S; X6 is T or V; and / or X7 is K or V (SEQ ID NO: 1107).

[0043] In some embodiments, the insertion is selected from SEQ ID NOs: 101-600.

[0044] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NOs:101-600; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0045] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NOs:101-200; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0046] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NOs:101-150; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0047] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NOs:101-120; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0048] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NOs:101-115; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0049] In some embodiments, the capsid protein comprises, in order from N-terminus to C-terminus, one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3; one polypeptide selected from SEQ ID NO:103 or 110; and one polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2 and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.

[0050] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to one of SEQ ID NOs: 601-1100, or a functional fragment thereof.

[0051] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to one of SEQ ID NOs: 601-615, or a functional fragment thereof.

[0052] In some embodiments, the insertion comprises an amino acid sequence selected from RKVHIEV (SEQ ID NO:81), RKYQSDL (SEQ ID NO:82), PLTNTVK (SEQ ID NO:83), LKYHGPP (SEQ ID NO:84), RKYQGDM (SEQ ID NO:85), RKFHSTD (SEQ ID NO:86), RKHHGLE (SEQ ID NO:87), PGTNVTK (SEQ ID NO:88), RKMHMPD (SEQ ID NO:89), PLKKIVQ (SEQ ID NO:90), PLGKKTS (SEQ ID NO:91), PRGVKVT (SEQ ID NO:92), PLAKSKS (SEQ ID NO:93), PRTKGAV (SEQ ID NO:94), and PSGRKAT (SEQ ID NO:95).

[0053] In some embodiments, the insertion comprises an amino acid sequence selected from ARKVHIEVA (SEQ ID NO:101), ARKYQSDLA (SEQ ID NO:102), APLTNTVKA (SEQ ID NO:103), ALKYHGPPA (SEQ ID NO:104), ARKYQGDMA (SEQ ID NO:105), ARKFHSTDA (SEQ ID NO:106), ARKHHGLEA (SEQ ID NO:107), APGTNVTKA (SEQ ID NO:108), ARKMHMPDA (SEQ ID NO:109), APLKKIVQA (SEQ ID NO:110), APLGKKTSA (SEQ ID NO:111), APRGVKVTA (SEQ ID NO:112), APLAKSKSA (SEQ ID NO:113), APRTKGAVA (SEQ ID NO:114), APSGRKATA (SEQ ID NO:115).

[0054] In some embodiments, the insertion comprises any one of SEQ ID NOs: 101-600. In some embodiments, the sequence comprises no more than 1, 2, 3, or 4 amino acid substitutions compared to any one of SEQ ID NOs: 101-600.

[0055] In some embodiments, the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO:83) {clone CR17}, or a sequence containing at most 1, 2, 3, or 4 amino acid substitutions compared to PLTNTVK (SEQ ID NO:83).

[0056] In some embodiments, the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO:90) {clone CR23}, or a sequence containing at most 1, 2, 3, or 4 amino acid substitutions compared to PLKKIVQ (SEQ ID NO:90).

[0057] In some embodiments, the insertion comprises the amino acid sequence APLTNTVKA (SEQ ID NO: 103) {clone CR17} or a sequence containing at most 1, 2, 3, or 4 amino acid substitutions compared to APLTNTVKA (SEQ ID NO: 103).

[0058] In some embodiments, the insertion comprises the amino acid sequence APLKKIVQA (SEQ ID NO:110) {clone CR23}, and the insertion further comprises 1, 2, 3, or 4 conservative amino acid substitutions.

[0059] In some embodiments, the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO:83) {clone CR17}, and the insertion further comprises 1, 2, 3, or 4 conservative amino acid substitutions.

[0060] In some embodiments, the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO:90) {clone CR23}, and the insertion further comprises 1, 2, 3, or 4 conservative amino acid substitutions.

[0061] In some embodiments, the insertion comprises the amino acid sequence APLTNTVKA (SEQ ID NO: 103) {clone CR17}, and the insertion further comprises 1, 2, 3, or 4 conservative amino acid substitutions.

[0062] In some embodiments, the insertion comprises at least the amino acid sequence APLKKIVQA (SEQ ID NO:110) {clone CR23}, and the insertion further comprises 1, 2, 3, or 4 conservative amino acid substitutions.

[0063] Conservative substitutions include substitutions of polar residues with polar residues, non-polar residues with non-polar residues, hydrophobic residues with hydrophobic residues, small residues with small residues, and large residues with large residues. Conservative substitutions further include substitutions within the following groups: {S, T}, {A, G}, {F, Y}, {R, H, K, N, E}, {S, T, N, Q}, {C, U, G, P; A}, and {A, V, I, L, M, F, Y, W}.

[0064] Transduction efficiency can be determined using methods known in the art or described in the Examples. In some embodiments, the rAAV virions exhibit increased transduction efficiency in cardiac cells compared to AAV virions containing the parental sequence.

[0065] In some embodiments, rAAV virions exhibit increased transduction efficiency in human cardiac fibroblasts (hCFs) compared to AAV virions containing the parental sequence.

[0066] In some embodiments, the rAAV virions exhibit at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold increase in transformation efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virions exhibit about a 2 to about 16-fold, about a 2 to about 14-fold, about a 2 to about 12-fold, about a 2 to about 10-fold, about a 2 to about 8-fold, about a 2 to about 6-fold, about a 2 to about 4-fold, or about a 2 to about 3-fold increase in transformation efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virions exhibit at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold increase in transformation efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virions exhibit an increase in transduction efficiency of about 20%-30%, about 30%-40%, about 40%-50%, about 50%-80%, about 80%-100%, about 100%-125%, about 125%-150%, about 150%-175%, or about 175%-200% at a multiplicity of infection (MOI) of 100,000 in hCF cells.

[0067] In some embodiments, the rAAV virions exhibit at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold increase in transformation efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virions exhibit about a 2- to about 16-fold, about a 2- to about 14-fold, about a 2- to about 12-fold, about a 2- to about 10-fold, about a 2- to about 8-fold, about a 2- to about 6-fold, about a 2- to about 4-fold, or about a 2- to about 3-fold increase in transformation efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virions exhibit an increase in transduction efficiency of about 20%-30%, about 30%-40%, about 40%-50%, about 50%-80%, about 80%-100%, about 100%-125%, about 125%-150%, about 150%-175%, or about 175%-200% at a multiplicity of infection (MOI) of 1,000 in hCF cells.

[0068] In some embodiments, the rAAV virions exhibit increased transduction efficiency in induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) compared to AAV virions containing the parental sequence.

[0069] In some embodiments, the rAAV virions exhibit at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold increase in transformation efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virions exhibit about a 2- to about 16-fold, about a 2- to about 14-fold, about a 2- to about 12-fold, about a 2- to about 10-fold, about a 2- to about 8-fold, about a 2- to about 6-fold, about a 2- to about 4-fold, or about a 2- to about 3-fold increase in transformation efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, rAAV virions exhibit an increase in transformation efficiency of about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% at a multiplicity of infection (MOI) of 100,000 in iPS-CM cells.

[0070] In some embodiments, the rAAV virions exhibit at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold increase in transformation efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virions exhibit about a 2- to about 16-fold, about a 2- to about 14-fold, about a 2- to about 12-fold, about a 2- to about 10-fold, about a 2- to about 8-fold, about a 2- to about 6-fold, about a 2- to about 4-fold, or about a 2- to about 3-fold increase in transformation efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, rAAV virions exhibit an increase in transformation efficiency of about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% at a multiplicity of infection (MOI) of 1,000 in iPS-CM cells.

[0071] Cell type selectivity is increased when the ratio of transduction efficiency of one cell type relative to another is increased by the capsid protein compared to the parental sequence, hi some embodiments, the rAAV virions exhibit increased selectivity of the rAAV virions for hCF cells over iPS-CM cells.

[0072] In some embodiments, the rAAV virions exhibit increased selectivity of the rAAV virions for iPS-CM cells over hCF cells.

[0073] In some embodiments, the capsid protein comprises a mutation selected from S651A, T578A, T582A, K251R, Y709F, Y693F, or S485A. In some embodiments, the capsid protein comprises a mutation selected from K251R, Y709F, Y693F, or S485A.

[0074] The polynucleotide encoding the capsid protein may comprise a sequence that includes either the native codons of the wild-type cap gene or alternative codons selected to encode the same protein. The codon usage of the insertion may vary. Representative nucleotide sequences of the cap genes encoding the capsid proteins of the present disclosure are provided as SEQ ID NOs: 51-72. In some embodiments, the present disclosure provides polynucleotides encoding AAV5 capsid proteins that include a sequence that is at least 95%, 99%, or 100% identical to one of SEQ ID NOs: 51-72. It is within the skill of the art to select an appropriate nucleotide sequence and derive an alternative nucleotide sequence to encode any capsid protein of the present disclosure. Reverse translation of the protein sequence can be performed using a codon usage table of the host organism, i.e., eukaryotic codon usage for humans.

[0075] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:51 {CR2 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:101 or ARKVHIEVA (SEQ ID NO:101).

[0076] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:52 {CR5 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:102 or ARKYQSDLA (SEQ ID NO:102).

[0077] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:53 {CR8 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:107 or ARKHHGLEA (SEQ ID NO:107).

[0078] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:54 {CR17 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:103 or APLTNTVKA (SEQ ID NO:103).

[0079] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:55 {CR18 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:104 or ALKYHGPPA (SEQ ID NO:104).

[0080] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:56 {CR19 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:105 or ARKYQGDMA (SEQ ID NO:105).

[0081] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:57 {CR20 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:106 or ARKFHSTDA (SEQ ID NO:106).

[0082] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:58 {CR21 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:108 or APGTNVTKA (SEQ ID NO:108).

[0083] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:59 {CR22 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:109 or ARKMHMPDA (SEQ ID NO:109).

[0084] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:60 {CR23 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains no more than one, two, three, or four amino acid substitutions compared to SEQ ID NO:110 or APLKKIVQA (SEQ ID NO:110).

[0085] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:61 {CR24 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:111 or APLGKKTSA (SEQ ID NO:111).

[0086] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:62 {CR25 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:112 or APRGVKVTA (SEQ ID NO:112).

[0087] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:63 {CR26 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:113 or APLAKSKSA (SEQ ID NO:113).

[0088] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:64 {CR27 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:114 or APRTKGAVA (SEQ ID NO:114).

[0089] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:65 {CR28 polynucleotide sequence}, wherein the AAV5 capsid protein comprises a sequence that contains at most one, two, three, or four amino acid substitutions compared to SEQ ID NO:115 or APSGRKATA (SEQ ID NO:115).

[0090] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:66, wherein the AAV5 capsid protein comprises the substitution K251R with respect to SEQ ID NO:1.

[0091] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:67, wherein the AAV5 capsid protein comprises a substitution S485A relative to SEQ ID NO:1.

[0092] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:68, wherein the AAV5 capsid protein comprises a substitution S651A relative to SEQ ID NO:1.

[0093] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:69, wherein the AAV5 capsid protein comprises a substitution T578A relative to SEQ ID NO:1.

[0094] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising an sequence at least 95%, 99%, or 100% identical to SEQ ID NO:70, wherein the AAV5 capsid protein comprises a substitution T582A relative to SEQ ID NO:1.

[0095] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to SEQ ID NO:71, wherein the AAV5 capsid protein comprises a substitution Y709F with respect to SEQ ID NO:1.

[0096] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 capsid protein comprising a sequence at least 95%, 99%, or 100% identical to SEQ ID NO:72, wherein the AAV5 capsid protein comprises a substitution Y693F with respect to SEQ ID NO:1.

[0097] In some embodiments, the disclosure provides an AAV5 capsid protein comprising a sequence that is at least 95%, 99%, or 100% identical to a modified capsid selected from SEQ ID NOs:9-34, where substitutions are present in the modified capsid from the reference capsid SEQ ID NO:1, and where other substitutions, optionally conservative substitutions, are permitted at the specified percent identity levels. In some embodiments, the insertion site is between any two, optionally adjacent, amino acids in the parent sequence corresponding to positions 420-460 of SEQ ID NO: 1. In some embodiments, the insertion site is between adjacent amino acids in the parent sequence corresponding to 444 and 445 of SEQ ID NO: 1. An exemplary embodiment is the AAV5 capsid disclosed as SEQ ID NO:31.

[0098] In some embodiments, a recombinant adeno-associated virus (rAAV) virion disclosed herein comprises an AAV5 capsid protein that comprises one or more substitutions selected from S651G, S651V, S651L, S651I, T578A, T578G, T578V, T578L, T578I, T582A, T582G, T582V, T582L, or T582I compared to the parent AAV5 capsid, and a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0099] In some embodiments, a recombinant adeno-associated virus (rAAV) virion disclosed herein comprises an AAV5 capsid protein that comprises one or more substitutions selected from S651A, T578A, or T582A compared to the parent AAV5 capsid, and a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0100] The AAV5 capsid protein may include any two substitutions selected from S651A or S651V; T578A or T578V; or T582A or T582V, or all three of S651A or S651V; T578A or T578V; or T582A or T582V. The following combinations of mutations are expressly contemplated by this disclosure: a) T578G+S651G; b) T578V+S651V; c) T578L+S651L; d) T578I+S651I; e) T582A+S651A; f) T582G+S651G; g) T582V+S651V; h) T582L+S651L; i) T582I+S651I; j) T578A+T582A+S651A; k) T578G+T582G+S651G; l) T578V+T582V+S651V; m) T578L+T582L+S651L; and n) T578I+T582I+S651I.

[0101] The AAV5 capsid protein can include any two substitutions selected from S651A, T578A, or T582A; or all three of S651A, T578A, or T582A. For example, the AAV5 capsid protein can be any one of SEQ ID NOs: 74-80, or a sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. [ka] [ka] [ka] [ka]

[0102] In any of the foregoing sequences, the alanine substitution can instead be a substitution of valine (V), glycine (G), leucine (L), or isoleucine (I).

[0103] In some embodiments, one or more amino acid residues are preserved to the residues present in wild-type AAV5 capsid.For example, in some embodiments, T581 is preserved as tyrosine (T) or semi-preserved as serine (S).In contrast, US Patent No. 10,046,016 teaches that the infectivity of lung epithelial cells is increased by mutation with T581A mutation.

[0104] definition Unless the context indicates otherwise, the features of the invention can be used in any combination. Any feature or combination of features described can be excluded or omitted. Certain features of the invention described in separate embodiments can also be provided in combination in a single embodiment. Features of the invention described in a single embodiment can also be provided individually or in any suitable subcombination. All combinations of embodiments are disclosed herein as if each and every combination were individually disclosed. All subcombinations of embodiments and elements are disclosed herein as if every such subcombination were individually disclosed.

[0105] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The detailed description is divided into sections merely for the convenience of the reader, and the disclosure found in any section may be combined with the disclosure found in another section. Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Reference to a publication does not constitute an admission that the publication is prior art.

[0106] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a recombinant AAV virion" includes a plurality of such virions, and reference to "the cardiac cell" includes one or more cardiac cells.

[0107] The conjunction "and / or" means both "and" and "or," and a list connected by "and / or" includes all possible combinations of one or more of the listed items.

[0108] The term "vector" refers to a macromolecule or molecular complex that contains a polynucleotide or protein to be delivered to a cell.

[0109] "AAV" is an abbreviation for adeno-associated virus. This term covers all subtypes of AAV, including both naturally occurring and recombinant forms, unless subtype is indicated. The abbreviation "rAAV" refers to recombinant adeno-associated virus. "AAV" includes AAV or any subtype. "AAV5" refers to AAV subtype 5. The genomic sequences of various serotypes of AAV, as well as the sequences of the naturally occurring inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, can be found in the literature or in public databases, such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8).Publications describing AAV include Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; International Patent Application Publication Nos. WO2018 / 222503A1, WO2012 / 145601A2, WO2000 / 028061A2, WO1999 / 61601A2, and WO1998 / 11244A2; U.S. Patent Application Nos. 15 / 782,980 and 15 / 433,322; and U.S. Patent Nos. 10,036,016, 10,790,472 ... ,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517, and 6,156,303.

[0110] As used in the art, "AAV vector" or "rAAV vector" refers to either the DNA packaged in rAAV virion or the rAAV virion itself, depending on the context. As used herein, unless otherwise clear from the context, rAAV vector refers to a nucleic acid (typically a plasmid) that contains a polynucleotide sequence that can be packaged in rAAV virion together with the capsid or other proteins of rAAV virion. In general, rAAV vectors contain a heterologous polynucleotide sequence (i.e., a polynucleotide that is not of AAV origin) and one or two AAV inverted terminal repeats (ITRs) that flank the heterologous polynucleotide sequence. Only one of the two ITRs may be packaged in rAAV, and still maintain the infectivity of the resulting rAAV virion. See Wu et al. (2010) Mol Ther. 18:80. rAAV vectors can be designed to generate either single-stranded (ssAAV) or self-complementary (scAAV). See McCarty D. (2008) Mo. Ther. 16:1648-1656; WO2001 / 11034; WO2001 / 92551; WO2010 / 129021.

[0111] "rAAV virion" refers to an extracellular viral particle that contains at least one viral capsid protein (e.g., VP1) and an encapsidated rAAV vector (or a fragment thereof) that contains capsid proteins.

[0112] For the sake of brevity and clarity, the present disclosure refers to a "capsid protein" or "capsid proteins." Those skilled in the art will understand that such a reference refers to VP1, VP2, or VP3, or a combination of VP1, VP2, and VP3. As in the case of wild-type AAV and most recombinant expression systems, VP1, VP2, and VP3 are expressed from the same open reading frame, and manipulation of the sequence encoding VP3 necessarily alters the sequence of the C-terminal domains of VP1 and VP2. Similarly, capsid proteins may be expressed from different open reading frames, in which case the resulting rAAV virion capsid may contain a mixture of wild-type and engineered capsid proteins, and a mixture of different engineered capsid proteins.

[0113] The term "inverted terminal repeat" or "ITR" as used herein refers to the AAV virus cis elements so named due to their symmetry. These elements are essential for efficient replication of the AAV genome. Without wishing to be bound by theory, it is believed that the minimum elements essential for ITR function are the Rep binding site and the terminal resolution site plus a variable palindrome that allows hairpin formation. The present disclosure contemplates that alternative means of generating AAV genomes may exist or may be prospectively developed to be compatible with the capsid protein of the present disclosure.

[0114] "Helper virus functions" refer to functions encoded in the helper virus genome that enable AAV replication and packaging.

[0115] "Packaging" refers to a series of intracellular events that result in the assembly of rAAV virions, including the encapsidation of rAAV vectors. AAV "rep" and "cap" genes refer to polynucleotide sequences that code for the replication and encapsidation proteins of adeno-associated viruses. AAV rep and cap are referred to herein as AAV "packaging genes". Packaging requires either the helper virus itself or, more commonly in recombinant systems, helper virus functions (i.e., one or more helper plasmids) provided by a helper-free system.

[0116] AAV "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. The helper virus can be an adenovirus, a herpesvirus, or a poxvirus, such as vaccinia.

