Modified cardiophilic aav capsid polypeptides and vectors

Abstract

This invention provides a variant AAV capsid polypeptide that, when present in the capsid of an AAV vector, promotes efficient transduction of cardiac cells, particularly human cardiac cells. A method for preparing AAV vectors, particularly a method for preparing vectors for transducing cardiac cells, especially a method for preparing AAV vectors for gene therapy purposes, particularly a method for preparing those vectors targeting the heart or cardiac cells and tissues, is also provided.

Description

Technical Field

[0001] This invention generally relates to a modified adeno-associated virus (AAV) capsid polypeptide and a nucleic acid-encoding molecule. It also relates to an AAV vector comprising the capsid polypeptide and a nucleic acid vector (e.g., a plasmid) comprising a nucleic acid-encoding molecule, and a host cell comprising the vector. Furthermore, this invention relates to methods and uses of the polypeptide, the nucleic acid-encoding molecule, the vector, and the host cell. Background Technology

[0002] Gene therapy is most commonly conducted using viral vectors, and significant progress has recently been made using adeno-associated virus (AAV) vectors. AAV is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb long. The AAV genome contains inverted terminal repeats (ITRs) at both ends of the molecule, flanked by two open reading frames (rep and cap). The cap gene encodes three structural capsid proteins: VP1, VP2, and VP3. These three capsid proteins typically assemble in a 1:1:8-10 ratio 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 an assembly activation protein (AAP) from another open reading frame. AAP promotes capsid assembly by targeting capsid proteins to the nucleolus and facilitating 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, genome integration, and other processes. Recently, an X gene was discovered at 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] ITRs participate in multiple functions, particularly the integration of 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, leading to latent infection. Therefore, AAV can be used to introduce heterologous sequences into cells. In nature, helper viruses (such as adenoviruses or herpesviruses) provide protein factors that allow AAV viruses to replicate and package new viral particles in infected cells. In the case of adenoviruses, genes E1A, E1B, E2A, E4, and VA provide helper functions. After infection with a helper virus, the AAV provirus is rescued and amplified, resulting in both AAV and helper viruses.

[0004] AAV vectors (also known as recombinant AAV, rAAV) have been successfully used in gene therapy settings. These AAV vectors contain a genome lacking part, most, or all of the natural AAV genome, and instead contain one or more heterologous sequences flanked by ITRs. These AAV vectors are widely used to deliver heterologous nucleic acids into an individual's cells for therapeutic purposes; in many cases, it is the expression of the heterologous nucleic acid that confers the therapeutic effect. Although several AAV vectors are now used clinically, only a limited number of vectors exhibit the required in vivo transduction efficiency in different types of primary human cells / tissues to achieve adequate expression of the heterologous nucleic acid for therapeutic applications. Therefore, there is a need to develop alternative AAV vectors containing capsid proteins to facilitate efficient transduction in host cells (including in vivo transduction). Summary of the Invention

[0005] This invention is partly based on the generation of modified AAV capsid peptides. In a specific embodiment, when the capsid is contained in an AAV vector, the capsid peptide promotes transduction of human cells (e.g., human heart cells). Generally, AAV vectors containing the capsid peptides of this invention exhibit improved or enhanced transduction (including in vivo transduction) compared to AAV vectors containing other AAV capsid peptides (e.g., the prototype AAV capsids listed in SEQ ID NO:1-4). Therefore, the capsid peptides of this invention are particularly useful in the preparation of AAV vectors (especially AAV vectors for gene therapy purposes). Similarly, AAV vectors containing the capsid peptides of this invention (i.e., having a capsid containing the capsid peptides of this invention or a capsid composed of the capsid peptides of this invention) are particularly useful in gene therapy applications (e.g., for delivering heterologous nucleic acids to treat various diseases and conditions).

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

[0007] In another aspect, the present invention provides a capsid polypeptide comprising the 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 therewith; wherein the amino acid sequence comprises: (a) amino acid residues 1-204 of SEQ ID NO:9 corresponding to SEQ ID NO:1; or an amino acid sequence having at least about 92% sequence identity therewith, and / or (b) amino acid residues 205-737 of SEQ ID NO:9 corresponding to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity therewith.

[0008] Also provided is a capsid polypeptide comprising: (i) an amino acid sequence having at least about 85% sequence identity with any of the sequences in SEQ ID NO: 5-8; (ii) amino acid residues 1-204 of SEQ ID NO: 5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; and / or (iii) amino acid residues 205-736 of SEQ ID NO: 5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; and wherein the capsid polypeptide comprises amino acids selected from those in SEQ ID NO: 5-8. At least one amino acid modification at a position in the group consisting of the following amino acid positions numbered NO:1: 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, wherein the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion (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 148 missing (148del); Q151 or Q151_E15 Two insertions are made between R(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 the amino acid position relative to SEQ ID NO:1.

[0009] In one embodiment, the capsid polypeptide comprises M1 deletion (M1 deletion); M2; T3; V19; A24; Q26; L129; A141; P148; intercalation R between Q151 and E152; S152; T157; K162; R168; S224; E418 and F584, with amino acid positions numbered relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises L129; L146; 148 deletion (148del); K162; K164; R168 and E418, with amino acid positions numbered 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; an insertion R between Q151 and E152; S152; T157; K162; and R168, with amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises 1 deletion (1del); M2; T3; V19; A24; Q26; L129; A141; P148; an insertion R between Q151 and E152; S152; T157; K162; R168; S224; E418; and F584, with amino acid positions relative to SEQ ID NO:1.

[0010] A capsid polypeptide is also provided, comprising: (i) the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity with it; (ii) amino acid residues 1-204 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity with it; and / or (iii) amino acid residues 205-736 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity with it; wherein the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of the following amino acid positions relative to the number of SEQ ID NO:1: 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; 1 64; 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, 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; T23 3; M235; A263; T264; R310; N312; Q326; T330; T345; E411; S447; S451; T451; T 452; 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, amino acid positions relative to SEQ ID NO:1. ;

[0011] An AAV vector comprising the capsid polypeptide of the present invention is also provided. In some instances, the vector further comprises a heterologous coding sequence, such as a heterologous coding sequence encoding a peptide, polypeptide, or polynucleotide (e.g., a therapeutic peptide, polypeptide, or polynucleotide).

[0012] In another aspect, an isolated nucleic acid molecule encoding the capsid polypeptide of the present invention is provided. In yet another aspect, a vector comprising the above-described nucleic acid molecule is provided. In some instances, the vector is selected from plasmids, granules, bacteriophages, and transposons.

[0013] A host cell is also provided, which contains the AAV vector, nucleic acid molecule or vector of the present invention.

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

[0015] In another aspect, a method for preparing an AAV vector is provided, comprising culturing host cells under conditions suitable for promoting AAV vector assembly, the host cells comprising a nucleic acid molecule encoding the capsid polypeptide of the present invention, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeat sequences, and auxiliary functions for generating productive AAV infection, the AAV vector comprising a capsid containing the capsid polypeptide of the present invention, wherein the capsid encapsulates the heterologous coding sequence. In some instances, the host cell is a cardiac cell.

[0016] Also provided is a method for preparing a modified AAV vector, wherein when the vector contains a transgene, the modified AAV vector enhances transgene expression in human heart cells, the method comprising: a) identifying a reference capsid polypeptide for in vivo transduction of human heart cells; b) selecting from the group corresponding to SEQ ID NO. The sequence of the reference capsid polypeptide is modified at at least one of the following amino acid positions encoded by NO:1: 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, thereby preparing a modified capsid polypeptide comprising at least one amino acid modification selected from the group consisting of: M1 or 1 deletion; 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 148 missing; Insert R between Q151 or Q151_E152; 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, amino acid positions relative to SEQ ID NO:1; and c) carrier the modified capsid polypeptide to prepare a modified AAV carrier.

[0017] Also provided is a method for preparing a modified AAV vector, wherein when the vector contains a transgene, the modified AAV vector enhances transgene expression in human heart cells, the method comprising: a) identifying a reference capsid polypeptide for in vivo transduction of human heart cells; b) selecting from the group corresponding to SEQ ID NO. The sequence of the reference capsid polypeptide is modified at at least one of the following amino acid positions corresponding to SEQ ID NO:1: 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, thereby preparing a modified capsid polypeptide comprising L129; L146; 148 deletion; K162; K164; R168; E418, amino acid positions relative to SEQ ID NO:1; and c) the modified capsid polypeptide is carrier-mediated, thereby preparing a modified AAV carrier.

[0018] A method for preparing a modified AAV vector is also provided, wherein when the vector contains a transgene, the modified AAV vector enhances cell entry into human heart cells, the method comprising: a) identifying a reference capsid polypeptide for in vivo transduction of human heart cells; b) modifying the amino acid sequence of the reference capsid polypeptide at at least one position selected from the following amino acid positions relative to the number of SEQ ID NO:1: 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, 59 8 and 642, thereby preparing a modified capsid polypeptide comprising one or more of the following: M1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152 intercalation of R; S152; T157; K162; R168; S224; E418; L584; F584; A598 and N642, amino acid positions relative to SEQ ID NO:1; and c) carrier-encapsulating the modified capsid polypeptide to prepare a modified AAV vector. In one embodiment, the modified capsid polypeptide comprises amino acid modifications including M1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; intercalation R between Q151 and E152; S152; T157; K162; R168; S224; L584; A598; and N642, with amino acid positions corresponding to SEQ ID NO:1. In another embodiment, the modified capsid polypeptide further comprises amino acid modifications E418 and F584, with amino acid positions corresponding to SEQ ID NO:1. Attached Figure Description

[0019] This document describes exemplary embodiments of the invention by way of non-limiting examples only, with reference to the following accompanying drawings.

[0020] Figure 1This is a schematic diagram of the cardiogenic rAAV variants obtained through directed evolution. Directed evolution was performed using the hiPSC-CM cell line WTCWT. After six rounds of screening, the enriched novel AAVs were sequenced and phylogenetic analyses were performed for comparison with parental AAVs. (A) Amino acid sequence clustering analysis from the novel variants (red) to the wild-type AAV parental serotype (black), with the relationship presented in the form of a phylogenetic tree. (B) A heatmap showing the percentage of identity between the selected parental AAVs and the enriched novel variants. (C) Analysis of parental sequence contribution to variants separated after directed evolution. The black line represents the most likely composition of each shuffled clone based on the longest identical sequence with the parental variant in the 5' to 3' direction. (D) Schematic diagram of the surface of the novel capsid variants KK01, KK02, KK03, and KK05. Colored areas (yellow, orange, purple) represent surface residues different from the parental AAV6 capsid. Amino acid variations not on the capsid surface are listed below (D). The capsid variant KK04 is described as shown in (E). The colored areas (red and purple) represent surface residues of the capsid that differ from the parent AAV3b.

[0021] Figure 2 These are the sequence alignment results for rAAV.KK01, rAAV.KK02, rAAV.KK03, rAAV.KK04, rAAV.KK05, AAV6, and AAV3b.

[0022] Figure 3 This is a comparative graph of the transduction efficiency of cardiotropic rAAV capsids in hiPSC-CM. (A) Four hiPSC-CM cell lines (WTCWT, SCVI 8, SCVI 100, and SCVI 480) were transduced with the rAAV.CBA.GFP vector at MOT 1000, and flow cytometry was used to quantify GFP (n=3 per group) on day 5 post-transduction. Flow cytometry scatter plots quantify the proportion of GFP-positive cardiomyocytes (cTnT+ cells). (B) Alignment of AAV1 and AAV6 capsids, showing amino acid differences in the VP3 region. (C) Alignment results of all novel variants except rAAV.KK04 with the parental AAV6. (D) The amino acid sequence of rAAV.KK04 is aligned with the parental AAV3.

[0023] Figure 4 This is a schematic diagram showing the viral titers of viral preparations of rAAV.KK01 to rAAV.KK05 and rAAV6 packaged with CBA-GFP expression cassettes.

[0024] Figure 5This is a schematic diagram of transducing hiPSC-CM cells using a barcoded rAAV library. Three hiPSC-CM cell lines were competitively transduced using a barcoded rAAV.CBA.GFP library at MOT 100, 1000, and 10000, and cells were collected on day 5 post-transduction. Cells were imaged to observe GFP autofluorescence before DNA / RNA harvesting.

[0025] Figure 6 This is a schematic diagram of rAAV.KK01 to rAAV.KK05 and gene delivery to hiPSC-CM. HiPSC-CM cells from SCVI 100 were competitively transduced using a barcode-encoded library of the rAAV.CBA.GFP vector, and cells were collected on day 5 post-transduction. DNA / RNA extraction was performed, followed by analysis using next-generation sequencing (n=3 per group). For transduction multiplicity (MOT) of 100, 1000, and 10000, the relative proportion of barcode reads after NGS analysis is given at the levels of cell entry (gDNA) and gene expression (mRNA). Results are expressed as a percentage of total reads for each cell line.

