Modified cardiotropic aav capsid polypeptides and vectors

Abstract

The present disclosure provides variant AAV capsid polypeptides that, when present in the capsid of an AAV vector, facilitate efficient transduction of cardiac cells, in particular human cardiac cells. Also provided are methods for preparing AAV vectors, in particular vectors that transduce cardiac cells, and in particular AAV vectors for gene therapy uses, in particular those targeting the heart or cardiac cells and tissue.

Description

MODIFIED CARDIOTROPIC AAV CAPSID POLYPEPTIDES AND VECTORS FIELD OF THE INVENTION

[0001] The present disclosure relates generally to modified adeno-associated virus (AAV) capsid polypeptides and encoding nucleic acid molecules. The present disclosure also relates to AAV vectors comprising the capsid polypeptides, and nucleic acid vectors (e.g. plasmids) comprising the encoding nucleic acids molecules, as well as to host cells comprising the vectors. The disclosure also relates to methods and uses of the polypeptides, encoding nucleic acids molecules, vectors and host cells. BACKGROUND OF THE INVENTION

[0002] Gene therapy has most commonly been investigated and achieved using viral vectors, with notable recent advances being based on adeno-associated viral vectors. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length. The AAV genome includes inverted terminal repeat (ITRs) at both ends of the molecule, flanking two open reading frames: rep and cap. The cap gene encodes three structural capsid proteins: VP1, VP2 and VP3. The three capsid proteins typically assemble in a ratio of 1:1:8-10 to form the AAV capsid, although AAV capsids containing only VP3, or VP1 and VP3, or VP2 and VP3, have been produced. The cap gene also encodes the assembly activating protein (AAP) from an alternative open reading frame. AAP promotes capsid assembly, acting to target the capsid proteins to the nucleolus and promote capsid formation. The rep gene encodes four known regulatory proteins: Rep78, Rep68, Rep52 and Rep40. These Rep proteins are involved in AAV genome replication, packaging, genomic integration and other processes. More recently, an X gene has been identified in the 3' end of the AAV2 genome (Cao et al. PLoS One, 2014, 9:e104596). The encoded X protein appears to be involved in the AAV life cycle, including DNA replication.

[0003] The ITRs are involved in several functions, in particular integration of the AAV DNA into the host cell genome, as well as genome replication and packaging. When AAV infects a host cell, the viral genome can integrate into the host's chromosomal DNA resulting in latent infection of the cell. Thus, AAV can be exploited to introduce heterologous sequences into cells. In nature, a helper virus (for example, adenovirus or herpesvirus) provides protein factors that allow for replication of AAV virus in the infected cell and packaging of new virions. In the case of adenovirus, genes E1A, E1B, E2A, E4and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and the helper virus are produced.

[0004] AAV vectors (also referred to as recombinant AAV, rAAV) that contain a genome that lacks some, most or all of the native AAV genome and instead contain one or more heterologous sequences flanked by the ITRs, have been successfully used in gene therapy settings. These AAV vectors are widely used to deliver heterologous nucleic acid to cells of a subject for therapeutic purposes, and in many instances, it is the expression of the heterologous nucleic acid that imparts the therapeutic effect. Although several AAV vectors have now been used in the clinic, there are a limited number that exhibit the required in vivo transduction efficiency for the different types of primary human cells / tissues to facilitate adequate expression of the heterologous nucleic acid for therapeutic applications. There is therefore a need to develop alternative AAV vectors that contain capsid proteins that facilitate efficient transduction of host cells, including in vivo. SUMMARY OF THE INVENTION

[0005] The present disclosure is predicated in part on the generation of modified AAV capsid polypeptides. In particular embodiments, the capsid polypeptides facilitate transduction of human cells (such as human cardiac cells) when the capsid is contained in an AAV vector. Typically, the transduction of AAV vectors comprising a capsid polypeptide of the present disclosure, including in vivo transduction, is improved or enhanced compared to AAV vectors comprising other AAV capsid polypeptides (e.g. the prototypic AAV capsids set forth in SEQ ID NOs:1-4). The capsid polypeptides of the present disclosure are therefore particularly useful in preparing AAV vectors, and in particular, AAV vectors for gene therapy uses. Similarly, AAV vectors comprising a capsid polypeptide of the present disclosure (i.e. having a capsid comprising or consisting of a capsid polypeptide of the present disclosure) are of particular use in gene therapy applications, such as for delivery of heterologous nucleic acids for the treatment of various diseases and conditions.

[0006] In one aspect, the disclosure provides a capsid polypeptide comprising an amino acid sequence of any one of SEQ ID NOs:5-8, or an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing; wherein the amino acid sequence comprises: (a) amino acid residues 1-204 of SEQ ID NO:5, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at leastabout 97% sequence identity thereto; (b) amino acid residues 1-204 of SEQ ID NO:6, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 98% sequence identity thereto; (c) amino acid residues 1-204 of SEQ ID NO:7, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 95% sequence identity thereto; or (d) amino acid residues 1-204 of SEQ ID NO:8, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity thereto.

[0007] In another aspect, the disclosure provides a capsid polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least about 95%, 96%, 97%, 98% or 99% sequence identity thereto; wherein the amino acid sequence comprises: (a) amino acid residues 1-204 of SEQ ID NO:9, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 92% sequence identity thereto, and / or (b) amino acid residues 205-737 of SEQ ID NO:9, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity thereto.

[0008] Also provided is a capsid polypeptide, comprising: (i) an amino acid sequence having at least about 85% sequence identity to any one of SEQ ID NOs:5-8; (ii) amino acid residues 1-204 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to a of the foregoing; and / or (iii) amino acid residues 205-736 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to any of the foregoing; and wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, wherein the at least one amino acid modification is selected from the group consisting of M1 or 1del; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K38 or H38; D41 or N41; G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; E134 or Q134; G135 or A135; A136 or G136; K137 or E137; G141 or A141; V146 or L146; Q148, P148 or 148del; Q 151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; K168 or R168; A224 or S224; D418 or E418; L584 or F584; V598 or A598; and H642 or N642, with numbering relative to SEQ ID NO:1.

[0009] In one embodiment, the capsid polypeptide comprises M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418 and F584, with numbering relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises L129; L146; 148del; K162; K164; R168 and E418, with numbering relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises A24; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151_E152insR; S152; T157; K162 and R168, with numbering relative to SEQ ID NO:1. In yet another embodiment, the capsid polypeptide comprises 1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; and F584, with numbering relative to SEQ ID NO:1.

[0010] Also provided is a capsid polypeptide, comprising:(i) the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity thereto; (ii) amino acid residues 1-204 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity thereto; and / or (iii) amino acid residues 205-736 of any one of SEQ ID NO:9 or a sequence having at least about 85% sequence identity thereto; wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 14; 21; 24; P29; 31; 34; 35; 36; 37; 39; 41; 42; 56; 67; 92; 125; 129; 134; 135; 136; 137; 141; 146; 147; 148; 151; 152; 157; 159; 162; 164; 168; 194; 196; 197; 198; 200; 205; 207; 224; 233; 235; 263; 264; 310; 312; 326; 330; 345; 411; 447; 451; 451; 452; 453; 455; 456, 457, 458, 459, 460; 465; 466; 467; 470; 473; 475; 489; 493; 494; 502; 505; 510; 515; 517; 518; 522; 531; 532; 538; 540; 547; 548; 549; 553; 554; 567, 568; 576; 577; 582; 584; 588; 590, 592; 594; 595; 597; 598; 599; 642; 648; 660; 661; 664; 665; 699; 706; 709; 717; 720; 722; 735, with numbering relative to SEQ ID NO:1, wherein the at least one amino acid modification is selected from the group consisting of T14; Q21; K24; P29; P31; P34; A35; E36; R37; K39; D41; S42; F56; A67; R92; V125; L129; E 134; G135; A136; K137; G141; V146; E147; P148; Q151_E152insR; S152; T157; I159; K162; Q164; R168; A194; S196; G197; V198; T200; T205; S207; S224; T233; M235; A263;; T264; R310; N312; Q326; T330; T345; E411; S447; S451; T451; T452; G453; T455; Q456, G457, T458, Q459, Q460; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H510; D515; L517; V518; P522; E531; E532; H538; N540; G547; T548; T549; A553; E554; R567, T568; Q576; Y577; N582; L584; N588; A590, T592; R594; T595; N597; D598; Q599; H642; M648; T660; T661; P664; A665; I699; N706; V709; T717; V720; S722; N735, with numbering relative toSEQ ID NO:1, with numbering relative to SEQ ID NO:1.

[0011] Also provided is an AAV vector, comprising a capsid polypeptide of the present disclosure. In some examples, the vector further comprises a heterologous coding sequence, such as a heterologous coding sequence that encodes a peptide, polypeptide or polynucleotide (e.g. a therapeutic peptide, polypeptide or polynucleotide).

[0012] In another aspect, there is provided an isolated nucleic acid molecule encoding a capsid polypeptide of the present disclosure. In a further aspect, provided is a vector comprising the aforementioned nucleic acid molecule. In some examples, the vector is selected from among a plasmid, cosmid, phage and transposon.

[0013] Also provided is a host cell, comprising an AAV vector, nucleic acid molecule or vector of the present disclosure.

[0014] In a further aspect, there is provided a method for introducing a heterologous coding sequence into a host cell, comprising contacting a host cell with an AAV vector of the present disclosure. In some examples, the host cell is a cardiac cell. In one embodiment, contacting a host cell with the AAV vector comprises administering the AAV vector to a subject. In another embodiment, the method is in vitro or ex vivo.

[0015] In another aspect, there is provided a method for producing an AAV vector, comprising culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide of the present disclosure, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising a capsid polypeptide of the present disclosure, wherein the capsid encapsidates the heterologous coding sequence. In some examples, the host cell is a cardiac cell.

[0016] Also provided is a method for producing a modified AAV vector that exhibits enhanced transgene expression in a human cardiac cell when the vector comprises a transgene, comprising: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce amodified capsid polypeptide that comprises at least one amino acid modification selected from the group consisting of M1 or 1del; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K38 or H38; D41 or N41; G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; E134 or Q134; G135 or A135; A136 or G136; K137 or E137; G141 or A141; V146 or L146; Q148, P148 or 148del; Q 151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; K168 or R168; A224 or S224; D418 or E418; L584 or F584; V598 or A598; and H642 or N642, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.

[0017] Also provided is a method for producing a modified AAV vector that exhibits enhanced transgene expression in a human cardiac cell when the vector comprises a transgene, comprising: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the amino acid sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises L129; L146; 148del; K162; K164; R168; E418, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.

[0018] Also provided is a method for producing a modified AAV vector that exhibits enhanced cell entry into a human cardiac cell when the vector comprises a transgene, comprising: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the amino acid sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises one or more of M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; L584; F584; A598 and N642, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector. In one embodiment, the the modified capsid polypeptide comprises amino acid modifications M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162;R168; S224; L584; A598 and N642, with numbering relative to SEQ ID NO:1. In another embodiment, the modified capsid polypeptide further comprises amino acid modifications E418 and F584, with numbering relative to SEQ ID NO:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Exemplary embodiments of the present disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0020] Figure 1 is a schematic representation of cardiotropic rAAV variants obtained by directed evolution. Directed evolution was performed using the hiPSC-CM line WTCWT. After six rounds of selection, the enriched novel AAVs were sequenced and phylogenetic analysis performed to compare to parental AAVs. (A) Clustering of amino acid sequences from novel variants (red) to wildtype AAV parental serotypes (black), with relationships depicted as a phylogenetic tree. (B) Heatmap illustrating the percentage identity among select parental AAVs and enriched novel variants. (C) Parental sequence contribution analysis of variants isolated after directed evolution. Black line represents the most probable composition of individual shuffled clones based on the longest sequence of identity to parental variants in a 5’ to 3’ direction.(D) Surface representations of the new capsid variants KK01, KK02, KK03, and KK05. The coloured regions (yellow, orange, purple) denote surface residues which differ from the parental AAV6 capsid. Amino acid changes not located on the capsid surface are listed below. (D) Depiction as in (E) for the capsid variant KK04. The coloured regions (red and purple) denote surface residues which differ from the parental AAV3b capsid.

