Recombinant adeno-associated viral gene therapy vectors with reduced liver tropism and enhanced cardiac cell transduction for treatment of heart disease and diseases associated with cardiac dysfunction

Abstract

The present invention provides recombinant adeno-associated viral vectors which are enriched in the heart but have reduced enrichment in other organs, particularly the liver and CNS cells, for use in the treatment of acquired and genetic left and right heart diseases as well as other diseases associated directly or indirectly with cardiac dysfunction.

Description

[0001] The present invention provides recombinant adeno-associated virus vectors that are enriched in the heart but reduced in other organs, particularly the liver and CNS cells, for the treatment of acquired and hereditary left and right heart diseases and other diseases directly or indirectly associated with cardiac dysfunction. Background Art

[0002] Recombinant adeno-associated virus (rAAV) vectors are widely used for in vivo gene transfer. The rAAV vector is a non-enveloped vector composed of a capsid with a diameter of 25 nm and a single-stranded DNA of 4.7 kb. The genome carries two genes, rep and cap, flanked by two palindromic regions called inverted terminal repeats (ITRs). The cap gene encodes three structural proteins, VP1, VP2, and VP3, that make up the AAV capsid. VP1, VP2, and VP3 share the same C-terminus, which is the entire VP3. With AAV2 as a reference, VP1 has a 735 amino acid sequence (GenBank YP_680426); VP2 (598 amino acids) starts at threonine 138 (T138), and VP3 (533 amino acids) starts at methionine 203 (M203). The AAV serotype is defined by its capsid. There are different serotypes, each showing its own tissue targeting specificity.

[0003] By exchanging fragments of the capsid sequence between the capsids of different naturally occurring AAV serotypes, chimeric AAV serotypes or hybrid AAV serotypes have been generated to increase AAV transduction efficiency or increase AAV tropism for a cell or tissue type of interest. For example, by combining domains of the capsid of the AAV8 serotype and domains of the capsid of an AAV serotype isolated from the primate brain, a hybrid AAV capsid was generated. The resulting AAV hybrid serotype can transduce human and mouse retinal tissues (Charbel Issa et al., PLOS ONE, 2013, 8, e60361). However, compared to AAV1, AAV8, and AAV9, one of the hybrid AAV serotypes showed improved transduction efficiency for adipose tissue (Liu et al., Molecular Therapy, 2014, 1, 8,).

[0004] WO 2015 / 191508 discloses recombinant hybrid AAV capsids generated by exchanging variable regions of AAV capsids from different species (human, primate, bird, snake, cow), particularly by exchanging AAV capsids with central nervous system tropism to generate CNS-specific chimeric capsids.

[0005] WO 2017 / 096164 discloses recombinant hybrid AAV capsids between AAV1 serotype, AAV2 serotype, AAV3b serotype, AAV6 serotype and AAV8 serotype, which exhibit enhanced human skeletal muscle tropism. However, all naturally occurring AAV serotypes and variants tested to date have a tendency to accumulate in the liver. As a result, liver accumulation of AAV elicits an inflammatory response in liver tissue through different parts of the innate immune system and the adaptive immune system in response to a) AAV capsid proteins, b) AAV genomes, and c) potentially expressed transgenes, which can lead to acute and progressive liver injury and destruction, and subsequent fatal liver failure, as reported in other clinical studies using, for example, AAV9 or AAV8. In particular, for non-engineered AAV and organotropically engineered AAV vectors, such as organotropically engineered AAV vectors for striated muscle type, both vectors retain their liver tropism, and hepatotoxicity and liver failure can be expected if a systemic administration route such as intravenous infusion of AAV vectors is used for both of them.

[0006] Tissue-specific promoters and microRNA-based gene regulation strategies have been used to isolate gene expression patterns in different tissue types. However, such regulation strategies do not prevent the sequestration of AAV vector capsids and genomes, and subsequent inflammatory responses in off-target organs, such as in the liver after systemic administration.

[0007] WO 2022 / 003211 discloses rAAV capsid protein mutants that have improved tropism for muscle and the CNS, but are de-targeted from the liver. As shown in Table 2 of WO 2022 / 003211, the mutants target the brain (CNS) with higher efficiency than the heart and AAV9.