[0026] Figure 7 This is a schematic diagram illustrating the efficiency of gene delivery of rAAV.KK01 to rAAV.KK05 in hiPSC-CM cells SCVI 8 and SCVI480. HiPSC-CM cells were competitively transduced with barcoded libraries of the rAAV.CBA.GFP vector at MOT 100, 1000, and 10000, and cells were collected on day 5 post-transduction. For (A) SCVI 8 and (B) SCVI 480, the relative proportions of barcoded reads after NGS analysis are shown at the levels of cellular entry (gDNA) and gene expression (mRNA). Results are expressed as a percentage of total reads for each cell line.

[0027] Figure 8This is a schematic diagram of GFP expression in hiPSC-CM cells transduced with rAAV.KK01 to rAAV.KK05. Three hiPSC-CM cell lines were transduced with MOT 1000 using the unbarcoded rAAV.CBA.GFP vector, and GFP was quantified by microscopy and flow cytometry on day 5 post-transduction (n=3 per group for SCVI 8 and SCVI 100, and n=4 per group for SCVI 480). (A) A fluorescence image of GFP autofluorescence in live cells (scale bar = 100 μM). (B) A flow cytometry scatter plot quantifies the proportion of GFP-positive cardiomyocytes (cTnT+ cells).

[0028] Figure 9 This is a schematic diagram illustrating the functional validation of a novel AAV variant in hiPSC-CM. Three hiPSC-CM cell lines were transduced with the unbarcoded rAAV.CBA.GFP vector at MOT 1000, and flow cytometry was used to quantify GFP (n=3 or 4 per group) on day 5 post-transduction. Flow cytometry scatter plots quantify the mean fluorescence intensity of GFP in (A) cardiomyocytes (cTnT+ cells). (B) Purity of each differentiation cell type (diff) was also measured by flow cytometry and expressed as cTnT%.

[0029] Figure 10 This is a schematic diagram of competitive transduction analysis and functional validation of novel AAV variants in hCO. (A) Human heart organoids were transduced with barcoded libraries of the rAAV.CBA.GFP vector at MOT 100, 1000, and 10000. Next-generation sequencing (n=8 per group) was then performed. The relative proportions of barcoded reads after NGS analysis for all cell lines are shown at the levels of cell entry (gDNA) and gene expression (cDNA). Results are expressed as a percentage of total reads for each cell line. (B) Human heart organoids were then transduced with the unbarcoded rAAV vector at MOT 10000, followed by microscopic analysis to quantify GFP (n=6 or 7 per group). Fluorescence images show GFP autofluorescence in live organoids. (C) GFP fluorescence intensity was quantified from images obtained from the microscope using MATLAB.

[0030] Figure 11 This is a schematic diagram of heart sections transduced with a barcoded AAV variant (which remained viable for up to two days post-slicing). Heart sections were taken from the left ventricular myocardium of normal pigs or pigs with infarcted hearts. (A) Viability was assessed by calcein staining on days 1 and 2 post-slicing. (B) GFP autofluorescence was observed on day 2 post-slicing / transduction, prior to tissue harvest.

[0031] Figure 12This is a schematic diagram of a competitive transduction assay used to compare the efficiency of barcoded AAV variants in porcine myocardial slices. Heart slices were taken from the left ventricular myocardium of normal pigs or pigs with infarcted hearts. The relative proportions of barcoded reads after NGS analysis are shown for normal and infarcted hearts at the levels of cell entry (A, C) and gene expression (B, D). Results are expressed as a percentage of total reads for each case. The p-value for each plot is presented as a heatmap.

[0032] Figure 13 This is a schematic diagram showing that the nuclear yield of non-myocellular cells is higher than that of myocytes in the heart of an infarcted pig. Nuclei were extracted from heart sections and sorted using an imaging hemocytometer. (A) Cardiomyocytes labeled with PCM1. Total nuclear yield of PCM1+ and PCM1- fractions is depicted in each sample from (B) a normal heart and (C) an infarcted heart.

[0033] Figure 14 This diagram illustrates the comparison of cardiomyocyte- and non-myocellular-specific transduction in normal porcine heart slices through analysis of sorted cell nuclei. For the PCM1+ve cardiomyocyte fraction (A and B) and the PCM1-ve non-myocellular fraction (C and D), the relative proportions of barcode reads after NGS analysis are provided in terms of gene expression and cellular-level entry. Results are expressed as a percentage of the total reads in each case.

[0034] Figure 15 This diagram illustrates the comparison of rAAV transduction in non-human primate heart sections through analysis of cardiac cell nuclei. Heart sections were generated from the left ventricular myocardium of non-human primates. Two days after transduction, DNA and RNA were extracted from the sections and analyzed using next-generation sequencing (NGS). The relative proportions of barcoded reads of DNA / RNA from the entire heart and nuclei after NGS analysis are presented at the levels of cell entry (A, C, E) and gene expression (B, D, F). Results are expressed as a percentage of total reads for each case.

[0035] Figure 16 This is a schematic diagram of a competitive transduction assay used to compare the efficiency of barcoded AAV variants in human myocardial slices. Heart slices were taken from the left ventricular myocardium of a donor heart. Heart slices were transduced and harvested on day 2 post-slicing for analysis. For the PCM1+ve cardiomyocyte fraction (A and B) and the PCM1-ve non-myocellular fraction (C and D), the relative proportions of barcoded reads after NGS analysis are provided at gene expression and cell entry levels. Results are expressed as a percentage of total reads under each condition.

[0036] Figure 17 is a schematic diagram of the results of an in vivo competitive transduction assay (in pig heart) comparing gene entry (gDNA) and gene expression (cDNA) of different AAV capsids. Figure 17A It is a heatmap showing gene entry and expression in each cardiac segment. Figure 17B This is a bar chart showing the summary results for each AAV capsid (for each capsid, from left to right: cDNA, gDNA, c / g ratio). Detailed Implementation

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, all patents, patent applications, published applications and publications, databases, websites and other published materials referenced throughout this invention are incorporated herein by reference in their entirety. Where terms have multiple definitions, the definition in this section shall prevail. When URLs or other such identifiers or addresses are mentioned, it is understood that such identifiers may change, and specific information on the Internet may disappear at any time, but equivalent information can be found by searching the Internet. The mention of identifiers demonstrates the availability and public dissemination of such information.

[0038] In this specification and the following claims, unless the context otherwise requires, the word “comprising” and variations thereof, such as “including” and “containing”, are to imply the inclusion of the said element or step or group of elements or steps, but do not exclude any other element or step or group of elements or steps.

[0039] As used herein, the term “derived from” should be considered as indicating that a particular element or group of elements is derived from the specified species, but not necessarily directly from the specified source. Furthermore, as used herein, unless the context clearly indicates otherwise, the singular forms of “a,” “an,” and “the” include plural references.

[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 pertains.

[0041] In the context of this specification, the term “about” is understood to mean a range of values ​​that a person skilled in the art would consider equivalent to the value in achieving the same function or result.

[0042] As used herein, "vector" includes polynucleotide vectors and viral vectors, each capable of delivering transgenes contained within the vector into host cells. Vectors can be free-living (i.e., not integrated into the host cell genome) or integrated into the host cell genome. Vectors can also be replicative or replication-deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, granules, and transposons. Exemplary viral vectors include, for example, AAV vectors, lentiviral vectors, retroviral vectors, adenovirus vectors, herpesvirus vectors, and hepatitis virus vectors.

[0043] As used herein, "adeno-associated virus vector" or "AAV vector" refers to a vector in which the capsid is derived from adeno-associated virus, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, AAV vectors from other clades or isolates, or vectors in which the capsid is derived from synthetic AAV capsid proteins, bioengineered AAV capsid proteins, or modified AAV capsid proteins (including chimeric capsid proteins). In specific embodiments, the AAV vector has a capsid containing the capsid polypeptide of the present invention. When referring to an AAV vector, both the source of the genome and the source of the capsid can be identified, wherein the first number indicates the source of the genome and the second number indicates the source of the capsid. Thus, for example, a vector in which both the capsid and the genome are derived from AAV2 is more accurately referred to as AAV2 / 2. Vectors containing an AAV6-derived capsid and an AAV2-derived genome are most accurately referred to as AAV2 / 6. Vectors containing a bioengineered DJ capsid and an AAV2-derived genome are most accurately referred to as AAV2 / DJ. For simplicity, since most vectors use an AAV2-derived genome, it is understood that references to AAV6 vectors usually refer to AAV2 / 6 vectors, references to AAV2 vectors usually refer to AAV2 / 2 vectors, and so on. AAV vectors may also be referred to herein by the terms “recombinant AAV,” “rAAV,” “recombinant AAV-viral particle,” “rAAV viral particle,” “AAV variant,” “recombinant AAV variant,” and “rAAV variant,” which are used interchangeably and refer to replication-defective viruses containing an AAV capsid encapsulating the AAV genome. The AAV vector genome (also called the vector genome, recombinant AAV genome, or rAAV genome) comprises transgenes flanked by functional AAV ITRs. Typically, one or more wild-type AAV genes, preferably the rep gene and / or the cap gene, have been wholly or partially deleted from the genome. Functional ITR sequences are essential for the rescue, replication, and packaging of vector genomes into rAAV viral particles.

[0044] The term "ITR" refers to the inverted terminal repeat sequences located at both ends of the AAV genome. These sequences can form hairpin structures and participate in AAV DNA replication and rescue or excision from prokaryotic plasmids. The ITRs used in this invention do not need to be wild-type nucleotide sequences and can be altered, for example, by nucleotide insertion, deletion, or substitution, as long as these sequences enable the functional rescue, replication, and packaging of rAAV.

[0045] As used herein, the term "functional" for a capsid polypeptide means that the polypeptide can self-assemble or assemble with different capsid polypeptides to prepare the protein coat (capsid) of AAV viral particles. It should be understood that not all capsid polypeptides in a given host cell will assemble into an AAV capsid. Preferably, at least 25%, at least 50%, at least 75%, at least 85%, at least 90%, and at least 95% of all AAV capsid polypeptide molecules will assemble into an AAV capsid. Suitable detection methods for measuring this bioactivity are described, for example, in Smith-Arica and Bartlett (2001), Curr Cardiol Rep 3(1): 43-49.

[0046] "AAV helper function" or "helper function" refers to the function that allows AAV to be replicated and packaged by host cells. AAV helper function can be provided in any of a variety of forms, including but not limited to helper virus forms or helper virus genes that assist AAV replication and packaging. Helper virus genes include, but are not limited to, adenovirus helper genes, such as E1A, E1B, E2A, E4, and VA. Helper viruses include, but are not limited to, adenoviruses, herpesviruses, and poxviruses (e.g., vaccinia virus and baculovirus). Adenoviruses comprise many different subgroups, although the most commonly used is subgroup C, type 5 adenovirus (Ad5). Many human, non-human mammalian, and avian adenoviruses are known to be available from collections such as the ATCC. Viruses of the Herpesviridae family are also available from collections such as the ATCC, including, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). The baculoviruses obtained from the collection include Autographa californica nuclear polyhedrosisvirus.

[0047] As used herein, the term "transduction" refers to the entry of an AAV vector into one or more specific cell types and the transfer of DNA contained in the AAV vector into the cells. Transduction can be assessed by measuring the amount of AAV DNA or RNA expressed by AAV DNA in a cell or cell population and / or by assessing the number of cells in a cell population containing AAV DNA or RNA expressed by DNA. When assessing the presence or amount of RNA, the type of transduction assessed herein is referred to as "functional transduction," which is the ability of AAV to transfer DNA into cells and express that DNA. The term "transduction efficiency" and its grammatical variations refer to the ability of an AAV vector to transduce into host cells, and more specifically, the efficiency with which an AAV vector transduces into host cells. In a specific implementation, transduction efficiency is in vivo transduction efficiency, referring to the ability of an AAV vector to transduce into host cells in vivo after administration of the vector to an individual. Transduction efficiency can be assessed in a variety of ways known to those skilled in the art, including assessing the number of transduced host cells after exposure to or administration of a given number of vector particles (e.g., by assessing the expression of reporter genes (e.g., GFP or eGFP) in the vector genome using microscopy or flow cytometry); assessing the amount of vector DNA (e.g., the amount of vector genome) in the host cell population after exposure to a given number of vector particles; assessing the amount of vector RNA in the host cell population after exposure to a given number of vector particles; and assessing the protein expression level of reporter genes (e.g., GFP or eGFP) in the vector genome in the host cell population after exposure to or administration of a given number of vector particles. The host cell population can represent a specific number of host cells, a tissue of a certain volume or weight, or an entire organ (e.g., the heart). In vivo transduction efficiency can reflect the ability of the AAV vector to enter host cells (e.g., myocytes in the heart); the ability of the AAV vector to enter host cells; and / or the expression of heterologous coding sequences contained in the vector genome upon entry into host cells.