[0021] Figure 2 is a sequence alignment of rAAV.KK01, rAAV.KK02, rAAV.KK03, rAAV.KK04, rAAV.KK05, AAV6 and AAV3b.

[0022] Figure 3 is a representation of a comparison of the transduction efficiency of cardiotropic rAAV capsids in hiPSC-CMs. (A) Four lines of hiPSC-CMs (WTCWT, SCVI 8, SCVI 100, SCVI 480) were transduced with rAAV.CBA.GFP vectors at MOT 1000, followed by analysis using flow cytometry to quantify GFP (n = 3 per group) on day 5 post transduction. Flow cytometry dot plots quantifying the proportions of GFP positive cardiomyocytes (cTnT+ cells). (B) Alignment of AAV1 and AAV6 capsids, showing amino acid differences within the VP3 region. (C) All novel variants except rAAV.KK04 were aligned to parental AAV6. (D) The amino acid sequence of rAAV.KK04 was aligned to parental AAV3.

[0023] Figure 4 is a representation of viral titres from virus preps of rAAV.KK01- rAAV.KK05 and rAAV6 packaged with a CBA-GFP cassette (AAV.CBA.GFP).

[0024] Figure 5 is a representation of the transduction of hiPSC-CMs with the barcoded rAAV library. Three lines of hiPSC-CMs were competitively transduced with a barcoded library of rAAV.CBA.GFP vectors at MOT 100, 1000 and 10,000, then harvested at D5 post-transduction. The cells were imaged to observe GFP auto-fluorescence prior to harvest of DNA / RNA.

[0025] Figure 6 is a representation of rAAV.KK01-rAAV.KK05 and gene delivery to hiPSC-CMs. hiPSC-CMs from SCVI 100 were competitively transduced with a barcoded library of rAAV.CBA.GFP vectors, then harvested at D5 post-transduction. Extraction of DNA / RNA was performed, followed by analysis using next generation sequencing (n = 3 per group). The relative proportions of barcode reads post NGS analysis were given at the level of cell entry (gDNA) and gene expression (mRNA) for multiplicity of transduction (MOT) 100, 1000 and 10,000. Results were expressed as percentage of total reads for each cell line.

[0026] Figure 7 is a representation of rAAV.KK01-rAAV.KK05 efficiency at gene delivery to hiPSC-CMs SCVI 8 and SCVI480. hiPSC-CMs were competitively transduced with a barcoded library of rAAV.CBA.GFP vectors at MOT 100, 1000 and 10,000, then harvested at D5 post-transduction. The relative proportions of barcode reads post NGS analysis were given at the level of cell entry (gDNA) and gene expression (mRNA) for (A) SCVI 8 and (B) SCVI 480. Results were expressed as percentage of total reads for each cell line

[0027] Figure 8 is a representation of GFP expression in hiPSC-CMs that were transduced with rAAV.KK01-rAAV.KK05. Three lines of hiPSC-CMs were transduced with unbarcoded rAAV.CBA.GFP vectors at MOT 1000, followed by analysis using microscopy and flow cytometry to quantify GFP (n = 3 per group for SCVI 8 and 100, n = 4 per group for SCVI 480) on day 5 post transduction. (A) Fluorescence images showing GFP auto-fluorescence in live cells (scale bar = 100 uM). (b) Flow cytometry dot plots quantifying proportions of GFP positive cardiomyocytes (cTnT+ cells).

[0028] Figure 9 is a representation of the functional validation of novel AAV variants in hiPSC-CMs. Three lines of hiPSC-CMs were transduced with unbarcoded rAAV.CBA.GFP vectors at MOT 1000, followed by analysis using flow cytometry toquantify GFP (n = 3 or 4 per group) on day 5 post transduction. Flow cytometry dot plots quantifying (A) the mean fluorescence intensity of GFP in cardiomyocytes (cTnT+ cells). (B) The purity of each diff was also measured by flow cytometry and expressed as % cTnT.

[0029] Figure 10 is a representation of competitive transduction assay and functional validation of novel AAV variants in hCO (A) Human cardiac organoids were transduced with a barcoded library of rAAV.CBA.GFP vectors at MOT 100, 1000 and 10,000. This was followed by analysis using next generation sequencing (n = 8 per group). The relative proportions of barcode reads post NGS analysis were given for all cell lines at the level of cell entry (gDNA) and gene expression (cDNA). Results were expressed as percentage of total reads for each cell line. (B) Human cardiac organoids were then transduced with unbarcoded rAAV vectors at MOT 10,000, followed by analysis by microscopy to quantify GFP (n = 6 or 7 per group). Fluorescence images showing GFP auto-fluorescence in live organoids. (C) GFP fluorescence intensity was quantified from microscopy acquired imaging by MATLAB.

[0030] Figure 11 is a representation of cardiac slices transduced with barcoded AAV variants remained viable up to two days post slicing. Cardiac slices were generated from the left ventricular myocardium of normal pigs or those with infarcted hearts. (A) Viability was assessed at D1 and D2 post slicing by calcein staining. (B) GFP autofluorescence was observed at D2 post slicing / transduction prior to tissue harvest.

[0031] Figure 12 is a representation of competitive transduction assay to compare efficiency of barcoded AAV variants in pig myocardial slices. Cardiac slices were generated from the left ventricular myocardium of normal pigs or those with infarcted hearts. The relative proportions of barcode reads post NGS analysis were given for normal and infarct hearts at the level of cell entry (A, C) and gene expression (B, D). Results were expressed as percentage of total reads for each condition. The p-values were expressed as a heat map for each graph.

[0032] Figure 13 is a representation of the nuclear yield of non-myocytes was higher than myocytes from infarcted pig hearts. Nuclei were extracted from cardiac slices and sorted using an imaging cytometer. (A) Cardiomyocytes were labelled with PCM1. Total nuclear yield from PCM1+ and PCM1- fractions in each sample is depicted for (B) normal and (C) infarct hearts respectively.

[0033] Figure 14 is a representation of a comparison of cardiomyocyte vs non-myocyte specific transduction in normal pig cardiac slices by analysis of sorted nuclei. The relative proportions of barcode reads post NGS analyses are provided, at the level of gene expression and cell entry for the PCM1+ve cardiomyocyte fraction (A and B) and the PCM1-ve non-myocyte fraction (C and D). The results are expressed as percentage of total reads for each condition.

[0034] Figure 15 is a representation of the comparison of rAAV transduction in non- human primate cardiac slices by analysis of cardiac nuclei. Cardiac slices were generated from the left ventricular myocardium of non-human primates. Two days post transduction, DNA and RNA were extracted from the slices and analysed by next generation sequencing. The relative proportions of barcode reads post NGS analysis were given for whole heart and nuclear DNA / RNA at the level of cell entry (A, C, E) and gene expression (B, D, F). Results were expressed as percentage of total reads for each condition.

[0035] Figure 16 is a representation of competitive transduction assay to compare efficiency of barcoded AAV variants in human myocardial slices. Cardiac slices were generated from the left ventricular myocardium of donor human hearts. Heart slices were transduced and harvested for analysis at D2 post slicing. The relative proportions of barcode reads post NGS analyses are provided, at the level of gene expression and cell entry for the PCM1+ve cardiomyocyte fraction (A and B) and the PCM1-ve non-myocyte fraction (C and D). The results are expressed as percentage of total reads for each condition.

[0036] Figure 17 is a representation of results from an in vivo competitive transduction assay (in a pig heart) comparing gene entry (gDNA) and expression (cDNA) for different AAV capsids. Figure 17A is a heat map showing gene entry and expression in each cardiac segments, and Figure 17B is a bar graph showing collective results for each AAV capsid (from left to right for each capsid: cDNA, gDNA, c / g ratio). DETAILED DESCRIPTION OF THE INVENTION

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In theevent that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0038] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0039] As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0042] As used herein, a "vector" includes reference to both polynucleotide vectors and viral vectors, each of which are capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids and transposons. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors.

[0043] As used herein, "adeno-associated viral vector" or “AAV vector” refers to a vector in which the capsid is derived from an adeno-associated virus, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, AAV12 or AAV13, AAV from other clades or isolates, or is derived from synthetic, bioengineered or modified AAV capsid proteins, including chimeric capsid proteins. In particular embodiments, the AAV vector has a capsid comprising a capsid polypeptide of the present disclosure. When referring to AAV vectors, both the source of the genome and the source of the capsid can be identified, where the source of the genome is the first number designated and the source of the capsid is the second number designated. Thus, for example, a vector in which both the capsid and genome are derived from AAV2 is more accurately referred to as AAV2 / 2. A vector with an AAV6-derived capsid and an AAV2-derived genome is most accurately referred to as AAV2 / 6. A vector with the bioengineered DJ capsid and an AAV2-derived genome is most accurately referred to as AAV2 / DJ. For simplicity, and because most vectors use an AAV2-derived genome, it is understood that reference to an AAV6 vector generally refers to an AAV2 / 6 vector, reference to an AAV2 vector generally refers to an AAV2 / 2 vector, etc. An AAV vector may also be referred to herein as "recombinant AAV", "rAAV", "recombinant AAV virion", "rAAV virion", “AAV variant”, “recombinant AAV variant”, and “rAAV variant” terms which are used interchangeably and refer to a replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. The AAV vector genome (also referred to as vector genome, recombinant AAV genome or rAAV genome) comprises a transgene flanked on both sides by functional AAV ITRs. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and / or cap genes. Functional ITR sequences are necessary for the rescue, replication and packaging of the vector genome into the rAAV virion.

[0044] The term “ITR” refers to an inverted terminal repeat at either end of the AAV genome. This sequence can form hairpin structures and is involved in AAV DNA replication and rescue, or excision, from prokaryotic plasmids. ITRs for use in the present disclosure need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging of rAAV.

[0045] As used herein, "functional" with reference to a capsid polypeptide means that the polypeptide can self-assemble or assemble with different capsid polypeptides to produce the proteinaceous shell (capsid) of an AAV virion. It is to be understood that not all capsid polypeptides in a given host cell assemble into AAV capsids. Preferably, at least 25%, atleast 50%, at least 75%, at least 85%, at least 90%, at least 95% of all AAV capsid polypeptide molecules assemble into AAV capsids. Suitable assays for measuring this biological activity are described e.g. in Smith-Arica and Bartlett (2001), Curr Cardiol Rep 3(1): 43-49.

[0046] "AAV helper functions" or "helper functions" refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, as a helper virus or as helper virus genes which aid in AAV replication and packaging. Helper virus genes include, but are not limited to, adenoviral helper genes such as E1A, E1B, E2A, E4 and VA. Helper viruses include, but are not limited to, adenoviruses, herpesviruses, poxviruses such as vaccinia, and baculovirus. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein- Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.