[0008] Attenuating heparin binding by mutating the basic residues R585 or R588 of the capsid protein has been shown to eliminate heparan sulfate binding and reduce the liver tropism of AAV2-derived vectors (Asokan et al., Nat. Biotechnol., 2010, 28, 79-82).

[0009] WO 2021 / 165544 A1 provides AAV2-based viral vector particles that contain, at the C-terminus of the amino acid at position 587, and / or at the C-terminus of the amino acid at position 588, and / or at the C-terminus of the amino acid at position 453 of the wild-type amino acid sequence of the capsid protein (CAP), a peptide sequence 6 to 7 amino acids in length inserted therein. The inserted sequences are shown in SEQ ID NOs: 1 to 53 of WO 2021 / 165544 A1. The relative expression of a test gene (EGFP) by the resulting recombinant viral vector particles has been tested in several tissues. Except for a reduced expression in hepatocytes compared to AAV9, the expression in the liver is much higher than the expression in cardiomyocyte cell types, and thus significant hepatotoxicity of the engineered vector can be expected.

[0010] Perabo et al., Molecular Therapy, Vol. 8, No. 1, 151 - 157 (2003) describe the insertion of a random 7 - amino acid sequence at position 587 of the VP1 capsid protein of AAV2 virions into the capsid protein and the selection of AAV2 mutants from the human megakaryocyte cell line M - 07e or the B - cell chronic lymphocytic leukemia cell line Mecl co - infected with adenovirus. As a result, the sequences of the mutant capsid proteins were identified, which confer receptor specificity rather than cell specificity to the viral vector.

[0011] Ying et al., Gene Therapy 17, 980 - 990 (2010) describe the selection of vectors with higher specificity for heart tissue by screening an AAV2 display peptide library in three rounds by injecting the AAV2 library into mice, isolating heart tissue sections from the mice 3 days later, and secondarily infecting the heart tissue sections in vitro with Ad5 under culture conditions. Using the native AAV2 serotype and the AAV9 serotype as biological controls, two AAV2 variants were identified that showed increased specificity for heart tissue. However, the biodistribution data of AAV2 presented in the supplement Figure 4A severely deviated from the data uniformly reported in the literature, such as Asokan et al., Nat. Biotechnol., 2010, 28, 79 - 82, Figure 2 B, regarding the biodistribution of wild - type AAV2, where AAV2 - mediated liver transduction exceeds heart transduction. In addition, the data in the supplement Figure 4A showed a significant increase in heart transduction relative to AAV2. However, the data also clearly showed that no significant detargeting from the liver relative to AAV2 was achieved, which is apparently contradictory to other data in the same publication.

[0012] Consistent with this, Zincarelli et al., Molecular Therapy, Vol.16, No.6, 1073-1080(2008) have demonstrated that among the natural serotypes of AAV, AAV9 shows the highest transgene expression in the mouse heart after systemic injection. However, AAV9 also targets other organs, including the liver.

[0013] Currently, in clinical applications, there are multiple gene therapies using AAV-derived vectors for different indications: Alipogene tiparvovec Voretigene neparvovec Onasemnogene abeparvovec Eladocagen exuparvovec Valoctocogene roxaparvovec and Etranacogene dezaparvovec They are all based on wild-type AAV capsid sequences because the wild-type tropisms shown by these capsids are suitable for the respective administration routes and indications of these treatments. However, recently, hepatotoxicity events of multiple systemic high-dose gene therapies based on wild-type AAV have been described (Kishimoto et al., Expert Opinion Biological Therapy 2022, pp.1-5).

[0014] As described by Hajjar and Ishikawa, Circulation Res, Vol.120, No.1, 33-35(2017), AAV9 is a vector with high cardiac and muscle tropism. However, this has not prevented the AAV9-based gene therapy drug for the treatment of spinal muscular atrophy from causing substantial hepatotoxicity, especially in children. In the summer of 2022, two children died due to liver failure after receiving treatment. Similar to AAV9, AAV8 shows rhabdomyotrophic tropism, and its use for the treatment of X-linked myotubular myopathy through the systemic administration route has also led to progressive liver dysfunction, and two patients died due to AAV-based hepatocyte damage.