[0117] An "infectious" virion or virus particle is one that contains a competently assembled viral capsid and is capable of delivering a polynucleotide component to a cell for which the virion is a tropic. This term does not necessarily imply the replicative capacity of the virus.

[0118] "Infectivity" refers to a measurement of the ability of virions to regulate a cell. Infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Infectivity is generally determined for a particular cell type. It can be measured both in vivo or in vitro. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (TCID 50 (1999) Gene Ther. 6:973; see also Wang et al. (1999) (description of an infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0119] The term "parent capsid" or "parent sequence" refers to the reference sequence from which a particle capsid or sequence is derived. Unless otherwise specified, the parent sequence refers to the sequence of a wild-type capsid protein of the same serotype as the engineered capsid protein.

[0120] A "replication competent" virus (e.g., replication competent AAV) refers to a virus that is infectious and capable of replicating in an infected cell (i.e., in the presence of a helper virus or helper virus functions). In some embodiments, the rAAV virions of the present disclosure comprise a genome that lacks the rep gene, or both the rep and cap genes, and are therefore replication incompetent.

[0121] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition; Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5 thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and refer to Sell (2013) Stem Cells Handbook.

[0122] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0123] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms also encompass amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0124] The term "peptide" refers to short polypeptides, for example peptides having between about 4 and 30 amino acid residues.

[0125] The term "isolated" means that a virion, cell, tissue, polynucleotide, peptide, polypeptide, or protein is separated from cellular and other components with which it is normally associated in nature. For example, an isolated cell is a cell separated from tissues or cells of a different phenotype or genotype.

[0126] As used herein, "sequence identity" or "identity" refers to the percentage of the number of amino acids that are identical between a sequence of interest and a reference sequence. In general, identity is determined by aligning a sequence of interest with a reference sequence, determining the number of amino acids that are identical between the aligned sequences, dividing the number by the total number of amino acids in the reference sequence, and multiplying the result by 100 to obtain a percentage. Sequences can be aligned using various computer programs, such as BLAST, available at ncbi.nlm.nih.gov. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996); and Meth. Mol. Biol. 70: 173-187 (1997); J. Mol. Biol. 48: 44. Those skilled in the art can select a suitable alignment method depending on various factors, including sequence length, differences, and the presence or absence of insertions or deletions with respect to the reference sequence.

[0127] "Recombinant," as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps and other procedures that result in a construct that differs from that found in nature, or that the polynucleotide is assembled from synthetic oligonucleotides. A "recombinant" protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that contains a recombinant polynucleotide and / or a recombinant protein, such as a recombinant capsid protein.

[0128] "Gene" refers to a polynucleotide that contains at least one open reading frame that can encode a specific protein after being transcribed and translated. "Gene product" is a molecule resulting from the expression of a specific gene. Gene products can include, but are not limited to, polypeptides, proteins, aptamers, interfering RNA, or mRNA. Gene editing systems (e.g., CRISPR / Cas systems) can be described as one gene product or several gene products (e.g., Cas proteins and guide RNAs) required to create the system.

[0129] "Short hairpin RNAs" or shRNAs are polynucleotide constructs used to express siRNAs.

[0130] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of the process and can be enhancing or inhibitory in nature. Control elements include transcriptional regulatory sequences such as promoters and / or enhancers.

[0131] A "promoter" is a DNA sequence capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region normally located downstream (in the 3' direction) from the promoter. The term "tissue-specific promoter," as used herein, refers to a promoter that is operable in the cells of a particular organ or tissue, such as cardiac tissue.

[0132] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows the elements to operate in their expected manner. By way of example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues between the promoter and the coding region may be present so long as this functional relationship is maintained.

[0133] An "expression vector" is a vector containing a coding sequence that encodes a gene product of interest that is used to affect expression of the gene product in a target cell. Expression vectors contain control elements operably linked to the coding sequence to facilitate expression of the gene product.

[0134] The term "expression cassette" refers to a heterologous polynucleotide that comprises a coding sequence that encodes a gene product of interest that is used to affect expression of the gene product in a target cell. Unless otherwise specified, an expression cassette in an AAV vector comprises a polynucleotide between (and not including) the ITRs.

[0135] The term "gene delivery" or "gene transfer," as used herein, refers to a method or system for reliably inserting a foreign nucleic acid sequence, e.g., DNA, into a host cell. Such methods may result in transient expression of unintegrated transferred DNA, extrachromosomal replication and expression of a transferred replicon (e.g., episome), or integration of the transferred genetic material into the genomic DNA of the host cell.

[0136] "Heterologous" means that it is derived from an entity that is genotypically different from the genotype of the rest of the entity to which it is compared.For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide.A promoter that is removed from its native coding sequence and operably linked to a coding sequence with which it is not found in nature is a heterologous promoter.Thus, for example, a rAAV that contains a heterologous nucleic acid is a rAAV that contains a nucleic acid that is not normally included in naturally occurring AAV.

[0137] The terms "genetic modification" and "genetic alteration" (and grammatical variants thereof) are used interchangeably herein and refer to a process in which a genetic element (e.g., a polynucleotide) is introduced into a cell by other than mitosis or meiosis. The element may be heterologous to the cell, or may be an additional copy or improved version of an element already present in the cell. Genetic modification may be effected, for example, by transfecting a recombinant plasmid or other polynucleotide into the cell through any process known in the art, such as electroporation, calcium phosphate precipitation, or by contacting a polynucleotide-liposome complex. Genetic modification may also be effected by transduction or infection with a vector.

[0138] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a polynucleotide sequence if the sequence is available to perform its function during long-term culture of the cell in vitro. Generally, such cells are "genetically" altered (genetically modified) in that a genetic alteration has been introduced that can also be inherited by progeny of the altered cell.

[0139] The term "transfection" as used herein refers to the uptake of exogenous nucleic acid molecules by cells. A cell is "transfected" when an exogenous nucleic acid is introduced into the interior of the cell membrane. Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous nucleic acid molecules can be introduced into a suitable host cell.

[0140] The term "transduction," as used herein, refers to the transfer of exogenous nucleic acid into a cell by a recombinant virion, as opposed to "infection" by a wild-type virion. When infection is used in reference to a recombinant virion, the terms "transduction" and "infectivity" are synonymous, and thus "infectivity" and "transformation efficiency" are equivalent and can be determined using similar methods.

[0141] Unless otherwise specified, all medical terms are given their ordinary meaning as used by the medical profession, e.g., as described in Harrison's Principles of Internal Medicine, 15th ed., particularly the chapter on cardiac or cardiovascular diseases, disorders, conditions, and dysfunctions, which is incorporated herein by reference in its entirety for all purposes.

[0142] "Treatment," "treating," and "treat" are defined as acting on a disease, disorder, or condition with an agent that reduces or ameliorates the harmful or any other undesirable effects of the disease, disorder, or condition, and / or its symptoms.

[0143] "Administration," "administering," and the like, when used in connection with the compositions of the present invention, refer to both direct administration (administration to a subject by a medical professional or self-administration by the subject) and / or indirect administration (prescribing the composition to a patient). Typically, an effective amount is administered, which can be determined by one of skill in the art. Any method of administration may be used. Administration to a subject may be accomplished, for example, by intravenous, intraarterial, intramuscular, intravascular, or intramyocardial delivery.

[0144] As used herein, the term "effective amount" and the like with respect to the amount of a composition refers to an amount that is sufficient to induce a desired physiological outcome (e.g., cell reprogramming or disease treatment). An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on multiple variables, including the period for which the individual dosage units are used, the bioavailability of the composition, the route of administration, and the like. However, it is understood that the specific amount of a composition (e.g., rAAV virions) for any particular subject will depend on a variety of factors, including the activity of the particular agent used, the age, weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the combination of compositions, the severity of the particular disease being treated, and the form of administration.

[0145] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates (e.g., monkeys); sports mammals (e.g., horses); farm mammals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0146] The terms "cardiac pathology" or "cardiac dysfunction" are used interchangeably and refer to any defect in the pumping function of the heart, including, for example, contractile defects, relaxing defects (sometimes called diastolic dysfunction), abnormal or improper function of the heart valves, myocardial disease (sometimes called cardiomyopathies), diseases such as angina, myocardial ischemia, and / or infarction characterized by inadequate blood supply to the myocardium, infiltrative diseases such as amyloidosis and hemochromatosis, generalized or regional hypertrophy (as may occur, for example, in certain cardiomyopathies or systemic hypertension), and abnormal conduction between the ventricles.

[0147] As used herein, the term "cardiomyopathy" refers to any disease or dysfunction that directly affects the myocardium. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibrogenic, hematological, genetic, or of unknown origin. Two fundamental forms are recognized: (1) the first type, consisting of myocardial disease of unknown etiology, and (2) the second type, consisting of myocardial disease of known etiology or associated with disease involving other organ systems. "Specific cardiomyopathies" refer to cardiac diseases associated with certain systemic or cardiac disorders; examples include hypertensive and metabolic cardiomyopathies. Cardiomyopathy includes dilated cardiomyopathy (DCM), a disorder in which the contractile pump function of the left and / or right ventricles is impaired, resulting in progressive cardiac hypertrophy; hypertrophic cardiomyopathy, characterized by left ventricular hypertrophy without obvious causes such as hypertension or aortic stenosis; and restrictive cardiomyopathies, characterized by abnormal diastolic function and overly rigid ventricular walls that impede ventricular filling. Cardiomyopathy also includes left ventricular noncompaction, arrhythmogenic right ventricular cardiomyopathy, and arrhythmogenic right ventricular dysplasia.

[0148] "Heart failure" refers to a pathological condition in which abnormalities in cardiac function cause the heart to be unable to pump blood at a rate that meets the requirements of metabolic tissues, and / or the heart can only do so if the diastolic capacity is abnormally elevated. Heart failure includes systolic and diastolic dysfunction. Heart failure patients are classified into those with low cardiac output (typically secondary to ischemic heart disease, hypertension, dilated cardiomyopathy, and / or valvular or pericardial disease) and those with elevated cardiac output (typically due to hyperthyroidism, anemia, pregnancy, arteriovenous fistula, beriberi, and Paget's disease). Heart failure includes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF).

[0149] The phrase "pharmacologically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without causing undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0150] The term "purified," as used herein, refers to material that has been isolated under conditions that reduce or eliminate the presence of extraneous materials, i.e., impurities, including the natural materials from which the material was derived. For example, purified rAAV vector DNA is preferably substantially free of cells and culture components, including tissue culture components, contaminants, and the like.

[0151] The terms "regenerate," "regeneration," and the like, when used herein in the context of damaged cardiac tissue, have their ordinary meaning, and also refer to the process of growing and / or developing new cardiac tissue in a heart or cardiac tissue that has been damaged, e.g., damaged by ischemia, infarction, reperfusion, or other disease. In some embodiments, cardiac tissue regeneration includes the generation of cardiomyocytes.

[0152] The term "therapeutic gene," as used herein, refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it is present, or on the mammal in which it is expressed. Examples of beneficial effects include ameliorating the signs or symptoms of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include genes that partially or fully correct a genetic defect in a cell or mammal.

[0153] As used herein, the term "functional cardiomyocyte" refers to a differentiated cardiomyocyte that is capable of sending or receiving electrical signals. In some embodiments, the cell is capable of generating action potentials and / or Ca 2+ Cardiomyocytes are said to be functional cardiomyocytes if they exhibit electrophysiological properties such as transients.

[0154] As used herein, a "differentiated non-cardiac cell" may refer to a cell that is not capable of differentiating into all cell types of an adult organism (i.e., is not a pluripotent cell) and is a cell lineage other than the cardiac lineage (e.g., neural lineage, or connective tissue lineage). Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In certain embodiments, a cell with a lower differentiation potential is considered "differentiated" with reference to a cell with a higher differentiation potential.

[0155] A "somatic cell" is a cell that forms the body of an organism. Somatic cells include the cells that make up the organs, skin, blood, bone, and connective tissue in an organism, but are not embryonic cells.

[0156] As used herein, the term "totipotency" refers to the ability of a cell to form all cell lineages of an organism. For example, in mammals, only fertilized eggs and first cleavage stage blastocysts are totipotent.

[0157] As used herein, the term "pluripotency" refers to the ability of cells to form all the lineages of the body or body.For example, embryonic stem cells are a type of pluripotent stem cells that can form cells from each of the three germ layers, namely ectoderm, mesoderm, and endoderm.Pluripotent cells can be recognized by their marker expression, such as Nanog and Rex1.

[0158] As used herein, the term "multipotency" refers to the ability of adult stem cells to form multiple cell types of one lineage.For example, hematopoietic stem cells can form all cells of blood cell lineage, such as lymphoid cells and myeloid cells.

[0159] As used herein, the term "oligopotency" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.

[0160] As used herein, the term "unipotent" refers to the ability of a cell to form a single cell type, for example, a spermatogonial stem cell can only form sperm cells.

[0161] As used herein, the term "reprogramming" or "transdifferentiation" refers to the generation of cells of a particular lineage (e.g., cardiac cells) from cells of a different cell type (e.g., fibroblasts) without the intermediate process that causes the cells to redifferentiate into cells that exhibit pluripotent stem cell characteristics.

[0162] As used herein, the term "cardiac cells" refers to any cell present in the heart that provides cardiac functions such as contraction or blood supply, or otherwise acts to maintain the structure of the heart. Cardiac cells, as used herein, include cells present in the epithelium, muscular membrane, or endothelium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiovascular system, such as cells of the coronary arteries or veins. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conduction cells, and cardiac pacemaker cells, which constitute the cardiac cells that support the heart muscle, blood vessels, and structure. Cardiac cells can be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0163] The term "cardiomyocyte" or "cardiomyocytes", as used herein, refers to sarcomere-containing striated muscle cells naturally found in mammalian hearts compared to skeletal muscle cells. Cardiomyocytes are characterized by the expression of specialized molecules, such as proteins like myosin heavy chain, myosin light chain, cardiac α-actin. The term "cardiomyocyte", as used herein, is a generic term that includes any cardiomyocyte subpopulation or cardiomyocyte subtype, such as atrial, ventricular, and pacemaker cardiomyocytes.

[0164] The term "cardiomyocyte-like cells" is intended to mean cells that share characteristics of cardiomyocytes, but may not share all characteristics. For example, cardiomyocyte-like cells may differ from cardiomyocytes in the expression of certain cardiac genes.

[0165] The term "culture" or "cell culture" refers to maintaining cells in an artificial in vitro environment. "Cell culture system" as used herein refers to a culture condition in which a cell population can grow in monolayer or suspension. "Culture medium" as used herein refers to a nutrient solution for the culture, growth, or proliferation of cells. Culture media can be characterized by functional properties, such as, but not limited to, the ability to maintain cells in a particular state (e.g., pluripotent state, quiescent state, etc.), the ability to mature cells, and in some cases specifically the ability to promote differentiation of progenitor cells into cells of a particular lineage (e.g., cardiomyocytes).

[0166] As used herein, the term "expression" or "expressing" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is then translated into a peptide, polypeptide, or protein.If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample.

[0167] The term "induced cardiomyocytes" or the abbreviation "iCM" refers to non-cardiomyocytes (and their progeny) that have been transformed into cardiomyocytes (and / or cardiomyocyte-like cells). The methods of the present disclosure can be used with any method now known or later discovered to generate induced cardiomyocytes, e.g., to augment other techniques.

[0168] The term "induced pluripotent stem cell derived cardiomyocytes" as used herein refers to human induced pluripotent stem cells that are differentiated into cardiomyocyte-like cells. An exemplary method for preparing iPS-CM cells is provided by Karakikes et al. Circ Res. 2015 Jun 19; 117(1): 80-88.

[0169] The terms "human cardiac fibroblasts" and "mouse cardiac fibroblasts," as used herein, refer to primary cells isolated from adult human or mouse ventricles and maintained in ex vivo culture, respectively.

[0170] The term "non-cardiomyocytes" as used herein refers to any cell or population of cells in a cell preparation that does not meet the criteria of a "cardiomyocyte" as defined and used herein. Non-limiting examples of non-cardiomyocytes include somatic cells, cardiac fibroblasts, non-cardiac fibroblasts, cardiac progenitor cells, and stem cells.

[0171] As used herein, "reprogramming" includes transdifferentiation, dedifferentiation, and the like.

[0172] As used herein, the term "reprogramming efficiency" refers to the number of cells in a sample that are successfully reprogrammed into cardiomyocytes compared to the total number of cells in the sample.

[0173] The term "reprogramming factor" as used herein includes the factor that is introduced into cells to be expressed, to help reprogram cells from one cell type to another.For example, reprogramming factor can include transcription factor that can be combined with other transcription factors and / or small molecules to reprogram cardiac fibroblasts into induced cardiomyocytes.Unless otherwise clear from the context, reprogramming factor refers to the polypeptide that can be encoded by the polynucleotide that AAV delivers.Reprogramming factor can also include small molecules.

[0174] The term "stem cell" refers to a cell that has the ability to self-renew and generate differentiated progeny. The term "pluripotent stem cell" refers to a stem cell that can give rise to cells of all three germ layers (endoderm, mesoderm, and ectoderm), but does not have the ability to give rise to a complete organism.

[0175] As used herein, the term "equivalent thereof" when referring to a polypeptide or nucleic acid sequence refers to a polypeptide or nucleic acid that differs from a reference polypeptide or nucleic acid sequence but retains essential properties (e.g., biological activity). A typical variant of a polypeptide differs in nucleotide sequence from another reference polynucleotide. The change in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. The change in the nucleotide sequence may result in amino acid substitutions, deletions, additions, fusions, and truncations in the polypeptide encoded by the reference sequence. Generally, the difference is limited so that the sequences of the reference polypeptide and the variant are overall very similar and in many regions identical.

[0176] As used herein, the term "progenitor cell" refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue. Like stem cells, progenitor cells can further differentiate into one or more types of cells, but are more mature than stem cells such that they have a more limited / committed differentiation potential.

[0177] The term "genetic modification" refers to a permanent or transient genetic change introduced into a cell after the introduction of a new nucleic acid (i.e., a nucleic acid exogenous to the cell). Genetic modification can be achieved by the incorporation of the new nucleic acid into the genome of the cardiac cell, or by the transient or stable maintenance of the new nucleic acid as an extrachromosomal element. If the cell is a eukaryotic cell, permanent genetic modification can be achieved by the introduction of a nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc.

[0178] The term "stem cell" refers to a cell that has the ability to self-renew and generate differentiated progeny. The term "pluripotent stem cell" refers to a stem cell that can give rise to cells of all three germ layers (endoderm, mesoderm, and ectoderm), but does not have the ability to give rise to a complete organism. In some embodiments, the composition for inducing cardiomyocyte phenotype can be used in the cell population to induce reprogramming. In other embodiments, the composition induces cardiomyocyte phenotype.