[0047] As used herein, the term “transduction” refers to entry of AAV vector into one or more particular cell types and transferal of the DNA contained within the AAV vector into the cell. Transduction can be assessed by measuring the amount of AAV DNA or RNA expressed from the AAV DNA in a cell or population of cells, and / or by assessing the number of cells in a population that contain AAV DNA or RNA expressed from the DNA. Where the presence or amount of RNA is assessed, the type of transduction assessed is referred to herein as "functional transduction", i.e. the ability of the AAV to transfer DNA to the cell and have that DNA expressed. The term "transduction efficiency" and grammatical variations thereof refers to the ability of an AAV vector to transduce host cells, and more particularly the efficiency with which an AAV vector transduces host cells. In particular embodiment, the transduction efficiency is in vivo transduction efficiency, and refers to the ability of an AAV vector to transduce host cells in vivo following administration of the vector to the subject. Transduction efficiency can be assessed in a number of ways known to those in the art, including assessing the number of host cellstransduced following exposure to, or administration of, a given number of vector particles (e.g. as assessed by expression of a reporter gene from the vector genome, such as GFP or eGFP, using microscopy or flow cytometry techniques); the amount of vector DNA (e.g. number of vector genomes) in a population of host cells following exposure to a given number of vector particles; the amount of vector RNA in population of host cells following exposure to a given number of vector particles; and the level of protein expression from a reporter gene (e.g. GFP or eGFP) in the vector genome in a population of host cells following exposure to, or administration of, a given number of vector particles. The population of host cells can represent a particular number of host cells, a volume or weight of tissue, or an entire organ (e.g. heart). In vivo transduction efficiency can reflect the ability of an AAV vector to access host cells, such as myocytes in the heart; the ability of an AAV vector to enter host cells; and / or expression of a heterologous coding sequence contained in the vector genome upon host cell entry.

[0048] As used herein, "corresponding nucleotides", "corresponding amino acid residues" or “corresponding positions”, "with numbering relative to" and the like refer to nucleotides, amino acids or positions that occur at aligned loci or positions. The sequences of related or variant polynucleotides or polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches (e.g. identical nucleotides or amino acids at positions), and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTN, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, etc) and others known to those of skill in the art. By aligning the sequences of polynucleotides, one skilled in the art can identify corresponding nucleotides. For example, by aligning two AAV capsid polypeptides (e.g. as shown in Figure 3), one of skill in the art can identify regions or amino acids residues within one AAV polypeptide that correspond to various regions or residues in the other AAV polypeptide. For example, the serine at position 179 of the AAV6 capsid polypeptide set forth in SEQ ID NO: 1 is the corresponding amino acid of, or corresponds to, the serine at position 180 of the rAAV.KK03 capsid polypeptide set forth in SEQ ID NO:7 (see the alignment of the capsid polypeptides of AAV6 and rAAV.KK03 in Figure 2). In another example, and with reference to the same alignment, the serine at position 538 of the AAV6 capsid polypeptide aligns with, or corresponds to the serine at position 537 of the rAAV.KK02 capsid polypeptide, and the histidine at position 539 of the rAAV.KK04 capsid polypeptide. Thus, when amino acid residues orpositions are referred to herein with respect to a particular capsid polypeptide, it is understood that, where appropriate, the reference is also to the corresponding amino acid residue or position in another capsid polypeptide. Table 4 disclosed herein sets out the amino acid modifications identified in the variant AAV capsid proteins KK01-KK05 with numbering relative to the prototypic AAV6 amino acid sequence of SEQ ID NO:1.

[0049] A "heterologous coding sequence" as used herein refers to nucleic acid sequence present in a polynucleotide, vector, or host cell that is not naturally found in the polynucleotide, vector, or host cell or is not naturally found at the position that it is at in the polynucleotide, vector, or host cell, i.e. is non-native. A "heterologous coding sequence" can encode a peptide or polypeptide, or a polynucleotide that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, and shRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous sequence and the genomic DNA can occur. In one example, the heterologous coding sequence is a functional copy of a gene for introduction into a cell that has a defective / mutated copy.

[0050] As used herein, the term “operably-linked” with reference to a promoter and a coding sequence means that the transcription of the coding sequence is under the control of, or driven by, the promoter.

[0051] The term "host cell" refers to a cell, such as a mammalian cell, that has introduced into it the exogenous DNA, such as a vector or other polynucleotide. The term includes the progeny of the original cell into which the exogenous DNA has been introduced. Thus, a "host cell" as used herein generally refers to a cell that has been transfected or transduced with exogenous DNA.

[0052] As used herein, "isolated" with reference to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the polynucleotide or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

[0053] The term "subject" as used herein refers to an animal, in particular a mammal andmore particularly a primate including a lower primate and even more particularly, a human who can benefit from the present invention. A subject, regardless of whether a human or non-human animal or embryo, may be referred to as an individual, subject, animal, patient, host or recipient. The present disclosure has both human and veterinary applications. For convenience, an "animal" specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as domestic animals, such as dogs and cats. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. In some embodiments, the subject is human.

[0054] As used herein, the term “conservative sequence modifications” or “conservative substitution” refers to amino acid modifications that do not significantly affect or alter the characteristics of a vector containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a vector that are compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a capsid can be replaced with other amino acid residues from the same side chain family and the altered capsid can be tested for tropism and / or the ability to deliver a payload using the functional assays described herein.

[0055] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting. Capsid polypeptides

[0056] The present disclosure is predicated in part on the generation of variant AAV capsid polypeptides that, when present in the capsid of an AAV vector, facilitate efficient transduction of a host cell, in particular human cardiac cell. The transduction of cells by the AAV vectors described herein having a capsid comprising a capsid polypeptide of the present disclosure, including in vivo transduction, is generally increased or enhanced when compared to AAV vectors comprising a reference or wildtype AAV capsid polypeptide (e.g. the prototypic AAV6, AAV3b, AAV1 or AAV9 capsids as set forth in SEQ ID NOs:1, 2, 3, or 4, respectively). Transduction or transduction efficiency of AAV vectors can be increased by at least or about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, e.g. an AAV vector comprising a capsid polypeptide of the present disclosure can be at least or about 1.1x, 1.2x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x or more efficient at transducing cells in vivo compared to a reference AAV capsid polypeptide (e.g. one set forth in SEQ ID NO:1, 2, 3 or 4). In particular examples, the increased transduction or transduction efficiency is observed in human cardiac cells, including myocytes.

[0057] The capsid polypeptides of the present disclosure are therefore particularly useful in preparing AAV vectors, and in particular AAV vectors for gene therapy uses. In exemplary embodiments, the capsid polypeptides of the present disclosure are particularly useful in preparing AAV vectors that transduce cardiac cells, and in particular, human cardiac cells, including myocytes, and are thus useful for gene therapy applications targeting the heart or cardiac cells and tissue.

[0058] Provided herein are polypeptides, including isolated polypeptides, comprising all or a portion of an AAV capsid polypeptide set forth in any one of SEQ ID NOs: 5-9, including all or a portion of the VP1 protein (comprising amino acid residues corresponding to those at positions 1-736 of SEQ ID NO:1), VP2 protein (comprising amino acid residues corresponding to those at positions 138-736 of SEQ ID NO:1) and / or the VP3 protein (comprising amino acid residues corresponding to those at positions 205- 736 of SEQ ID NO:1), and variants thereof, including variants comprising at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP1, VP2 or VP3 proteins described herein.

[0059] Table 1 provides a brief description of the capsid polypeptides described herein,and exemplary nucleotide sequences. Table 1. Brief Description of the Sequences

[0060] Capsid polypeptides of the disclosure include those comprising (i) an amino acid sequence having at least about 85% sequence identity to any one of SEQ ID NOs:5-8; (ii) amino acid residues 1-204 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to a of the foregoing; and / or (iii) amino acid residues 205-735 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to any of the foregoing; and wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1. In an embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K38 or H38; D41 or N41; G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; E134 or Q134; G135 or A135; A136 or G136; K137 or E137; G141 or A141; V146 or L146; Q148, P148 or 148del; Q 151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; K168 or R168; A224 or S224; D418 or E418; L584 or F584; V598 orA598; and H642 or N642, with numbering relative to SEQ ID NO:1.

[0061] Capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO: 5 (also referred to herein as rAAV.KK01) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the amino acids 1- 204 of SEQ ID NO: 5 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids 205-737 of SEQ ID NO: 5 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO: 1. In one embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1. In a particular embodiment, the capsid polypeptide comprises at least one of the amino acid modifications M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418 and F584, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418 and F584, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications M1del; M2; T3; V19; A24; Q26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168, S224; D418; F584; A598 and N642, with numbering relative to SEQ ID NO: 1. In a preferred embodiment, the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In another embodiment, the capsid polypeptide consists of amino acid sequence of SEQ ID NO: 5.

[0062] Capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO: 6 (also referred to herein as rAAV.KK02) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the amino acids 1- 204 of SEQ ID NO: 6 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids 205-737 of SEQ ID NO: 6 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 ,597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO: 1. In one embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553 or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1. In a particular embodiment, the capsid polypeptide comprises at least one of the amino acid modifications L129; L146; 148del; K162; K164; R168 and E418, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications L129; L146; 148del; K162; K164; R168 and E418, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications M1; A2; A3; I19; D24; K26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; G141; L146; 148del; Q151; E152; S157; K162; K164; R168; A224; E418; L584; V598; and H642, with numbering relative to SEQ ID NO: 1. In a preferred embodiment, the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In another embodiment, the capsid polypeptide consists of amino acid sequence of SEQ IDNO: 6.

[0063] Capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO: 7 (also referred to herein as rAAV.KK03) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the amino acids 1- 204 of SEQ ID NO: 7 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids 205-737 of SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO: 1. In one embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1. In a particular embodiment, the capsid polypeptide comprises at least one of the amino acid modifications comprises A24; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151_E152insR; S152; T157; K162 and R168, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications A24; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151_E152insR; S152; T157; K162 and R168, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications M1; A2; A3; I19; A24; K26; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; G141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168; A224; D418; L584; V598; and H642, with numbering relative to SEQ ID NO: 1. In a preferred embodiment, the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In another embodiment, the capsid polypeptide consists of amino acid sequence of SEQ ID NO: 7.

[0064] Capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO: 8 (also referred to herein as rAAV.KK05) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the amino acids 1- 204 of SEQ ID NO: 8 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids 205-737 of SEQ ID NO: 8 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 ,547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO: 1. In one embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553 or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1. In a particular embodiment, the capsid polypeptide comprises at least one of the amino acid modifications 1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; and F584, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications 1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; and F584, with numbering relative to SEQ ID NO: 1. In another embodiment, the capsid polypeptide comprises the amino acid modifications 1del; M2; T3; V19; A24; Q26; A29; Q31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168; S224; D418; F584; V598; and H642,with numbering relative to SEQ ID NO: 1. In a preferred embodiment, the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In another embodiment, the capsid polypeptide consists of amino acid sequence of SEQ ID NO: 8.

[0065] Capsid polypeptides of the disclosure include those comprising (i) an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 9 (also referred to herein as rAAV.KK04) (ii) amino acid residues 1-204 of any one of SEQ ID NO: 9 or an amino acid sequence having at least about 85% sequence identity to a of the foregoing; and / or (iii) amino acid residues 205-736 of any one of SEQ ID NO: 9 or an amino acid sequence having at least about 85% sequence identity to any of the foregoing; and wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO: 1. In one embodiment, the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1. In a particular embodiment, the capsid polypeptide comprises at least one of the amino acid modifications selected from the group consisting of T14; Q21; K24; P29; P31; P34; A35; E36; R37; H38; K39; D41; S42; F56; A67; R92; V125; L129; E 134; G135; A136; K137; G141; V146; E147; P148; Q151_E152insR; S152; T157; I159; K162; Q164; R168; A194; S196; G197; V198; T200; N201; T205; S207; S224; T233; M235; A263; T264; R310; N312; Q326; T330; T345; E411; S447; S451; T452; G453; T455; Q456, G457, T458, Q459, Q460; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H510; D515; L517; V518; P522; E531; E532; H538; N540; G547; T548; T549; A553; E554; R567, T568; Q576; Y577; N582; L584; N588; A590, T592; R594; T595; N597; D598; Q599; H642; M648; T660; T661; P664; A665; I699; N706; V709; T717; V720; S722; N735, with numbering relative to SEQ ID NO:1. In a preferred embodiment, the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 9. In another embodiment, the capsid polypeptide consists of amino acid sequence of SEQ ID NO: 9.