[0015] Takashi Kei kishi moto and Richard Jude samul ski (2022), Expert Opinion on Biological Therapy doi:10.1080 / 14712598.2022.2060737 reported that "adverse events associated with elevated liver transaminases have now been widely reported in AAV gene therapy clinical trials, with an increased prevalence at higher vector doses. AAV gene therapy for neuromuscular diseases typically requires a dose of 1 - 3E14 vg / kg. Onasemnogene abeparvovec is an AAV9 therapy for spinal muscular atrophy (SMA) and is also the first systemic AAV gene therapy approved by the FDA, having been administered to over 1400 patients. Approximately one - third of patients receiving a dose of 1.1E14 vg / kg experienced at least one hepatotoxic adverse event."

[0016] Therefore, there is a need to increase the target - tissue selectivity of rAAV vectors while avoiding the risk of off - target adverse events.

[0017] Object of the Invention

[0018] For the treatment of primary or secondary cardiac indications, there are no known AAV capsid sequences with tropism suitable for peripheral intravenous injection. Therefore, current attempts to target the heart with AAV - derived vectors rely on invasive percutaneous catheterization of the heart as the administration route. However, this administration route is expensive, invasive, limits the market penetration of the corresponding products, and is inefficient.

[0019] Therefore, there is a need for new AAV vectors with reduced off - target tropism, particularly reduced off - target tropism for the liver, and concomitant increased cardiac transduction.

[0020] Accordingly, an object of the present invention is to provide AAV - based viral vector particles having improved specificity, also referred to as tropism, for human cardiomyocytes and a lower preference for liver tissue, e.g., a lower preference compared to wild - type serotype AAV2. For use in gene therapy, viral vector particles with good specificity for cardiomyocytes should allow the nucleic acid coding sequence contained within the viral vector particles to be expressed in cardiomyocytes. Summary of the Invention

[0021] The inventors have solved the problem of insufficient cardiac specificity by engineering new recombinant AAV - derived vectors that exhibit cardiac - specific tropism, enabling the development of gene therapies that can be delivered by peripheral intravenous injection without percutaneous catheterization. Importantly, the cardiac specificity of these vectors is high enough to avoid hepatotoxic events.

[0022] Based on wild-type AAV capsids, the inventors have engineered a recombinant capsid library and screened for variants in the library that display cardiac tropism in mice ( Figure 1 and Figure 2 ), non-human primates, and pigs (screened in a manner similar to that shown for non-human primates and pigs in Figure 1 and Figure 2 ), which tropism enables peripheral intravenous gene transfer. AAV capsid variants have been identified in non-human primates (Rhesus macaques, Macaca mulatta), pigs (Sus scrofa, German landrace), and mice (Mus musculus) that display cardiac enrichment but reduced enrichment in other organs, particularly liver and CNS cells. Primarily, these new cardiac-tropic AAV capsid variants are characterized by cardiac enrichment and liver detargeting. This makes the inventors' solution for cardiac gene therapy particularly valuable because there is a well-known high risk of hepatotoxicity when high doses of AAV are administered systemically in humans, and liver detargeting vectors are needed.

[0023] As shown in Figure 1 (1), an initial screening library of novel recombinant AAVs was established based on eight previously described wild-type AAV cap gene sequences (parental cap gene sequences). All AAV capsid variants in this initial screening library were generated by a random recombination process, resulting in new and previously undescribed capsid variants. (2) The initial screening library was injected into mice via the tail vein of the peripheral vein. (3) A secondary screening library containing cardiac-tropic AAV capsid variants was established from the AAV capsid sequences recovered from the heart. (4) The secondary screening library was injected into mice via the tail vein of the peripheral vein. (5) AAV capsid sequences were recovered from all tissues, and next-generation sequencing (NGS) allowed for biodistribution analysis and identification of those AAV capsid variants that were enriched in the heart and detargeted in all other organs (off-target organs), particularly in the liver and CNS. The initial screening library was also injected into non-human primates and pigs, and cardiac AAV capsid variants were screened as described for mice. The new capsid variants can be used for gene therapy of diseases affecting cardiac tissue.

[0024] In different animal models such as mice, pigs, or non-human primates (NHPs), AAV capsid variants show cardiac enrichment relative to the liver. In addition, AAV capsid variants show cardiac enrichment relative to the CNS (e.g., the brain). In particular, compared to AAV9, the AAV capsid variants show increased cardiac enrichment relative to the liver and increased cardiac enrichment relative to the CNS. In addition, AAV capsid variants are provided that exhibit cardiac enrichment and de-targeting to other tissues such as the adrenal gland, kidney, pancreas, spleen, lymph nodes, testis, fat, bone marrow, and aorta. Detailed Description

[0025] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear nucleic acids and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers. Unless otherwise specified or required, any polynucleotide embodiment of the invention described herein includes both double-stranded forms and one of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0026] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term also includes amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation to a label component.