[0179] The term "induced pluripotent stem cell" has its usual meaning and also refers to differentiated mammalian somatic cells (e.g., adult somatic cells, e.g., skin) that are reprogrammed to show at least one characteristic of pluripotency.See, for example, Takahashi et al. (2007) Cell 131(5):861-872, Kim et al. (2011) Proc. Natl. Acad. Sci. 108(19): 7838-7843, Sell (2013) Stem Cells Handbook.

[0180] The term "transduction efficiency" refers to the percentage of cells transduced by at least one AAV genome. For example, 1×10 6 cells were exposed to the virus, and 0.5 × 10 6 When it is determined that each cell contains at least one copy of AAV genome, the transduction efficiency is 50%.An exemplary method for determining transduction efficiency is flow cytometry.For example, when AAV genome contains a polynucleotide that codes for green fluorescent protein (GFP), the percentage of GFP+ cells is a measure of transduction efficiency.

[0181] The term "selectivity" refers to the ratio of transduction efficiency of one cell type relative to another or all other cell types.

[0182] The term "infectivity" refers to the ability of an AAV virion to infect a cell, particularly a cell in vivo. Infectivity is therefore at least a function of biodistribution and neutralizing antibody escape.

[0183] Unless otherwise stated, the abbreviations used throughout this specification have the following meanings: AAV, adeno-associated virus; rAAV, recombinant adeno-associated virus; AHCF, adult human cardiac fibroblasts; APCF, adult porcine cardiac fibroblasts; a-MHC-GFP, alpha-myosin heavy chain green fluorescent protein; CF, cardiac fibroblasts; cm, centimeters; CO, cardiac output; EF, ejection fraction; FACS, fluorescence activated cell sorting; GFP, green fluorescent protein; GMT, Gata4, Mef2c, and Tbx5; GMTc, Gata4, Mef2c, and Tbx5; Tbx5, TGF-βi, WNTi; GO, gene ontology; hCF, human cardiac fibroblasts; iCM, induced cardiomyocytes; kg, kilogram; μg, microgram; μl, microliter; mg, milligram; ml, milliliter; MI, myocardial infarction; msec, millisecond; min, minute; MyAMT, myocardin, Ascl1, Mef2c, and Tbx5; MyA, myocardin, and Ascl1; MyMT, myocardin, Mef2c, and Tbx5; MyMTc, myocardin, Mef2c, Tbx 5, TGF-βi, WNTi;MRI, nuclear magnetic resonance imaging;PBS, phosphate-buffered saline;PBST, phosphate-buffered saline, triton;PFA, paraformaldehyde;qPCR, quantitative polymerase chain reaction;qRT-PCR, quantitative reverse transcription polymerase chain reaction;RNA, ribonucleic acid;RNA-seq, RNA sequencing;RT-PCR, reverse transcription polymerase chain reaction;sec, seconds;SV, stroke volume;TGF-β, transforming growth factor beta;TGF-βi, transforming growth factor beta inhibitor;WN T, wingless-Int; WNTi, wingless-Int inhibitor; YFP, yellow fluorescent protein; 4F, Gata4, Mef2c, TBX5, and myocardin; 4Fc, Gata4, Mef2c, TBX5, and myocardin + TGF-βi and WNTi; 7F, Gata4, Mef2c, and Tbx5, Essrg, myocardin, Zfpm2, and Mesp1; 7Fc, Gata4, Mef2c, and Tbx5, Essrg, myocardin, Zfpm2, and Mesp1 + TGF-β, and WNTi.

[0184] EMBODIMENTS AND VARIATIONS composition Efforts to identify capsid variants with properties useful for gene therapy include DNA shuffling of AAV2 and AAV5 cap genes as described in U.S. Patent No. 9,233,131, and directed evolution methods as described in International Patent Application Nos. WO2012 / 145601A2 and WO2018 / 222503A1, the disclosures of which are incorporated herein by reference for all purposes, and in particular with respect to methods of making and using AAV virions and the polynucleotide sequences and gene products disclosed therein, and transcription factor combinations useful for treating cardiac diseases or disorders.

[0185] The AAV capsid is encoded by the AAV cap gene, which is also called the right open reading frame (ORF) (as opposed to the left ORF, rep). Representative AAV capsid structures have been described in various publications, including Xie et al. (2002) Proc. Natl. Acad. Sci USA 99:10405-1040 (AAV2); Govindasamy et al. (2006) J. Virol. 80:11556-11570 (AAV4); Nam et al. (2007) J. Virol. 81:12260-12271 (AAV8) and Govindasamy et al. (2013) J. Virol. 87:11187-11199 (AAV5).

[0186] The AAV capsid contains 60 copies (total) of three viral proteins (VPs), VP1, VP2, and VP3, arranged in T=1 icosahedral symmetry with an expected ratio of 1:1:10. The three VPs are translated from the same mRNA, with VP1 containing the complete VP2 sequence at its C-terminal region plus a unique N-terminal domain. VP2 contains VP3 plus an extra N-terminal sequence at its C-terminus. In most crystal structures, only the C-terminal polypeptide sequence (approximately 530 amino acids) common to all capsid proteins is observed. The unique N-terminal region of VP1, the VP1-VP2 overlap region, and the first 14-16 N-terminal residues of VP3 appear to be primarily disturbed. Cryo-electron microscopy and image reconstruction data suggest that in intact AAV capsids, the N-terminal regions of VP1 and VP2 proteins are located within the capsid and are inaccessible for receptor and antibody binding. Thus, receptor binding and transduction phenotypes are generally determined by amino acid sequences within the common C-terminal domain of VP1, VP2, and VP3.

[0187] In some embodiments, one or more amino acid insertions, substitutions, or deletions are present in the GH loop or loop IV of AAV capsid protein, for example, in the solvent-accessible portion of the GH loop or loop IV of AAV capsid protein.For the GH loop / loop IV of AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15: 1955.In some embodiments, the "parent" AAV capsid protein is a wild-type AAV5 capsid protein.In some embodiments, the "parent" AAV capsid protein is a chimeric AAV capsid protein.The amino acid sequences of various AAV capsid proteins are known in the art. See, e.g., GenBank Accession No. NP_049542 for AAV1; GenBank Accession No. NP_044927 for AAV4; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95450 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No. AAS99264 for AAV9, and GenBank Accession No. AAT46337 for AAV10. See, e.g., Santiago-Ortiz et al. (2015) Gene Ther. 22:934 for predicted ancestral AAV capsids.

[0188] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is about 4.7 kb long and contains two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the AAV5 genome is provided in GenBank Accession No. AF085716. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). Exemplary AAV vectors are provided in US7,105,345; US15 / 782,980; US7,259,151; US6,962,815; US7,718,424; US6,984,517; US7,718,424; US6,156,303; US8,524,446; US7,790,449; US7,906,111; US9,737,618; US application Ser. No. 15 / 433,322; and US7,198,951, each of which is incorporated by reference in its entirety for all purposes.

[0189] The rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. The gene product may be a polypeptide or RNA, or both. If the gene product is a polypeptide, the nucleotide sequence encodes a messenger RNA that is translated into the gene product polypeptide, optionally with one or more introns. The nucleotide sequence may encode one, two, three, or more gene products (the number is limited by the packaging capacity of the rAAV virion, but is typically about 5.2 kb). The gene products may be operably linked to one promoter (for a single transcription unit) or more than one promoter. Multiple gene products may also be produced using an internal ribosome entry site (IRES) or a self-cleaving peptide (e.g., 2A peptide).

[0190] In some embodiments, the gene product is a polypeptide. In some embodiments, the polypeptide gene product is a polypeptide that induces reprogramming of cardiac fibroblasts to generate induced cardiomyocyte-like cells (iCMs). In some embodiments, the polypeptide gene product is a polypeptide that enhances cardiac cell function. In some embodiments, the polypeptide gene product is a polypeptide that provides a missing or defective function in cardiac cells. In some embodiments, the polypeptide gene product is a genome editing endonuclease.

[0191] In some embodiments, the gene product comprises a fusion protein fused to a heterologous polypeptide. In some embodiments, the gene product comprises a genome editing nuclease fused to an amino acid sequence that provides subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLS) for targeting the nucleus, two or more NLSs, three or more NLSs, etc.).

[0192] Generally, viral vectors are produced by introducing viral DNA or RNA constructs into a "producer cell" or "packaging cell" line. Packaging cell lines include, but are not limited to, any easily transfectable cell line. Packaging cell lines can be based on HEK291, 293T cells, NIH3T3, COS, HeLa, or Sf9 cell lines. Examples of packaging cell lines include, but are not limited to, Sf9 (ATCC® CRL-1711™). Exemplary packaging cell lines and methods for producing rAAV virions are provided in International Patent Application Nos. WO2017075627, WO2015 / 031686, WO2013 / 063379, WO2011 / 020710, WO2009 / 104964, WO2008 / 024998, WO2003 / 042361, and WO1995 / 013392; U.S. Patent Nos. 9441206B2, US8679837, and US7091029B2.

[0193] In some embodiments, the gene product is a functional cardiac protein. In some embodiments, the gene product is a genome editing endonuclease (optionally together with a guide RNA, a single guide RNA, and / or a repair template) that replaces or repairs a non-functional cardiac protein into a functional cardiac protein.Functional cardiac proteins include cardiac troponin T; 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; titin; 5'-AMP-activated protein kinase subunit gamma-2; and 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); sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal-derived factor-1 (S DF-1); adenylate cyclase-6 (AC6); beta-ARKct (beta-adrenergic receptor kinase C-terminal); 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); merusin; apolipoprotein-E (ApoE); superoxide dismutase (SOD); and S100A1 (small calcium-binding protein; see, e.g., Ritterhoff and Most (2012) Gene Ther. 19:613; Kraus et al. (2009) Mol. Cell. Cardiol. 47:445).

[0194] In some embodiments, the gene product is a gene product whose expression compensates for a defect in a gene that is the cause of a genetic disorder. The present disclosure provides rAAV virions comprising polynucleotides encoding one or more of the following: TAZ (Barth syndrome); FXN (Friedreich's ataxia); CASQ2 (CPVT); FBN1 (Marfan); RAF1 and SOS1 (Noonan); SCN5A (Vulgareta); KCNQ1 and KCNH2 (long QT syndrome); DMPK (myotonic dystrophy type 1); LMNA (limb-girdle muscular dystrophy type 1B); JUP (Naxos); TGFBR2 (Loeys-Dietz); EMD (X-linked EDMD); and ELN (SV aortic stenosis), for use in, but not limited to, the disorders listed in parentheses or for other disorders caused by each. In some embodiments, the rAAV virion comprises a polynucleotide encoding one or more of cardiac troponin T (TNNT2); BAG family molecular chaperone regulatory factor 3 (BAG3); 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 type 1 (ACTC1); potassium voltage-dependent channel, KQT-like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

[0195] In some embodiments, the gene product of the present disclosure is a polypeptide reprogramming factor.Reprogramming factor is desirable as a means to convert one cell type into another cell type.Non-cardiomyocytes can be differentiated into cardiomyocytes in vitro or in vivo using any method available to those skilled in the art. See, e.g., the methods described in Ieda et al. (2010) Cell 142:375-386; Christoforou et al. (2013) PLoS ONE 8:e63577; Addis et al. (2013) J. Mol. Cell Cardiol. 60:97-106; Jayawardena et al. (2012) Circ. Res. 110: 1465-1473; Nam Y et al. (2003) PNAS USA 110:5588-5593; Wada R et al. (2013) PNAS USA 110: 12667-12672; and Fu J et al. (2013) Stem Cell Reports 1:235-247.

[0196] In the cardiac context, reprogramming factors may be capable of converting cardiac fibroblasts into cardiomyocytes, either directly or through intermediate cell types. In particular, reprogramming can be performed directly or by first converting fibroblasts into pluripotent or totipotent stem cells. Such pluripotent stem cells are called induced pluripotent stem cells (iPS). iPS cells that are then converted into cardiomyocyte (CM) cells are called iPS-CM cells. In this example, iPS-CM derived from cardiac fibroblasts in vitro are used in vivo to select a capsid protein of interest. The present disclosure also contemplates using the capsid proteins of the present disclosure to generate iPS-CM cells in turn in vitro, but particularly in vivo as part of a therapeutic gene therapy regimen. Induced cardiomyocyte-like (iCM) cells refer to cells that are directly reprogrammed into cardiomyocytes.

[0197] The induced cardiomyocytes express one or more cardiomyocyte-specific markers, including, but not limited to, cardiac troponin I, cardiac troponin-C, tropomyosin, caveolin-3, myosin heavy chain, myosin light chain-2a, myosin light chain-2v, ryanodine receptor, sarcomeric a-actinin, Nkx2.5, connexin 43, and atrial natriuretic factor. The induced cardiomyocytes can also exhibit sarcomere structures. Induced cardiomyocytes show increased expression of cardiomyocyte-specific genes ACTC1 (cardiac a-actin), ACTN2 (actinin a2), MYH6 (a-myosin heavy chain), RYR2 (ryanodine receptor 2), MYL2 (myosin regulatory light chain 2, ventricular isoform), MYL7 (myosin regulatory light chain, atrial isoform), TNNT2 (troponin T type 2, cardiac), and NPPA (natriuretic peptide precursor type A), PLN (phospholamban). Expression of fibroblast markers, such as Colla2 (collagen la2), is downregulated in induced cardiomyocytes compared to the fibroblasts from which iCMs are derived.

[0198] Reprogramming methods involving polypeptide reprogramming factors (in some instances supplemented by small molecule reprogramming factors supplied in combination with rAAV) include those described in US2018 / 0112282A1, WO2018 / 005546, WO2017 / 173137, US2016 / 0186141, US2016 / 0251624, US2014 / 0301991, and US2013 / 0216503A1, which are incorporated by reference in their entireties, particularly with respect to the reprogramming methods and factors disclosed.

[0199] In some embodiments, cardiac cells are reprogrammed into induced cardiomyocyte-like (iCM) cells using one or more reprogramming factors that modulate the expression of one or more polynucleotides or proteins of interest, such as Achaete-scute homolog 1 (ASCL1), myocardin (MYOCD), myocyte-specific enhancer factor 2C (MEF2C), and / or T-box transcription factor 5 (TBX5). In some embodiments, the one or more reprogramming factors are provided as polynucleotides (e.g., RNA, mRNA, or DNA polynucleotides) that encode the one or more polynucleotides or proteins of interest. In some embodiments, the one or more reprogramming factors are provided as proteins.

[0200] In some embodiments, the reprogramming factor is a microRNA or a microRNA antagonist, an siRNA, or a small molecule capable of increasing the expression of one or more polynucleotides or proteins of interest. In some embodiments, the expression of the polynucleotide or protein of interest is increased by the expression of a microRNA or a microRNA antagonist. For example, the endogenous expression of Oct polypeptide can be increased by the introduction of microRNA-302 (miR-302) or by increasing the expression of miR-302. See, for example, Hu et al., Stem Cells 31(2): 259-68 (2013), which is incorporated herein by reference in its entirety. Thus, miRNA-302 can be an inducer of endogenous Oct polypeptide expression. miRNA-302 can be introduced alone or together with a nucleic acid encoding an Oct polypeptide. In some embodiments, the suitable nucleic acid gene product is a microRNA. Suitable microRNAs include, for example, mir-1, mir-133, mir-208, mir-143, mir-145, and mir-499.

[0201] In some embodiments, the methods of the present disclosure include administering a rAAV virion of the present disclosure before, during, or after administration of a small molecule reprogramming factor. In some embodiments, the small molecule reprogramming factor is a small molecule selected from the group consisting of SB431542, LDN-193189, dexamethasone, LY364947, D4476, myricetin, IWR1, XAV939, docosahexaenoic acid (DHA), S-nitroso-TV-acetylpenicillamine (SNAP), Hh-Agl.5, alprostadil, cromakalim, MNITMT, A769662, retinoic acid p-hydroxyanilide, decamethonium dibromide, nifedipine, piroxicam, bacitracin, aztreonam, halmalol hydrochloride, amide-C2 (A7), Ph-C12 (CIO), mCF3-C-7 (J5), G856-7272 (A473), 5475707, or any combination thereof.

[0202] In some embodiments, the gene product comprises a reprogramming factor that modulates expression of one or more proteins of interest selected from ASCL1, MYOCD, MEF2C, and TBX5. In some embodiments, the gene product comprises one or more reprogramming factors selected from ASCL1, MYOCD, MEF2C, and TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, ZFPM2, and miR-133.

[0203] In some embodiments, the gene products include GATA4, MEF2C, and TBX5 (i.e., GMT). In some embodiments, the gene products include MYOCD, MEF2C, and TBX5 (i.e., MyMT). In some embodiments, the gene products include MYOCD, ASCL1, MEF2C, and TBX5 (i.e., MyAMT). In some embodiments, the gene products include MYOCD and ASCL1 (i.e., MyA). In some embodiments, the gene products include GATA4, MEF2C, TBX5, and MYOCD (i.e., 4F). In other embodiments, the gene products include GATA4, MEF2C, TBX5, ESSRG, MYOCD, ZFPM2, and MESP1 (i.e., 7F). In some embodiments, the gene products include one or more of ASCL1, MEF2C, GATA4, TBX5, MYOCD, ESRRG, and MESPL.

[0204] In some embodiments, rAAV virions generate cardiac myocytes in vitro or in vivo. Cardiomyocytes or cardiac myocytes are muscle cells that make up the cardiac muscle. Each cardiomyocyte contains myofibrils, which are long chains of sarcomeres, the contractile units of muscle cells. Cardiomyocytes exhibit striations similar to those of skeletal muscle cells, but differ from multinucleated skeletal cells in that they contain only one nucleus. Cardiomyocytes have high mitochondrial density, which allows them to rapidly produce ATP and makes them highly resistant to fatigue. Mature cardiomyocytes can express one or more of the following cardiac markers: α-actinin, MLC2v, MY20, cMHC, NKX2-5, GATA4, cTNT, cTNI, MEF2C, MLC2a, or any combination thereof. In some embodiments, mature cardiomyocytes express NKX2-5, MEF2C, or a combination thereof. In some embodiments, the cardiac progenitor cells express early cardiac progenitor markers, such as GATA4, ISL1, or a combination thereof.

[0205] In some embodiments, the gene product is a polynucleotide. In some embodiments, the gene product is a guide RNA capable of binding to an RNA-guided endonuclease, as described below. In some embodiments, the gene product is an inhibitory nucleic acid capable of reducing the level of, for example, an mRNA and / or polypeptide gene product in cardiac cells. For example, in some embodiments, the polynucleotide gene product is an interfering RNA capable of selectively inactivating the transcript encoded by an allele that causes a cardiac disease or disorder. As an example, the allele is a myosin heavy chain 7, myocardial, beta (MYH7) allele that contains a mutation that causes hypertrophic cardiomyopathy. Other examples include interfering RNAs that selectively inactivate transcripts encoded by alleles that cause hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or left ventricular noncompaction (LVNC), where the alleles are MYL3 (myosin light chain 3, alkaline, ventricular, skeletal slow muscle type), MYH7, TNNI3 (troponin I type 3 (heart)), TNNT2 (troponin T type 2 (heart)), TPM1 (tropomyosin 1 (alpha)), or ACTC1 alleles that contain mutations that cause HCM, DCM, or LVNC. For examples of mutations that cause cardiac disease, see, for example, US Patent Application Publication No. 2016 / 0237430.