[0066] Also provided are nucleic acid molecules, including isolated nucleic acid molecules, encoding a capsid polypeptide described herein. Thus, for example, amongst the nucleic acid molecules provided herein are those encoding any one of the capsid polypeptides described herein. Non-limiting examples of nucleic acid molecules include those set forth in SEQ ID NOs:10-18, those having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the foregoing, and those that hybridize with medium or high stringency to nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs:10-18. Vectors

[0067] The present disclosure also provides vectors comprising a nucleic acid molecule that encodes a capsid polypeptide described herein, and vectors comprising a capsid polypeptide described herein. The vectors include nucleic acid vectors that comprise a nucleic acid molecule that encodes a capsid polypeptide described herein, and AAVvectors that have a capsid comprising a capsid polypeptide described herein. Nucleic acid vectors

[0068] Vectors of the present disclosure include nucleic acid vectors that comprise a polynucleotide that encodes all or a portion of a capsid polypeptide described herein, e.g. that encodes a capsid polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:5-9 or an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:5-9, or a fragment thereof (e.g. all or a portion of the VP2 or VP3 protein), as described herein. The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell) or can be vectors that integrate into the host cell genome. Exemplary vectors that comprise a nucleic acid molecule encoding a capsid polypeptide include, but are not limited to, plasmids, cosmids, transposons and artificial chromosomes. In particular examples, the vectors are plasmids.

[0069] Vectors, such as plasmids, suitable for use in bacterial, insect and mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that vectors of the present disclosure may also contain additional sequences and elements useful for the replication of the vector in prokaryotic and / or eukaryotic cells, selection of the vector and the expression of a heterologous sequence in a variety of host cells. For example, the vectors of the present disclosure can include a prokaryotic replicon (that is, a sequence having the ability to direct autonomous replication and maintenance of the vector extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In some embodiments, the vectors can include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistance gene, which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. The nucleic acid vectors will likely also comprise other elements, including any one or more of those described below. Most typically, the vectors will comprise a promoter operably linked to the nucleic acid encoding the capsid protein.

[0070] The nucleic acid vectors of the present disclosure can be constructed using known techniques, including, without limitation, the standard techniques of restrictionendonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors of the present disclosure may be introduced into a host cell using any method known in the art. Accordingly, the present disclosure is also directed to host cells comprising a vector or nucleic acid described herein. AAV vectors

[0071] Provided herein are AAV vectors comprising a capsid polypeptide described herein, such as a polypeptide comprising all or a portion of an AAV capsid polypeptide (e.g. a capsid polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:5-9 or an amino acid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:5-9, or a fragment thereof (e.g. all or a portion of the VP1 protein).

[0072] Methods for vectorizing a capsid protein are well known in the art and any suitable method can be employed for the purposes of the present disclosure. For example, the cap gene can be recovered (e.g. by PCR or digest with enzymes that cut upstream and downstream of cap) and cloned into a packaging construct containing rep. Any AAV rep gene may be used, including, for example, a rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 and any variants thereof. Typically, the cap gene is cloned downstream of rep so the rep p40 promoter can drive cap expression. This construct does not contain ITRs. This construct is then introduced into a packaging cell line with a second construct containing ITRs, typically flanking a heterologous coding sequence. Helper function or a helper virus are also introduced, and recombinant AAV comprising a capsid generated from capsid proteins expressed from the cap gene, and encapsidating a genome comprising the transgene flanked by the ITRs, is recovered from the supernatant of the packaging cell line. Various types of cells can be used as the packaging cell line. For example, packaging cell lines that can be used include, but are not limited to, HEK293 cells, HeLa cells, and Vero cells, for example as disclosed in US20110201088. The helper functions may be provided by one or more helper plasmids or helper viruses comprising adenoviral helper genes. Non-limiting examples of the adenoviral helper genes include E1A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging. Helper viruses of AAV areknown in the art and include, for example, viruses from the family Adenoviridae and the family Herpesviridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US20110201088, helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[0073] In some instances, rAAV virions are produced using a cell line that stably expresses some of the necessary components for AAV virion production. For example, a plasmid (or multiple plasmids) comprising the nucleic acid containing a cap gene identified as described herein and a rep gene, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of a cell (the packaging cells). The packaging cell line can then be transfected with an AAV vector and a helper plasmid or transfected with an AAV vector and co-infected with a helper virus (e.g., adenovirus providing the helper functions). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce the nucleic acid encoding the capsid polypeptide, and optionally the rep gene, into packaging cells. As yet another non-limiting example, the AAV vector is also stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.

[0074] In still further instances, the AAV vectors are produced synthetically, by synthesising AAV capsid proteins and assembling and packaging the capsids in vitro.

[0075] Typically, the AAV vectors of the present disclosure also comprise a heterologous coding sequence. The heterologous coding sequence may be operably linked to a promoter to facilitate expression of the sequence. The heterologous coding sequence can encode a peptide or polypeptide, such as a therapeutic peptide or polypeptide, or can encode a polynucleotide or transcript that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, and shRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and thegenomic DNA can occur. As would be appreciated, the nature of the heterologous coding sequence is not essential to the present disclosure. In particular embodiments, the vectors comprising the heterologous coding sequence(s) will be used in gene therapy.

[0076] In particular examples, the heterologous coding sequence encodes a peptide or polypeptide, or polynucleotide, whose expression is of therapeutic use, such as, for example, for the treatment of a disease or disorder. For example, expression of a therapeutic peptide or polypeptide may serve to restore or replace the function of the endogenous form of the peptide or polypeptide that is defective (i.e. gene replacement therapy). In other examples, expression of a therapeutic peptide or polypeptide, or polynucleotide, from the heterologous sequence serves to alter the levels and / or activity of one or more other peptides, polypeptides or polynucleotides in the host cell. Thus, according to particular embodiments, the expression of a heterologous coding sequence introduced by a vector described herein into a host cell can be used to provide a therapeutic amount of a peptide, polypeptide or polynucleotide to ameliorate the symptoms of a disease or disorder. In other instance, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and the genomic DNA can occur. Accordingly, the introduction of a heterologous sequence by an AAV vector described herein into a host cell can be used to correct mutations in genomic DNA, which in turn can ameliorate the symptoms of a disease or disorder.

[0077] In non-limiting examples, the heterologous coding sequence encodes an expression product that, when delivered to a subject, and in particular the heart of a subject, treats a heart-associated disease or condition, including heart injury, such as myocardial infarction, congenic heart disease, heart disease associated with aging, and other heart failure, as described herein.

[0078] The heart-associated disease or condition can be related to cardiac pathology or cardiac dysfunction, or any impairment to the heart’s pumping function contractility, impairments in the ability to relax (sometimes referred to as diastolic dysfunction), abnormal or improper functioning of the heart’s valves, diseases of the heart muscle (sometimes referred to as cardiomyopathies), diseases such as angina pectoris, myocardial ischemia and / or infarction characterized by inadequate blood supply to the heart muscle,infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (such as may occur in some kinds of cardiomyopathy or systemic hypertension), and abnormal communications between chambers of the heart. The heart disease may be related to cardiomyopathy, refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and / or stiffened. As a result, the heart muscle’s ability to pump blood is usually weakened. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematological, genetic, or unknown in origin.

[0079] Heart failure (HF) is a complex clinical syndrome that can result from any structural or functional cardiovascular disorder causing systemic perfusion inadequate to meet the body’s metabolic demands without excessively increasing left ventricular filling pressures. It is characterized by specific symptoms, such as dyspnea and fatigue, and signs, such as fluid retention. Chronic heart failure or congestive heart failure refer to an ongoing or persistent forms of heart failure.

[0080] Those skilled in the art would readily be able to select an appropriate heterologous coding sequence useful for treating such diseases, illustrative examples of which are described, for example, in Hulot et al. (European Heart Journal, 2016, 37:1651-1658), the entire contents of which is incorporated herein by reference. In some examples, the heterologous coding sequence comprises all or a part of a gene that is associated with the disease. Introduction of such a sequence to the heart can be used for gene replacement or gene editing / correction, e.g. using CRISPR-Cas9. In particular examples, the heterologous coding sequence encodes a protein encoded by a gene that is associated with the disease, illustrative examples of which are set forth in Table 3.

[0081] The heterologous coding sequence in the AAV vector is flanked by 3' and 5' AAV ITRs. AAV ITRs used in the vectors of the disclosure need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. Such ITRs are well known in the art.

[0082] As will be appreciated by a skilled artisan, any method suitable for purifying AAV can be used in the embodiments described herein to purify the AAV vectors, and such methods are well known in the art. For example, the AAV vectors can be isolated andpurified from packaging cells and / or the supernatant of the packaging cells. In some embodiments, the AAV is purified by separation method using a CsCl or iodixanol gradient centrifugation. In other embodiments, AAV is purified as described in US20020136710 using a solid support that includes a matrix to which an artificial receptor or receptor-like molecule that mediates AAV attachment is immobilized. Additional elements in the vectors

[0083] The vectors of the present disclosure can comprise promoters. In instances where the vector is a nucleic acid vector comprising nucleic acid encoding the capsid polypeptide, the promoter may facilitate expression of the nucleic acid encoding the capsid polypeptide. In instances where the vector is an AAV vector, the promoter may facilitate expression of a heterologous coding sequence, as described above.

[0084] In some examples, the promoters are AAV promoters, such as the p5, p19 or p40 promoter. In other examples, the promoters are derived from other sources. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the human alpha 1-antitrypsin (hAAT) promoter and the EF1α promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Non-limiting examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin- inducible system. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. In some embodiments, tissue specific promoters are used. Non-limiting examples of such promoters include the heart cell-specific promoters, illustrative examples of which include cardiac troponin I and T (cTnI and cTnT), alpha-myosin heavy chain (a-MHC) and myosinlight chain (MLC-2v). The selection of an appropriate promoter is well within the ability of one of ordinary skill in the art.

[0085] The vectors can also include transcriptional enhancers (e.g., an ApoE enhancer, translational signals, and transcriptional and translational termination signals. Examples of transcriptional termination signals include, but are not limited to, polyadenylation signal sequences, such as bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit beta-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence.

[0086] The vectors can include various posttranscriptional regulatory elements. In some embodiments, the posttranscriptional regulatory element can be a viral posttranscriptional regulatory element. Non-limiting examples of viral posttranscriptional regulatory element include woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), hepatitis B virus posttranscriptional regulatory element (HBVPRE), RNA transport element, and any variants thereof. The RTE can be a rev response element (RRE), for example, a lentiviral RRE. A non-limiting example is bovine immunodeficiency virus rev response element (RRE). In some embodiments, the RTE is a constitutive transport element (CTE). Examples of CTE include, but are not limited to, Mason-Pfizer Monkey Virus CTE and Avian Leukemia Virus CTE.

[0087] A signal peptide sequence can also be included in the vector to provide for secretion of a polypeptide from a mammalian cell. Examples of signal peptides include, but are not limited to, the endogenous signal peptide for HGH and variants thereof; the endogenous signal peptide for interferons and variants thereof, including the signal peptide of type I, II and III interferons and variants thereof; and the endogenous signal peptides for known cytokines and variants thereof, such as the signal peptide of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and variants thereof. Typically, the nucleotide sequence of the signal peptide is located immediately upstream of the heterologous sequence (e.g., fused at the 5′ of the coding region of the protein of interest) in the vector.