[0048] As used herein, "corresponding nucleotide," "corresponding amino acid residue," or "corresponding position," "relative to," etc., refers to the nucleotide, amino acid, or position appearing at the aligned site or position. The sequences of related or variant polynucleotides or polypeptides are aligned using any method known to those skilled in the art. These methods typically maximize matching (e.g., identical nucleotides or amino acids at multiple positions), including, for example, manual alignment and the use of various available alignment programs (e.g., BLASTN, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, etc.) and other methods known to those skilled in the art. By aligning the sequences of polynucleotides, those skilled in the art can identify the corresponding nucleotides. For example, by aligning two AAV capsid polypeptides (e.g., ... Figure 3 As shown), those skilled in the art can identify regions or amino acid residues in one AAV polypeptide that correspond to different regions or residues in another AAV polypeptide. For example, the serine at position 179 of the AAV6 capsid polypeptide listed in SEQ ID NO:1 is the corresponding amino acid of the serine at position 180 of the rAAV.KK03 capsid polypeptide listed in SEQ ID NO:7, or the serine at position 179 of the AAV6 capsid polypeptide listed in SEQ ID NO:1 corresponds to the serine at position 180 of the rAAV.KK03 capsid polypeptide listed in SEQ ID NO:7 (see [link to SEQ ID NO:1]). Figure 2 (The alignment results of the AAV6 capsid peptide and the rAAV.KK03 capsid peptide are shown). In another example, referring to the same alignment, the serine at position 538 of the AAV6 capsid peptide is aligned with the serine at position 537 of the rAAV.KK02 capsid peptide and the histidine at position 539 of the rAAV.KK04 capsid peptide, or the serine at position 538 of the AAV6 capsid peptide corresponds to the serine at position 537 of the rAAV.KK02 capsid peptide and the histidine at position 539 of the rAAV.KK04 capsid peptide. Therefore, when this document refers to a specific capsid peptide by referring to an amino acid residue or position, it is understood that, where appropriate, the reference may also refer to the corresponding amino acid residue and position in another capsid peptide. Table 4 disclosed herein lists the amino acid modifications identified in variant AAV capsid proteins KK01 to KK05 relative to the prototype AAV6 amino acid sequence number of SEQ ID NO:1.

[0049] As used herein, a "heterologous coding sequence" refers to a nucleic acid sequence present in a polynucleotide, vector, or host cell that is not naturally present in the polynucleotide, vector, or host cell, or is not naturally present at its location in the polynucleotide, vector, or host cell; that is, it is non-natural. A "heterologous coding sequence" can encode a peptide or polypeptide, or a polynucleotide that is functional or active itself, such as an antisense oligonucleotide or repressive oligonucleotide, including antisense DNA and RNA (e.g., miRNA, siRNA, and shRNA). In some instances, a heterologous coding sequence is a nucleic acid segment that is substantially homologous to a segment of nucleic acid in an animal's genomic DNA, such that homologous recombination can occur between the heterologous sequence and the genomic DNA when the heterologous coding sequence is introduced into an animal cell. In one instance, the heterologous coding sequence is a functional copy of a gene intended for introduction into a cell with a defective / mutated copy.

[0050] As used in this article, the term "operable link" in relation to promoters and coding sequences refers to the transcription of coding sequences being controlled or driven by a promoter.

[0051] The term "host cell" refers to a cell (e.g., a mammalian cell) into which foreign DNA (such as a vector or other polynucleotide) has been introduced. This term includes the progeny of the original cell into which the foreign DNA has been introduced. Therefore, as used herein, "host cell" generally refers to a cell transfected or transduced with foreign DNA.

[0052] As used herein, the term “isolated” in relation to polynucleotides or polypeptides means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cell from which it is derived, or, in the case of chemical synthesis, “isolated” means that the polynucleotide or polypeptide is substantially free of chemical precursors or other chemical substances.

[0053] As used herein, the term "individual" refers to an animal, specifically a mammal, more specifically a primate (including lower primates), and even more specifically a human, that can benefit from the present invention. An individual, whether human or a non-human animal or embryo, may be referred to as a subject, individual, animal, patient, host, or recipient. The present invention is applicable to both human and veterinary applications. For convenience, "animal" specifically includes livestock such as cattle, horses, sheep, pigs, camels, goats, and donkeys, as well as domestic animals such as dogs and cats. Regarding horses, this includes horses used in the racing industry, as well as horses used for recreational or livestock purposes. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs, and hamsters. Rabbits and rodents (e.g., rats and mice), as well as primates and lower primates, provide a convenient testing system or animal model. In some embodiments, the individual is a human.

[0054] As used herein, the term "conserved sequence modification" or "conserved substitution" refers to an amino acid modification that does not significantly affect or alter the properties of a vector containing an amino acid sequence. Such conserved modifications include amino acid substitution, addition, and deletion. Modifications compatible with various embodiments can be introduced into vectors using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution refers to the replacement of an amino acid residue 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 having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the capsid can be replaced with other amino acid residues from the same side chain family, and the functional assays described herein can be used to test the tropism and / or payload delivery capability of the modified capsid.

[0055] It should be understood that the terms and related definitions mentioned above are for explanatory purposes only and are not intended to be restrictive.

[0056] Capsid polypeptide This invention is partly based on the generation of variant AAV capsid peptides, which, when present in the capsid of an AAV vector, promote efficient transduction in host cells, particularly human heart cells. AAV vectors having a capsid containing the capsid peptide of this invention typically increase or enhance cellular transduction (including in vivo transduction) compared to AAV vectors containing a reference AAV capsid peptide or a wild-type AAV capsid peptide (e.g., the prototype AAV6 capsid, AAV3b capsid, AAV1 capsid, or AAV9 capsid listed in SEQ ID NO: 1, 2, 3, or 4, respectively). The transduction or transduction efficiency of the AAV vector 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, for example, compared to a reference AAV capsid peptide (e.g., SEQ ID NO: 1, 2, 3, or 4). Compared to any one listed in NO: 1, 2, 3, or 4, the AAV vector containing the capsid polypeptide of the present invention can achieve an in vivo cell transduction efficiency of 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, or 100x or higher. In specific examples, increased transduction or transduction efficiency has been observed in human cardiac cells, including myocytes.

[0057] Therefore, the capsid peptides of the present invention are particularly useful in the preparation of AAV vectors (especially AAV vectors for gene therapy purposes). In an exemplary embodiment, the capsid peptides of the present invention are particularly useful in the preparation of AAV vectors for transducing cardiac cells (including myocytes, particularly human cardiac cells), and thus can be used for gene therapy applications targeting the heart or cardiac cells and tissues.

[0058] This document provides polypeptides, including isolated polypeptides comprising all or part of an AAV capsid polypeptide comprising any sequence of SEQ ID NO:5-9; including VP1 protein (containing amino acid residues corresponding to amino acid residues at positions 1 to 736 of SEQ ID NO:1), VP2 protein (containing amino acid residues corresponding to amino acid residues at positions 138 to 736 of SEQ ID NO:1) and / or VP3 protein (containing amino acid residues corresponding to amino acid residues at positions 205 to 736 of SEQ ID NO:1) and all or part of 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 with the VP1, VP2, or VP3 protein described herein.

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

[0060] Table 1 provides a brief description of the sequences.

[0061] The capsid polypeptides of the present invention comprise those comprising (i) an amino acid sequence having at least about 85% sequence identity with any of the sequences in SEQ ID NO: 5-8; (ii) amino acid residues 1-204 of any of the sequences in SEQ ID NO: 5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; and / or (iii) amino acid residues 205-735 of any of the sequences in SEQ ID NO: 5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; and wherein the capsid polypeptide comprises those selected relative to SEQ ID NO: 5-8. At least one amino acid modification at the position of the group consisting of the following amino acid positions numbered NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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 148 deletion; Q151 or Q151-E152 insertion of R; 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, amino acid positions relative to SEQ ID NO:1.

[0062] The capsid polypeptides of the present invention include those capsid polypeptides comprising all or a portion of the VP1 protein listed in SEQ ID NO:5 (also referred to herein as rAAV.KK01), or polypeptides having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Therefore, the present invention also includes capsid polypeptides comprising all or a portion of amino acids 1-204 of SEQ ID NO:5, or comprising sequences having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with amino acids 205-737 of SEQ ID NO:5 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises a sequence selected relative to SEQ ID NO:5. At least one amino acid modification at the position of the group consisting of the following amino acid positions encoded by NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing or Q148; Q151 or R inserted between Q151 and E152; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A1 96; 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; P 502 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; A55 3 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 The amino acid positions are relative to SEQ ID NO:1. In one specific embodiment, the capsid polypeptide comprises at least one of the following amino acid modifications: M1 deletion; M2; T3; V19; A24; Q26.L129; A141; P148; Q151-E152 inter-insertion R; S152; T157; K162; R168; S224; E418 and F584, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: M1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152 inter-insertion R; S152; T157; K162; R168; S224; E418 and F584, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: M1 deletion; M2; T3; V19; A24; Q26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; an insertion of R between Q151 and E152; S152; T157; K162; Q164; R168, S224; D418; F584; A598; and N642, the amino acid positions 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 the amino acid sequence of SEQ ID NO:5.

[0063] The capsid polypeptides of the present invention include those capsid polypeptides comprising all or a portion of the VP1 protein listed in SEQ ID NO:6 (also referred to herein as rAAV.KK02), or polypeptides having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Therefore, the present invention also includes capsid polypeptides comprising all or a portion of amino acids 1-204 of SEQ ID NO:6, or comprising sequences having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with amino acids 205-737 of SEQ ID NO:6 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises a sequence selected relative to SEQ ID NO:6. At least one amino acid modification at the position in the group consisting of the following amino acid positions numbered NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing 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 A1 96; 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; P 502 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; A55 3 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 amino acid positions relative to SEQ ID NO:1. In one specific embodiment, the capsid polypeptide comprises at least one of the following amino acid modifications: L129; L146; 148 deletion; K162; K164;R168 and E418, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: L129; L146; 148 deletion; K162; K164; R168 and E418, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following 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, the amino acid positions corresponding 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 the amino acid sequence of SEQ ID NO:6.

[0064] The capsid polypeptides of the present invention comprise those capsid polypeptides comprising all or a portion of the VP1 protein listed in SEQ ID NO:7 (also referred to herein as rAAV.KK03), or polypeptides having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Therefore, the present invention also includes capsid polypeptides comprising all or a portion of amino acids 1-204 of SEQ ID NO:7, or comprising sequences having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with amino acids 205-737 of SEQ ID NO:7 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises a sequence selected relative to SEQ ID NO:7. At least one amino acid modification at the position in the group consisting of the following amino acid positions numbered NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing, or Q148; Q151 or R inserted between Q151 and E152; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A1 96; 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; P 502 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; A55 3 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 The amino acid positions are relative to SEQ ID NO:1. In one specific embodiment, the capsid polypeptide comprises at least one of the following amino acid modifications: A24; V29; Q31; H38; N41; R42; 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.G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151-E152 interpolated with R; S152; T157; K162 and R168, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: A24; V29; Q31; H38; N41; R42; G56; E67; K92; L129; Q134; A135; G136; E137; P148; Q151-E152 interpolated with R; S152; T157; K162 and R168, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following 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-E152 interposition of R; S152; T157; K162; Q164; R168; A224; D418; L584; V598; and H642, the amino acid positions 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 the amino acid sequence of SEQ ID NO:7.