[0027] The term "peptide" refers to short polypeptides, e.g., peptides having from about 4 to 20 amino acid residues.

[0028] The term "vector" refers to a vehicle, macromolecule, or molecular complex that contains a polynucleotide or protein to be delivered to a cell. An "expression vector" is a vector that contains a coding sequence encoding a gene product of interest for the purpose of achieving expression of the gene product in a target cell. The expression vector contains control elements operably linked to the coding sequence to facilitate expression of the gene product.

[0029] A "gene" refers to a polynucleotide that contains at least one open reading frame that is capable of encoding a specific protein after being transcribed and translated. A "gene product" is a molecule produced by the expression of a specific gene. Gene products can include, but are not limited to, polypeptides, proteins, aptamers, interfering RNAs, or mRNAs. Gene editing systems (e.g., CRISPR / Cas systems) can be described as a gene product or several gene products required to constitute the system (e.g., Cas proteins and guide RNAs).

[0030] The term "isolated" refers to being separated from the components of cells and other constituents, where virions, cells, tissues, polynucleotides, peptides, polypeptides, or proteins are normally associated in nature. For example, an isolated cell is a cell separated from tissues or cells of different phenotypes or genotypes.

[0031] The term "genetic modification" refers to a permanent or transient genetic change induced in a cell after the introduction of a new nucleic acid (i.e., a nucleic acid exogenous to the cell). The genetic change can be achieved by integrating the new nucleic acid into the genome of the cardiomyocyte or by the transient or stable maintenance of the new nucleic acid as an extrachromosomal element. When the cell is a eukaryotic cell, a permanent genetic change can be achieved by introducing the nucleic acid into the genome of the cell. Suitable genetic modification methods include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc.

[0032] "Sequence identity" or "identity" refers to the percentage of the number of identical amino acids between a sequence of interest and a reference sequence. "Identity" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or the same amino acid residue, then the corresponding molecules are identical at that position. The percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, the comparison is made when the two sequences are aligned to give the maximum identity. Identity can be calculated by performing an alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, 7th Edition, Madison, Wisconsin) pileup program, or any sequence comparison algorithm such as FASTA, CLUSTALW, Clustal Omega, or BLAST (available from ncbi.nlm.nih.gov).

[0033] The term "equivalent" with respect to a polypeptide or nucleic acid sequence refers to a polypeptide or nucleic acid that is different from a reference polypeptide or nucleic acid sequence but retains the basic properties (e.g., biological activity). Typical variants of polynucleotides differ in their nucleotide sequences from another reference polynucleotide. The nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, deletions, additions, fusions, and truncations in the polypeptide encoded by the reference sequence. Generally, the differences are limited, so the sequences of the reference polypeptide and the variant are overall very similar and are identical in many regions.

[0034] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, and other procedures that generate a construct different from a naturally occurring polynucleotide, or that the polynucleotide is assembled from synthetic oligonucleotides. A "recombinant" protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that contains a recombinant polynucleotide and / or a recombinant protein, such as a recombinant capsid protein.

[0035] The term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample.

[0036] A "gene of interest" is a gene that is useful for a particular application, such as, but not limited to, diagnosis, reporting, modification, treatment, and genome editing. For example, a gene of interest can be a therapeutic gene, a reporter gene, or a genome editing enzyme. As used herein, the term "therapeutic gene" refers to a gene that, when expressed, has a beneficial effect on the cell or tissue in which the gene is located, or on a mammal that expresses the gene. Examples of beneficial effects include improving the signs or symptoms of a disorder or disease, preventing or suppressing a disorder or disease, or conferring a desired characteristic. Therapeutic genes include genes that partially or fully correct a genetic defect in a cell or a mammal.

[0037] A "therapeutic gene" may also be referred to as an "effector molecule" and can be selected from any wild-type sequence, such as for complementing a defective gene in a viral vector particle receptor. Exemplary effector molecules are native genes, including genes from any species, preferably human genes.