[0088] In further examples, the vectors can contain a regulatory sequence that allows, for example, the translation of multiple proteins from a single mRNA. Non-limiting examples of such regulatory sequences include internal ribosome entry site (IRES) and 2A self-processing sequence, such as a 2A peptide site from foot-and-mouth disease virus (F2A sequence). Host cells

[0089] Also provided herein are host cells comprising a nucleic acid molecule or vector or of the present disclosure. In some instances, the host cells are used to amplify, replicate, package and / or purify a polynucleotide or vector. In other examples, the host cells are used to express a heterologous sequence, such as one packaged within AAV vector. Exemplary host cells include prokaryotic and eukaryotic cells. In some instances, the host cell is a mammalian host cell. It is well within the skill of a skilled artisan to select an appropriate host cell for the expression, amplification, replication, packaging and / or purification of a polynucleotide, vector or rAAV virion of the present disclosure. Exemplary mammalian host cells include, but are not limited to, HEK293 cells, HeLa cells, Vero cells, HuH-7 cells, and HepG2 cells. In particular examples, the host cell is a cardiac cell or cell-line derived from a cardiac cell.

[0090] In some embodiments, the host cell is a mammalian cardiac cell. In another embodiment, the cardiac cell is a primate cardiac cell. In a preferred embodiment, the cardiac cell is a human cardiac cell. In another embodiment, the cardiac cell is a myocyte.

[0091] In another embodiment, the cardiac cell is comprised in a cardiac organoid. In an embodiment, the cardiomyocyte is a primary cardiomyocte. In another embodiment, the cardiac cell is an iPSC-derived cardiomyocyte. Compositions

[0092] Also provided are compositions comprising the nucleic acid molecules, polypeptides and / or vectors of the present disclosure. In particular examples, provided are pharmaceutical compositions comprising the AAV vectors disclosed herein and a pharmaceutically acceptable carrier. The compositions can also comprise additional ingredients such as diluents, stabilizers, excipients, and adjuvants.

[0093] The carriers, diluents and adjuvants can include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum aAAVC.umin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG). In some embodiments, the physiologically acceptable carrier is an aqueous pH buffered solution. Methods

[0094] The AAV vectors of the present disclosure, and compositions containing the AAV vectors, may be used in methods for the introduction of a heterologous coding sequence into a host cell. Such methods involve contacting the host cell with the AAV vector. This may be performed in vitro, ex vivo or in vivo. In particular embodiments, the host cell is a myocyte (e.g. a cardiac cell).

[0095] When the methods are performed ex vivo or in vivo, typically the introduction of the heterologous sequence into the host cell is for therapeutic purposes, whereby expression of the heterologous sequence results in the treatment of a disease or condition. Thus, the AAV vectors disclosed herein can be administered to a subject (e.g., a human) in need thereof, such as subject with a disease or condition amendable to treatment with a protein, peptide or polynucleotide encoded by a heterologous sequence described herein.

[0096] When used in vivo, titers of AAV vectors to be administered to a subject will vary depending on, for example, the particular recombinant virus, the disease or disorder to be treated, the mode of administration, the treatment goal, the individual to be treated, and the cell type(s) being targeted, and can be determined by methods well known to those skilled in the art. Although the exact dosage will be determined on an individual basis, in most cases, typically, recombinant viruses of the present disclosure can be administered to a subject at a dose of between 1×1010genome copies of the recombinant virus per kg of the subject and 1×1014genome copies per kg. In other examples, less than 1×1010genome copies may be sufficient for a therapeutic effect. In other examples, more than 1×1014genome copies may be required for a therapeutic effect.

[0097] The route of the administration is not particularly limited. For example, a therapeutically effective amount of the AAV vector can be administered to the subject via, for example, intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, epicutaneous, intradermal, rectal, intraocular, pulmonary, intracranial, intraosseous, oral, buccal, or nasal routes. The AAV vector can be administrated as a single dose or multipledoses, and at varying intervals.

[0098] Also provided are methods for producing an AAV vector described above and herein, i.e., one comprising a capsid polypeptide of the present disclosure. Such methods comprise culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide the present disclosure, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid polypeptide of the present disclosure, wherein the capsid encapsidates the heterologous coding sequence.

[0099] In further aspects, provided are methods for enhancing the in vivo human cardiac cell transduction efficiency of an AAV vector. As demonstrated herein, modifications in certain amino acid positions in the VP1 (comprising amino acid residues corresponding to 1-204 amino acids of SEQ ID NO:1) and / or VP3 (comprising amino acid residues corresponding to those at positions 205-736 of SEQ ID NO:1) regions unexpectedly provide for efficient transduction of human cardiac cells and / or enhanced DNA to RNA conversion (i.e. efficient expression despite less efficient vector uptake) by an AAV vector.

[0100] Thus, provided herein are methods for producing a modified AAV vector that exhibits enhanced transgene expression in a human cardiac cell, where the methods include the steps of a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises at least one amino acid modification selected from the group consisting of M1 or 1del; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K38 or H38; D41 or N41; G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; E134 or Q134; G135 or A135; A136 or G136; K137 or E137; G141 or A141; V146 or L146; Q148, P148 or 148del; Q 151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; K168 or R168; A224 or S224; D418 or E418; L584 or F584; V598 or A598; and H642 or N642, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector. In an embodiment, the amino acid sequence of thereference capsid polypeptide is modified at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises L129; L146; 148del; K162; K164; R168; E418. In one embodiment, the reference capsid polypeptide comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:1.

[0100] Also provided herein are methods for producing a modified AAV vector that exhibits enhanced transgene expression in a human cardiac cell, where the methods include the steps of: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the amino acid sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises one or more of M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; L584; F584; A598 and N642, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector. In an embodiment, the amino acid sequence of the reference capsid polypeptide is modified to comprise M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; L584; A598 and N642, with numbering relative to SEQ ID NO:1. In another embodiment, the modified capsid polypeptide further comprises amino acid modifications E418 and F584, with numbering relative to SEQ ID NO:1.

[0101] The methods disclosed herein may suitably include an initial step of first identifying a reference capsid polypeptide for transducing a host cell, including in vivo. The reference capsid polypeptide may be any AAV polypeptide, such as an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 capsid polypeptide, or a synthetic or chimeric capsid polypeptide.

[0102] It will be understood that any modification or combination of modifications, e.g. amino acid replacement or substitution, amino acid deletion and / or amino acid insertion, will result in a change of amino acid sequence in the modified capsid polypeptidecompared to the reference capsid polypeptide. Thus, for example, reference to modification does not include within its scope amino acid substitutions where one amino acid residue is substituted with the same amino acid residue, or modifications when an amino acid deletion is accompanied by an insertion of that deleted amino acid, such that there is no difference in the amino acid sequence of the modified capsid polypeptide compared to the reference capsid polypeptide sequence, i.e. the amino acid sequence of the modified capsid polypeptide can not be the same as (or must be different to) the amino acid sequence of the reference capsid polypeptide sequence.

[0103] Methods for modifying the sequence of a reference capsid polypeptide or polynucleotide so as to produce a modified capsid polypeptide or polynucleotide are well known in the art, and any such method can be utilised so as to perform the methods of the present disclosure. For example, the modification of the sequence of the reference capsid polynucleotide to produce a modified capsid polynucleotide can be performed using any method known in the art, including recombinant and synthetic methods, performed (either in part or in whole) in silico and / or in vitro. In a particular example, the modification of the sequence is performed in silico, followed by de novo synthesis of the modified capsid polynucleotide having the modified sequence (e.g. by gene synthesis methods such as those involving the chemical synthesis of overlapping oligonucleotides following by gene assembly).

[0104] The modified capsid polynucleotides may be contained in nucleic acid vector, such as a plasmid, for subsequent expression, replication, amplification and / or manipulation. Vectors suitable for use in bacterial, insect and mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that the vectors may also contain additional sequences and elements useful for the replication of the vector in prokaryotic and / or eukaryotic cells, selection of the vector and the expression of a heterologous sequence in a variety of host cells. For example, the vectors can include a prokaryotic replicon, which is a sequence having the ability to direct autonomous replication and maintenance of the vector extrachromosomally in a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In some embodiments, the vectors can include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistancegene, which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. The nucleic acid vectors will likely also comprise other elements, including any one or more of those described below. Most typically, the vectors will comprise a promoter operably linked to the nucleic acid encoding the capsid protein.

[0105] The nucleic acid vectors can be constructed using known techniques, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors comprising a modified capsid polynucleotide may be introduced into a host cell using any method known in the art.

[0106] Following modification, the modified capsid are then vectorised. Methods for vectorising a capsid polypeptide are well known in the art and non-limiting examples are described above.

[0107] The AAV vector produced by these methods typically has a level of transgene expression per cell that is enhanced compared to a reference AAV vector having a capsid comprising the reference capsid polypeptide. The level of transgene expression can be enhanced by at least or about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% 1000%, or more, e.g. the transgene expression of the AAV vector can be at least or about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x or more compared to transgene expression from an unmodified AAV vector (i.e. an AAV vector comprising the reference capsid) in vivo. In some examples, this is assessed in myocytes, such as hiPSC-myocytes, primary myocytes, cardiac slices or cardiac organoids.

[0108] Thus, also provided are AAV vectors produced by the methods of the present disclosure.

[0109] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

[0110] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (orinformation derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. EXAMPLES Example 1. Materials and Methods AAV capsid library construction and directed evolution

[0111] A library of AAV capsids was constructed as previously described (Cabanes- Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). Differentiated hiPSC-CMs in GeltrexTM(Gibco, #A14133-02, Massachusetts, USA) coated 24 well plates from the WTC iPSC line were obtained at >90% purity based on cardiac troponin (cTnT) expression. Cardiomyocytes were infected with four 10-fold dilutions of AAV library from 3.6 x 1010vg. The following day, cells were washed with 1xPBS, then co-infected with WT human Adenovirus 5 (Ad5; ATCC VR-5, Manassas, VA, USA), propagated as described previously (Cabanes-Creus, M., et al., 2022, Mol Ther Methods Clin Dev 24: 88- 101). After four days, the cells were harvested and lysed by three freeze-thaw cycles. The clarified supernatant was then analysed for AAV amplification by qPCR as previously described (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). Primers specific to AAV rep2 were used after each round of selection to verify AAV amplification and to select the appropriate dilution to be used for subsequent rounds of selection. The library dilution selected for the next round of selection was heated to 65oC for 30 min to inactivate the Ad5, before a dilution series was applied to the next batch of cells. In total, six rounds of selection were performed on fresh batches of hiPSC-CMs.Phylogenetic analysis and vectorization of enriched shuffled AAV variants

[0112] After selection, AAV capsid sequences were recovered by PCR as described previously (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). Twenty clones were sequenced after each round to track the progress of selection. The amino acid sequences of enriched AAV variants were aligned to known parental AAVs using Geneious Prime 2022.2.1 (https: / / www.geneious.com). A phylogenetic tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Amino acid substitutions were also displayed using a heat map generated from GraphPad Prism version 8.2.1 for Windows (GraphPad Software, USA). Vectorization was then performed as previously described (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). The novel capsid variants AAV.KK01-KK05 were then used to package one set of non-barcoded GFP expressing vectors, and another set of barcoded GFP expressing vectors. AAV1, AAV6 and AAV9 were also included as control vectors. Vector packaging and titration was done as described previously (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). Maintenance of hiPS cells and differentiation into cardiomyocytes

[0113] Four different hiPSC lines were used in this study: the WTCWT iPSC line obtained from The J. David Gladstone Institutes and SCVI 8, 100 and 480 from Stanford Cardiovascular Institute. All lines were maintained on Matrigel (Corning, #354277, New York, USA) coated 60mm cell culture dishes using the mTeSRTMPlus kit (STEMCELL Technologies, #05825, Vancouver, Canada). Upon confluence, cells were passaged as colonies using gentle cell dissociation reagent (STEMCELL Technologies, #07174, Vancouver, Canada) every 6-7 days.