[0206] In some embodiments, the gene product is a polypeptide-encoding RNA. In some embodiments, the gene product is an interfering 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 therapeutic polypeptide, e.g., a polypeptide that provides a clinical benefit. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function. In some embodiments, the gene product is an RNA-guided endonuclease that provides modification of a target nucleic acid. In some embodiments, the gene product is: i) an RNA-guided endonuclease that provides modification of a target nucleic acid; and ii) a guide RNA that includes: i) an RNA-guided endonuclease that provides modification of a target nucleic acid; and a second segment that binds to the RNA-guided endonuclease. In some embodiments, the gene product is: i) an RNA-guided endonuclease that provides modification of a target nucleic acid; ii) a first guide RNA comprising a first segment that binds to a first target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease; and iii) a first guide RNA comprising a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease.

[0207] The nucleotide sequence encoding the heterologous gene product in the rAAV virion of the present disclosure can be operably linked to a promoter.For example, the nucleotide sequence encoding the heterologous gene product in the rAAV virion of the present disclosure can be operably linked to a constitutive promoter, a regulated promoter, or a cardiac cell-specific promoter.Suitable constitutive promoters include human elongation factor 1 alpha subunit (EF1α) promoter, β-actin promoter, α-actin promoter, β-glucuronidase promoter, CAG promoter, supercore promoter, and ubiquitin promoter.In some embodiments, the nucleotide sequence encoding the heterologous gene product in the rAAV virion of the present disclosure is operably linked to a cardiac-specific transcriptional regulator element (TRE), and the cardiac-specific TRE comprises a promoter and an enhancer. Suitable cardiac-specific TREs include, but are not limited to, TREs derived from the following genes: myosin light chain-2 (MLC-2), a-myosin heavy chain (a-MHC), desmin, AE3, cardiac troponin C (cTnC), and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. NY. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14: 1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051. See also Pacak et al. (2008) Genet Vaccines Ther. 6:13. In some embodiments, the promoter is an alpha-MHC promoter, an MLC-2 promoter, or a cTnT promoter.

[0208] The polynucleotide encoding the gene product is operably linked to a promoter and / or enhancer to facilitate expression of the gene product. Depending on the host / vector system utilized, any number of suitable transcriptional and translational control elements may be used in the rAAV virion, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, and the like (see, for example, Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0209] Separate promoters and / or enhancers can be used for each of the polynucleotides. In some embodiments, the same promoter and / or enhancer is used for two or more polynucleotides in a single open reading frame. Vectors using this configuration of genetic elements are called "polycistronic". An illustrative example of a polycistronic vector includes an enhancer and promoter operably linked to a single open reading frame that includes two or more polynucleotides linked by 2A region(s), whereby expression of the open reading frame co-translationally produces multiple polypeptides. Although the 2A region is believed to mediate the production of multiple polypeptide sequences through codon skipping, the present disclosure also relates to polycistronic vectors that use post-translational cleavage to produce two or more proteins of interest from the same polynucleotide. Exemplary 2A sequences, vectors, and related methods are provided in US20040265955A1, which is incorporated herein by reference.

[0210] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include CMV from retrovirus, CMV immediate early, HSV thymidine kinase, early and late SV40, long terminal repeat (LTR) from retrovirus, and mouse metallothionein-I. In some embodiments, a promoter capable of conferring cardiac specific expression is used. Non-limiting examples of suitable cardiac specific promoters include desmin (Des), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT), and cardiac troponin C (cTnC). Non-limiting examples of suitable neuron specific promoters include synapsin I (SYN), calcium / calmodulin dependent protein kinase II, tubulin alpha I, neuron specific enolase and platelet derived growth factor beta chain promoters, and hybrid promoters by fusing the cytomegalovirus enhancer (E) to these neuron specific promoters.

[0211] Examples of promoters suitable for driving expression of reprogramming factors include, but are not limited to, retroviral long terminal repeat (LTR) elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, β-actin, phosphoglycerol kinase (PGK); inducible promoters such as promoters containing Tet-operator elements; cardiac specific promoters such as desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT), and cardiac troponin C (cTnC); neural specific promoters such as nestin, neuronal nuclear (NeuN), microtubule associated protein 2 (MAP2), beta III tubulin, neuron specific enolase (NSE), oligodendrocyte lineage (Oligl / 2), and glial fibrillary acidic protein (GFAP); and pancreatic specific promoters such as Pax4, Nkx2.2, Ngn3, insulin, glucagon, and somatostatin.

[0212] In some embodiments, the polynucleotide is operably linked to a cell type-specific transcriptional regulatory element (TRE), the TRE comprising a promoter and an enhancer. Suitable TREs include, but are not limited to, TREs from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. NY Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) PNAS USA 89:4047-4051.

[0213] The promoter can be the promoter naturally associated with the gene or nucleic acid segment. Similarly, for RNA (e.g., microRNA), the promoter can be the promoter naturally associated with the microRNA gene (e.g., miRNA-302 gene). Such naturally associated promoters can be called "natural promoters" and can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. Similarly, the enhancer can be the enhancer naturally associated with the nucleic acid sequence. However, the enhancer can be located either downstream or upstream of the sequence.

[0214] Alternatively, certain advantages are obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid in its natural environment. Recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers that are "not naturally occurring", i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences may be produced using nucleic acid amplification techniques, including recombination cloning and / or PCR™, with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each of which is incorporated herein by reference).

[0215] The promoter used can be constitutive, inducible, development-specific, tissue-specific, and / or can be useful under appropriate conditions to direct high-level expression of nucleic acid segment.For example, the promoter can be a constitutive promoter, such as CMV promoter, CMV cytomegalovirus immediate early promoter, CAG promoter, EF-1α promoter, HSV1-TK promoter, SV40 promoter, β-actin promoter, PGK promoter, or combinations thereof.Examples of eukaryotic cell promoters that can be used include, but are not limited to, constitutive promoters, such as viral promoters, such as CMV, SV40, and RSV promoters, and regulated promoters, such as inducible or repressible promoters, such as tet promoter, hsp70 promoter, and synthetic promoters regulated by CRE.In certain embodiments, cell type-specific promoters are used to drive the expression of reprogramming factors in specific cell types. Examples of suitable cell type specific promoters useful for the methods described herein include, but are not limited to, the synthetic macrophage specific promoter described in He et al (2006), Human Gene Therapy 17:949-959; the granulocyte and macrophage specific lysozyme M promoter (see, e.g., Faust et al (2000), Blood 96(2):719-726); and the myeloid specific CD11b promoter (see, e.g., Dziennis et al (1995), Blood 85(2):319-329). Other examples of promoters that can be used include the human EF1α elongation factor promoter, the CMV cytomegalovirus immediate early promoter, the CAG chicken albumin promoter, viral promoters associated with any of the viral vectors described herein, or promoters that are homologous to any of the promoters described herein (e.g., from another species).Examples of prokaryotic promoters that can be used include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters.

[0216] In some embodiments, an internal ribosome entry site (IRES) element can be used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, Nature 334(6180):320-325 (1988)). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis viruses) have been described (Pelletier and Sonenberg, Nature 334(6180):320-325 (1988)), as well as IRESs from mammalian messages (Macejak & Samow, Nature 353:90-94 (1991)). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed together to create polycistronic messages. The IRES element allows each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see, e.g., U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0217] In some embodiments, the nucleotide sequence is operably linked to a polyadenylation sequence.Suitable polyadenylation sequences include bovine growth hormone polyA signal (bGHpolyA) and short polyA signal.Optionally, the rAAV vector of the present disclosure comprises a woodchuck posttranscriptional regulatory element (WPRE).In some embodiments, the polynucleotide encoding the gene product is linked by a sequence that includes a so-called self-cleaving peptide, such as the P2A peptide.

[0218] In some embodiments, the gene product comprises a site-specific endonuclease that provides site-specific knockdown of gene function, for example, the endonuclease knocks out the allele associated with cardiac disease or disorder.For example, if the dominant allele encodes a defective copy of a gene that is a cardiac structural protein and / or provides normal cardiac function when wild type, the site-specific endonuclease can target the defective allele and knock out the defective allele.In some embodiments, the site-specific endonuclease is an RNA-guided endonuclease.

[0219] In addition to knocking out the defective allele, the site-specific nuclease can also be used to stimulate homologous recombination with the donor DNA that encodes a functional copy of the protein encoded by the defective allele. For example, the subject rAAV virion can be used to deliver both the site-specific endonuclease that knocks out the defective allele, the functional copy of the defective allele (or a fragment thereof), resulting in the repair of the defective allele, thereby providing the production of a functional cardiac protein (e.g., functional troponin, etc.). In some embodiments, the rAAV virion of the present invention comprises a heterologous nucleotide sequence that encodes the site-specific endonuclease and a heterologous nucleotide sequence that encodes a functional copy of the defective allele that encodes a functional cardiac protein. Functional cardiac proteins include, for example, troponin, chloride channel, etc.

[0220] Suitable site-specific endonucleases for use include, for example, zinc finger nucleases (ZFNs); meganucleases; and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are not naturally occurring and are engineered to target specific genes. Such site-specific nucleases can be engineered to cleave specific locations in the genome, and then non-homologous end joining can repair the cleavage while inserting or deleting some nucleotides. Such site-specific nucleases (also called "INDELs") then release the protein out of frame, effectively knocking out the gene. See, for example, US Patent Application Publication No. 2011 / 0301073. Suitable site-specific endonucleases include engineered meganucleases, re-engineered homing endonucleases. Suitable endonucleases include I-Tevl nuclease. Suitable meganucleases include I-Scel (see, e.g., Bellaiche et al. (1999) Genetics 152: 1037); and I-Crel (see, e.g., Heath et al. (1997) Nature Structural Biology 4: 468). Suitable site-specific endonucleases for use include the CRISPRi system and the Cas9-based SAM system.

[0221] In some embodiments, the gene product is an RNA-guided endonuclease. In some embodiments, the gene product comprises an RNA that comprises a nucleotide sequence that codes for an RNA-guided endonuclease. In some embodiments, the gene product is a guide RNA, for example, a single guide RNA. In some embodiments, the gene product is: 1) a guide RNA; and 2) an RNA-guided endonuclease. The guide RNA may comprise: a) a protein binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid. The RNA-guided endonuclease is also referred to herein as "genome editing nuclease".

[0222] An example of a suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., a class 2 CRISPR / Cas endonuclease, such as a type II, type V, or type VI CRISPR / Cas endonuclease). A suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., a class 2 CRISPR / Cas endonuclease, such as a type II, type V, or type VI CRISPR / Cas endonuclease). In some embodiments, the gene product comprises a class 2 CRISPR / Cas endonuclease. In some embodiments, the gene product comprises a class 2 type II CRISPR / Cas endonuclease (e.g., a Cas9 protein). In some embodiments, the gene product comprises a class 2 type V CRISPR / Cas endonuclease (e.g., a Cpfl protein, a C2cl protein, or a C2c3 protein). In some embodiments, the gene product comprises a class 2 type VI CRISPR / Cas endonuclease (e.g., a C2c2 protein; also referred to as a "Casl3a" protein). In some embodiments, the gene product comprises a CasX protein. In some embodiments, the gene product comprises a CasY protein.

[0223] How to use In some embodiments, the present disclosure provides a method for identifying AAV capsid proteins that cause rAAV virions to increase transduction efficiency in target cells. The method includes the steps of providing a population of rAAV virions, the rAAV genome of which comprises a library of cap polynucleotides encoding variant AAV capsid proteins; optionally contacting non-target cells with the population for a sufficient period of time to allow binding of undesired rAAV virions to the non-target cells; contacting target cells with the population for a sufficient period of time to allow transduction of cap polynucleotides into the target cells by the rAAV virions; and sequencing the cap polynucleotides from the target cells, thereby identifying AAV capsid proteins that cause increased transduction efficiency in the target cells. In some embodiments, the method further includes depleting the population of rAAV virions by contacting non-target cells with the population for a sufficient period of time to allow binding of rAAV virions to the non-target cells. Non-limiting examples of such identification methods are provided in the Examples.

[0224] The present disclosure provides a method for generating cardiomyocytes and / or cardiomyocyte-like cells in vitro using rAAV virions. Selected starter cells are transduced with rAAV and, if necessary, exposed to small molecule reprogramming factors (before, during, or after transduction) for a time and under conditions sufficient to convert the starter cells across lineage and / or differentiation boundaries to form cardiac progenitor cells and / or cardiomyocytes. In some embodiments, the starter cells are fibroblasts. In some embodiments, the starter cells express one or more markers indicative of a differentiation phenotype. The time for converting the starter cells into cardiac progenitor cells and cardiomyocytes can vary. For example, the starter cells may be incubated after treatment with one or more polynucleotides or proteins of interest until cardiac cell or cardiomyocyte markers are expressed. Such cardiac or cardiomyocyte markers may include any of the following markers: α-GATA4, TNNT2, MYH6, RYR2, NKX2-5, MEF2C, ANP, Actinin, MLC2v, MY20, cMHC, ISL1, cTNT, cTNI, and MLC2a, or any combination thereof. In some embodiments, the induced cardiomyocyte is negative for one or more neuronal cell markers. Such neuronal cell markers may include any of the following markers: DCX, TUBB3, MAP2, and ENO2.

[0225] Incubation can proceed until cardiac progenitor markers are expressed by the starting cells. Such cardiac progenitor markers include GATA4, TNNT2, MYH6, RYR2, or combinations thereof. Cardiac progenitor markers, such as GATA4, TNNT2, MYH6, RYR2, or combinations thereof, can be expressed by about 8 days, or by about 9 days, or by about 10 days, or by about 11 days, or by about 12 days, or by about 14 days, or by about 15 days, or by about 16 days, or by about 17 days, or by about 18 days, or by about 19 days, or by about 20 days after the start of incubation of cells in the composition described herein. Further incubation of cells can be performed until expression of late cardiac progenitor markers, such as NKX2-5, MEF2C, or combinations thereof, occurs.

[0226] Reprogramming efficiency can be measured as a function of cardiomyocyte marker. Such pluripotency markers include, but are not limited to, the expression of cardiomyocyte marker protein and mRNA, cardiomyocyte morphology, and electrophysiology phenotype. Non-limiting examples of cardiomyocyte markers include a-sarcoglycan, atrial natriuretic peptide (ANP), bone morphogenetic protein 4 (BMP4), connexin 37, connexin 40, crypt, desmin, GATA4, GATA6, MEF2C, MYH6, myosin heavy chain, NKX2.5, TBX5, and troponin T.

[0227] The expression of various markers specific for cardiomyocytes can be detected by conventional biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay, immunohistochemical assay, etc.). Alternatively, the expression of nucleic acids encoding cardiomyocyte-specific markers can be evaluated. The expression of cardiomyocyte-specific marker-encoding nucleic acids in cells can be confirmed by reverse transcription polymerase chain reaction (RT-PCR) or hybridization analysis, molecular biology methods that have been commonly used to amplify, detect, and analyze mRNA encoding any marker protein. Nucleic acid sequences encoding markers specific for cardiomyocytes are known and available from public databases such as GenBank. Thus, the marker-specific sequences required for use as primers or probes are easily determined.

[0228] Cardiomyocytes exhibit several cardiac-specific electrophysiological properties. One electrical feature is the action potential, a short-lived event in which the electrical potential difference between the inside and outside of each cardiac cell rises and falls according to a consistent trajectory. Another electrophysiological feature of cardiomyocytes is the Ca 2+ Transients of free Ca in the cytoplasm 2+ These characteristics are periodic fluctuations in the concentration of mitochondrial cells, which are used to regulate the contraction and relaxation of cardiomyocytes. These characteristics can be detected and evaluated to assess whether a population of cells has been reprogrammed into cardiomyocytes.

[0229] The present disclosure provides a method of delivering a gene product to a cardiac cell, such as a cardiac fibroblast. The method generally involves infecting a cardiac cell (e.g., a cardiac fibroblast) with a rAAV virion, and a gene product(s) encoded by a heterologous nucleic acid present in the rAAV virion is produced in the cardiac cell (e.g., a cardiac fibroblast). The delivery of the gene product(s) to the cardiac cell (e.g., a cardiac fibroblast) can provide treatment for a cardiac disease or disorder. The delivery of the gene product(s) to the cardiac cell (e.g., a cardiac fibroblast) can provide generation of an induced cardiomyocyte-like (iCM) cell from the cardiac fibroblast. The delivery of the gene product(s) to the cardiac cell (e.g., a cardiac fibroblast) can provide editing of the genome of the cardiac cell (e.g., a cardiac fibroblast).

[0230] In some embodiments, infection or transduction of cardiac cells (e.g., cardiac fibroblasts) is performed in vitro. In some embodiments, infection or transduction of cardiac cells (e.g., cardiac fibroblasts) is performed in vitro; the infected / transduced cardiac cells (e.g., cardiac fibroblasts) are introduced (e.g., transfused or implanted) into an individual in need thereof, e.g., directly into the cardiac tissue of an individual in need thereof. For in vitro transduction, an effective amount of rAAV virions delivered to cells is about 10 of the rAAV virions. 5 ~about 10 13 Other effective dosages can be readily established by one of ordinary skill in the art through routine testing to establish dose-response curves.

[0231] In some embodiments, infection of cardiac cells (e.g., cardiac fibroblasts) is performed in vivo. For example, in some embodiments, an effective amount of the rAAV virions of the present disclosure is administered directly to the cardiac tissue of an individual in need thereof. An "effective amount" falls within a relatively broad range that can be determined through experimentation and / or clinical trials. For example, for in vivo injection, i.e., direct injection into cardiac tissue, a therapeutically effective amount is about 10 6 ~about 10 15Approximately 10 rAAV virions 5 ~10 12 In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via vascular delivery through a coronary artery. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0232] For example, about 10 4 ~about 10 5 , about 10 5~ about 10 6 , about 10 6 ~about 10 7 , about 10 6 ~about 10 7 , about 10 7 ~about 10 8 , about 10 8 ~about 10 9 , about 10 9 ~about 10 10 、 about 10 10 ~about 10 11 , about 10 11 (to about 10 11 ), about 10 11 ~about 10 12 , about 10 12 ~about 10 13 , about 10 13 ~about 10 14 , about 10 14 ~about 10 15 Genome copies or 10 15 More than one genome copy of the rAAV virion of the present disclosure is administered to an individual, for example, directly to the individual's cardiac tissue or via another route. The number of rAAV virions administered to an individual can be expressed as viral genomes (vg) per kilogram (kg) of the individual's body weight. In some embodiments, an effective amount of the rAAV virion of the present disclosure is about 10 2 vg / kg~10 4 vg / kg, approx. 104 vg / kg~about 10 6 vg / kg, approx. 10 6 vg / kg~about 10 8 vg / kg, approx. 10 8 vg / kg~about 10 10 vg / kg, approx. 10 10 vg / kg~about 10 12 vg / kg, approx. 10 12 vg / kg~about 10 14 vg / kg, approx. 10 14 vg / kg~about 10 16 vg / kg, approx. 10 16 vg / kg~about 10 18 vg / kg or 10 18 More than vg / kg.