[0038] A "control element" or "control sequence" is a nucleotide sequence that participates in molecular interactions that contribute to the functional regulation of a polynucleotide, including replication, reiteration, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of the process and can be either enhancing or inhibitory in nature. Control elements include transcriptional regulatory sequences, such as promoters and / or enhancers.

[0039] A "promoter" is a DNA sequence that, under certain conditions, is capable of binding RNA polymerase and initiating the transcription of a coding region that is typically located downstream (3' direction) of the promoter. As used herein, the term "tissue-specific promoter" refers to a promoter that is operable in cells of a particular organ or tissue, such as cells of heart tissue.

[0040] "Effectively linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to operate in the intended manner. For example, if a promoter contributes to initiating the transcription of a coding sequence, the promoter is operably linked to the coding region. Inserted residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0041] The term "expression cassette" refers to a heterologous polynucleotide containing a coding sequence that encodes a gene product of interest for effecting the expression of the gene product in a target cell. Unless otherwise specified, the expression cassette of an AAV vector includes the polynucleotide between (but not including) the ITRs.

[0042] "AAV" is an abbreviation for adeno-associated virus. AAV consists of an icosahedral protein capsid and a single-stranded DNA genome of approximately 4.7 kb. The capsid contains three types of subunits, VP1, VP2, and VP3, a total of 60 copies, in a ratio of 1:1:10 (VP1:VP2:VP3). There are two T-shaped inverted terminal repeats (ITRs) at both ends of the genome, which mainly serve as the origin of viral replication and packaging signals. The rep gene encodes four proteins required for viral replication. The cap gene encodes three capsid subunits by alternative splicing and translation from different start codons. In addition, a fourth and fifth gene encoding an assembly activation protein (AAP) and a membrane-associated accessory protein (MAAP) are encoded in different reading frames within the cap coding sequence and have been shown to facilitate virion assembly. The term "AAV" includes all subtypes or serotypes of AAV, unless a serotype is specified, and includes both naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus. For example, "AAV9" refers to AAV serotype 9. The genomic sequences of various AAV serotypes, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, can be found in the literature or public databases such as GenBank. See, for example, GenBank accession numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAVS), AF028704.1 (AAV6), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AX753250 (AAV9).

[0043] As used in the art, an "AAV vector" or "rAAV vector" refers to DNA packaged in an rAAV virion or the rAAV virion itself, depending on the context. As used herein, unless otherwise specified in the context, an rAAV vector refers to a nucleic acid (usually a plasmid) containing a polynucleotide sequence that can be packaged into an rAAV virion, but carrying the capsid or other proteins of the rAAV virion. Generally, an rAAV vector contains a heterologous polynucleotide sequence (i.e., a polynucleotide not of AAV origin) and one or two AAV inverted terminal repeats (ITRs) flanking the heterologous polynucleotide sequence. Only one of the two ITRs can be packaged into rAAV, and the infectivity of the resulting rAAV virion can still be maintained. See Wu et al. (2010) Mol Ther. 18, p. 80ff.

[0044] An "rAAV virion" refers to an extracellular viral particle containing at least one viral capsid protein (e.g., VP1) and a packaged rAAV vector (or a fragment thereof), containing the capsid protein.

[0045] "Capsid protein" refers to VP1, VP2, or VP3, or a combination of VP1, VP2, and VP3. As in wild-type AAV and most recombinant expression systems, VP1, VP2, and VP3 are expressed from the same open reading frame, and engineering of the sequence encoding VP3 inevitably alters the sequences of the C-terminal domains of VP1 and VP2. Capsid proteins from different open reading frames can also be expressed, in which case the capsid of the resulting rAAV virion can contain a mixture of wild-type and engineered capsid proteins, as well as a mixture of different engineered capsid proteins.

[0046] The term "inverted terminal repeat" or "ITR" as used herein refers to an AAV viral cis-element. These elements are essential for the efficient multiplication of the AAV genome.

[0047] An "auxiliary virus" of AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. The auxiliary virus can be an adenovirus, a herpesvirus, or a poxvirus, such as vaccinia virus. "Auxiliary virus function" refers to the function encoded in the auxiliary virus genome that allows AAV replication and packaging.