[0065] The capsid polypeptides of the present invention include those capsid polypeptides comprising all or a portion of the VP1 protein listed in SEQ ID NO:8 (also referred to herein as rAAV.KK05), or polypeptides having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. Therefore, the present invention also includes capsid polypeptides comprising all or a portion of amino acids 1-204 of SEQ ID NO:8, or comprising sequences having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with amino acids 205-737 of SEQ ID NO:8 or a functional fragment thereof. In one embodiment, the capsid polypeptide comprises a sequence selected relative to SEQ ID NO:8. At least one amino acid modification at the position in the group consisting of the following amino acid positions numbered NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing, or Q148; Q151 or R inserted between Q151 and E152; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K168; A194 or T194; S196 or A1 96; 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; P 502 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; A55 3 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 amino acid positions relative to SEQ ID NO:1. In one specific embodiment, the capsid polypeptide comprises at least one of the following amino acid modifications: 1 deletion; M2; T3; V19; A24; Q26;L129; A141; P148; Q151-E152 (intercalation R); S152; T157; K162; R168; S224; E418; and F584, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: 1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152 (intercalation R); S152; T157; K162; R168; S224; E418; and F584, amino acid positions relative to SEQ ID NO:1. In another embodiment, the capsid polypeptide comprises the following amino acid modifications: 1 deletion; M2; T3; V19; A24; Q26; A29; Q31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151-E152 interposition of R; S152; T157; K162; Q164; R168; S224; D418; F584; V598; and H642, the amino acid positions being 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 the amino acid sequence of SEQ ID NO:8.

[0066] The capsid polypeptide of the present invention comprises (i) an amino acid sequence having at least about 85% sequence identity with SEQ ID NO:9 (also referred to herein as rAAV.KK04), (ii) amino acid residues 1-204 of SEQ ID NO:9 or an amino acid sequence having at least about 85% sequence identity with the aforementioned sequence; and / or (iii) amino acid residues 205-736 of SEQ ID NO:9 or an amino acid sequence having at least about 85% sequence identity with the aforementioned sequence; and wherein the capsid polypeptide comprises amino acids selected from those of SEQ ID NO:9. At least one amino acid modification at the position in the group consisting of the following amino acid positions numbered NO:1: 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. In one embodiment, the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing, or Q148; R inserted between Q151 or Q151_E152; 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; P 502 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; A55 3 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, amino acid positions relative to SEQ ID NO:1. In one specific embodiment, the capsid polypeptide comprises at least one of the following amino acid modifications: T14; Q21; K24; P29; P31; P34; A35; E36; R37; H38; K39; D41; S42; F56; A67; R92; V125; L129; E134; G135; A136; K137; G141; V146; E147; P148; Q151-E152 intercalation R; 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; S 447; S451; T452; G453; T455; Q456, G457, T458, Q459, Q460; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H51 0; 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, amino acid positions 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 the amino acid sequence of SEQ ID NO:9.

[0067] Nucleic acid molecules encoding capsid polypeptides described herein are also provided, comprising isolated nucleic acid molecules. Thus, for example, among the nucleic acid molecules provided herein, there are nucleic acid molecules encoding any of the capsid polypeptides described herein. Non-limiting examples of nucleic acid molecules include those listed in SEQ ID NO:10-18, those nucleic acid molecules having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the aforementioned sequences, and those nucleic acid molecules that hybridize with nucleic acid molecules containing sequences listed in any of SEQ ID NO:10-18 with moderate or high stringency.

[0068] carrier The present invention also provides a vector comprising a nucleic acid molecule encoding the capsid polypeptide described herein, and a vector comprising the capsid polypeptide described herein. The vectors include nucleic acid vectors and AAV vectors, wherein the nucleic acid vector comprises a nucleic acid molecule encoding the capsid polypeptide described herein, and the AAV vector has a capsid comprising the capsid polypeptide described herein.

[0069] Nucleic acid vector The vectors of the present invention include nucleic acid vectors containing a polynucleotide encoding all or a portion of the capsid polypeptide described herein, such as polynucleotides encoding a capsid polypeptide comprising the following sequences: amino acid sequences listed in any of the sequences in SEQ ID NO:5-9, or amino acid sequences having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the sequences in SEQ ID NO:5-9 or fragments thereof (e.g., all or a portion of VP2 or VP3 protein). The vector can be a free vector (i.e., a vector not integrated into the host cell genome) or a vector integrated into the host cell genome. Exemplary vectors containing nucleic acid molecules encoding capsid polypeptides include, but are not limited to, plasmids, viscera, transposons, and artificial chromosomes. In a particular instance, the vector is a plasmid.

[0070] Vectors (e.g., plasmids) suitable for bacterial, insect, and mammalian cells are widely described and well-known in the art. Those skilled in the art will understand that the vectors of the present invention may also include additional sequences and elements for vector replication, vector selection, and heterologous sequence expression in prokaryotic and / or eukaryotic cells. For example, the vectors of the present invention may include prokaryotic replicons (i.e., sequences having the ability to direct autonomous extrachromosomal replication and maintenance of the vector in prokaryotic host cells (e.g., bacterial host cells). Such replicons are well-known in the art. In some embodiments, the vector may include a shuttle element that adapts the vector for replication and integration in prokaryotes and eukaryotes. Furthermore, the vector may include its expression of a gene conferring a detectable marker (e.g., a drug resistance gene) for host cell selection and maintenance. The vector may also have a reportable marker (e.g., a gene encoding a fluorescent protein or other detectable protein). Nucleic acid vectors may also include other elements, including any one or more elements described below. Most typically, the vector will include a promoter operatively linked to a nucleic acid encoding a capsid protein.

[0071] The nucleic acid vectors of the present invention can be constructed using known techniques, including but not limited to standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors of the present invention can be introduced into host cells using any method known in the art. Therefore, the present invention also relates to host cells comprising the vectors or nucleic acids described herein.

[0072] AAV carrier This document provides AAV vectors comprising the capsid polypeptides described herein, such as polypeptides comprising all or a portion of the AAV capsid polypeptide (e.g., capsid polypeptides comprising the amino acid sequences listed in any of the sequences in SEQ ID NO: 5-9, or capsid polypeptides comprising 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 with any of the sequences in SEQ ID NO: 5-9 or fragments thereof (e.g., all or a portion of the VP1 protein)).

[0073] Methods for carrier-based capsid proteins are well known in the art, and any suitable method can be used for the purposes of this invention. For example, the capsid protein can be recycled. cap Genes (e.g., through PCR or by cutting) cap (The upstream and downstream enzymes are used for digestion) and then cloned into a cell containing... rep Within the wrapper construct. Any AAV can be used. rep Genes, including, for example, those from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 and any variants thereof. rep Genes. Usually in rep downstream cloning cap Genes make rep The p40 promoter drives cap expression. This construct does not contain an ITR. This construct, along with a second construct containing an ITR (ITRs are typically flanking the heterologous coding sequence), was then introduced into a packaging cell line. Helper functions or helper viruses were also introduced, and recombinant AAV, containing [a specific component], was recovered from the supernatant of the packaging cell line. capA capsid formed by the gene-expressed capsid protein encapsulates the genome, including transgenes flanked by ITRs. Various cell types can be used as packaging cell lines. For example, packaging cell lines that can be used include, but are not limited to, HEK293 cells, HeLa cells, and Vero cells, as disclosed in US20110201088. Helper functions can be provided by one or more helper plasmids or helper viruses containing adenoviral helper genes. Non-limiting examples of adenoviral helper genes include E1A, E1B, E2A, E4, and VA, which can provide helper functions for AAV packaging. Helper viruses for AAV are known in the art, including, for example, viruses from the Adenoviridae and Herpesviridae families. Examples of helper viruses for AAV include, but are not limited to, the SAdV-13 helper virus and SAdV-13-like helper viruses described in US20110201088, and the helper vector pHELP (Applied Viromics). Those skilled in the art will understand that any helper virus or helper plasmid capable of providing sufficient helper functions for AAV can be used herein.

[0074] In some cases, rAAV viral particles are prepared using cell lines that stably express some components necessary for the production of AAV viral particles. For example, plasmids (or plasmids) containing nucleic acids can be integrated into the genome of a cell (packaging cell) containing the nucleic acids identified herein. cap Genes and rep Genes, and selective markers (e.g., neomycin resistance genes). Packaging cell lines can then be transfected with an AAV vector and helper plasmids, or transfected with an AAV vector and co-infected with a helper virus (e.g., adenovirus providing helper function). The advantage of this method is that the cells are selectable, making it suitable for large-scale production of recombinant AAV. As another non-limiting example, adenovirus or baculovirus can be used instead of plasmids to encode the nucleic acid encoding the capsid polypeptide and optional [unclear text - possibly a gene or marker]. rep Gene delivery into packaging cells. As another non-limiting example, AAV vectors are also stably integrated into the DNA of production cells, and wild-type adenoviruses can provide auxiliary functions to prepare recombinant AAV.

[0075] In another case, AAV vectors are artificially prepared by synthesizing AAV capsid proteins and assembling and packaging the capsid in vitro.

[0076] Typically, the AAV vector of the present invention also contains a heterologous coding sequence. The heterologous coding sequence can be operatively linked to a promoter to promote sequence expression. The heterologous coding sequence can encode a peptide or polypeptide, such as a therapeutic peptide or polypeptide; or it can encode a polynucleotide or transcript that is functional or active in itself, such as an antisense oligonucleotide or repressive oligonucleotide, including antisense DNA and antisense RNA (e.g., miRNA, siRNA, and shRNA). In some instances, the heterologous coding sequence is a nucleic acid segment that is substantially homologous to a nucleic acid segment in the animal genomic DNA, such that homologous recombination can occur between the heterologous coding sequence and the genomic DNA when the heterologous coding sequence is introduced into animal cells. It should be understood that the nature of the heterologous coding sequence is not essential to the present invention. In specific embodiments, the vector containing the heterologous coding sequence will be used for gene therapy.

[0077] In specific instances, the heterologous coding sequence encodes a peptide, polypeptide, or polynucleotide whose expression has therapeutic uses, such as for treating diseases or disorders. For example, the expression of a therapeutic peptide or polypeptide can be used to restore or replace the function of a defective endogenous form of the peptide or polypeptide (i.e., gene replacement therapy). In other instances, the expression of a therapeutic peptide, polypeptide, or polynucleotide derived from a heterologous sequence is used to alter the level and / or activity of one or more other peptides, polypeptides, or polynucleotides in host cells. Therefore, according to specific embodiments, the expression of a heterologous coding sequence introduced into host cells via the vector described herein can be used to provide therapeutic amounts of peptides, polypeptides, or polynucleotides to improve symptoms of a disease or disorder. In another case, the heterologous coding sequence is a segment of nucleic acid substantially homologous to a segment of nucleic acid in the animal genomic DNA, such that homologous recombination can occur between the heterologous coding sequence and the genomic DNA when the heterologous sequence is introduced into animal cells. Therefore, the introduction of a heterologous sequence into host cells via the AAV vector described herein can be used to correct mutations in the genomic DNA, thereby improving symptoms of a disease or disorder.

[0078] In a non-limiting instance, the heterologous coding sequence encodes an expression product that, when delivered to an individual (specifically, the individual's heart), treats heart-related diseases or conditions including cardiac damage, such as myocardial infarction, congenital heart disease, age-related heart disease, and other heart failures as described herein.

[0079] Heart-related diseases or conditions can be associated with cardiopathology or cardiac dysfunction, or with any impairment of the heart's pumping function (systolic), impaired diastolic capacity (sometimes called diastolic dysfunction), valvular abnormalities or dysfunctions, myocardial diseases (sometimes called cardiomyopathy), diseases characterized by insufficient blood supply to the myocardium such as angina, myocardial ischemia and / or myocardial infarction, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (which may occur in certain types of cardiomyopathy or systemic hypertension), and abnormal connectivity between heart chambers. Heart disease may be associated with cardiomyopathy, which is any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened, and / or stiffened. Therefore, the heart muscle's ability to pump blood is usually impaired. The causes of this disease or condition can be, for example, inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematologic, genetic, or of unknown cause.

[0080] Heart failure (HF) is a complex clinical syndrome that can be caused by any structural or functional cardiovascular disease that results in insufficient systemic perfusion, failing to meet the body's metabolic needs without excessively increasing left ventricular filling pressure. The condition is characterized by specific symptoms such as dyspnea and fatigue, and signs such as fluid retention. Chronic heart failure, or congestive heart failure, refers to a progressive or persistent form of heart failure.

[0081] Those skilled in the art will be able to readily select appropriate heterologous coding sequences for treating such diseases, for example, in Hulot. et al . ( European Heart Journal Illustrative examples of this are described in [reference to a document, 2016, 37:1651-1658](the entire contents of which are incorporated herein by reference). In some instances, the heterologous coding sequence contains all or part of a disease-related gene. Introducing such a sequence into the heart can be used for gene replacement or gene editing / correction, for example, using CRISPR-Cas9. In specific instances, the heterologous coding sequence encodes a protein encoded by a disease-related gene; illustrative examples are listed in Table 3.