[0233] In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0234] In some embodiments, more than one dose (e.g., two, three, four or more doses) may be used to achieve a desired level of gene expression. In some embodiments, more than one dose is administered at various intervals, such as daily, weekly, twice monthly, monthly, every three months, every six months, yearly, etc. In some embodiments, multiple doses are administered over a period of 1-2 months, 2-4 months, 4-8 months, 8-12 months, 1-2 years, 2-5 years, or longer than 5 years.

[0235] The present disclosure provides methods for reprogramming cardiac fibroblasts to generate induced cardiomyocyte-like cells (iCMs). The methods generally involve infecting cardiac fibroblasts with a rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors.

[0236] The expression of various markers specific to cardiomyocytes is detected by conventional biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay; immunohistochemical assay, etc.). Alternatively, the expression of nucleic acids encoding cardiomyocyte-specific markers can be evaluated. The expression of cardiomyocyte-specific marker-encoding nucleic acids in cells can be confirmed by reverse transcription polymerase chain reaction (RT-PCR) or hybridization analysis, molecular biology methods that have been commonly used to amplify, detect, and analyze mRNA encoding any marker protein. Nucleic acid sequences encoding markers specific to cardiomyocytes are known and available through public databases such as GenBank; thus, the marker-specific sequences required for use as primers or probes can be easily determined.

[0237] Induced cardiomyocytes can also exhibit spontaneous contractions. Whether induced cardiomyocytes exhibit spontaneous contractions can be determined using standard electrophysiological methods (e.g., patch clamp).

[0238] In some embodiments, the induced cardiomyocytes are capable of spontaneous Ca 2+ It can show oscillations. 2+ Oscillations can be detected using standard methods, for example using any of a variety of calcium-sensitive dyes, such as intracellular Ca 2+Ion detection dyes include fura-2, bis-fura 2, indo-1, Quin-2, Quin-2 AM, benzothiaza-1, benzothiaza-2, indo-5F, Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, rhod-3, fura-4F, fura-5F, fura-6F, fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, calcium green, calcein, Fura-C18, calcium green-C18, calcium orange, calcium crimson, calcium green-5N, magnesium green, Oregon Green 488 BAPTA-1, Oregon Green 488 BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N, Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4, aequorin, dextran conjugates or any other derivatives of any of these dyes, and others (see, for example, the catalog or internet site of Molecular Probes, Eugene, as well as Nuccitelli, ed., Methods in Cell Biology, Volume 40: A Practical Guide to the Study of Calcium in Living Cells, Academic Press (1994); Lambert, ed., Calcium Signaling Protocols (Methods in Molecular Biology Volume 114), Humana Press (1999); WT Mason, ed., Fluorescent and Luminescent Probes for Biological Activity.A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed, Academic Press (1999); see also Calcium Signaling Protocols (Methods in Molecular Biology), 2005, DG Lamber, ed., Humana Press).

[0239] In some embodiments, the iCM is generated in vitro; the iCM is introduced into an individual, e.g., the iCM is implanted into cardiac tissue of an individual in need thereof. The methods of the disclosure can include infecting a population of cardiac fibroblasts in vitro to generate a population of iCMs; the population of iCMs is implanted into cardiac tissue of an individual in need thereof.

[0240] In some embodiments, the iCM is generated in vivo. For example, in some embodiments, the rAAV virion of the present disclosure comprising a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors is administered to an individual. In some embodiments, the rAAV virion is administered directly to the cardiac tissue of an individual in need thereof. In some embodiments, about 10% of the rAAV virion of the present disclosure comprising a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors is administered to an individual. 6 ~about 10 5 , about 10 5 ~about 10 9 , about 10 9 ~about 10 10 , about 10 10 ~about 10 11 , about 10 11 ~about 10 12 , about 10 12 ~about 10 13 , about 10 13 ~about 10 14 , about 10 14 ~about 10 15 genome copies, or 10 15More than one genome copy may be administered to an individual, for example, directly into the individual's cardiac tissue or via another route of administration. The number of rAAV virions administered to an individual may be expressed as viral genomes (vg) per kilogram (kg) of the individual's body weight. In some embodiments, an effective amount of the rAAV virions of the present disclosure is about 10 2 vg / kg~10 4 vg / kg, approx. 10 4 vg / kg~about 10 6 vg / kg, approx. 10 6 vg / kg~about 10 8 vg / kg, approx. 10 8 vg / kg~about 10 10 vg / kg, approx. 10 10 vg / kg~about 10 12 vg / kg, approx. 10 12 vg / kg~about 10 14 vg / kg, approx. 10 14 vg / kg~about 10 14 vg / kg, approx. 10 14 vg / kg~about 10 16 vg / kg or 10 16 vg / kg. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via vascular delivery through a coronary artery. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0241] The present disclosure provides a method of modifying ("editing") the genome of a cardiac cell. The present disclosure provides a method of modifying ("editing") the genome of a cardiac fibroblast. The present disclosure provides a method of modifying ("editing") the genome of a cardiomyocyte. The method generally involves infecting a cardiac cell (e.g., a cardiac fibroblast or a cardiomyocyte) with a rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising a nucleotide sequence encoding a genome editing endonuclease. In some embodiments, the method comprises infecting a cardiac fibroblast or a cardiomyocyte with a rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising a nucleotide sequence encoding a RNA-guided genome editing endonuclease. In some embodiments, the method comprises infecting a cardiac fibroblast or a cardiomyocyte with a rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising i) an RNA-guided genome editing endonuclease, and ii) one or more guide RNAs. In some embodiments, the method comprises infecting cardiac fibroblasts or cardiomyocytes with a rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising a nucleotide sequence encoding i) an RNA-guided genome editing endonuclease, ii) a guide RNA, and iii) a donor template DNA. Suitable RNA-guided genome editing endonucleases are described above.

[0242] In some embodiments, the step of infecting cardiac cells (e.g., cardiac fibroblasts; cardiomyocytes) is performed in vitro. In some embodiments, the step of infecting cardiac cells (e.g., cardiac fibroblasts; cardiomyocytes) is performed in vitro; the infected cardiac cells (e.g., cardiac fibroblasts) are introduced (e.g., implanted) into an individual in need thereof, e.g., directly into the cardiac tissue of an individual in need thereof. For in vitro transduction, the effective amount of rAAV virions delivered to cells is about 10 s ~about 10 13 Other effective dosages can be readily established by one of ordinary skill in the art through routine testing to establish dose-response curves.

[0243] In some embodiments, the step of infecting cardiac cells (e.g., cardiac fibroblasts; cardiomyocytes) is performed in vivo. For example, in some embodiments, an effective amount of the rAAV virions of the present disclosure is administered directly to the cardiac tissue of an individual in need thereof. An "effective amount" falls within a relatively broad range that can be determined through experimentation and / or clinical trials. For example, in the case of in vivo injection, i.e., direct injection into cardiac tissue, a therapeutically effective dose is about 10 of the rAAV virions of the present disclosure. 6 ~about 10 15 Approximately 10 rAAV virions 11 ~10 12 In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via vascular delivery through a coronary artery. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of the rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0244] For example, about 10 of the rAAV virions of the present disclosure 6 ~about 10 7 , about 10 7 ~about 10 8 , about 10 8 ~about 10 9 , about 10 9 ~about 10 10 , about 10 10 ~about 10 11 , about l0 11 ~about 10 12 , about 10 12 ~about 10 13 , about 10 13 ~about 10 14 , about 10 14 ~about 10 15 genome copies, or 10 15More than one genome copy is administered to an individual, for example, directly into the cardiac tissue of an individual. The number of rAAV virions administered to an individual can be expressed as viral genomes (vg) per kilogram (kg) of body weight of the individual. In some embodiments, an effective amount of the rAAV virions of the present disclosure is about 10 2 vg / kg~10 4 vg / kg, approx. 10 4 vg / kg~about 10 6 vg / kg, approx. 10 6 vg / kg~about 10 8 vg / kg, approx. 10 8 vg / kg~about 10 10 vg / kg, approx. 10 10 vg / kg~about 10 12 vg / kg, approx. 10 12 vg / kg~about 10 14 vg / kg, approx. 10 14 vg / kg~about 10 16 vg / kg, approx. 10 16 vg / kg~about 10 18 vg / kg or 10 18 vg / kg. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via vascular delivery through a coronary artery. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of a rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0245] In some embodiments, genome editing comprises homologous recombination repair (HDR). In some embodiments, HDR corrects the defect of endogenous target nucleic acid in cardiac fibroblasts or cardiomyocytes, and the defect is related to or causes the defect of cardiac fibroblasts or cardiomyocytes structure and / or function or the defect of cardiac fibroblasts or cardiomyocytes components.

[0246] In some embodiments, genome editing comprises non-homologous end joining (NHEJ).In some embodiments, NHEJ causes the endogenous target nucleic acid defect in cardiac fibroblasts or cardiomyocytes to be deleted, and the defect is related to or causes the structural and / or functional defect of cardiac fibroblasts or cardiomyocytes or the defect of components of cardiac fibroblasts or cardiomyocytes.

[0247] The disclosed method for editing the genome of cardiac cells can be used to correct any of a variety of genetic defects that cause cardiac disease or disorders. Mutations of interest include mutations in one or more of the following genes: 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-dependent channel, KQT-like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3). Particular mutations of interest include, but are not limited to, MYH7 R663H mutation; TNNT2 R173W; PKP2 2013delC mutation; PKP2 Q617X mutation; and KCNQ1 G269S missense mutation.Mutations of interest include mutations in one or more of the following genes: MYH6, ACTN2, SERCA2, GATA4, TBX5, MYOCD, NKX2-5, NOTCH1, MEF2C, HAND2, and HAND1.In some embodiments, mutations of interest include mutations in the following genes: MEF2C, TBX5, and MYOCD.Heart diseases and disorders that can be treated by the method of the present disclosure include coronary heart disease, cardiomyopathy, pericarditis, congenital cardiovascular defects, and congestive heart failure. Cardiac diseases and disorders that can be treated by the methods of the present disclosure include hypertrophic cardiomyopathy; valvular heart disease; myocardial infarction; congestive heart failure; long QT syndrome; atrial arrhythmias; ventricular arrhythmias; diastolic heart failure; systolic heart failure; valvular heart disease; valvular calcification; left ventricular noncompaction; ventricular septal defect; and ischemia.

[0248] Treatment Method The present disclosure provides a method of treating a cardiac condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a rAAV virion, wherein the rAAV virion transduces cardiac tissue.

[0249] Subjects in need of treatment using the compositions and methods of the present disclosure include, but are not limited to, individuals with congenital heart defects, individuals suffering from myodegenerative diseases, individuals suffering from conditions that lead to ischemic heart tissue (e.g., individuals with coronary artery disease), etc. In some examples, the method is useful for treating myodegenerative diseases or conditions (e.g., familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease that leads to ischemic cardiomyopathy). In some examples, the methods of the invention are useful for treating individuals with a cardiac or cardiovascular disease or disorder, such as cardiovascular disease, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease coronary, artery disease dilated, systolic dysfunction, endocarditis, high blood pressure (hypertension), cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease resulting in ischemic cardiomyopathy, mitral valve prolapse, myocardial infarction (heart attack), or venous thromboembolism.

[0250] Subjects suitable for treatment using the compositions, cells, and methods of the present disclosure include individuals (e.g., mammalian subjects, e.g., humans, non-human primates, domestic mammals, experimental non-human mammalian subjects, e.g., mice, rats, etc.) having a cardiac condition, including but not limited to conditions that result in ischemic cardiac tissue (e.g., individuals having coronary artery disease), and the like.

[0251] In some cases, the individual suitable for treatment suffers from heart or cardiovascular disease or condition, such as cardiovascular disease, aneurysm, angina pectoris, arrhythmia, atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, coronary valve disease, arterial dilatation, diastolic dysfunction, endocarditis, high blood pressure (hypertension), cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease leading to ischemic cardiomyopathy, mitral valve prolapse, myocardial infarction (heart attack), or venous thromboembolism.In some cases, the individual suitable for treatment by the method of the present invention includes the individual with myodegenerative disease, such as familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease leading to ischemic cardiomyopathy.

[0252] For example, the cardiac condition can be selected from the group consisting of congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis, and arrhythmia. In some embodiments, the subject is diabetic. In some embodiments, the subject is non-diabetic. In some embodiments, the subject suffers from diabetic cardiomyopathy.

[0253] For treatment, the rAAV virions and / or pharmaceutical compositions of the present disclosure can be administered locally or systemically. The rAAV virions can be introduced by injection, catheter, implantable device, and the like. The rAAV virions can be administered in any physiologically acceptable excipient or carrier that does not adversely affect cells. For example, the rAAV virions and / or pharmaceutical compositions of the present disclosure can be administered through an intravenous or intracardiac route (e.g., intraepicardially or intramyocardially). Methods of administering the rAAV virions and / or pharmaceutical compositions of the present disclosure to a subject, particularly a human subject, include injection or infusion of a pharmaceutical composition (e.g., a composition comprising rAAV virions). Injection may include direct intramuscular injection, and infusion may include intravenous infusion. The rAAV virions or pharmaceutical compositions can be inserted into a delivery device that facilitates introduction by injection into the subject. Such delivery devices include tubes, e.g., catheters, for injecting cells or fluids into the body of a recipient subject. The tubes may further include needles, e.g., syringes, through which the cells of the present invention can be introduced into the subject at a desired location.

[0254] In some embodiments, the rAAV virion is administered by subcutaneous, intravenous, intramuscular, intraperitoneal, or intracardiac injection or intracardiac catheterization. In some embodiments, the rAAV virion is administered by direct intramyocardial injection or transvascular administration. In some embodiments, the rAAV virion is administered by direct intramyocardial injection, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculation delivery (MCARD).

[0255] rAAV virions can be inserted into such delivery devices in different forms, for example, syringes. rAAV virions can be provided in the form of pharmaceutical compositions. Such compositions can include isotonic excipients prepared under sufficiently sterile conditions for administration in humans. For general principles in pharmaceutical preparations, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The choice of excipients and any accompanying components of the composition can be adapted to optimize administration by the route and / or device used.

[0256] Recombinant AAV can be administered locally or systemically. Recombinant AAV can be engineered to target specific cell types by selecting the appropriate capsid protein of the present disclosure. To determine the suitability of various therapeutic administration regimens and dosages of AAV virion compositions, rAAV virions can first be tested in suitable animal models. At one level, recombinant AAV is evaluated for its ability to infect target cells in vivo. Recombinant AAV can also be evaluated for whether it migrates to target tissues, whether they induce immune responses in the host, or to ensure the determination of the appropriate number or dosage of rAAV virions to be administered. Depending on the disease that the recombinant AAV is to treat, it may or may not be desirable to generate an immune response. In general, if repeated administration of virions is required, it is advantageous for the virions to be non-immunogenic. For testing purposes, rAAV virion compositions can be administered to immunodeficient animals (e.g., nude mice, or animals that have been rendered immunodeficient by chemical or radiation irradiation). The target tissue or cells are harvested after the infection period and evaluated to determine whether the tissue or cells are infected and whether the desired phenotype (e.g., induced cardiomyocytes) has been induced in the target tissue or cells.

[0257] Recombinant AAV virions can be administered by various routes, including but not limited to direct injection into the heart or cardiac catheterization. Alternatively, rAAV virions can be administered systemically, such as by intravenous infusion. When using direct injection, it can be performed by either open chest surgery or minimally invasive surgery. In some embodiments, the recombinant virus is delivered to the pericardial cavity by injection or infusion. The injected or injected recombinant virus can be tracked by a variety of methods. For example, recombinant AAV that is labeled by or expresses a detectable label (e.g., green fluorescent protein or beta-galactosidase) can be easily detected. Recombinant AAV can be engineered to cause target cells to express a marker protein, such as a surface-expressed protein or a fluorescent protein. Alternatively, infection of target cells by recombinant AAV can be detected by its expression of a cell marker that is not expressed by the animal used for testing (e.g., a human-specific antigen when cells are injected into a laboratory animal). The presence and phenotype of target cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), or by immunohistochemistry (e.g., using antibodies against human antigens), ELISA (using antibodies against human antigens), or by RT-PCR analysis using primers and hybridization conditions that allow amplification specific for RNA indicative of the cardiac phenotype.

[0258] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising the rAAV virion of the present disclosure. The pharmaceutical composition may include one or more of pharmaceutically acceptable carriers, diluents, excipients, and buffers. In some embodiments, the pharmaceutically acceptable carriers, diluents, excipients, or buffers are suitable for use in humans. Such excipients, carriers, diluents, and buffers include any agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts, such as salts of inorganic acids, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like, may be included therein. In addition, auxiliary substances, such as pH buffer substances, may be present in such media. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd and is fully described in a variety of publications, including ed. Amer. Pharmaceutical Assoc.

[0259] To prepare the composition, rAAV virions are produced and purified if necessary or desired. The rAAV can be mixed with or suspended in a pharma- ceutically acceptable carrier. These rAAV can be adjusted to an appropriate concentration and combined with other agents if necessary. The concentration of rAAV virions and / or other agents in a unit dose can vary widely. The dose and number of administrations can be optimized by those skilled in the art. For example, about 10 2 ~10 10 In some embodiments, the dose is at least about 10 2 vg, about 10 3 vg, about 10 4 vg, about 10 5 vg, about 10 6 vg, about 10 7 vg, about 10 8 vg, about 10 9 vg, about 10 10 vg, or more vector genomes. The daily dose of the compound may vary as well. Such a daily dose may be, for example, at least about 10 2 vg / day, about 10 3 vg / day, about 10 4 vg / day to about 10 5 vg / day, about 10 6 vg / day, about 10 7 vg / day, about 10 8 vg / day, about 10 9 vg / day, about 10 10 vg / day, or even more vector genomes / day.

[0260] In certain embodiments, the method of treatment is augmented by administration of one or more anti-inflammatory agents, such as an anti-inflammatory steroid or a nonsteroidal anti-inflammatory drug (NSAID).

[0261] Anti-inflammatory steroids for use in the present invention include corticosteroids, particularly steroids with glucocorticoid activity, such as dexamethasone and prednisone. Nonsteroidal anti-inflammatory drugs (NSAIDs) for use in the present invention generally act by blocking the production of prostaglandins, cyclooxygenase-1 (COX-1) and / or cyclooxygenase-2 (COX-2), which cause inflammation and pain. Traditional NSAIDs act by blocking both COX-1 and COX-2. COX-2 selective inhibitors block only the COX-2 enzyme. In certain embodiments, the NSAID is a COX-2 selective inhibitor, such as celecoxib (Celebrex®), rofecoxib (Vioxx), and valdecoxib (B extra). In certain embodiments, the anti-inflammatory agent is an NSAID prostaglandin inhibitor, such as piroxicam.