[0114] For cardiomyocyte differentiation, WTCWT cells were maintained and differentiated as previously described (Lian, X., et al., 2012, Proc Natl Acad Sci USA, 109: E1848-57; Burridge, P.W., et al., 2014, Nat Methods, 11: 855-60). The SCVI 8, 100 and 480 lines were differentiated as follows. Cells were dissociated from confluent dishes on D-2 (two days prior to diff start on D0) using TrypLETMExpress Enzyme (ThermoFisher Scientific, 12604-021, Massachusetts, USA), then seeded into Matrigel coated 12 well plates at 700,000 cells / well using mTeSRTMPlus supplemented with Y-27632 (STEMCELL Technologies, #72304, Vancouver, Canada). On D-1, a media change was performed to remove the Y-27632. On D0, the differentiation was then commenced usingthe STEMdiff Cardiomyocyte Differentiation Kit according to manufacturer’s instructions until the point of maintenance in STEMdiff Cardiomyocyte Maintenance Medium (CMM). On D11, beating cells were subjected to two days of metabolic selection using lactate medium composed of glucose free DMEM (ThermoFisher Scientific, #A14430-01, Massachusetts, USA) supplemented with L-(+)-Lactic acid (Sigma-Aldrich, #L1750-10G, St. Louis, MO, USA) to final concentration of 4 mM and bovine serum albumin (Sigma, #A9418-10G, St. Louis, MO, USA) to 15 µM. On D13, cells were then returned to CMM and maintained until differentiation completion on D15.

[0115] After 1 hour of incubation with 10µM Y-27632, differentiated cardiomyocytes were dissociated using TrypLETMExpress supplemented with 2 µg / mL DNase I (STEMCELL Technologies, #07900, Vancouver, Canada). Dissociated cells were then replated into GeltrexTMcoated 24 well plates at 500,000 cells / well using RPMI 1640 media (ThermoFisher Scientific, 21870076, Massachusetts, USA) and B27 supplement (ThermoFisher Scientific, 17504-001, Massachusetts, USA) accompanied with Y-27632. Transduction of hiPSC-CM

[0116] One well of cells was used for counting to determine viability at D2 post- replating. Subsequently, cells were transduced with rAAV.GFP vectors in RPMI 1640 + B27 at MOT 1000. For competitive transduction assays, barcoded AAV variants were mixed at equimolar ratio to total MOT of 100, 1000 or 10,000. On D5 post-transduction, cells were imaged using the Zeiss Axiovert 200M Live Cell Imaging Microscope (Zeiss, Oberkochen, Germany), then harvested for nucleic acid extraction using the AllPrep DNA / RNA Micro Kit (Qiagen, #80284, Hilden, Germany) or flow cytometry to quantify transduced cardiomyocytes. Flow cytometry

[0117] Cells were dissociated using TrypLETMExpress, then washed with Dulbecco’s Phosphate Buffered Saline (DPS) without calcium and magnesium (Lonza, #12001-664, Basel, Switzerland). Cells were then stained using the Zombie NIRTMFixable Viability Kit (Biolegend, #423105, San Diego, USA). After washing, cells were fixed in 4% PFA (w / v) for 30 min, then washed and further stained with BV421 conjugated mouse anti-cTnT antibody (BD Biosciences, #565618, San Diego, USA) for 2 hours. Upon further washing, cells were analysed on the FACSCantoTMII Cell Analyzer or LSRFortessaTMand data recorded using FACSDivaTMSoftware (BD Biosciences, Franklin Lakes, NJ, USA).Analysis was subsequently performed using FlowJo (FlowJo LLC, Oregon, USA) version 10. Embryonic stem cell culture

[0118] Female HES3 (WiCell) were maintained in mTeSR PLUS (Stem Cell Technologies) / Matrigel (Millipore) and passaged using ReLeSR (Stem Cell Technologies). Quality control was performed with Karyotyping and mycoplasma testing.

[0119] Cardiomyocyte and stromal cell differentiation was achieved using previously described protocols (Voges, H.K., et al., 2017, Development, 144: 1118-1127; Burridge, P.W., et al., 2014, Nat Methods, 11: 855-60). hPSCs were seeded on Matrigel-coated flasks at 2 x104cells / cm2and cultured in mTeSR PLUS for 3 days and then mTeSR-1 for 1 day prior to differentiation. To induce cardiac mesoderm, hPSCs were cultured in RPMI B27-medium (RPMI 1640 GlutaMAX+ 2% B27 supplement without insulin, 200 μM L- ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) and 1% penicillin / streptomycin (ThermoFisher Scientific), supplemented with 5 ng / ml BMP-4 (RnD Systems), 9 ng / ml Activin A (RnD Systems), 5 ng / ml FGF-2 (RnD Systems) and 1 μM CHIR99021 (Stem Cell Technologies), with daily medium exchanges for 3 days. Cardiac specification was performed using RPMI B27- containing 5 μM IWP-4 (Stem Cell Technologies) for another 3 days, and then further 7 days using 5 μM IWP-4 RPMI B27+ (RPMI1640 Glutamax + 2% B27 supplement with insulin, 200 μM L-ascorbic acid 2- phosphate sesquimagnesium salt hydrate and 1% Penicillin / Streptomycin) with media changes every 2-3 days. The cell were then cultured in another 2 days in RPMI B27+ before harvesting using 0.2% collagenase type I (Sigma) in 20% foetal bovine serum (FBS) in PBS (with Ca2+and Mg2+) at 37°C for 1 hour, and then washed before incubation in 0.25% trypsin-EDTA at 37°C for 10 minutes. Cells were neutralized in organoid formation medium (10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% penicillin / streptomycin), filtered through a 100 μm mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 minutes, and re-suspended in organoid formation medium. hCO Fabrication

[0120] The hCO culture inserts were fabricated using SU-8 photolithography and PDMS molding (Mills et al., 2017 PNAS 114: E8372-E8381). Acid-solubilized bovine collagen 1 (Devro) was salt balanced using 10X DMEM and pH neutralized using 0.1M NaOH beforecombining with Matrigel and then the cell suspension on ice. Each hCO contained 5 x 104cells, a final concentration of 2.6 mg / ml collagen I and 9% Matrigel.3.5 μL of suspension was pipetted into the hCO culture insert and incubated at 37°C with 5% CO2 for 45-60 minutes to gel. After gelling, organoid formation medium was added and hCO were cultured for 2 days. The hCO were subsequently cultured for 5 days in maturation medium (MM) - 4% B27– insulin (ThermoFisher Scientific), 1 mM glucose, 200 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 1% penicillin / streptomycin (ThermoFisher Scientific), 1% GlutaMAX (100x) (ThermoFisher Scientific), 33 µg / mL aprotinin (Sigma) and 100 µM palmitate (conjugated to bovine serum albumin in B27, Sigma) in DMEM without glucose, glutamine and phenol red (ThermoFisher Scientific) with a medium change after 2 days

[0024] . hCO were then cultured in weaning medium (WM) 4% B27– insulin (ThermoFisher Scientific), 5.5 mM glucose, 1 nM insulin (Sigma), 200 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 1% penicillin / streptomycin (ThermoFisher Scientific), 1% GlutaMAX (100x) (ThermoFisher Scientific), 33 µg / mL aprotinin (Sigma) and 100 µM palmitate (conjugated to bovine serum albumin in B27, Sigma) in DMEM without glucose, glutamine and phenol red (ThermoFisher Scientific) . hCO AAV Infection and Analysis

[0121] After 7 days of hCO culture they were infected with AAV at the specified MOT. The hCO were imaged using a Leica Thunder microscope after 2 days and then again after 5 days where substantial GFP was observed. Fluorescence images were captured at 5 days and intensity quantified using custom Matlab files

[0024] . The hCO were subsequently snap frozen for other downstream analyses. Generation of cardiac slices and transduction with AAV

[0122] Porcine left ventricular myocardium was obtained from tissues discarded by researchers at the Westmead Institute for Medical Research. Cardiac slices were generated as described previously (Liu, Z., et al., 2020 Journal of Translational Medicine, 18: 437). In brief, LV tissue was obtained and stored in cardioplegic solution within 30 minutes of euthanasia. Slices of 300 µm thickness were cut using a Leica vibratome (Leica VT1000S or VT1200S, Biosystems, Germany) within 4-6 hours of tissue collection. Slices were maintained in transwell plates using the culture-air-liquid interface method and transduced with rAAV vectors at MOT 10,000 on the day of slicing. Slices were maintained until day2 post slicing. Viability was measured as previously described (Liu, Z., et al., 2020 Journal of Translational Medicine, 18: 437).

[0123] All slices were then imaged using Zeiss Axiovert 200M Live Cell Imaging Microscope (Zeiss, Oberkochen, Germany), then snap frozen and stored at – 80oC.

[0124] For bulk DNA / RNA, tissues were lysed directly in lysis buffer for either nucleic acid extraction using the AllPrep DNA / RNA Micro Kit (Qiagen, #80284, Hilden, Germany). For nuclear isolation, tissues were lysed and processed as described previously with minor modifications (Au-Bergmann, O. and S. Au-Jovinge, 2012 JoVE, 65: e4205). Once tissues were lysed in the dounce homogeniser, the lysate was filtered through a 70 µm filter, followed by a 40 µm filter. The lysate was centrifuged at 700 x g for 10 min. Supernatant was removed and the pellet resuspended in 1 mL of NSB. The nuclear pellet was then stained for PCM1+ (pericentriolar material 1), washed, stained with goat anti- rabbit IgG (Sigma-Aldrich, #HPA023370-100UL, St. Louis, MO, USA) and washed again in NSB. The nuclear pellet was stained in DAPI, then sorted using the imaging cytometer to separate the PCM1+ cardiomyocyte fraction and the PCM1- non-myocyte fraction. The sorted nuclei were immediately used for nucleic acid extraction using the AllPrep DNA / RNA Micro Kit. Next generation sequencing analysis

[0125] For next generation sequencing analysis, DNA / RNA samples from hiPSC-CMs, organoids, and cardiac slices were prepared and analysed as previously described (Cabanes-Creus, M., et al., 2022, Mol Ther Methods Clin Dev 24: 88-101). In silico capsid structure prediction

[0126] 3D models of the VP monomers for the new capsid variants were generated from the protein sequence in the online tool SWISS-MODEL using the structure of AAV6 (PDB ID: 3OAH) or AAV3 (PDB ID: 3KIC) as templates, respectively (Schwede, T., et al., 2003 Nucleic Acids Res, 31:3381-5). These reference monomer models were used to generate 60mer capsids (based on 60 copies of the VP3 protein) with the VIPERdb2 oligomer generator (Ho, P.T., et al., 2018 Annu Rev Virol,.5: 477-488). The resulting 60mer models were imported into PyMol in order to generate surface maps and the amino acids changed compared to the parental AAV capsids highlighted.Statistical analysis

[0127] Depending on the data, different statistical analyses detailed in the figure legends were performed. For all used tests, significance was represented as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Example 2. Generation of modified AAV capsids by directed evolution in hiPSC-CMs

[0128] A shuffled AAV capsid library generated from WT AAV serotypes AAV1-12 (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84) was screened in hiPSC-CMs to identify novel cardiotropic AAV capsids.

[0129] After six rounds of selection via wild-type adenovirus mediated replication, the majority of the enriched AAV capsid variants (AAV.KK01, AAV.KK02, AAV.KK03 and AAV.KK05) were found to be most similar to AAV6 capsid (Figure 1A and C), comprising between 6 to 20 amino acid residue differences (Figure 1B). Variant AAV.KK04 was most similar to the AAV3b capsid (Figure 1A, B and C). A sequence alignment is provided in Figure 2.

[0130] Transduction experiments using four lines of hiPSC-CMs (WTCWT, SCVI 8, SCVI 100, SCVI 480) and rAAV.CBA.GFP vectors at MOT 1000, was analysed using flow cytometry to quantify GFP levels on day 5 post transduction. The results indicate that cells transduced with AAV6 demonstrated increased GFP expression when compared to cells transduced with AAV1 or AAV9 (Figure 3A). AAV1 and AAV6 differ by a number of amino acid residues, with a majority located in the VP3 region (Figure 3B).