[0082] The heterologous coding sequence in the AAV vector is flanked by 3' AAV ITR and 5' AAV ITR. The AAV ITR used in the vector of this invention does not need to have a wild-type nucleotide sequence and can be modified, for example, by inserting, deleting, or substituting nucleotides. Furthermore, the AAV ITR can be derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. These ITRs are well known in the art.

[0083] Those skilled in the art will understand that any suitable method for purifying AAV vectors can be used in the embodiments described herein, and such methods are well known in the art. For example, AAV vectors can be isolated and purified from packaging cells and / or the supernatant of packaging cells. In some embodiments, AAV can be purified by separation methods using CsCl or iodixanol gradient centrifugation. In other embodiments, AAV is purified using a solid-phase carrier comprising a matrix, as described in US20020136710, wherein an artificial receptor or receptor-like molecule mediating AAV attachment is immobilized on the matrix.

[0084] Additional elements in the carrier The vector of the present invention may contain a promoter. When the vector is a nucleic acid vector containing a nucleic acid encoding a capsid peptide, the promoter can promote the expression of the nucleic acid encoding the capsid peptide. As described above, when the vector is an AAV vector, the promoter can promote the expression of a heterologous coding sequence.

[0085] In some instances, the promoter is an AAV promoter, such as the p5, p19, or p40 promoter. In other instances, the promoter is from other sources. Examples of constitutive promoters include, but are not limited to, the retroviral Rouss sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, the human α1-antitrypsin (hAAT) promoter, and the EF1α promoter. Inducible promoters can regulate gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors (e.g., temperature), or specific physiological states (e.g., acute phase, specific differentiation state of the cell, or only in replicating cells). Non-limiting examples of inducible promoters regulated by exogenously provided promoters include zinc-inducible sheep metallothionein (MT) promoters, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoters, and T7 polymerase promoter systems; ecdysone insect promoters, tetracycline inhibition systems, tetracycline induction systems, RU486 induction systems, and rapamycin induction systems. Other types of inducible promoters that may be useful in this context are those regulated by specific physiological states (e.g., temperature, acute phase, specific differentiation state of the cell, or only in replicating cells). In some embodiments, tissue-specific promoters are used. Non-limiting examples of such promoters include cardiac cell-specific promoters, illustrative examples of which include cardiac troponin I and T (cTnI and cTnT), α-myosin heavy chain (a-MHC), and myosin light chain (MLC-2v). The selection of a suitable promoter is entirely within the capabilities of those skilled in the art.

[0086] The vector may also include transcriptional enhancers (e.g., the 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) sequences, rabbit β-globin (RBG) poly(A) sequences, thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the post-transcriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence.

[0087] Vectors may include various post-transcriptional regulatory elements. In some embodiments, the post-transcriptional regulatory element may be a viral post-transcriptional regulatory element. Non-limiting examples of viral post-transcriptional regulatory elements include the marmot hepatitis virus post-transcriptional regulatory element (WPRE), the hepatitis B virus post-transcriptional regulatory element (HBVPRE), RNA transport elements, and any variants thereof. An RNA transport element (RTE) may be a rev response element (RRE), such as a lentiviral RRE. A non-limiting example is the bovine immunodeficiency virus rev response element (RRE). In some embodiments, the RTE is a constitutive transport element (CTE). Examples of CTEs include, but are not limited to, the Mason-Pfizer Monkey Virus CTE and the Avian Leukemia Virus CTE.

[0088] Signal peptide sequences may also be included in the vector to facilitate the secretion of polypeptides from mammalian cells. Examples of signal peptides include, but are not limited to, endogenous signal peptides and variants of HGH; endogenous signal peptides and variants of interferons, including signal peptides and variants of type I, II, and III interferons; and endogenous signal peptides and variants of known cytokines, such as signal peptides and variants of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β. Typically, the nucleotide sequence of the signal peptide is located immediately upstream of the heterologous sequence in the vector (e.g., fused to the 5' end of the coding region of the protein of interest).

[0089] In other instances, the vector may contain regulatory sequences capable of translating multiple proteins from a single mRNA, for example. Non-restrictive examples of such regulatory sequences include internal ribosome entry sites (IRES) and 2A self-processing sequences, such as the 2A peptide site (F2A sequence) of foot-and-mouth disease virus.

[0090] host cells This document also provides host cells comprising the nucleic acid molecules or vectors of the present invention. In some cases, the host cell is used for amplification, replication, packaging, and / or purification of the polynucleotide or vector. In other instances, the host cell is used for expression of heterologous sequences, such as sequences packaged within an AAV vector. Exemplary host cells include prokaryotic and eukaryotic cells. In some cases, the host cell is a mammalian host cell. Selecting a suitable host cell for the expression, amplification, replication, packaging, and / or purification of the polynucleotides, vectors, or rAAV viral particles of the present invention is entirely within the capabilities of those skilled in the art. Exemplary mammalian host cells include, but are not limited to, HEK293 cells, HeLa cells, Vero cells, HuH-7 cells, and HepG2 cells. In specific instances, the host cell is a cardiac cell or a cell line derived from cardiac cells.

[0091] In some embodiments, the host cell is a mammalian heart cell. In another embodiment, the heart cell is a primate heart cell. In a preferred embodiment, the heart cell is a human heart cell. In another embodiment, the heart cell is a muscle cell.

[0092] In another embodiment, the heart cells are contained within a heart organoid. In one embodiment, the heart cells are primary cardiomyocytes. In another embodiment, the heart cells are iPSC-derived heart cells.

[0093] Composition Compositions comprising nucleic acid molecules, peptides, and / or carriers of the present invention are also provided. In specific examples, pharmaceutical compositions comprising AAV carriers disclosed herein and pharmaceutically acceptable carriers are provided. The compositions may also contain additional ingredients such as diluents, stabilizers, excipients, and adjuvants.

[0094] Carriers, diluents, and adjuvants may include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides (e.g., fewer 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions (e.g., sodium); and / or, for example, Tween. TM Pluronics TM Alternatively, a nonionic surfactant such as polyethylene glycol (PEG) may be used. In some embodiments, the physiologically acceptable carrier is an aqueous pH buffer solution.

[0095] method The AAV vector and compositions containing the AAV vector of the present invention can be used in methods for introducing heterologous coding sequences into host cells. These methods involve contacting host cells with the AAV vector. This can be performed in vitro, ex vivo, or in vivo. In a particular embodiment, the host cell is a muscle cell (e.g., a heart cell).

[0096] When this method is performed in vitro or in vivo, the introduction of the heterologous sequence into host cells is typically for therapeutic purposes, thereby leading to the treatment of the disease or condition through expression of the heterologous sequence. Therefore, the AAV vector disclosed herein can be administered to individuals in need (e.g., humans), such as those suffering from diseases or conditions that are treatable with proteins, peptides, or polynucleotides encoded by the heterologous sequences described herein.

[0097] When used in vivo, the titer of the AAV vector administered to an individual will vary depending on, for example, the specific recombinant virus, the disease or condition to be treated, the route of administration, the treatment target, the individual to be treated, and the targeted cell type, and can be determined by methods well known to those skilled in the art. While the exact dosage will be determined on an individual basis, in most cases, typically, the recombinant virus of the present invention can be administered at a dose of 1 × 10⁻⁶ per kg of individual. 10 One recombinant viral genome copy per kg of 1×10⁻⁶ 14 A dose of one genome copy is administered to an individual. In other instances, less than 1 × 10⁻⁶. 10 Genome copies may be sufficient to achieve a therapeutic effect. In other cases, a value greater than 1 × 10⁻⁶ may be required. 14 To achieve therapeutic effects, multiple copies of the genome are used.

[0098] There are no particular restrictions on the route of administration. For example, a therapeutically effective amount of AAV carrier can be administered to an individual via, for example, intramuscular, intravaginal, intraperitoneal, subcutaneous, epidermal, intradermal, rectal, intraocular, pulmonary, intracranial, intraosseous, oral, buccal, or nasal routes. AAV carrier can be administered in single or multiple doses, and at different intervals.

[0099] Methods for preparing the AAV vectors described above and herein, specifically methods for preparing AAV vectors comprising the capsid polypeptide of the present invention, are also provided. Such methods include culturing host cells under conditions suitable for promoting the assembly of AAV vectors comprising the capsid polypeptide of the present invention, said host cells comprising a nucleic acid molecule encoding the capsid polypeptide of the present invention, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeat sequences, and auxiliary functions for generating productive AAV infection, wherein the capsid encapsulates the heterologous coding sequence.

[0100] In other respects, methods for enhancing the in vivo transduction efficiency of AAV vectors in human cardiac cells are provided. As shown herein, modifications at certain amino acid positions in the regions of VP1 (containing amino acid residues corresponding to amino acids 1-204 of SEQ ID NO:1) and / or VP3 (containing amino acid residues corresponding to amino acid residues 205-736 of SEQ ID NO:1) unexpectedly provided efficient transduction and / or enhanced DNA-to-RNA conversion in human cardiac cells via AAV vectors (i.e., high-efficiency expression despite lower vector uptake efficiency).

[0101] Therefore, this article provides a method for preparing a modified AAV vector that exhibits enhanced transgenic expression in human heart cells, wherein the method comprises the following steps: a) identifying a reference capsid polypeptide for in vivo transduction of human heart cells; b) selecting from a subset of SEQ ID NO. The sequence of the reference capsid polypeptide is modified at at least one of the following 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, thereby preparing a modified capsid polypeptide comprising at least one amino acid modification selected from the group consisting of: M1 or 1 deletion; 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 148 missing; Insert R between Q151 or Q151_E152; 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, amino acid positions relative to SEQ ID NO:1; and c) carrier the modified capsid polypeptide to prepare a modified AAV vector. In one embodiment, the amino acid sequence of a reference capsid polypeptide is modified at at least one of the following positions selected relative to the SEQ ID NO:1: 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 to prepare a modified capsid polypeptide comprising L129; L146; 148 deletion; K162; K164; R168; E418.In one embodiment, the reference capsid polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:1.

[0102] This document also provides a method for preparing a modified AAV vector that exhibits enhanced transgenic expression in human heart cells, wherein the method comprises the following steps: a) identifying a reference capsid polypeptide for in vivo transduction of human heart cells; b) modifying the sequence of the reference capsid polypeptide at at least one of the following amino acid positions relative to SEQ ID NO:1: 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, thereby preparing a modified capsid polypeptide comprising one or more of the following: M1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152 intercalation R; S152; T157; K162; R168; S224; E418; L584; F584; A598 and N642, amino acid positions relative to SEQ ID NO:1; and c) carrier-encapsulating the modified capsid polypeptide to prepare a modified AAV vector. In one embodiment, the amino acid sequence of the reference capsid polypeptide is modified to include the deletions of M1; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152insR; S152; T157; K162; R168; S224; L584; A598; and N642, with amino acid positions relative to SEQ ID NO:1. In another embodiment, the modified capsid polypeptide further includes amino acid modifications E418 and F584, with amino acid positions relative to SEQ ID NO:1.

[0103] The methods disclosed herein may suitably include an initial step, including in vivo, of identifying a reference capsid polypeptide for transducing host cells. The reference capsid polypeptide may be any AAV polypeptide, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid polypeptides, or synthetic capsid polypeptides or chimeric capsid polypeptides.

[0104] It should be understood that any modification or combination of modifications, such as amino acid substitution, deletion, and / or insertion, will result in a change in the amino acid sequence of the modified capsid peptide compared to the reference capsid peptide. Therefore, for example, when referring to modification, the following situations are excluded: an amino acid residue is replaced by the same amino acid residue, or an amino acid deletion is accompanied by the insertion of the deleted amino acid, resulting in an amino acid sequence of the modified capsid peptide that is indistinguishable from that of the reference capsid peptide; that is, the amino acid sequence of the modified capsid peptide cannot be the same as that of the reference capsid peptide (or the two must be different).

[0105] Methods for modifying the sequence of a reference capsid polypeptide or polynucleotide to prepare a modified capsid polypeptide or polynucleotide are well known in the art, and any such method can be used to implement the methods of the present invention. For example, the modification of a reference capsid polynucleotide sequence to prepare a modified capsid polynucleotide can be performed (partially or entirely) by any method known in the art (including recombination and synthesis methods) performed on a computer and / or in vitro. In a particular example, the sequence modification is performed on a computer, and then the modified capsid polynucleotide having the modified sequence is resynthesized (e.g., by a gene synthesis method, such as a method involving the chemical synthesis of overlapping oligonucleotides followed by genome assembly).