[0262] The amount of rAAV virion used for treatment will vary not only depending on the specific carrier selected, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient. Ultimately, the attending medical provider may determine the appropriate dosage. The pharmaceutical composition may be formulated with the appropriate ratio of each compound in a single unit dosage form for administration with or without cells. The cells or vectors may be provided separately and mixed with the liquid solution of the compound composition, or may be administered separately.

[0263] The recombinant AAV may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion), and may be presented in unit dosage form in ampoules, prefilled syringes, low-volume injection containers, or multi-dose containers with added preservatives. The pharmaceutical compositions may take the form of suspensions, solutions, emulsions in oily or aqueous media, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Suitable carriers include saline solutions, phosphate-buffered saline, and other materials commonly used in the art.

[0264] The compositions may also contain other ingredients, such as agents useful for the treatment of cardiac diseases, conditions, and injuries, for example, anticoagulants (e.g., dalteparin (fragmin), danaparoid (orgaran), enoxaparin (lovenox), heparin, tinzaparin (innohep), and / or warfarin (coumadin)), antiplatelet agents (e.g., aspirin, ticlopidine, clopidogrel, or dipyridamole), angiotensin-converting enzyme inhibitors (e.g., benazepril (Lotensin), captopril (Capoten), enalapril (Vasotec), fosinopril (Monopril), lisinopril (Prinivil, Zestril), moexipril (Univasc), perindopril, and the like). pril (Aceon), quinapril (Accupril), ramipril (Altace), and / or trandolapril (Mavik)), angiotensin II receptor blockers (e.g., candesartan (Atacand), eprosartan (Teveten), irbesartan (Avapro), losartan (Cozaar), telmisartan (Micardis), and / or valsartan (Diovan)), beta-blockers (e.g., acebutolol (Sectral), atenolol (Tenormin), betaxolol (Kerlone), bisoprolol / hydrochlorothiazide (Ziac), bisoprolol (Zebeta), carteolol (Cartrol), metoprolol (Lopressor, ToprolXL), nadolol (Corgard), propranolol (Inderal), sotalol (Betapace), and / or timolol (Blocadren)), calcium channel blockers (e.g., amlodipine (Norvasc, Lotrel), bepridil (Vascor), diltiazem (Cardizem, Tiazac), felodipine (Plendil), nifedipine (Adalat, Procardia), nimodipine (Nimotop), nisoldipine (Sular), verapamil (Calan, Isoptin, Verelan), diuretics (e.g., amiloride (Midamor), bumeta Nido (Bumex), chlorothiazide (Diuril), chlorthalidone (Hygroton), furosemide (Lasix), hydrochlorothiazide (Esidrix, Hydrodiuril), indapamide (Lozol), and / or spironolactone (Aldactone)), vasodilators (e.g., isosorbide dinitrate (Isordil), nesiritide (Natrecor), hydrazine (Apresoline), nitrates and / or minoxidil), statins, nicotinic acid, gemfibrozil, clofibrate, digoxin, digitoxin, lanoxin, or any combination thereof.

[0265] Additional agents may be included, such as antibacterial agents, antimicrobial agents, antiviral agents, biological response modifiers, growth factors, immune modulators, monoclonal antibodies, and / or preservatives. The compositions of the invention may also be used in conjunction with other forms of therapy.

[0266] The rAAV virions described herein can be administered to a subject to treat a disease or disorder. Such compositions can be single doses, multiple doses, in a continuous or intermittent manner, depending, for example, on the physiological state of the recipient, whether the purpose of administration is in response to a traumatic injury or for a more sustained therapeutic purpose, and other factors known to the skilled practitioner. Administration of the compounds and compositions of the invention can be essentially continuous over a preselected period of time, or can be a series of spaced doses. Both local and systemic administration is contemplated. In some embodiments, local delivery of rAAV virions is achieved. In some embodiments, local delivery of rAAV virions is used to generate a cell population in the heart. In some embodiments, such a local population acts as a "pacemaker cell" for the heart. In some embodiments, rAAV virions are used to generate, regenerate, repair, replace, and / or rejunevate one or more of the sinoatrial (SA) node, the atrioventricular (AV) node, a bindle of His, and / or Purkinje fibers.

[0267] To control isotonicity, the aqueous pharmaceutical composition may contain a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, and calcium chloride.

[0268] The composition may include one or more buffers. Typical buffers include phosphate buffers, Tris buffers, borate buffers, succinate buffers, histidine buffers, or citrate buffers. Buffers are typically included at concentrations ranging from 5 to 20 mM. The pH of the composition is generally between 5 and 8, more typically between 6 and 8, for example between 6.5 and 7.5, or between 7.0 and 7.8.

[0269] The composition is preferably sterile.The composition is preferably gluten free.The composition is preferably non-pyrogenic.

[0270] In some embodiments, the composition comprising the cells may include a cryoprotectant, non-limiting examples of which include glycols (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combination thereof.

[0271] One or more of the following types of compounds can also be present in a composition with the rAAV virions: WNT agonists, GSK3 inhibitors, TGF-beta signaling inhibitors, epigenetic modifiers, LSD1 inhibitors, adenyl cyclase agonists, or any combination thereof.

[0272] kit A variety of kits are described herein that include any of the compositions described herein (e.g., rAAV virions). The kits may include any of the compositions described herein mixed together or packaged individually, and in dry or hydrated form. The rAAV virions and / or other agents described herein may be packaged individually in separate vials, bottles, or other containers. Alternatively, the rAAV virions and / or any of the agents described herein may be packaged together as a single composition, or as two or more compositions that can be used together or individually. The compounds and / or agents described herein may be packaged in appropriate ratios and / or amounts to facilitate the conversion of selected cells across differentiation boundaries to form cardiac progenitor cells and / or cardiomyocytes.

[0273] The kits may include instructions for administering the compositions, compounds, and / or agents. Such instructions may provide the information described throughout this application. The rAAV virions or pharmaceutical compositions may be provided in any kit in the form of a delivery device. Alternatively, the delivery device may be included separately in the kit, and the instructions may describe how to assemble the delivery device prior to administration to a subject.

[0274] Any kit may include syringes, catheters, scalpels, sterile containers for sample or cell collection, diluents, pharma- ceutically acceptable carriers, etc. The kit may provide other factors, such as any of the cofactors or drugs described herein with respect to the compositions in preceding sections or elsewhere in this application.

[0275] Numbered embodiments Embodiment I-1. A recombinant adeno-associated virus (rAAV) virion comprising: a) an AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product , rAAV virions.

[0276] Embodiment I-2. A rAAV virion according to embodiment I-1, which exhibits increased transduction efficiency in cardiac cells compared to an AAV virion containing the parent sequence.

[0277] Embodiment I-3. A rAAV virion as described in embodiment I-1, which exhibits increased transduction efficiency in human cardiac fibroblasts (hCFs) compared to an AAV virion containing the parent sequence.

[0278] Embodiment I-4. The rAAV virion of embodiment I-3, which exhibits at least a 10-fold increase in transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000.

[0279] Embodiment I-5. The rAAV virion of embodiment I-3, which exhibits at least a two-fold increase in transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000.

[0280] Embodiment I-6. A rAAV virion as described in embodiment I-1, which exhibits increased transduction efficiency in induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) compared to an AAV virion containing the parental sequence.

[0281] Embodiment I-7. The rAAV virion of embodiment I-6, which exhibits at least a 2-fold increase in transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000.

[0282] Embodiment I-8. The rAAV virion of embodiment I-6, which exhibits at least a four-fold increase in transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000.

[0283] Embodiment I-9. A rAAV virion described in embodiment I-1, which exhibits increased selectivity of the rAAV virion for hCF cells over iPS-CM cells.

[0284] Embodiment I-10. A rAAV virion as described in embodiment I-1, which exhibits increased selectivity of the rAAV virion for iPS-CM cells over hCF cells.

[0285] Embodiment I-11. The rAAV virion of embodiment I-1, wherein the insertion site is between any two, optionally adjacent, amino acids in the parent sequence corresponding to positions 560 to 594 of SEQ ID NO:1.

[0286] Embodiment I-12. The rAAV virion of embodiment I-11, wherein the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1.

[0287] Embodiment I-13. The insertion has the formula: -X0-(X) n -X10 - (In the formula, n is 5 to 9, X0 and X 10 are each independently A, S, G, or absent (SEQ ID NO: 1101). The rAAV virion of embodiment I-12, wherein the peptide is

[0288] Embodiment I-14. The insertion has the formula: -X0-X1-X2-X3-X4-X5-X6-X7-X8- (Wherein, X0 and X8 are each independently A, S, G, or absent) (SEQ ID NO: 1102). The rAAV virion of embodiment I-12, wherein the peptide is

[0289] Embodiment I-15. The insertion has the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (In the formula: a) X1 is P, R, or G; b) X2 is K, L, or R; c) X3 is any amino acid; d) X4 is N, H, K, or Q; e) X5 is any amino acid, or optionally X5 is G, K, or S; f) X6 is any amino acid, or optionally, X6 is T or V; g) X7 is any amino acid or, optionally, X7 is K or V (SEQ ID NO: 1103). The rAAV virion of embodiment I-12, wherein the peptide is

[0290] The rAAV virion of embodiment I-14, wherein the insertion comprises an amino acid sequence selected from SEQ ID NOs: 101-600.

[0291] Embodiment I-17. The rAAV virion of embodiment I-14, wherein the insertion comprises an amino acid sequence selected from RKVHIEV (SEQ ID NO:81), RKYQSDL (SEQ ID NO:82), PLTNTVK (SEQ ID NO:83), LKYHGPP (SEQ ID NO:84), RKYQGDM (SEQ ID NO:85), RKFHSTD (SEQ ID NO:86), RKHHGLE (SEQ ID NO:87), PGTNVTK (SEQ ID NO:88), RKMHMPD (SEQ ID NO:89), PLKKIVQ (SEQ ID NO:90), PLGKKTS (SEQ ID NO:91), PRGVKVT (SEQ ID NO:92), PLAKSKS (SEQ ID NO:93), PRTKGAV (SEQ ID NO:94), and PSGRKAT (SEQ ID NO:95).

[0292] Embodiment I-18. The rAAV virion of embodiment I-14, wherein the insertion comprises an amino acid sequence selected from ARKVHIEVA (SEQ ID NO:101), ARKYQSDLA (SEQ ID NO:102), APLTNTVKA (SEQ ID NO:103), ALKYHGPPA (SEQ ID NO:104), ARKYQGDMA (SEQ ID NO:105), ARKFHSTDA (SEQ ID NO:106), ARKHHGLEA (SEQ ID NO:107), APGTNVTKA (SEQ ID NO:108), ARKMHMPDA (SEQ ID NO:109), APLKKIVQA (SEQ ID NO:110), APLGKKTSA (SEQ ID NO:111), APRGVKVTA (SEQ ID NO:112), APLAKSKSA (SEQ ID NO:113), APRTKGAVA (SEQ ID NO:114), APSGRKATA (SEQ ID NO:115).

[0293] Embodiment I-19. The rAAV virion of embodiment I-14, wherein the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO: 83), or a sequence containing no more than one, two, three, or four amino acid substitutions compared to PLTNTVK (SEQ ID NO: 83).

[0294] Embodiment I-20. The rAAV virion of embodiment I-14, wherein the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO:90), or a sequence containing no more than one, two, three, or four amino acid substitutions compared to PLKKIVQ (SEQ ID NO:90).

[0295] Embodiment I-21. An AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence.

[0296] Embodiment I-22. The AAV5 capsid protein of embodiment I-21, wherein the AAV5 capsid protein exhibits increased transduction efficiency in cardiac cells compared to AAV virions comprising the parent sequence.

[0297] Embodiment I-23. The AAV5 capsid protein of embodiment I-21, wherein the AAV5 capsid protein exhibits increased transduction efficiency in human cardiac fibroblasts (hCF) compared to AAV virions containing the parental sequence.

[0298] Embodiment I-24. The AAV5 capsid protein of embodiment I-23, wherein the AAV5 capsid protein exhibits at least a 10-fold increase in transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000.

[0299] Embodiment I-25. The AAV5 capsid protein of embodiment I-23, wherein the AAV5 capsid protein exhibits at least a 2-fold increase in transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000.

[0300] Embodiment I-26. The AAV5 capsid protein of embodiment I-21, wherein the AAV5 capsid protein exhibits increased transduction efficiency in induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) compared to AAV virions containing the parental sequence.

[0301] Embodiment I-27. The AAV5 capsid protein of embodiment I-26, wherein the AAV5 capsid protein exhibits at least a 2-fold increase in transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000.

[0302] Embodiment I-28. The AAV5 capsid protein of embodiment I-26, wherein the AAV5 capsid protein exhibits at least a 4-fold increase in transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000.

[0303] Embodiment I-29. The AAV5 capsid protein of embodiment I-21, wherein the AAV5 capsid protein exhibits increased selectivity of the AAV5 capsid protein for hCF cells over iPS-CM cells.

[0304] Embodiment I-30. The AAV5 capsid protein of embodiment I-21, wherein the AAV5 capsid protein exhibits increased selectivity of the AAV5 capsid protein for iPS-CM cells over hCF cells.

[0305] Embodiment I-31. The AAV5 capsid protein of embodiment I-21, wherein the insertion site is between any two, optionally adjacent, amino acids in the parent sequence corresponding to positions 560 to 594 of SEQ ID NO:1.

[0306] Embodiment I-32. The AAV5 capsid protein of embodiment I-21, wherein the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1.

[0307] Embodiment I-33. The insertion has the formula: -X0-(X) n -X 10 - (In the formula, n is 5 to 9, X0 and X 10 are each independently A, S, G, or absent (SEQ ID NO: 1101). The AAV5 capsid protein of embodiment I-32, which is a peptide of

[0308] Embodiment I-34. The insertion has the formula: -X0-X1-X2-X3-X4-X5-X6-X7-X8- (Wherein, X0 and X8 are each independently A, S, G, or absent) (SEQ ID NO: 1102). The AAV5 capsid protein of embodiment I-32, which is a peptide of

[0309] Embodiment I-35. The insertion is of the formula: -A-X1-X2-X3-X4-X5-X6-X7-A- (In the formula: a) X1 is P, R, or G; b) X2 is K, L, or R; c) X3 is any amino acid; d) X4 is N, H, K, or Q; e) X5 is any amino acid, or optionally X5 is G, K, or S; f) X6 is any amino acid, or optionally, X6 is T or V; g) X7 is any amino acid or, optionally, X7 is K or V (SEQ ID NO: 1103). The AAV5 capsid protein of embodiment I-32, which is a peptide of

[0310] Embodiment I-36. The AAV5 capsid protein of embodiment I-34, wherein the insertion comprises an amino acid sequence selected from SEQ ID NOs: 101-600.

[0311] Embodiment I-37. The AAV5 capsid protein of embodiment I-34, wherein the insertion comprises an amino acid sequence selected from RKVHIEV (SEQ ID NO:81), RKYQSDL (SEQ ID NO:82), PLTNTVK (SEQ ID NO:83), LKYHGPP (SEQ ID NO:84), RKYQGDM (SEQ ID NO:85), RKFHSTD (SEQ ID NO:86), RKHHGLE (SEQ ID NO:87), PGTNVTK (SEQ ID NO:88), RKMHMPD (SEQ ID NO:89), PLKKIVQ (SEQ ID NO:90), PLGKKTS (SEQ ID NO:91), PRGVKVT (SEQ ID NO:92), PLAKSKS (SEQ ID NO:93), PRTKGAV (SEQ ID NO:94), and PSGRKAT (SEQ ID NO:95).

[0312] Embodiment I-38. The AAV5 capsid protein of embodiment I-34, wherein the insertion comprises an amino acid sequence selected from ARKVHIEVA (SEQ ID NO:101), ARKYQSDLA (SEQ ID NO:102), APLTNTVKA (SEQ ID NO:103), ALKYHGPPA (SEQ ID NO:104), ARKYQGDMA (SEQ ID NO:105), ARKFHSTDA (SEQ ID NO:106), ARKHHGLEA (SEQ ID NO:107), APGTNVTKA (SEQ ID NO:108), ARKMHMPDA (SEQ ID NO:109), APLKKIVQA (SEQ ID NO:110), APLGKKTSA (SEQ ID NO:111), APRGVKVTA (SEQ ID NO:112), APLAKSKSA (SEQ ID NO:113), APRTKGAVA (SEQ ID NO:114), APSGRKATA (SEQ ID NO:115).

[0313] Embodiment I-39. The AAV5 capsid protein of embodiment I-34, wherein the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO: 83), or a sequence containing no more than 1, 2, 3, or 4 amino acid substitutions compared to PLTNTVK (SEQ ID NO: 83).

[0314] Embodiment I-40. The AAV5 capsid protein of embodiment I-34, wherein the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO: 90), or a sequence containing no more than one, two, three, or four amino acid substitutions compared to PLKKIVQ (SEQ ID NO: 90).

[0315] Embodiment I-41. A polynucleotide encoding an AAV5 capsid protein according to any one of embodiments I-21 to I-40.

[0316] Embodiment I-42. A pharmaceutical composition comprising a rAAV virion according to any one of embodiments I-1 to I-20.

[0317] Embodiment I-43. A method for transducing cardiac cells, comprising contacting cardiac cells with a rAAV virion described in any one of embodiments I-1 to I-20, wherein the rAAV virion transduces the cardiac cells.

[0318] Embodiment I-44. A method of delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with a rAAV virion described in any one of embodiments I-1 to I-20, wherein the cardiac cell expresses the gene product.

[0319] Embodiment I-45. The method of embodiment I-44, wherein the one or more gene products comprise a short hairpin RNA or a microRNA.

[0320] Embodiment I-46. The method of embodiment I-44, wherein one or more gene products comprising a guide RNA, a Cas protein, and optionally a repair template, said gene products form a functional CRISPR / Cas system.

[0321] Embodiment I-47. The method of embodiment I-44, wherein the one or more gene products comprise a polypeptide.

[0322] Embodiment I-48. The polypeptide optionally comprises: cardiac troponin T; cardiac sarcomeric protein; beta-myosin heavy chain; ventricular myosin essential light chain 1; ventricular myosin regulatory light chain 2; cardiac alpha-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); sarcoplasmic reticulum calcium-ATPase 2a The method of embodiment I-47, wherein the compound is selected from the group consisting of: (SERCA2a); stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminal); 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); apolipoprotein-E (ApoE); and superoxide dismutase (SOD); S100A1.

[0323] Embodiment I-49. The method of embodiment I-44, wherein the one or more gene products comprise one or more reprogramming factors.

[0324] Embodiment I-50. The method of embodiment I-49, wherein the one or more reprogramming factors are one or more polypeptides selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2.

[0325] Embodiment I-51. The method of any one of embodiments I-43 to I-50, wherein cardiac cells are reprogrammed into cardiac cardiomyocytes.