[0131] The VP3 domains of the AAV variants (AAV.KK01-05) were analysed.. For AAV.KK01, AAV.KK02 and AAV.KK05, the changes to the capsid surface relative to the parental AAV6 (Figure 1D) were minor predicted. However, AAV.KK04 (compared to parental AAV3) was predicted to have significantly more changes to the capsid surface (Figure 1E).

[0132] AAV.KK01-05had differences in amino acid residues within VP1 and VP2, and were not in regions that would be expected to induce structural changes on the capsid surface. (Figure 3B and C).

[0133] To assess packaging efficiency, the five variants and AAV6 capsid were used to package rAAV.CBA.GFP. rAAV.KK01, rAAV.KK02 and rAAV.KK05 yieldedsignificantly more genome containing particles compared to AAV6; with rAAV.KK01, rAAV.KK02 and rAAV.KK05 yielding two times more genome containing particles compared to AAV6 (Figure 4). Example 3: Cell entry and gene expression in hiPSC-CMs transduced with rAAV.KK01-05 vectors

[0134] The novel capsids were vectorized and functionally compared to wildtype AAV1, AAV6 and AAV9 capsids using a competitive transduction assay. Each vector (rAAV.CBA.GFP-BC) was identified by a unique barcode, which allowed the pool of vectors to be mixed at equimolar ratio and used to simultaneously transduce hiPSC-CMs at various multiplicity of transduction (MOT) 100, 1000 and 10,000 (Figure 5),

[0135] Testing was performed in cardiomyocytes differentiated from three hiPSC lines, SCVI 100 (Figure 6) and SCVI 8 and SCVI 480 (Figure 7). Transduction efficiency was determined at the level of cell entry (DNA) and transgene expression (mRNA / cDNA). At all tested MOTs, rAAV.KK04 was the most efficient at cell entry and transgene expression in human cardiomyocytes. rAAV9 was inefficient at transduction at all MOTs and in cardiomyocytes derived from all three iPSC lines, rAAV6 was the best among the three wildtype AAV tested.

[0136] The five novel capsids were then tested individually. All vectors were used to package a non-barcoded transgene (rAAV.CBA.GFP). rAAV.KK04 induced the strongest GFP expression in cardiomyocytes across all hiPSC lines (Figure 8A). This was further quantified by flow cytometry, which showed a significantly higher proportion of transduced cardiomyocytes (Figure 8B). The mean fluorescence intensity was similar across all AAVs tested. (Figure 9A). All hiPSC-CM cultures were confirmed to have purity >90% prior to transduction (Figure 9B). Example 4: Cell entry and gene expression in cardiac organoids transduced with rAAV.KK01-05 vectors

[0137] hiPSC-CMs have a relatively foetal phenotype, when compared to primary cardiomyocytes. To evaluate the effect of cell maturity on transduction with the rAAV.KK01-rAAV.KK05 vectors, the maturity of hiPSC-CMs were enhanced by culturing into human cardiac organoids (hCOs) (Voges, H.K., et al., 2017, Development 144:1118-1127), to model a clinically relevant mature myocardium cell type.

[0138] The hCOs were transduced with barcoded rAAV.CBA.GFP-BC vectors in acompetitive transduction assay (MOT 100, 1000 and 10,000). Low transduction efficiency was observed for rAAV9. rAAV.KK05 gave the highest cell entry efficiency. rAAV.KK04 was less efficient that the other variants(Figure 10A). However, that rAAV.KK04 showed the gene expression (30.47% and 29.80% barcode mRNA reads) at MOT 100 and 1000, respectively.

[0139] The five novel capsids were also compared to the three wildtype AAVs, using the unbarcoded rAAV.CBA.GFP and GFP expression as a measure of efficiency. The highest GFP fluorescence was seen in hCOs transduced with rAAV6 and rAAV.KK02 (Figure 10B-C). Example 5: Cell entry and gene expression in myocytes of pig, primate and human cardiac slices transduced with rAAV.KK01-05 vectors

[0140] To assess the performance of the novel AAV variants in a model of primary cardiomyocytes, cardiac slices were generated from the left ventricular myocardium of normal pigs and animals with infarcted hearts, and transduced with the barcoded rAAV.CBA.GFP-BC vector mix on the day of slicing. GFP expression was observed at day 2 post-transduction (Figure 11).

[0141] Bulk DNA / RNA obtained from the cardiac slices and analysed by NGS revealed that in the normal heart, the five novel variants were comparable to rAAV6 at the level of cell entry. Wildtype AAV1 and AAV9 did not demonstrate significant cell entry, or gene expression. (Figure 12A-B). The five novel variants demonstrated improved gene expression, when compared to wildtype AAV1 and AAV9. However, rAAV6 showed superior transgene expression, with significantly higher barcode reads observed at the mRNA level (Figure 12B). Cardiac slices generated from the infarcted pig heart showed a similar trend as cardiac slices generated from normal heart.

[0142] Cardiac slice is known to contain a heterogeneous population of cells. To determine the best capsid for efficiently targeting cardiomyocytes, cardiomyocytes were identified from extracted nuclei by PCM1+ sorting (Figure 13A). Infarct heart tissue showed an increase in the non-myocyte population (Figure 13B and C). The extracted nuclei were then used for extraction of nuclear DNA / RNA, followed by analysis by next generation sequencing.

[0143] In extracted nuclei of normal hearts, we observed that rAAV.KK01 was most efficient at cell entry into cardiomyocytes (Figure 14B), though transgene expression washigh for rAAV6, rAAV.KK02, rAAV.KK03, rAAV.KK04 and rAAV.KK05, when compared to AAV1, AAV8, AAV9, AAV serotype rh10 and recombinant AAV serotype rh74 (Figure 14A). Similar trends were observed in the extracted nuclei of non-myocyte fractions (Figure 14C-D).

[0144] In cardiac slices of the non-human primate (NHP) heart, rAAV.KK01 and rAAV.KK04 were efficient at cell entry in the cardiac slice (Figure 15A). rAAV.KK01 showed a high level of gene expression, and did rAAV.KK05 andrAAV.KK04 (Figure 15B). However, when cardiomyocyte and non-myocyte populations were analysed, it was confirmed that rAAV.KK01 was efficient for cell entry in both myocytes and non- myocetes (Figure 15C), while rAAV.KK01, rAAV.KK02 and rAAV6 also drove strong transgene expression in NHP myocytes (Figure 15D). In the non-myocyte fraction, rAAV.KK01 was demonstrated the highest cell entry transgene expression of the vectors tested (Figure 15E-F).

[0145] Similar cardiac slice experiments were performed using cardiac slices generated from the left ventricular myocardium of donor human hearts. In extracted nuclei of human heart tissue, we observed that rAAV.KK01 was most efficient at cell entry into myocytes (Figure 16B). Of the natural variant AAVs, AAV1 and rh10 appeared to provide high transgene expression. rAAV.KK01, rAAV.KK02, rAAV.KK02, rAAV.KK03, rAAV.KK04 and rAAV.KK05 also provided transgene expression in human myocytes, but improved transgene expression was observed rAAV.KK01 and rAAV.KK03 (Figure 16A). Whereas in non-myocyte fractions, rAAV.KK01 and rAAV.KK04 were more efficient at cell entry (Figure 16D), while AAV1, AAV6 and rAAV.KK03 and rAAV.KK04 provided transgene expression in human non-myocytes (Figure 16C).

[0146] The data from the pig, NHP and human cardiac slices indicate that the modified adeno-associated virus (AAV) capsid polypeptides disclosed herein are advantageous for targeting cell entry and / or facilitating transgene expression in primary adult cardiomyocytes. Example 6: In vivo gene entry and gene expression in pig heart transduced with rAAV.KK01-05 and FDA approved AAVs

[0147] A kit of AAVs was created for an in vivo competitive transduction assay. The kit included rAAV.KK01-05 of the present disclosure and a series of known AAVs. For benchmarking purposes, the kit was designed to include AAVs currently approved by theFDA for gene therapy clinical trials in cardiac indications. These include AAV9, AAVrh74 and AAV2i8. The AAVs were added to the kit in equal amounts based on vector titre to produce 8.5 x1012vgs (vector genomes) in total. The entire amount was infused via the intracoronary route in the left anterior descending equivalent in a domestic pig. This delivery approach mimics clinical delivery of therapeutics in the cardiac catheter laboratory in humans. Six weeks later the heart was removed and segmented (anterior LV wall, apex, lateral LV wall, posterior LV wall, RV free wall and septum). DNA and RNA (converted to cDNA) were extracted for each segment. The AAV expression cassette for each AAV capsid had a unique 6-mer barcode. This region of the vector genome was PCR amplified from DNA and cDNA, and the amplicon analysed by next generation sequencing to enable quantification of gene entry (DNA) and expression (cDNA) for each AAV capsid type. The results are shown in the heat map (Figure 17A) for each cardiac segment and collectively ranked in the bar graph (Figure 17B). All five of the modified capsids of the present disclosure, rAAV.KK01, rAAV.KK02, rAAV.KK03, rAAV.KK04 and rAAV.KK05 outperformed AAV9, AAVrh74 and AAV2i8.

[0148] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Table 2. Capsid SequencesTable 3. Exemplary associated genesTable 4. Amino acid modifications across the variant AAV capsid proteins* Amino acid positions shown are relative to SEQ ID NO:1 (AAV6) ** Arginine (R) inserted between amino acid positions 151 and 152 relative to the amino acid sequence of SEQ ID NO:6

Claims

CLAIMS:

1. A capsid polypeptide comprising an amino acid sequence of any one of SEQ ID NOs:5-8, or an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoing; wherein the amino acid sequence comprises: a. amino acid residues 1-204 of SEQ ID NO:5, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity thereto; b. amino acid residues 1-204 of SEQ ID NO:6, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 98% sequence identity thereto; c. amino acid residues 1-204 of SEQ ID NO:7, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 95% sequence identity thereto; or d. amino acid residues 1-204 of SEQ ID NO:8, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity thereto.

2. A capsid polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least about 95%, 96%, 97%, 98% or 99% sequence identity thereto; wherein the amino acid sequence comprises: a. amino acid residues 1-204 of SEQ ID NO:9, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 92% sequence identity thereto, and / or b. amino acid residues 205-737 of SEQ ID NO:9, with numbering relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity thereto.

3. The capsid polypeptide of claim 1, comprising:(i) an amino acid sequence having at least about 85% sequence identity to any one of SEQ ID NOs:5-8; (ii) amino acid residues 1-204 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to a of the foregoing; and / or (iii) amino acid residues 205-735 of any one of SEQ ID NOs:5-8 or an amino acid sequence having at least about 85% sequence identity to any of the foregoing; and wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 207, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460, 465, 466, 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO:

1.

4. The capsid polypeptide of claim 3, wherein the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 orM541; S547 or G547; A548 or T548; G549 or T549; A553 or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:

1.

5. The capsid polypeptide of claim 3 or claim 4, wherein the at least one amino acid modification comprises M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418 and F584, with numbering relative to SEQ ID NO:

1.

6. The capsid polypeptide of any one of claims 3 to 5, wherein the at least one amino acid modification comprises M1del; M2; T3; V19; A24; Q26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168, S224; D418; F584; A598 and N642, with numbering relative to SEQ ID NO:

1.

7. The capsid polypeptide of any one of claims 3 to 6, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

5.

8. The capsid polypeptide of claim 7, wherein the capsid polypeptide consists of the amino acid sequence of SEQ ID NO:

5.

9. The capsid polypeptide of claim 3 or claim 4, wherein the at least one amino acid modification comprises L129; L146; 148del; K162; K164; R168 and E418, with numbering relative to SEQ ID NO:

1.