[0106] Modified capsid polynucleotides can be contained in nucleic acid vectors (e.g., plasmids) for subsequent expression, replication, amplification, and / or manipulation. Vectors suitable for bacterial, insect, and mammalian cells are widely described and well-known in the art. Those skilled in the art will understand that vectors can also contain additional sequences and elements for vector replication, vector selection, and heterologous sequence expression in prokaryotic and / or eukaryotic cells. For example, a vector may include a prokaryotic replicon, which is a sequence capable of directing autonomous extrachromosomal replication and maintenance of the vector in a prokaryotic host cell (e.g., a bacterial host cell). Such replicons are well-known in the art. In some embodiments, the vector may include elements that adapt the vector for replication and integration in prokaryotes and eukaryotes. Furthermore, the vector may include its expression of a gene conferring a detectable marker (e.g., a drug resistance gene), which enables selection and maintenance of host cells. The vector may also have a reportable marker (e.g., a gene encoding fluorescence or other detectable proteins). Nucleic acid vectors may also contain other elements, including any one or more elements described below. Most typically, the vector will contain a promoter that is operatively linked to the nucleic acid encoding the capsid protein.

[0107] Nucleic acid vectors can be constructed using known techniques, including but not limited to standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. Vectors containing modified capsid polynucleotides can be introduced into host cells using any method known in the art.

[0108] After modification, the modified capsid is carrier-based. Methods for carrier-based capsid peptides are well known in the art, and non-limiting examples have been described above.

[0109] Compared to reference AAV vectors with a capsid containing a reference capsid polypeptide, AAV vectors prepared by these methods typically exhibit enhanced transgene expression levels per cell. Transgene expression levels 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%, or more than 1000%, for example, compared to in vivo transgene expression in unmodified AAV vectors (i.e., AAV vectors containing a reference capsid), the transgene expression of AAV vectors 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, or more than 100x. In some instances, it was evaluated in myocytes (e.g., hiPSC myocytes, primary myocytes, heart slices, or heart organoids).

[0110] Therefore, an AAV carrier prepared by the method of the present invention is also provided.

[0111] To make the present invention easy to understand and put into practice, specific preferred embodiments will now be described by way of the following non-limiting examples.

[0112] Any prior publications (or information derived therefrom) or any known matters mentioned in this specification shall not constitute, nor should be construed as, an endorsement or acknowledgment, or in any way imply that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge of the field covered by this specification.

[0113] Example Example 1: Materials and Methods AAV Capsid Library Construction and Directed Evolution As mentioned earlier (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev12: ​​71-84), an AAV capsid library was constructed. The hiPSC-CM, a derivative of the WTC iPSC lineage, was then used in Geltrex. TM Obtained from 24-well plates coated with (Gibco, Massachusetts, USA, #A14133-02), based on cardiac troponin (cTnT) expression, with a purity >90%. Used from 3.6 x 10⁻⁶ wells. 10 Vg was used to infect cardiomyocytes with four 10-fold serial dilutions of the AAV library. The next day, cells were washed with 1xPBS and then co-infected with WT human adenovirus 5 (Ad5; ATCC VR-5, Manassas, VA, USA) for amplification as described previously (Cabanes-Creus, M., et al., 2022, Mol Ther Methods Clin Dev 24:88-101). Four days later, cells were collected and lysed using three freeze-thaw cycles. AAV amplification in the clear supernatant was then analyzed by qPCR as described previously (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84). AAV amplification was validated using AAV rep2-specific primers after each round of selection, and appropriate dilutions were selected for subsequent selections. Before serial dilutions of the next batch of cells, the library dilutions selected for the next round of screening were heated to 65°C and held for 30 minutes to inactivate Ad5. A total of six rounds of screening were performed on fresh batches of hiPSC-CM.

[0114] Phylogenetic analysis and vectorization of enriched truncation AAV variants Following screening, AAV capsid sequences were recovered by PCR as described previously (Cabanes-Creus, M., et al., 2018, Mol TherMethods Clin Dev 12: 71-84). Twenty clones were sequenced after each round to track screening progress. The amino acid sequences of enriched AAV variants were aligned with known parental AAVs using Geneious Prime 2022.2.1 (https: / / www.geneious.com). A phylogenetic tree was plotted to scale, with branch length measured by the number of substitutions at each site. A heatmap generated using GraphPad Prism version 8.2.1 for Windows (GraphPad software) was used to visualize amino acid substitutions. Vectorization was then performed as described previously (Cabanes-Creus, M., et al., 2018, MolTher Methods Clin Dev 12: 71-84). Then, novel capsid variants AAV.KK01-KK05 were used to package one set of GFP expression vectors without barcodes and another set of GFP expression vectors with barcodes. AAV1, AAV6, and AAV9 were also included as control vectors. Vector packaging and titration were performed as described previously (Cabanes-Creus, M., et al., 2018, MolTher Methods Clin Dev 12: 71-84).

[0115] Maintenance of hiPS cells and differentiation into cardiomyocytes This study used four different hiPSC lines: the WTCWTiPSC line obtained from the J. David Gladstone Institute and SCVI 8, SCVI 100, and SCVI 480 obtained from the Stanford Cardiovascular Institute. mTeSR was used. TM The Plus kit (STEMCELL Technologies, Vancouver, Canada, #05825) preserved all cell lines on 60mm cell culture dishes coated with Matrigel (Corning, New York, USA, #354277). After confluence, cells were passaged in clone form every 6–7 days using a mild cell dissociation reagent (STEMCELL Technologies, Vancouver, Canada, #07174).

[0116] For cardiomyocyte differentiation, as described above (Lian, X., et al., 2012, Proc Natl AcadSci USA, 109: E1848-57; Burridge, PW, et al., 2014, Nat Methods, 11: 855-60), WTCWT cells were maintained and differentiated. The following describes the differentiation of SCVI 8, SCVI 100, and SCVI 480 cell lines. TrypLE was used on day D-2 (two days before the differentiation start day D0). TM The cells were detached from the congregating culture dish using the Express enzyme (ThermoFisher Scientific, Massachusetts, USA, 12604-021), and then mTeSR supplemented with Y-27632 was used. TM Plus (STEMCELL Technologies, Vancouver, Canada, #72304) seeded the cells at 700,000 cells / well in Matrigel-coated 12-well plates. On day D-1, the medium was changed to remove Y-27632. On day D0, differentiation was initiated using the STEMdiff Cardiomyocyte Differentiation Kit according to the manufacturer's instructions until the maintenance point in STEMdiff Cardiomyocyte Maintenance Medium (CMM). On day 11, pulsatile cells underwent a two-day metabolic screening using lactate medium composed of glucose-free DMEM (Thermo Fisher Scientific, Massachusetts, USA, #A14430-01) supplemented with a final concentration of 4 mM L-(+)-lactic acid (Sigma-Aldrich, St. Louis, Missouri, USA, #L1750-10G) and a final concentration of 15 µM bovine serum albumin (Sigma, St. Louis, Missouri, USA, #A9418-10G). On day 13, the cells were then switched back to CMM and maintained until differentiation was complete on day 15.

[0117] After incubation for 1 hour with 10 µM Y-27632, TrypLE was administered with 2 µg / mL DNase I (STEMCELL Technologies, Vancouver, Canada, #07900). TMExpress cells were used to dissociate differentiated cardiac cells. The dissociated cells were then reseeded into Geltrex cells at a density of 500,000 cells / well using RPMI 1640 medium (Thermo Fisher Scientific, Massachusetts, 21870076, USA) and a B27 supplement with Y-27632 (Thermo Fisher Scientific, Massachusetts, 17504-001, USA). TM In a 24-well plate with a coating.

[0118] transduction of hiPSC-CM Cells were counted using a single well to determine viability on day 2 (D2) after replate formation. Cells were then transduced in RPMI 1640+B27 with the rAAV.GFP vector at a MOT of 1000. For competitive transduction assays, barcoded AAV variants were mixed in equimolar ratios to achieve total MOTs of 100, 1000, or 10000. On day 5 (D5) post-transduction, cells were imaged using a Zeiss Axiovert 200M live-cell imaging microscope (Zeiss, Oberkohen, Germany), and then harvested. Nucleic acid extraction was performed using the AllPrep DNA / RNA Micro Kit (Qiagen, Germany, #80284), or transduced cardiomyocytes were quantified using flow cytometry.

[0119] Flow cytometry Using TrypLE TM The cells were dissociated using Express and then washed with Dulbecco's Phosphate Buffered Saline (DPS) (Lonza, Basel, Switzerland, #12001-664), which is free of calcium and magnesium. Then, the cells were subjected to Zombie NIR. TM Cells were stained using the Fixable Viability Kit (Biolegend, San Diego, #423105). After washing, cells were fixed in 4% PFA (w / v) for 30 minutes, then washed and further stained with BV421-conjugated mouse anti-cTnT antibody (BD Biosciences, San Diego, #565618) for 2 hours. After further washing, cells were stained using FACSCanto... TM II Cell Analyzer or LSR Fortessa TM The cells were analyzed using FACSDiva. TMData was recorded using software (BD Biosciences, Franklin Lake, New Jersey, USA). Analysis was then performed using FlowJo (FlowJo Inc., Oregon, USA), version 10.

[0120] Embryonic stem cell culture Female HES3 (WiCell) were maintained in mTeSR PLUS (Stem Cell Technologies) / Matrigel (Millipore) and passaged using ReLeSR (Stem Cell Technologies). Quality control was performed through karyotype analysis and mycoplasma testing.

[0121] Cardiomyocyte and stromal cell differentiation was achieved using the experimental protocol described above (Voges, HK, et al., 2017, Development, 144: 1118-1127; Burridge, PW, et al., 2014, Nat Methods, 11:855-60). Differentiation of cardiomyocytes and stromal cells was achieved using 2x10- cells. 4 cells / cm 2hPSCs were seeded in Matrigel-coated culture flasks, cultured for 3 days in mTeSR PLUS before differentiation, and then cultured for 1 day in mTeSR-1. To induce cardiac mesodermal development, hPSCs were cultured in RPMI B27 medium (RPMI 1640 GlutaMAX + 2% insulin-free B27 supplement) 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 (STEMCELL Technologies), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate (Sigma), and 1% penicillin / streptomycin (Thermo Fisher Scientific), with the medium changed daily for 3 days. Cardiac specification was then performed for another 3 days using RPMI B27 supplemented with 5 μM IWP-4 (STEMCELL Technologies), followed by 5 μM IWP-4 RPMI B27 + (RPMI 1640 GlutaMAX + 2% insulin-free B27 supplement), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate (Sigma), and 1% penicillin / streptomycin (Thermo Fisher Scientific). Cells were cultured for 7 days with Glutamax + 2% insulin-containing B27 supplement, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, and 1% penicillin / streptomycin, with the medium changed every 2-3 days. Cells were then cultured for another 2 days in RPMI B27+, followed by collection at 37°C for 1 hour with 0.2% type I collagenase (Sigma) in PBS (containing Ca2+ and Mg2+) containing 20% ​​fetal bovine serum (FBS). After washing, cells were incubated at 37°C for 10 minutes in 0.25% trypsin-EDTA. The digestion reaction was terminated in organoid culture medium (10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium hydrate, and 1% penicillin / streptomycin), filtered through a 100 μm mesh cell filter (BD Biosciences), centrifuged at 300 x g for 3 minutes, and then resuspended in organoid culture medium.

[0122] hCO preparation hCO culture inserts were prepared using SU-8 photolithography and PDMS molding (Mills et al., 2017 PNAS 114:E8372-E8381). Acid-soluble bovine collagen 1 (Devro) was salt-equilibrated using 10X DMEM, and the pH was neutralized with 0.1M NaOH before mixing with Matrigel and then with cell suspension on ice. Each hCO contained 5 x 10⁻⁶ cells. 4One cell, with a final concentration of type I collagen and 9% Matrigel. 3.5 μL of the suspension was transferred into an hCO2 culture insert and incubated at 37°C and 5% CO2 for 45–60 minutes to allow gelation. After gelation, organoid formation medium was added, and the cells were cultured in hCO2 for 2 days. Subsequently, hCO was cultured in mature medium (MM) for 5 days, and the medium was changed after 2 days

[24] . The mature medium (MM) was DMEM (Thermo Fisher Scientific) without glucose, glutamine and phenol red, with 4% B27-insulin (Thermo Fisher Scientific), 1mM glucose, 200µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma), 1% penicillin / streptomycin (Thermo Fisher Scientific), 1% GlutaMAX (100x) (Thermo Fisher Scientific), 33µg / mL aprotinin (Sigma) and 100µM palmitate (conjugated with bovine serum albumin in B27, Sigma) added. Then, hCO was cultured in weaning medium (WM) which was DMEM (ThermoFisher Scientific) without glucose, glutamine, and phenol red, and supplemented with 4% B27-insulin (ThermoFisher Scientific), 5.5 mM glucose, 1 nM insulin (Sigma), 200 µM L-ascorbic acid 2-phosphate sesquimagnesium hydrate (Sigma), 1% penicillin / streptomycin (ThermoFisher Scientific), 1% GlutaMAX (100x) (ThermoFisher Scientific), 33 µg / mL aprotinin (Sigma), and 100 µM palmitate (conjugated with bovine serum albumin in B27, Sigma).