[0326] Embodiment I-52. A method for treating a cardiac condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in embodiment I-42, wherein the rAAV virions transduce cardiac tissue.

[0327] Embodiment I-53. The method of embodiment I-52, wherein the rAAV virions are administered by subcutaneous, intravenous, epicardial, intramuscular, intraperitoneal or intracardiac injection, or by intracardiac catheterization.

[0328] The method of embodiment I-52, wherein the rAAV virions are administered by direct intramyocardial injection or intravascular administration.

[0329] Embodiment I-55. The method of embodiment I-52, wherein the rAAV virion is administered by direct intramyocardial injection, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculatory delivery (MCARD).

[0330] Embodiment I-56. A method of identifying an AAV capsid protein that confers increased transduction efficiency to rAAV virions in a target cell, comprising: a) providing a population of rAAV virions, the rAAV genome comprising a library of cap polynucleotides encoding variant AAV capsid proteins; b) contacting the target cells with the population for a period of time sufficient to allow transduction of the cap polynucleotide into the target cells by the rAAV virions; and c) sequencing the cap polynucleotide from the target cells, thereby identifying AAV capsid proteins that confer increased transduction efficiency in the target cells. A method comprising:

[0331] Embodiment I-57. The method of embodiment I-56, further comprising, prior to step (b), depleting the population of rAAV virions by contacting the population with non-target cells for a period of time sufficient to allow binding of the rAAV virions to the non-target cells.

[0332] Embodiment I-58. A recombinant adeno-associated virus (rAAV) virion comprising: a) an AAV5 capsid protein that contains, relative to a parent AAV5 capsid, one or more substitutions selected from S651A, T578A, or T582A; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product , rAAV virions.

[0333] Embodiment I-59. The rAAV virion of embodiment I-58, wherein the AAV5 capsid protein comprises two or more substitutions selected from S651A, T578A or T582A.

[0334] Embodiment I-60. The rAAV virion of embodiment I-58, wherein the AAV5 capsid protein comprises a T582A and a S651A substitution.

[0335] Embodiment I-61. The rAAV virion of embodiment I-58, wherein the AAV5 capsid protein comprises a T578A and a T582A substitution.

[0336] Embodiment I-62. The rAAV virion of embodiment I-58, wherein the AAV5 capsid protein comprises a T578A and a S651A substitution.

[0337] Embodiment I-63. The rAAV virion of embodiment I-58, wherein the AAV5 capsid protein comprises S651A, T578A, and T582A substitutions.

[0338] Embodiment I-64. The rAAV virion of any one of embodiments I-58 to I-62, wherein the AAV5 capsid protein shares at least 95% identity with SEQ ID NO:73.

[0339] Embodiment I-65. The rAAV virion of any one of embodiments I-58 to I-62, wherein the AAV5 capsid protein shares at least 98% identity with SEQ ID NO:73.

[0340] Embodiment I-66. The rAAV virion of any one of embodiments I-58 to I-62, wherein the AAV5 capsid protein shares at least 99% identity with SEQ ID NO:73.

[0341] Embodiment I-67. The rAAV virion of any one of embodiments I-58 to I-62, wherein the AAV5 capsid protein comprises the polypeptide sequence SEQ ID NO: 73.

[0342] Embodiment I-68. An AAV5 capsid protein comprising one or more substitutions selected from S651A, T578A, or T582A compared to the parent AAV5 capsid.

[0343] Embodiment I-69. The AAV5 capsid protein of embodiment I-67, wherein the capsid protein comprises two or more substitutions selected from S651A, T578A or T582A.

[0344] Embodiment I-70. The AAV5 capsid protein of embodiment I-67, wherein the capsid protein comprises a T582A and a S651A substitution.

[0345] Embodiment I-71. The AAV5 capsid protein of embodiment I-68, wherein the capsid protein comprises a T578A and a T582A substitution.

[0346] Embodiment I-72. The AAV5 capsid protein of embodiment I-68, wherein the capsid protein comprises a T578A and a S651A substitution.

[0347] Embodiment I-73. The AAV5 capsid protein of embodiment I-68, wherein the capsid protein comprises S651A, T578A, and T582A substitutions.

[0348] Embodiment I-74. The AAV5 capsid protein of any one of embodiments I-68 to I-73, wherein the capsid protein shares at least 90% identity with SEQ ID NO: 73.

[0349] Embodiment I-75. The AAV5 capsid protein of any one of embodiments I-68 to I-73, wherein the capsid protein shares at least 95% identity with SEQ ID NO:73.

[0350] Embodiment I-76. An AAV5 capsid protein according to any one of embodiments I-68 to I-73, sharing at least 98% identity with SEQ ID NO: 73.

[0351] Embodiment I-77. An AAV5 capsid protein according to any one of embodiments I-68 to I-73, sharing at least 99% identity with SEQ ID NO: 73.

[0352] Embodiment I-78. A polynucleotide encoding an AAV5 capsid protein according to any one of embodiments I-68 to I-77.

[0353] Embodiment I-79. A pharmaceutical composition comprising a rAAV virion according to any one of embodiments I-58 to I-67.

[0354] Embodiment I-80. A method for transducing cardiac cells, comprising contacting cardiac cells with a rAAV virion described in any one of embodiments I-58 to I-67, wherein the rAAV virion transduces the cardiac cells.

[0355] Embodiment I-81. A method for delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with a rAAV virion described in any one of embodiments I-58 to I-67, wherein the cardiac cell expresses the gene product.

[0356] Embodiment I-82. The method of embodiment I-81, wherein the one or more gene products comprise a short hairpin RNA or a microRNA.

[0357] Embodiment I-83. The method of embodiment I-81, wherein one or more gene products comprising a guide RNA, a Cas protein, and optionally a repair template, said gene products form a functional CRISPR / Cas system.

[0358] Embodiment I-84. The method of embodiment I-81, wherein the one or more gene products comprise a polypeptide.

[0359] Embodiment I-85. The protein is selected from the group consisting of cardiac troponin T; cardiac sarcomeric protein; beta-myosin heavy chain; ventricular myosin essential light chain 1; ventricular myosin regulatory light chain 2; cardiac alpha-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); sarcoplasmic reticulum calcium-ATPase 2a The method of embodiment I-84, wherein the enzyme is selected from: (SERCA2a); stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminal); 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); apolipoprotein-E (ApoE); and superoxide dismutase (SOD); S100A1.

[0360] Embodiment I-86. The method of embodiment I-81, wherein the one or more gene products comprise one or more reprogramming factors.

[0361] Embodiment I-87. The method of embodiment I-86, wherein the one or more reprogramming factors are one or more polypeptides selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2.

[0362] Embodiment I-88. The method of any one of embodiments I-80 to I-87, wherein cardiac cells are reprogrammed into cardiac cardiomyocytes.

[0363] Embodiment I-89. A method for treating a cardiac condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in embodiment I-79, wherein the rAAV virions transduce cardiac tissue.

[0364] The method of embodiment I-89, wherein the rAAV virions are administered by subcutaneous, epicardial, intravenous, intramuscular, intraperitoneal or intracardiac injection, or by intracardiac catheterization.

[0365] The method of embodiment I-89, wherein the rAAV virion is administered by direct intramyocardial injection or intravascular administration.

[0366] Embodiment I-91. The method of embodiment I-89, wherein the rAAV virion is administered by direct intramyocardial injection, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculatory delivery (MCARD).

[0367] The following non-limiting examples illustrate some of the experimental work involved in the development of the present invention. EXAMPLES

[0368] Example 1 Identification of capsid protein sequences Library generation and AAV selection A library screening strategy incorporating an initial negative selection step for induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) followed by a positive selection step for human cardiac fibroblasts (hCFs) was performed as shown in Figure 1. Briefly, a library of cap gene sequences was generated by PCR using primers P1 (GTCGGCGGGCAGATGGCCACCAACAACCAGGCCNNKNNKNNKNNKNNKNNKNNKGCCAGCTCCACCACTGCCCCCGCGACCGGCACG) (SEQ ID NO: 6) and P2 (TCCCCAGCATGAGCGATGCATTTTATTGAGGGTATGCGAC) (SEQ ID NO: 7) {N=A, C, G, or T; K=G or T} and cloned via Gibson assembly into NotI- and NheI-linearized pCR-AAV-LIB575 (Figure 2A). pCR-AAV-LIB575 was packaged into rAAV virions using the HEK293T packaging cell line. The vector contains the AAV2 rep gene ("Rep2") and the AAV cap gene ("Cap5") flanked by 3' and 5' inverted repeats (ITRs). A random 7-mer polypeptide library (AXXXXXXA; SEQ ID NO: 100) flanked by alanine residues is inserted into the cap gene at position 575. Figure 2B shows the structure of the AAV5 capsid, with the black arrow indicating the insertion site shown in one of the 60 copies of the capsid protein present on the assembled AAV virion. Insertion into the cap gene results in the same polypeptide being inserted into all 60 copies at the equivalent position (not shown).

[0369] Virions with multiplicities of infection ranging from 50,000 (round 1) to 1,000 (round 3) were mixed in liquid culture medium and applied to iPS-CM cells isolated from a healthy 30-year-old male. Cells were incubated for 24 h at 37 °C and 5% CO2. DNA was isolated from iPS-CM cells, amplified by PCR using primers P2 (SEQ ID NO: 7) and P3 (CCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAG) (SEQ ID NO: 8), and sequenced using an Oxford NanoPore MiniION according to the manufacturer's instructions. Sequencing read data for this negative selection step were analyzed (data not shown).

[0370] Culture medium containing rAAV virions that were not attached to or infected with iPS-CM cells was then transferred to hCF cells and incubated for 24 hours at 37°C and 5% CO2. After 24 hours, the plate was washed with culture medium to remove attached hCF cells, and PCR amplification and sequencing of the cap gene was performed on DNA isolated from hCF cells infected with rAAV as described above. Representative data for the starting library of sequences and sequences after this first round of negative and positive selection are shown in Figure 3A and Figure 3B, respectively. The complexity of the library was reduced, which is consistent with the enrichment of selected sequences, as the percentage of unique reads decreased from 97.6% to 87.5%.

[0371] The library of cap genes obtained from the first round was again subcloned into pCR-AAV-LIB575 and the same negative and positive selection procedures were performed as round 2. This was repeated as round 3. A sustained enrichment of sequences was observed by a decrease in unique read data from 58.2% to 41.5% from round 2 (Figure 3C) to round 3 (Figure 3D).

[0372] Confirmation of increased transduction efficiency in an in vitro system The number of reads for each unique sequence correlates with the increased transduction efficiency of rAAV virions containing that insert, and thus read enrichment is an empirical measure of increased transduction efficiency. The 500 sequences with the highest abundance are shown in Table 2 below.

[0373] The sequences listed in Table 1 were selected for retesting in cultured hCF cells (FIGS. 4A-4D) and iPS-CM cells (FIGS. 5A-5D) using a GFP reporter assay. [Table 1-1] [Table 1-2]

[0374] Briefly, the peptides in Table 1 were each cloned into the cap gene at the same insertion site in a Rep-Cap plasmid lacking terminal inverted repeats (ITRs) rather than the AAV genome as in the selection experiments. These Rep-Cap plasmids were each used to express Rep2 and the respective Cap variant to generate rAAVs packaging reporter AAV genomes (AAV2 ITRs flanking a GFP transgene controlled by a CAG promoter). The resulting rAAV virions containing the GFP transgene with engineered capsid proteins as capsids were plated onto adherent hCF or iPS-CM cells at an MOI of 100,000 (Figure 4A, Figure 5A, Figure 5D) or 1,000 (Figure 4D). Transduction efficiency was measured by counting GFP fluorescent cells using automated digital wide-field microscopy with a Cytation5™ cell imaging multimode reader. Representative fluorescent micrographs are shown (Figure 4B; parental AAV5 capsid - SEQ ID NO: 1) and (Figure 4C; AAV5.CR17 capsid - SEQ ID NO: 103). Transduction efficiency of 15 AAV5 variants in hCF cells (Figure 4A) was benchmarked against unmodified AAV5 (right-most bar). Darker bars represent sequences that conferred increased transformation efficiency in hCF cells in this experiment.

[0375] In iPS-CM cells (Figures 5A-5D), an increase in transduction efficiency was similarly observed, and indeed the fold enhancement of transduction compared to parental AAV5 was higher than in hCF cells (Figures 5A and 5D). Representative fluorescent images are shown for hCF transduced with either unmodified AAV5 (Figure 5B) or AAV5.CR23 (Figure 5C).

[0376] Next, the tests with human cardiac fibroblasts were repeated with either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP at high (100,000), moderate (10,000), or low (1,000) MOI in mouse cardiac fibroblasts (mCFs). In addition to an increase in the total number of GFP+ cells in tests comparing AAV5.CR17 to AAV5, we observed that a high intensity GFP population was formed by AAV5.CR17, but not AAV5 (Figure 6A). Thus, measurements of the intensity of individual cells in Figures 6B-6E were gated on cell subsets of the total population and the high GFP+ population. When cells were transduced at high, moderate, or low MOI with AAV5.CR17 compared to unmodified AAV5, an increase in percent GFP+ (Figure 6B) and GFP intensity (Figure 6C) was observed in the "total GFP" population. An increase in percent GFP+ (FIG. 6D) and GFP intensity (FIG. 6E) was also observed in the "high GFP" population when cells were transduced with AAV5.CF17 compared to unmodified AAV5. The trend in FIG. 6E was less pronounced because gating on high GFP+ cells simply selected a cell population with high intensity GFP expression.

[0377] In vivo testing Next, the AAV5.CR17 capsid was tested in healthy and diseased animals in vivo. Specifically, CD-1® IGS mice (wild-type background) purchased from Charles River® were divided into healthy and diseased groups. The diseased group was subjected to induced myocardial infarction (MI) using the ligation method of Gao et al. Circulation Research (2010) 107:1445-1453. 1.2×10 64-cell cultures of either AAV5.CAG-GFP or AAV5.CR17.CAG-GFP were incubated for 12 hours at 4°C for 1 h. 11vg was injected via intramyocardial injection into 6-week-old CD-1 mice. GFP intensity and distribution were assessed 7 days after injection. All cardiac tissue sections were imaged using a 20x objective with equal gain and exposure. Anti-GFP immunostaining and nuclear counterstaining with DAPI are shown. Cardiomyocytes and fibroblasts were identified by morphology. Increased transduction of cardiomyocytes and fibroblasts is observed in AAV5.CR17 (Figures 7E-7F and Figures 7G-7H) compared to AAV5 (Figures 7A-7B and Figures 7C-7D) in both non-MI and MI animals.

[0378] Shared sequence motifs To identify residues shared among insertions that demonstrate enrichment after selection, sequence motifs were generated using all sequencing data or the top sequences after round 3. Briefly, sequence motifs were generated for the top 100 (Figure 8A), top 50 (Figure 8B), and top 20 (Figure 8C) sequences in Table 2, as well as the sequences retested in vitro in this example (Figure 8D). Sequence motifs were also generated for all sequences using the number of reads for each sequence to weight the importance of each amino acid in the motif (Figure 8E). Sequence motifs suggest that certain amino acids are preferred at certain positions in the insertion site.

[0379] The top sequences from the library are shown below in Table 2. The adjacent "A" residues at the beginning and end of the insertion are in some embodiments replaced with another amino acid, such as a flexible amino acid (G or S), or are absent. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13]

[0380] Example 2 Rational design of AAV5 capsid proteins with increased transduction efficiency In conjunction with the library-based approach described in Example 1, a rational design approach was used to identify AAV5 capsid proteins that result in increased transduction efficiency. Surface-exposed residues were selected. Substitutions of these amino acids were tested in hCF assays as described in Example 1. Substitutions S651A, T578A, or T582A in the AAV5 capsid resulted in improved human cardiac fibroblast transduction at 72 hours post-infection at a multiplicity of infection of 100,000 vg / cell ( FIG. 9 ).

[0381] In follow-up experiments, the substitutions identified in this rational design experiment are tested in combination with each other and with the insertions disclosed in Tables 1 and 2.

[0382] Example 3 Comparative study comparing AAVx1 to wild-type AAV5 AAV5 capsids (AAVx1) were generated with two or three of the S651A, T578A, and / or T582A substitutions. The capsid sequence was as follows: [ka]

[0383] To test the ability to transduce human cardiac fibroblasts (hCF), hCF were infected with AAV5.CAG-GFP or AAVx1(T582A+S651A) at MOIs ranging from 1,000 to 1,000,000. 48 hours after infection, cells were harvested and GFP expression was quantified using flow cytometry. At all MOIs tested, percent transduction (Figure 10A), GFP expression (Figure 10B), and relative transduction (Figure 10C) were increased compared to wild-type AAV5 capsid.

[0384] The ability of AAVx1 vectors to deliver functional cargo was tested by generating AAV5 and AAVx1 vectors containing expression cassettes encoding myocardin and ASCL1 (MyA). An internal deletion of myocardin, MyΔ3, described in International Patent Application No. PCT / US2019 / 049150 and U.S. Provisional Patent Application No. 62 / 788,479, each of which is incorporated herein by reference, was used. Notably, AAVx1 vectors performed on average 9-fold better than AAV5 vectors based on transgene and marker expression at day 7 post-infection (shown in Table 3). [Table 3]

[0385] Example 4 In vivo transduction of cardiac tissue with AAVx1 Testing in mice To evaluate in vivo transduction, AAV5 or AAVx1(T582A+S651A) capsids were used to deliver an expression cassette encoding a GFP reporter to mice using a LAD-ligated mouse myocardial infarction (MI) model, with injections of AAV vectors performed epicardially at the time of injury. A single vector dose of 1.2 × 10 11 Genome copies (GC) were delivered to the heart of each subject by intracardiac injection. Seven days after injection, animals were sacrificed and their hearts were dissociated by enzymatic digestion. Dissociated cells were stained with endothelial cell markers (CD31) and fibroblast markers (Thy-1) and sorted using fluorescence-activated cell sorting (FAC). GFP+, Thy-1+, and CD31- cells were classified as transduced cardiac fibroblasts, while GFP-, Thy-1+, and CD31- cells were classified as non-transduced cardiac fibroblasts. The number of transduced cells was normalized to AAV5. As shown in Figure 11, in vivo transduction of cardiac fibroblasts (CF) was 4-fold higher using AAVx1 vectors than AAV5 vectors.

[0386] For functional in vivo studies, AAVx1 (T582A+S651A) capsids were used to deliver expression cassettes encoding myocardin and ASCL1 (MyA). AAVx1 vectors were tested in mice using the LAD ligated mouse myocardial infarction (MI) model, with injection of AAV vectors into the epicardium at the time of injury. A single vector dose of 1.2 × 10 11 Genome copies (GC) were delivered to the heart of each subject by intracardiac injection.

[0387] Efficacy was assessed by echocardiography at 2, 4, 6, and 8 weeks post-injection. The AAVx1 vector significantly preserved cardiac function (ejection fraction) compared to the GFP-encoded control (Figure 12).