10. The capsid polypeptide of any one of claims 3 to 4 and 9, wherein the at least one amino acid modification comprises M1; A2; A3; I19; D24; K26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; G141; L146; 148del; Q151; E152; S157; K162; K164; R168; A224; E418; L584; V598; and H642, with numbering relative to SEQ ID NO:

1.

11. The capsid polypeptide of any one of claims 3 to 4, 9 and 10, wherein the capsidpolypeptide comprises the amino acid sequence of SEQ ID NO:

6.

12. The capsid polypeptide of claim 11, wherein the capsid polypeptide consists of the amino acid sequence of SEQ ID NO:

6.

13. The capsid protein of claim 3 or claim 4, wherein the at least one amino acid modification comprises A24; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151_E152insR; S152; T157; K162 and R168, with numbering relative to SEQ ID NO:

1.

14. The capsid polypeptide of any one of claims 3, 4 and 13, wherein the at least one amino acid modification comprises M1; A2; A3; I19; A24; K26; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; G141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168; A224; D418; L584; V598; and H642 with numbering relative to SEQ ID NO:

1.

15. The capsid polypeptide of any one of claims 3, 4, 13 and 14, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

16. The capsid polypeptide of claim 15, wherein the capsid polypeptide consists of the amino acid sequence of SEQ ID NO:

7.

17. The capsid polypeptide of claim 3 or claim 4, wherein the at least one amino acid modification comprises 1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; E418; and F584, with numbering relative to SEQ ID NO:

1.

18. The capsid polypeptide of any one of claims 3, 4 and 17, wherein the at least one amino acid modification comprises 1del; M2; T3; V19; A24; Q26; A29; Q31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151_E152insR; S152; T157; K162; Q164; R168; S224; D418; F584; V598; and H642, with numbering relative to SEQ ID NO:

1.

19. The capsid protein of any one of claims 3, 4, 17 and 18, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

8.

20. The capsid protein of claim 19, wherein the capsid polypeptide consists of the aminoacid sequence of SEQ ID NO:

8.

21. The capsid polypeptide of claim 2, comprising: (i) the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity thereto; (ii) amino acid residues 1-204 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity thereto; and / or (iii) amino acid residues 205-737 of any one of SEQ ID NO:9 or a sequence having at least about 85% sequence identity thereto; wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 207, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599 , 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO:

1.

22. The capsid polypeptide of claim 21, wherein the at least one amino acid modification is selected from the group consisting of M1 or 1del; A2 or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553 or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:

1.

23. The capsid polypeptide of claim 22, wherein the at least one amino acid modification is selected from the group consisting of T14; Q21; K24; P29; P31; P34; A35; E36; R37; H38; K39; D41; S42; F56; A67; R92; V125; L129; E 134; G135; A136; K137; G141; V146; E147; P148; Q151_E152insR; S152; T157; I159; K162; Q164; R168; A194; S196; G197; V198; T200; N201; T205; S207; S224; T233; M235; A263; T264; R310; N312; Q326; T330; T345; E411; S447; S451; T452; G453; T455; Q456, G457, T458, Q459, Q460; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H510; D515; L517; V518; P522; E531; E532; H538; N540; G547; T548; T549; A553; E554; R567, T568; Q576; Y577; N582; L584; N588; A590, T592; R594; T595; N597; D598; Q599; H642; M648; T660; T661; P664; A665; I699; N706; V709; T717; V720; S722; N735, with numbering relative to SEQ ID NO:

1.

24. The capsid polypeptide of any one of claims 21 to 23, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

9.

25. The capsid polypeptide of claim 24, wherein the capsid polypeptide consists of the amino acid sequence of SEQ ID NO:

9.

26. An AAV vector comprising the capsid polypeptide of any one of claims 1 to 25.

27. The AAV vector of claim 26, further comprising a heterologous coding sequence.

28. The AAV vector of claim 27, wherein the heterologous coding sequence encodes a peptide, polypeptide or polynucleotide.

29. The AAV vector of claim 28, wherein peptide, polypeptide or polynucleotide is a therapeutic peptide, polypeptide or polynucleotide.

30. An isolated nucleic acid molecule encoding the capsid polypeptide of any one of claims 1 to 25.

31. A vector comprising the nucleic acid molecule of claim 30.

32. The vector of claim 31, wherein the vector is selected from among a plasmid, cosmid, phage and transposon.

33. A host cell comprising the AAV vector of any one of claims 26 to 29, the nucleic acid molecule of claim 30, or the vector of claim 31 or claim 32.

34. A method for introducing a heterologous coding sequence into a host cell, comprising contacting a host cell with the AAV vector of claim 27 or claim 28.

35. A method for producing an AAV vector, comprising culturing a host cell comprising a nucleic acid molecule encoding the capsid polypeptide of any one of claims 1 to 25, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising the capsid polypeptide of any one of claim 1 to 25, wherein the capsid encapsidates the heterologous coding sequence.

36. The method of claim 34, wherein contacting the host cell with the AAV vector comprises administering the AAV vector to a subject.

37. The method of any one of claims 34 to 36, wherein the method is carried out in vitro or ex vivo.

38. The method of any one of claims 34 to 37, wherein the host cell is a cardiac cell.

39. The method of claim 38, wherein the cardiac cell is a mammalian cardiac cell.

40. The method of claim 38 or claim 39, wherein the cardiac cell is a primate cardiac cell.

41. The method of any one of claims 38 to 40, wherein the cardiac cell is a human cardiac cell.

42. The method of any one of claims 38 to 41, wherein the cardiac cell is in a cardiac organoid.

43. The method of any one of claims 38 to 42, wherein the cardiac cell is a cardiomyocte.

44. The method of claim 43, wherein the cardiomyocyte is a primary cardiomyocte.

45. The method of claim 43 or claim 44, wherein the cardiomyocyte is an iPSC-derived cardiomyocyte.

46. A method for producing a modified AAV vector that exhibits enhanced transgene expression in a human cardiac cell when the vector comprises a transgene, comprising: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 19, 24, 26, 29, 31, 38, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 224, 418, 584, 598 and 642, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises at least one amino acid modification selected from the group consisting of M1 or 1del; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K38 or H38; D41 or N41; G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; E134 or Q134; G135 or A135; A136 or G136; K137 or E137; G141 or A141; V146 or L146; Q148, P148 or 148del; Q 151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; K168 or R168; A224 or S224; D418 or E418; L584 or F584; V598 or A598; and H642 or N642, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.

47. A method for producing a modified AAV vector that exhibits enhanced transgeneexpression in a human cardiac cell, and wherein the vector comprises a transgene, wherein the method comprises: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the amino acid sequence of the reference capsid polypeptide at amino acid positions 129, 146, 148, 162, 164, 168 and 418, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises L129; L146; 148del; K162; K164; R168 and E418, with numbering relative to SEQ ID NO:1; and c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.

48. The method of claim 46 or claim 47, wherein the reference capsid polypeptide comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

6.

49. A method for producing a modified AAV vector that exhibits enhanced cell entry into a human cardiac cell when the vector comprises a transgene, comprising: a) identifying a reference capsid polypeptide for transducing human cardiac cells in vivo; b) modifying the amino acid sequence of the reference capsid polypeptide at at least one position selected from amino acid positions 1, 2, 3, 14, 19, 21, 24, 26, 29, 31, 34, 35, 36, 37, 38, 39, 41, 42, 56, 67, 92, 129, 134, 135, 136, 137, 141, 146, 148 151, 152, 157, 157, 162, 164, 168, 194, 196, 197, 200, 201, 205, 207, 224, 235, 326, 330, 345, 411, 418, 447, 451, 452, 453, 455, 456, 457, 458, 459, 460 , 465 , 466 , 467 , 470 , 473 , 475 , 489 , 493 , 494, 502 , 505 , 510 , 515 , 517 , 518 , 522 , 531 , 532 , 538 , 540 , 541 , 547 , 548 , 549 , 553 , 554 , 567, 568 , 576 , 577 , 582 , 584 , 588 , 590, 592 , 594 , 595 , 597 , 598, 599, 642, 648 , 660 , 661 , 664 , 665 , 699 , 706 , 709 , 717 , 720 , 722 , 735, with numbering relative to SEQ ID NO:1, to thereby produce a modified capsid polypeptide that comprises one or more of M1 or 1del; A2or M2; A3 or 3T, T14 or N14; V19 or I19; Q21 or E21; K24, A24 or D24; Q26 or K26; P29 or V29 or A29; P31, Q31 or K31; P34 or A34; A35 or N35; E36 or Q36; Q37 or R37; K38 or H38; K39 or Q39; D41 or N41; S42, G42 or R42; F56 or G56; A67 or E67; R92 or K92; F129 or L129; Q134 or E134; G135 or A135; G136 or A136; E137 or K137; A141 or G141; L146 or V146; P148, 148del, or Q148; Q151 or Q151_E152insR; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A196; A197 or G197; T200 or P200; N201 or T201; S205 or T205; S207 or G207; A224 or S224; M235 or L235; ; Q326 or T326; T330 or V330; S345 or T345; E411 or T411; D418 or E418; N447 or S447; N451 or S451; T452 or Q452; G453 or S453; S455 or T455; Q456 or A456, G457 or Q457, N458 or T458, Q459 or K459, Q460 or D460; Q465 or R465; A466 or G466; S467 or G467; N470 or G470; V473 or A473; A475 or P475; L489 or V489; A493 or K493; N494 or T494; P502 or T502; A505 or G505; H510 or N510; D515 or E515; L517 or I517; I518 or V518; P522 or T522; E531 or K531; E532 or D532; H538 or S538; N540 or V540; L541 or M541; S547 or G547; A548 or T548; G549 or T549; A553 or T553; A554 or E554; K567 or R567, A568 or T568; R576 or Q576; F577 or Y577; V582 or N582; F584 or L584; S588 or N588; D590 or A590, A592 or T592; G594 or R594; D595 or T595; H597 or N597; V598, A598 or D598; M599 or Q599; H642 or N642; M648 or L648; A660 or T660; E661 or T661; A664 or P664; T665 or A665; V699 or I699; A706 or N706; A709 or V709; N717 or T717; L720 or V720; T722 or S722; P735 or N735, with numbering relative to SEQ ID NO:1; c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.

50. The method of claim 49, wherein the modified capsid polypeptide comprises amino acid modifications M1del; M2; T3; V19; A24; Q26; L129; A141; P148; Q151_E152insR; S152; T157; K162; R168; S224; L584; A598 and N642, with numbering relative to SEQ ID NO:

1.

51. The method of claim 50, wherein the modified capsid polypeptide further comprises amino acid modifications E418 and F584, with numbering relative to SEQ ID NO:

1.

52. The method of claim 49, wherein the modified capsid polypeptide comprises amino acid modifications of T14; Q21; K24; P29; P31; P34; A35; E36; R37; H38; K39; D41;S42; F56; A67; R92; V125; L129; E 134; G135; A136; K137; G141; V146; E147; P148; Q151_E152insR; S152; T157; I159; K162; Q164; R168; A194; S196; G197; V198; T200; N201; T205; S207; S224; T233; M235; A263; T264; R310; N312; Q326; T330; T345; E411; S447; S451; T452; G453; T455; Q456, G457, T458, Q459, Q460; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H510; D515; L517; V518; P522; E531; E532; H538; N540; G547; T548; T549; A553; E554; R567, T568; Q576; Y577; N582; L584; N588; A590, T592; R594; T595; N597; D598; Q599; H642; M648; T660; T661; P664; A665; I699; N706; V709; T717; V720; S722; N735, with numbering relative to SEQ ID NO:

1.

53. The method of any one of claims 46 to 52, wherein the modified AAV vector comprises a transgene.

54. The method of any one of claims 46 to 53, further comprising assessing the transgene expression of the modified AAV vector in an in vitro system that utilises human cardiac cells.

55. The method of claim 54, wherein the in vitro system comprises a cardiac organoid comprising human cardiac cells.

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