[0123] hCO AAV infection and analysis After 7 days of hCO culture, they were infected with AAV under specific MOT conditions. The hCOs were imaged using a Leica Thunder microscope 2 days later and again 5 days later, in which abundant GFP was observed. Fluorescence images were captured at 5 days and the intensity was quantified using a custom Matlab file

[24] . The hCOs were then rapidly frozen for further downstream analysis.

[0124] Preparation of cardiac slices and transduction with AAV Porcine left ventricular myocardium was obtained from tissue discarded by researchers at the Westmead Institute for Medical Research. Heart sections were prepared as described previously (Liu, Z., et al., 2020 Journal of Translational Medicine, 18: 437). Briefly, left ventricular tissue was obtained within 30 minutes of euthanasia and preserved in cardioplegic solution. Within 4 to 6 hours of tissue collection, 300 µm thick sections were cut using a Leicavibratome (Leica VT1000S or VT1200S, Biosystems, Germany). Sections were cultured in Transwell plates using a gas-liquid interface method and transduced with rAAV vector using MOT 10000 on the day of sectioning. Sections were cultured until day 2 post-section. Viability was measured as described previously (Liu, Z., et al., 2020 Journal of Translational Medicine, 18: 437).

[0125] All sections were then imaged using a Zeiss Axiovert 200M live-cell imaging microscope (Zeiss GmbH, Oberkohen, Germany), and then rapidly frozen and stored at -80°C.

[0126] For total DNA / RNA, tissues were directly lysed in lysis buffer using the AllPrep DNA / RNA Micro Kit (Qiagen, #80284, Germany) to extract nucleic acids. For nuclear isolation, tissues were lysed and processed according to the method described above (with slight modifications) (Au-Bergmann, O. and S. Au-Jovinge, 2012 JoVE, 65: e4205). Once lysed in a dounce homogenizer, the lysate was filtered through a 70µm filter, followed by a 40µm filter. The lysate was centrifuged at 700x g for 10 minutes. The supernatant was removed, and the precipitate was resuspended in 1 mL NSB. The nuclear pellet was then stained with PCM1+ (centriole peripheral material 1), washed, stained with goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, Missouri, USA, #HPA023370-100UL), and washed again in NSB. The nuclear pellet was stained with DAPI and then sorted using an imaging cytometer to separate the PCM1+ cardiomyocyte fraction and the PCM1- non-myocellular fraction. Nucleic acid extraction was immediately performed on the sorted nuclei using the AllPrep DNA / RNA Micro Kit.

[0127] Next-generation sequencing analysis For next-generation sequencing analysis, as described above (Cabanes-Creus, M., et al., 2022, MolTher Methods Clin Dev 24: 88-101), DNA / RNA samples from hiPSC-CM, organoids, and heart slices were prepared and analyzed.

[0128] Computer simulation prediction of capsid structure For novel capsid variants of VP monomers, 3D models were generated based on their protein sequences using the online tool SWISS-MODEL, with the structures of AAV6 (PDB ID: 3OAH) or AAV3 (PDB ID: 3KIC) as templates (Schwede, T., et al., 2003 Nucleic Acids Res, 31:3381-5). These reference monomer models were then used to generate 60-mer capsids (based on 60 copies of the VP3 protein) using the VIPERdb2 oligomer generator (Ho, PT, et al., 2018 Annu Rev Virol, 5: 477-488). The resulting 60-mer models were imported into PyMol to generate surface maps, highlighting amino acid changes compared to the parental AAV capsid.

[0129] Statistical analysis Based on the data, detailed statistical analyses were performed as shown in the legend. The significance levels for all tests used are as follows: p<0.05, p<0.01, p<0.001, p<0.0001.

[0130] Example 2: Preparation of modified AAV capsids via directed evolution in hiPSC-CM Reorganized AAV capsid libraries prepared from WT AAV serotypes AAV1-12 were screened in hiPSC-CM (Cabanes-Creus, M., et al., 2018, Mol Ther Methods Clin Dev 12: 71-84) to identify novel cardiogenic AAV capsids.

[0131] After six rounds of screening via wild-type adenovirus-mediated replication, most enriched AAV capsid variants (AAV.KK01, AAV.KK02, AAV.KK33, and AAV.KK05) were found to be most similar to the AAV6 capsid. Figure 1 A and Figure 1 The C), containing 6 to 20 amino acid residue differences ( Figure 1 The variant AAV.KK04 is most similar to the AAV3b capsid. Figure 1 A, Figure 1 B and Figure 1(C). Sequence alignment results are as follows: Figure 2 As shown.

[0132] Flow cytometry was used to analyze transduction experiments using the MOT1000 rAAV.CBA.GFP vector in four hiPSC-CM lines (WTCWT, SCVI 8, SCVI 100, and SCVI 480) to quantify GFP levels on day 5 post-transduction. Results showed that cells transduced with AAV6 exhibited increased GFP expression compared to cells transduced with AAV1 or AAV9. Figure 3 AAV1 and AAV6 differ in many amino acid residues, most of which are located in the VP3 region (A). Figure 3 (B).

[0133] The VP3 domain of AAV variants (AAV.KK01-05) was analyzed. For AAV.KK01, AAV.KK02, and AAV.KK05, compared to the parent AAV6, smaller variations in their capsid surface were predicted. Figure 1 (D). However, the changes in the capsid surface of AAV.KK04 (compared to the parent AAV3) were significantly more numerous (D). Figure 1 (E).

[0134] AAV.KK01-05 exhibits differences in amino acid residues within VP1 and VP2, and these differences are not located in regions where changes in the capsid surface structure are expected. Figure 3 B and Figure 3 (C).

[0135] To evaluate packaging efficiency, these five variants and the AAV6 capsid were used to package rAAV.CBA.GFP. Compared to AAV6, rAAV.KK01, rAAV.KK02, and rAAV.KK05 produced significantly more genome-containing particles; rAAV.KK01, rAAV.KK02, and rAAV.KK05 produced twice as many genome-containing particles as AAV6. Figure 4 ).

[0136] Example 3: Cell entry and gene expression in hiPSC-CM transduced with rAAV.KK01-05 vector Novel capsid vectors were developed, and their functions were compared with wild-type AAV1, AAV6, and AAV9 capsids using competitive transduction assays. Each vector (rAAV.CBA.GFP-BC) was identified by a unique barcode, enabling the vector pools to be mixed in equimolar ratios and used for simultaneous transduction of hiPSC-CM at different transduction coefficients (MOTs) of 100, 1000, and 10000. Figure 5 ).

[0137] Cardiomyocytes differentiated from three hiPSC lineages (SCVI 100) Figure 6 ), SCVI 8 and SCVI 480 ( Figure 7 The transduction efficiency was tested in [the study]. Transduction efficiency was determined at the levels of cell entry (DNA) and transgene expression (mRNA / cDNA). At all tested MOTs, rAAV.KK04 showed the highest transduction efficiency in human cardiomyocytes for both cell entry and transgene expression. At all MOTs, rAAV9 showed lower transduction efficiency in cardiomyocytes from all three iPSC lines, while rAAV6 showed the best transduction efficiency among the three wild-type AAVs tested.

[0138] Five novel capsids were then tested. All vectors were used to package barcode-free transgenes (rAAV.CBA.GFP). rAAV.KK04 induced the strongest GFP expression in all hiPSC cardiomyocyte lines. Figure 8 (A). Further quantification using flow cytometry revealed a significantly higher proportion of transduced cardiomyocytes. Figure 8 (B). The average fluorescence intensity of AAV in all tests was similar ( Figure 9 (A). All hiPSC-CM cultures were confirmed to be >90% pure prior to transduction. Figure 9 (B).

[0139] Example 4: Cell entry and gene expression in cardiac organoids transduced with rAAV.KK01-05 vector.

[0140] Compared to primary cardiomyocytes, hiPSC-CM exhibits a relatively fetal phenotype. To evaluate the effect of cell maturity on transduction using the rAAV.KK01-rAAV.KK05 vector, hiPSC-CM was cultured into human heart organoids (hCO) to enhance its maturity (Voges, HK, et al. , 2017, Development (144:1118-1127) to simulate clinically relevant mature cardiomyocyte types.

[0141] In competitive transduction assays, hCO was transduced using the barcoded rAAV.CBA.GFP-BC vector (MOT100, 1000, and 10000). Lower transduction efficiency was observed with rAAV9. rAAV.KK05 showed the highest cell entry efficiency. rAAV.KK04 was less efficient than other variants. Figure 10(A). However, rAAV.KK04 showed gene expression (30.47% and 29.80% of barcoded mRNA reads) at MOT values ​​of 100 and 1000, respectively.

[0142] We also used rAAV.CBA.GFP without barcodes and compared five novel capsids with three wild-type AAVs using GFP expression as a measure of efficiency. The highest GFP fluorescence was observed in hCO transduced with rAAV6 and rAAV.KK02. Figure 10 B to Figure 10 (C).

[0143] Example 5: Cell entry and gene expression in myocytes from porcine, primate, and human heart slices transduced with rAAV.KK01-05 vector To evaluate the performance of the novel AAV variant in primary cardiomyocyte models, cardiac sections were prepared from left ventricular myocardium of normal pigs and animals with infarcted hearts, and transduced using a mixture of rAAV.CBA.GFP-BC vectors (mix) with barcodes on the day of sectioning. GFP expression was observed on day 2 post-transduction. Figure 11 ).

[0144] Total DNA / RNA obtained from heart sections and analyzed by NGS showed that, in normal hearts, the five novel variants were comparable to rAAV6 in terms of cell entry levels. Wild-type AAV1 and AAV9 did not show significant cell entry or gene expression. Figure 12 A to Figure 12 (B). Compared to wild-type AAV1 and AAV9, gene expression was improved in these five novel variants. However, rAAV6 showed superior transgene expression, with significantly higher barcode reads observed at the mRNA level. Figure 12 (B). Heart slices prepared from the hearts of infarcted pigs showed a similar trend to myocardial slices prepared from normal hearts.

[0145] Cardiac sections are known to contain heterogeneous cell populations. To determine the optimal capsid for effective targeting of cardiomyocytes, cardiomyocytes were identified from the extracted nuclei using PCM1+ sorting. Figure 13 A). Infarcted cardiac tissue showed an increase in non-myocellular populations ( Figure 13 B and Figure 13 (C). The extracted cell nuclei were then used to extract nuclear DNA / RNA, which was then analyzed by next-generation sequencing.

[0146] In the extracted nuclei of normal cardiac cells, we observed that rAAV.KK01 was most effective at cell entry into muscle cells. Figure 14 Although the transgene expression of rAAV6, rAAV.KK02, rAAV.KK03, rAAV.KK04, and rAAV.KK05 was higher compared to AAV1, AAV8, AAV9, AAV serotype rh10, and recombinant AAV serotype rh74 (B), Figure 14 A similar trend was observed in the nuclei extracted from non-muscle cell portions. Figure 14 (CD).

[0147] In heart sections of non-human primate (NHP) hearts, rAAV.KK01 and rAAV.KK04 were effective in terms of cell entry. Figure 15 (A). rAAV.KK01 showed high levels of gene expression, as did rAAV.KK05 and AAV.KK04. Figure 15 (B). However, analysis of cardiomyocytes and non-myoblasts confirmed that rAAV.KK01 was effective in both cardiomyocytes and non-myoblasts for cell entry. Figure 15 (C), while rAAV.KK01, rAAV.KK02, and rAAV6 also drive strong transgene expression in NHP muscle cells (C). Figure 15 (D). In the non-muscle cell portion, among the vectors tested, rAAV.KK01 exhibited the highest cell entry and transgene expression (D). Figure 15 E to Figure 15 (F).

[0148] Similar cardiac section experiments were performed using cardiac sections prepared from left ventricular myocardium from donor hearts. In the nuclei of cells extracted from human heart tissue, we observed that rAAV.KK01 was most effective at cell entry into muscle cells. Figure 16 (B). Among the natural variants AAV, AAV1 and rh10 appear to provide higher transgene expression. rAAV.KK01, rAAV.KK02, rAAV.KK02, rAAV.KK03, rAAV.KK04, and rAAV.KK05 also provided transgene expression in human muscle cells, but improved transgene expression was observed in rAAV.KK01 and rAAV.KK03. Figure 16 (A). In non-myocellular components, rAAV.KK01 and rAAV.KK04 are more effective in cell entry (A). Figure 16 (D), while AAV1, AAV6, rAAV.KK03, and rAAV.KK04 provided transgene expression in human non-muscle cells (D). Figure 16 (C).