[0388] Testing in pigs The AAVx1 vector was further tested in a porcine myocardial infarction (MI) model. MyA was packaged into AAVx1 capsids and injected epicardially into the border zone of the porcine heart 28 days after induction of ischemic injury by balloon occlusion for 90 minutes. In parallel, the infarcted pigs were also injected with formulation buffer, with each group consisting of 10 animals. Echocardiography was performed 3, 5, 7, and 9 weeks after injection. Pigs administered AAVx1 vectors demonstrated significant improvement in ejection fraction compared to control injections at 5, 7, and 9 weeks (Figure 13). Thus, treatment with AAVx1 vectors resulted in an average of 10% improvement in cardiac performance over pre-treatment baseline. In certain embodiments, for example, the following are provided: (Item 1) A recombinant adeno-associated virus (rAAV) virion comprising: a) an AAV5 capsid protein that contains one or more substitutions selected from S651A, S651G, S651V, S651L, S651I, T578A, T578G, T578V, T578L, T578I, T582A, T582G, T582V, T582L, or T582I compared to a parent AAV5 capsid; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product , rAAV virions. (Item 2) 3. The rAAV virion of item 2, wherein the AAV5 capsid protein comprises two or more substitutions selected from S651A or S651V; T578A or T578V; and / or T582A or T582V. (Item 3) 3. The rAAV virion of item 2, wherein the AAV5 capsid protein comprises a T582A and an S651A substitution. (Item 4) 4. The rAAV virion of item 3, wherein the AAV5 capsid protein comprises a T578A and a T582A substitution. (Item 5) 4. The rAAV virion of item 3, wherein the AAV5 capsid protein comprises a T578A and an S651A substitution. (Item 6) 4. The rAAV virion of item 3, wherein the AAV5 capsid protein comprises S651A, T578A, and T582A substitutions. (Item 7) 7. The rAAV virion of any one of items 1 to 6, wherein the AAV5 capsid protein shares at least 95% identity, at least 98% identity, or at least 99% identity with SEQ ID NO:73. (Item 8) An AAV5 capsid protein that contains one or more substitutions selected from S651A, S651G, S651V, S651L, S651I, T578A, T578G, T578V, T578L, T578I, T582A, T582G, T582V, T582L, or T582I compared to the parent AAV5 capsid. (Item 9) 9. The AAV5 capsid protein of item 8, comprising two or more substitutions selected from S651A or S651V; T578A or T578V; or T582A or T582V. (Item 10) 10. The AAV5 capsid protein of item 9, comprising a T582A and S651A substitution. (Item 11) 10. The AAV5 capsid protein of item 9, comprising a T578A and a T582A substitution. (Item 12) 10. The AAV5 capsid protein of item 9, comprising a T578A and S651A substitution. (Item 13) 10. The AAV5 capsid protein of item 9, comprising S651A, T578A, and T582A substitutions. (Item 14) 14. The AAV5 capsid protein of any one of items 8 to 13, which shares at least 90% identity with SEQ ID NO: 73. (Item 15) 14. The AAV5 capsid protein of any one of items 8 to 13, which shares at least 95% identity, at least 98% identity, or at least 99% identity with SEQ ID NO: 73. (Item 16) A polynucleotide encoding the AAV5 capsid protein of any one of items 8 to 15. (Item 17) 8. A pharmaceutical composition comprising the rAAV virion of any one of items 1 to 7. (Item 18) 8. A method for transducing cardiac cells, comprising contacting the cardiac cells with a rAAV virion described in any one of items 1 to 7, wherein the rAAV virion transduces the cardiac cells. (Item 19) 8. A method for delivering one or more gene products to a cardiac cell, comprising contacting said cardiac cell with an rAAV virion described in any one of items 1 to 7, wherein said cardiac cell expresses said gene product. (Item 20) Optionally, the one or more gene products are selected from the group consisting of cardiac troponin T; cardiac sarcomeric protein; beta-myosin heavy chain; ventricular myosin essential light chain 1; ventricular myosin regulatory light chain 2; cardiac alpha-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); sarcoplasmic reticulum calcium-ATPase 2a (SERCA The method of claim 18 or 19, comprising a polypeptide selected from the group consisting of stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminal); 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); apolipoprotein-E (ApoE); and superoxide dismutase (SOD); S100A1. (Item 21) 20. The method of claim 18 or 19, wherein the one or more gene products optionally comprise one or more reprogramming factors selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2. (Item 22) 22. The method of claim 21, wherein the cardiac cells are reprogrammed into cardiac cardiomyocytes. (Item 23) 18. A method for treating a cardiac condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of item 17, wherein the rAAV virions transduce cardiac tissue. (Item 24) 24. The method of claim 23, wherein the rAAV virion is administered by epicardial injection. (Item 25) 24. The method of claim 23, wherein the rAAV virion is administered by intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculation delivery (MCARD). (Item 26) A recombinant adeno-associated virus (rAAV) virion comprising: a) an AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product , rAAV virions. (Item 27) 27. The rAAV virion of item 26, wherein the insertion site is between any two, optionally adjacent, amino acids in the parent sequence corresponding to positions 560 to 594 of SEQ ID NO:1. (Item 28) 28. The rAAV virion of item 27, wherein the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1. (Item 29) The insertion has the formula: -X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 - (In the formula, X 0 and X 8 are each independently A, S, G, or absent (SEQ ID NO: 1102). 29. The rAAV virion of any one of items 26 to 28, wherein the peptide is (Item 30) The insertion has the formula: -AX 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -A- (In the formula: a)X 1 is P, R, or G; b) X 2 is K, L, or R; c) X 3 is any amino acid; d) X 4 is N, H, K, or Q; e)X 5 is any amino acid or, optionally, X 5 is G, K, or S; f)X 6 is any amino acid or, optionally, X 6 is T or V; g) X 7 is any amino acid or, optionally, X 7 is K or V) (SEQ ID NO: 1103) 30. The rAAV virion of item 29, wherein the peptide is (Item 31) 31. The rAAV virion of item 30, wherein the insertion comprises an amino acid sequence selected from SEQ ID NOs: 101 to 600. (Item 32) 31. The rAAV virion of claim 30, wherein the insertion comprises an amino acid sequence selected from RKVHIEV (SEQ ID NO: 81), RKYQSDL (SEQ ID NO: 82), PLTNTVK (SEQ ID NO: 83), LKYHGPP (SEQ ID NO: 84), RKYQGDM (SEQ ID NO: 85), RKFHSTD (SEQ ID NO: 86), RKHHGLE (SEQ ID NO: 87), PGTNVTK (SEQ ID NO: 88), RKMHMPD (SEQ ID NO: 89), PLKKIVQ (SEQ ID NO: 90), PLGKKTS (SEQ ID NO: 91), PRGVKVT (SEQ ID NO: 92), PLAKSKS (SEQ ID NO: 93), PRTKGAV (SEQ ID NO: 94), and PSGRKAT (SEQ ID NO: 95). (Item 33) 31. The rAAV virion of claim 30, wherein the insertion comprises an amino acid sequence selected from ARKVHIEVA (SEQ ID NO: 101), ARKYQSDLA (SEQ ID NO: 102), APLTNTVKA (SEQ ID NO: 103), ALKYHGPPA (SEQ ID NO: 104), ARKYQGDMA (SEQ ID NO: 105), ARKFHSTDA (SEQ ID NO: 106), ARKHHGLEA (SEQ ID NO: 107), APGTNVTKA (SEQ ID NO: 108), ARKMHMPDA (SEQ ID NO: 109), APLKKIVQA (SEQ ID NO: 110), APLGKKTSA (SEQ ID NO: 111), APRGVKVTA (SEQ ID NO: 112), APLAKSKSA (SEQ ID NO: 113), APRTKGAVA (SEQ ID NO: 114), and APSGRKATA (SEQ ID NO: 115). (Item 34) 31. The rAAV virion of claim 30, wherein the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO: 83), or a sequence containing at most one, two, three, or four amino acid substitutions compared to PLTNTVK (SEQ ID NO: 83). (Item 35) 31. The rAAV virion of claim 30, wherein the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO: 90), or a sequence containing at most one, two, three, or four amino acid substitutions compared to PLKKIVQ (SEQ ID NO: 90). (Item 36) 36. The rAAV virion of any one of items 26 to 35, wherein the capsid protein comprises one or more substitutions selected from S651A, T578A, or T582A compared to the parent AAV5 capsid. (Item 37) 37. The rAAV virion of item 36, wherein the AAV5 capsid protein contains two or more substitutions selected from S651A, T578A, or T582A. (Item 38) 37. The rAAV virion of item 36, wherein the AAV5 capsid protein comprises a T582A and an S651A substitution. (Item 39) 37. The rAAV virion of item 36, wherein the AAV5 capsid protein comprises a T578A and a T582A substitution. (Item 40) 37. The rAAV virion of item 36, wherein the AAV5 capsid protein comprises a T578A and an S651A substitution. (Item 41) 37. The rAAV virion of item 36, wherein the AAV5 capsid protein comprises S651A, T578A, and T582A substitutions. (Item 42) 42. The rAAV virion of any one of paragraphs 26 to 41, which exhibits increased transduction efficiency in cardiac cells compared to an AAV virion containing the parent sequence. (Item 43) 43. The rAAV virion of items 26 to 42, which exhibits increased transduction efficiency in human cardiac fibroblasts (hCF) compared to an AAV virion containing the parent sequence. (Item 44) An AAV5 capsid protein comprising an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence. (Item 45) 45. The AAV5 capsid protein of claim 44, wherein the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1. (Item 46) The insertion has the formula: -X 0 -(X) n -X 10 - (In the formula, n is 5 to 9, X 0 and X 10 are each independently A, S, G, or absent (SEQ ID NO: 1101). 46. ​​The AAV5 capsid protein according to claim 44 or 45, which is a peptide of (Item 47) The insertion has the formula: -X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8- (In the formula, X 0 and X 8 are each independently A, S, G, or absent (SEQ ID NO: 1102). 46. ​​The AAV5 capsid protein according to claim 44 or 45, which is a peptide of (Item 48) The insertion has the formula: -AX 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -A- (In the formula: a)X 1 is P, R, or G; b) X 2 is K, L, or R; c) X 3 is any amino acid; d) X 4 is N, H, K, or Q; e)X 5 is any amino acid or, optionally, X 5 is G, K, or S; f)X 6 is any amino acid or, optionally, X 6 is T or V; g) X 7 is any amino acid or, optionally, X 7 is K or V) (SEQ ID NO: 1103) 48. The AAV5 capsid protein according to item 47, which is a peptide of (Item 49) 49. The AAV5 capsid protein of item 48, wherein the insertion comprises an amino acid sequence selected from SEQ ID NOs: 101 to 600. (Item 50) 49. The AAV5 capsid protein of claim 48, wherein the insertion comprises an amino acid sequence selected from RKVHIEV (SEQ ID NO: 81), RKYQSDL (SEQ ID NO: 82), PLTNTVK (SEQ ID NO: 83), LKYHGPP (SEQ ID NO: 84), RKYQGDM (SEQ ID NO: 85), RKFHSTD (SEQ ID NO: 86), RKHHGLE (SEQ ID NO: 87), PGTNVTK (SEQ ID NO: 88), RKMHMPD (SEQ ID NO: 89), PLKKIVQ (SEQ ID NO: 90), PLGKKTS (SEQ ID NO: 91), PRGVKVT (SEQ ID NO: 92), PLAKSKS (SEQ ID NO: 93), PRTKGAV (SEQ ID NO: 94), and PSGRKAT (SEQ ID NO: 95). (Item 51) 49. The AAV5 capsid protein of claim 48, wherein the insertion comprises an amino acid sequence selected from ARKVHIEVA (SEQ ID NO: 101), ARKYQSDLA (SEQ ID NO: 102), APLTNTVKA (SEQ ID NO: 103), ALKYHGPPA (SEQ ID NO: 104), ARKYQGDMA (SEQ ID NO: 105), ARKFHSTDA (SEQ ID NO: 106), ARKHHGLEA (SEQ ID NO: 107), APGTNVTKA (SEQ ID NO: 108), ARKMHMPDA (SEQ ID NO: 109), APLKKIVQA (SEQ ID NO: 110), APLGKKTSA (SEQ ID NO: 111), APRGVKVTA (SEQ ID NO: 112), APLAKSKSA (SEQ ID NO: 113), APRTKGAVA (SEQ ID NO: 114), and APSGRKATA (SEQ ID NO: 115). (Item 52) 49. The AAV5 capsid protein of claim 48, wherein the insertion comprises the amino acid sequence PLTNTVK (SEQ ID NO: 83) or a sequence containing at most one, two, three, or four amino acid substitutions compared to PLTNTVK (SEQ ID NO: 83). (Item 53) 49. The AAV5 capsid protein of claim 48, wherein the insertion comprises the amino acid sequence PLKKIVQ (SEQ ID NO: 90) or a sequence containing at most one, two, three, or four amino acid substitutions compared to PLKKIVQ (SEQ ID NO: 90). (Item 54) 54. The AAV5 capsid protein of any one of items 44 to 53, comprising one or more substitutions selected from S651A, T578A, or T582A compared to the parent AAV5 capsid. (Item 55) 55. The AAV5 capsid protein of item 54, comprising two or more substitutions selected from S651A, T578A, or T582A. (Item 56) A polynucleotide encoding the AAV5 capsid protein of any one of items 44 to 55. (Item 57) 44. A pharmaceutical composition comprising the rAAV virion of any one of items 26 to 43. (Item 58) A method for transducing cardiac cells, comprising contacting the cardiac cells with a rAAV virion described in any one of items 26 to 43, wherein the rAAV virion transduces the cardiac cells. (Item 59) 44. A method for delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with an rAAV virion described in any one of items 26 to 43, wherein the cardiac cell expresses the gene product. (Item 60) Optionally, the one or more gene products are selected from the group consisting of cardiac troponin T; cardiac sarcomeric protein; beta-myosin heavy chain; ventricular myosin essential light chain 1; ventricular myosin regulatory light chain 2; cardiac alpha-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); sarcoplasmic reticulum calcium-ATPase 2a (SERCA 2a); stromal-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminal); 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); apolipoprotein-E (ApoE); and superoxide dismutase (SOD); The method of claim 58 or 59, comprising a polypeptide selected from the group consisting of S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7, S100A8, S100A9, S100A1 ... (Item 61) 60. The method of claim 58 or 59, wherein the one or more gene products optionally comprise one or more reprogramming factors selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2. (Item 62) 62. The method of claim 61, wherein the cardiac cells are reprogrammed into cardiac cardiomyocytes. (Item 63) 58. A method for treating a cardiac condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of item 57, wherein the rAAV virions transduce cardiac tissue. (Item 64) 64. The method of claim 63, wherein the rAAV virion is administered by epicardial injection. (Item 65) 64. The method of claim 63, wherein the rAAV virion is administered by intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculation delivery (MCARD). (Item 66) 1. A method for identifying an AAV capsid protein that confers increased transduction efficiency to a rAAV virion in a target cell, comprising: a) providing a population of rAAV virions, the rAAV genome comprising a library of cap polynucleotides encoding variant AAV capsid proteins; b) contacting the target cells with the population for a period of time sufficient to allow transduction of the cap polynucleotide into the target cells by the rAAV virions; and c) sequencing the cap polynucleotide from the target cells, thereby identifying AAV capsid proteins that confer increased transduction efficiency in the target cells. A method comprising: (Item 67) 67. The method of claim 66, further comprising, prior to step (b), depleting the population of rAAV virions by contacting the population with non-target cells for a period of time sufficient to allow binding of the rAAV virions to the non-target cells. (Item 68) 68. The method of claim 66 or 67, wherein the variant AAV capsid protein is an AAV5 capsid protein. (Item 69) 69. The method of any one of items 66 to 68, wherein the variant capsid proteins each individually comprise an insertion of 5 to 11 amino acids relative to the parent sequence at an insertion site in the GH loop of the parent sequence. (Item 70) 70. The method according to item 69, wherein the insertion site is between any two, optionally adjacent, amino acids in the parent sequence corresponding to positions 560 to 594 of SEQ ID NO:1. (Item 71) 70. The method of claim 69, wherein the insertion site is between adjacent amino acids in the parent sequence corresponding to 574 and 575 of SEQ ID NO:1. (Item 72) The insertion has the formula: -X 0 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 - (In the formula, X 0 and X 8 are each independently A, S, G, or absent (SEQ ID NO: 1102). 72. The method according to any one of items 69 to 71, wherein the peptide is

Claims

1. A recombinant adeno-associated virus (rAAV) virion comprising: a) an AAV5 capsid protein that, compared to a parent AAV5 capsid comprising SEQ ID NO:1, comprises only one or more substitutions selected from: (i) T582A, or (ii) T582A and S651A; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; 3. An rAAV virion comprising:

2. The rAAV virion of claim 1 , wherein the AAV5 capsid protein comprises SEQ ID NO:

73.

3. An AAV5 capsid protein that, compared to a parent AAV5 capsid comprising SEQ ID NO:1, comprises only one or more substitutions selected from: (i) T582A, or (ii) T582A and S651A.

4. 4. The AAV5 capsid protein of claim 3, comprising SEQ ID NO:

73.

5. A polynucleotide encoding the AAV5 capsid protein of claim 3 or 4.

6. A pharmaceutical composition comprising the rAAV virion of claim 1 or 2.

7. 11. An in vitro method of transducing a cardiac cell, comprising contacting the cardiac cell with a rAAV virion described in claim 1 or 2, wherein the rAAV virion transduces the cardiac cell.

8. An in vitro method of delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with an rAAV virion described in claim 1 or 2, wherein the cardiac cell expresses the gene product.

9. The gene products include cardiac troponin T; cardiac sarcomeric protein; β-myosin heavy chain; ventricular myosin essential light chain 1; ventricular myosin regulatory light chain 2; cardiac α-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); sarcoplasmic reticulum calcium-ATPase 2a (SERCA 2a); stromal derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminal); 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); apolipoprotein-E (ApoE); and superoxide dismutase (SOD); S100A1.

10. 9. The in vitro method of claim 7 or claim 8, wherein the gene product comprises one or more reprogramming factors selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2.

11. The in vitro method of claim 10, wherein the cardiac cells are reprogrammed into cardiac cardiomyocytes.

12. Use of the pharmaceutical composition described in claim 6 in the manufacture of a medicament for treating a cardiac condition in a subject in need thereof, wherein the treatment comprises a step of administering a therapeutically effective amount of the pharmaceutical composition to the subject, wherein the rAAV virions transduce cardiac tissue.

13. The use according to claim 12, characterized in that the rAAV virions are administered by epicardial injection.

14. 13. The use according to claim 12, characterized in that the rAAV virions are administered by intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendocardial injection, or molecular cardiac surgery with recirculation delivery (MCARD).

15. 7. The pharmaceutical composition of claim 6 for treating a cardiac condition in a subject in need thereof, wherein the rAAV virion transduces cardiac tissue.

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