[0149] Data from pig, NHP, and human heart slices indicate that the modified adeno-associated virus (AAV) capsid peptide disclosed in this paper facilitates targeted entry into cells and / or promotes transgene expression in primary adult cardiomyocytes.

[0150] Example 6: In vivo gene entry and expression in porcine hearts transduced with rAAV.KK01-05 and FDA-approved AAV An AAV kit for in vivo competitive transduction assays was created. The kit includes rAAV.KK01-05 of this invention and a range of known AAVs. For benchmark testing, the kit was designed to include AAVs currently approved by the FDA for gene therapy clinical trials for cardiac indications. These AAVs include AAV9, AAVrh74, and AAV2i8. AAVs were added in equal amounts to the kit according to the vector titer, resulting in a total preparation of 8.5 x 10⁻⁶ AAVs. 12 VGS (vector genome). The entire dose was injected into the equivalent site of the left anterior descending coronary artery in domestic pigs via an intracoronary route. This delivery method mimics the clinical delivery of therapeutic drugs in human cardiac catheterization laboratories. Six weeks later, the heart was removed and partitioned (left ventricular anterior wall, apex, left ventricular lateral wall, left ventricular posterior wall, right ventricular free wall, and interventricular septum). DNA and RNA (converted to cDNA) were extracted from each segment. Each AAV capsid has a unique 6-mer barcode for its AAV expression cassette. This region of the vector genome was amplified by PCR from the DNA and cDNA, and the amplicons were analyzed by next-generation sequencing to quantify gene entry (DNA) and expression (cDNA) for each AAV capsid type. Results for each cardiac segment are displayed in a heatmap ( Figure 17A The data is presented in the column chart and summarized in the column chart. Figure 17B In this study, all five modified capsids (rAAV.KK01, rAAV.KK02, rAAV.KK03, rAAV.KK04, and rAAV.KK05) of the present invention outperformed AAV9, AAVrh74, and AAV2i8.

[0151] Those skilled in the art will understand that the invention described herein is readily adaptable to variations and modifications beyond the specific description. It should be understood that the invention encompasses all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned in this specification, as well as any and all combinations of any two or more of the stated steps or features.

[0152] Table 2 Capsid Sequence

[0153] Table 3. Examples of relevant genes

[0154] Table 4. Amino acid modifications of variant AAV capsid protein

[0155] The amino acid position relative to SEQ ID NO:1 (AAV6) is indicated Arginine (R) is inserted between amino acid positions 151 and 152 relative to the amino acid sequence of SEQ ID NO:6.

Claims

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

2. A capsid polypeptide comprising the 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 amino acid positions relative to SEQ ID NO:1; or an amino acid sequence having at least about 92% sequence identity with it, and / or b. Amino acid residues 205-737 of SEQ ID NO:9, with amino acid positions relative to SEQ ID NO:1; or an amino acid sequence having at least about 97% sequence identity with it.

3. The capsid polypeptide according to claim 1, wherein the capsid polypeptide comprises: (i) An amino acid sequence that has at least about 85% sequence identity with any of SEQ ID NO:5-8; (ii) Amino acid residues 1-204 of any of SEQ ID NO: 5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; and / or (iii) Amino acid residues 205-735 of any of SEQ ID NO:5-8 or an amino acid sequence having at least about 85% sequence identity with any of the aforementioned sequences; And the capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of the following amino acid positions relative to the number of SEQ ID NO:1: 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.

4. The capsid polypeptide according to claim 3, wherein the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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, 148 missing or Q148; Q151 or Q151-E152 inserted R; E152 or S152; S157 or T157; T162 or K162; Q164 or K164; R168 or K16 8; 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; E41 1 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 R4 65; 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; L 517 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, amino acid positions relative to SEQ ID NO:

1.

5. The capsid polypeptide according to claim 3 or claim 4, wherein the at least one amino acid modification comprises: M1 is missing; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152 are interposed with R; S152; T157; K162; R168; S224; E418 and F584, amino acid positions relative to SEQ ID NO:

1.

6. The capsid polypeptide according to any one of claims 3 to 5, wherein the at least one amino acid modification comprises: M1 is missing; M2; T3; V19; A24; Q26; A29; K31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151-E152 are interpolated with R; S152; T157; K162; Q164; R168, S224; D418; F584; A598 and N642, amino acid positions relative to SEQ ID NO:

1.

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

5.

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

1.

10. The capsid polypeptide according to 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; 148 missing; Q151; E152; S157; K162； K164; R168; A224; E418; L584; V598; and H642, amino acid positions relative to SEQ ID NO:

1.

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

6.

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

6.

13. The capsid protein according to 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-E152 are interpolated with R; S152; T157; K162 and R168, the amino acid positions relative to SEQ ID NO:

1.

14. The capsid polypeptide according to 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-E152 are interpolated with R; S152; T157; K162; Q164; R168; A224; D418; L584; V598; and H642, the amino acid positions relative to SEQ ID NO:

1.

15. The capsid polypeptide according to 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 according to claim 15, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

17. The capsid polypeptide according to claim 3 or claim 4, wherein the at least one amino acid modification comprises:

1. Deletion; M2; T3; V19; A24; Q26; L129; A141; P148; Q151-E152. Insertion of R; S152; T157; K162; R168; S224; E418; and F584, amino acid positions relative to SEQ ID NO:

1.

18. The capsid polypeptide according to any one of claims 3, 4, and 17, wherein the at least one amino acid modification comprises:

1. Deletion; M2; T3; V19; A24; Q26; A29; Q31; K38; D41; G42; F56; A67; R92; L129; E134; G135; A136; K137; A141; V146; P148; Q151-E152; Insertion of R; S152; T157; K162; Q164; R168; S224; D418; F584; V598; and H642, amino acid positions relative to SEQ ID NO:

1.

19. The capsid protein according to 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 comprises the amino acid sequence of SEQ ID NO:

8.

21. The capsid polypeptide according to claim 2, wherein the capsid polypeptide comprises: (i) The amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 85% sequence identity with it; (ii) amino acid residues 1-204 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity with it; and / or (iii) Amino acid residues 205-737 of SEQ ID NO:9 or a sequence having at least about 85% sequence identity with it; The capsid polypeptide comprises at least one amino acid modification at a position selected from the group consisting of the following amino acid positions relative to the number of SEQ ID NO:1: 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.

22. The capsid polypeptide of claim 21, wherein the at least one amino acid modification is selected from the group consisting of: M1 or 1 deletion; 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; A3 5 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 G1 41; L146 or V146; P148, 148 missing or Q148; Q151 or Q151_E152 inserted R; 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; N 201 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, amino acid positions 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-E152 Insert R; 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, Q46 0; Q465; A466; G467; N470; A473; A475; L489; A493; N494; P502; A505; H510; D515; L517; V518; P522; E531; E532; H538; N540; G547; T548; T 549; A553; E554; R567, T568; Q576; Y577; N582; L584; N588; A590, T592; R594; T595; N597; D598; Q599; H642; I699; N706; V709; T717; V720; S722; N735, amino acid positions relative to SEQ ID NO:

1.

24. The capsid polypeptide according to 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 according to claim 24, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO:

9.

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

27. The AAV carrier according to claim 26, wherein the AAV carrier further comprises a heterogeneous coding sequence.

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

29. The AAV carrier according to claim 28, wherein the peptide, polypeptide, or polynucleotide is a therapeutic peptide, therapeutic polypeptide, or therapeutic polynucleotide.

30. An isolated nucleic acid molecule, said nucleic acid molecule encoding a capsid polypeptide according to any one of claims 1 to 25.

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

32. The vector according to claim 31, wherein the vector is selected from plasmids, granules, bacteriophages and transposons.

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, the method comprising contacting the host cell with the AAV vector of claim 27 or claim 28.

35. A method for preparing an AAV vector, the method comprising culturing host cells under conditions suitable for promoting AAV vector assembly, said host cells comprising a nucleic acid molecule encoding a capsid polypeptide according to any one of claims 1 to 25, and an AAV vector. rep The AAV vector comprises a gene, a heterologous coding sequence flanked by AAV inverted terminal repeat sequences, and an auxiliary function for generating productive AAV infection, the AAV vector comprising a capsid containing a capsid polypeptide according to any one of claims 1 to 25, wherein the capsid encapsulates 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 an individual.

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

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

39. The method of claim 38, wherein the heart cells are mammalian heart cells.

40. The method according to claim 38 or claim 39, wherein the heart cell is a primate heart cell.

41. The method according to any one of claims 38 to 40, wherein the heart cells are human heart cells.

42. The method according to any one of claims 38 to 41, wherein the cardiac cells are located in cardiac organoids.

43. The method according to any one of claims 38 to 42, wherein the cardiac cells are cardiomyocytes.

44. The method according to claim 43, wherein the cardiomyocytes are primary cardiomyocytes.

45. The method of claim 43 or claim 44, wherein the cardiomyocytes are iPSC-derived cardiomyocytes.

46. ​​A method for preparing a modified AAV vector, wherein when the vector contains a transgene, the modified AAV vector exhibits enhanced transgene expression in human heart cells, the method comprising: a) Identify reference capsid peptides for in vivo transduction of human cardiac cells; b) Modifying the sequence of the reference capsid polypeptide at at least one of the following amino acid positions relative to SEQ ID NO:1: 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, thereby preparing a modified capsid polypeptide comprising at least one amino acid modification selected from the group consisting of: M1 or 1 deletion; M2 or A2; A3 or T3; I19 or V19; D24 or A24; K26 or Q26; A29 or V29; K31 or Q31; K3 8 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 148 missing ; R inserted between Q151 or Q151_E152; 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, amino acid positions relative to SEQ ID NO:1; and c) The modified capsid polypeptide is carrier-mediated to prepare a modified AAV carrier.

47. A method for preparing a modified AAV vector, said modified AAV vector exhibiting enhanced transgene expression in human heart cells, wherein said vector contains a transgene, wherein said method comprises: a) Identify reference capsid peptides for in vivo transduction of human cardiac cells; b) Modify the amino acid sequence of the reference capsid polypeptide at amino acid positions 129, 146, 148, 162, 164, 168 and 418 relative to the SEQ ID NO:1 to prepare a modified capsid polypeptide comprising the deletion of L129; L146; 148. K162； K164; R168 and E418, amino acid positions relative to SEQ ID NO:1; and c) The modified capsid polypeptide is carrier-mediated to prepare a modified AAV carrier.

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

6.

49. A method for preparing a modified AAV vector, wherein when the vector contains a transgene, the modified AAV vector exhibits enhanced cell entry into human heart cells, the method comprising: a) Identify reference capsid peptides for in vivo transduction of human cardiac cells; b) Modify the amino acid sequence of the reference capsid polypeptide at at least one of the following amino acid positions relative to SEQ ID NO:1: 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, 4 67, 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, 5 94, 595, 597, 598, 599, 642, 648, 660, 661, 664, 665, 699, 706, 709, 717, 720, 722, 735, thereby preparing modified capsid peptides, wherein the modified capsid peptides comprise one or more of the following: M1 or 1 deletion; 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; P 148, 148 missing or Q148; R inserted between Q151 or Q151_E152; 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; L 489 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; A54 8 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, amino acid positions relative to SEQ ID NO:1; c) The modified capsid polypeptide is carrier-mediated to prepare a modified AAV carrier.

50. The method of claim 49, wherein the modified capsid polypeptide comprises the following amino acid modifications: M1 deletion; M2; T3; V19; A24; Q26; L129; A141; P148; R inserted between Q151 and E152; S152; T157; K162; R168; S224; L584; A598 and N642, the amino acid positions being relative to SEQ ID NO:

1.

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

1.

52. The method of claim 49, wherein the modified capsid polypeptide comprises the following amino acid modifications: T14; Q21; K24; P29; P31; P34; A35; E36; R37; H38; K39; D41; S42; F56; A67; R92; V125; L129; E134; G135; A136; K137; G141; V146; E147; P148; Insert R between Q151 and E152; 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; A5 53; 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, amino acid positions relative to SEQ ID NO:

1.

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

54. The method according to any one of claims 46 to 53, further comprising evaluating the transgenic expression of the modified AAV vector in an in vitro system utilizing human heart cells.

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

Images