[0048] As used herein, the term "tropism" refers to the specificity of an AAV capsid protein present in an AAV viral particle for infecting or transducing a particular type of cell or tissue. The tropism of AAV is mediated by the capsid and is thus a result of the capsid sequence. Despite many efforts, the relationship between the capsid sequence and tissue tropism remains unknown. Thus, tropism cannot be inferred from a given capsid sequence, nor can the sequence for a desired tissue tropism be predicted. For only a few individual amino acids in the capsid has their role in targeting or detargeting certain tissues mainly the liver been described (Becker et al., Pathogens 2022, 11, p. 756; Zinn et al., Cell Reports Medicine 2022, p. 100803).

[0049] The tropism of an AAV capsid for a particular type of cell or tissue can be determined by measuring the ability of an AAV vector particle to infect or transduce a particular type of cell or tissue, using standard assays well known in the art, such as those disclosed in the examples of the present application.

[0050] As used herein, the term "liver tropism" or "hepatotropism" refers to tropism for the liver or hepatic tissues and cells, including hepatocytes.

[0051] As used herein, the terms "cardiotropism", "cardiotropic", or "cardiophilic" refer to tropism for cardiac tissues and cells, including cardiomyocytes.

[0052] As used herein, the term "non-human primate" refers to any mammal of the order Primates in zoology, but not including humans. Primates are a distinct order of mammals. They are divided into the suborder Strepsirrhini, including lemurs, galagos, and lorises, and the suborder Haplorhini, including tarsiers and simians (monkeys and apes). According to the present invention, they preferably include rhesus monkeys and macaques. Many characteristics of primates represent adaptations to living in challenging environments, including large brains, visual acuity, color vision, a shoulder girdle that permits a wide range of motion at the shoulder joint, and dexterous hands. Primates range in size from 30 grams (lemurs) to over 200 kilograms (gorillas).

[0053] "Packaging" refers to the series of intracellular events that result in the assembly of rAAV virions, including the encapsidation of an rAAV vector. The AAV "rep" and "cap" genes refer to the polynucleotide sequences encoding the adeno-associated virus replication and encapsidation proteins. AAV rep and AAV cap are referred to herein as AAV packaging genes. Packaging requires the helper virus itself, or more commonly, in a recombinant system, the helper virus functions provided by a helper virus-free system (i.e., one or more helper plasmids).

[0054] An "infectious" virion or viral particle is a virion or viral particle that contains a properly assembled viral capsid and is capable of delivering a polynucleotide component to a cell for which the virion has tropism. The term does not necessarily imply any replicative ability of the virus. "Infectivity" refers to a measure of the ability of a virion to infect a cell. Infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Infectivity is typically determined by a particular cell type. It can be measured in vivo or in vitro by methods known in the art, such as Zolotukhin et al., (1999) Gene Ther. 6:973.

[0055] The terms "parental capsid" or "parental sequence" refer to the reference sequence from which a particle capsid or sequence is derived. Unless otherwise stated, the parental sequence refers to the wild-type capsid protein sequence that is of the same serotype as the engineered capsid protein.

[0056] A "fully replication-competent" virus (e.g., fully replication-competent AAV) refers to a virus that is infectious and is also capable of replicating in an infected cell (i.e., in the presence of a helper virus or helper virus functions).

[0057] The term "transfection" as used herein refers to the uptake of exogenous nucleic acid molecules by a cell. A cell is "transfected" when exogenous nucleic acid is introduced across the cell membrane. Many transfection techniques are well known in the art. See, e.g., Graham et al., (1973) Virology, 52:456, Sambrook et al (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al (1981) Gene 13:197. These techniques can be used to introduce one or more exogenous nucleic acid molecules into a suitable host cell.

[0058] The term "transduction" as used herein refers to the transfer of exogenous nucleic acid into a cell by a recombinant virion, as opposed to "infection" by a wild-type virion. When infection is used with respect to a recombinant virion, the terms "transduction" and "infection" are synonymous, and thus "infectivity" and "transduction efficiency" are equivalent and can be determined using similar methods.

[0059] As used herein, the term "gene delivery" or "gene transfer" refers to a method or system for the reliable insertion of an exogenous nucleic acid sequence, such as DNA, into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication, and expression of an episomal replicon, or integration of the transferred genetic material into the genomic DNA of the host cell.

[0060] "Treatment" is defined as the action of a reagent on a disease, disorder, or condition to alleviate or ameliorate the harmful or any other undesirable effects of the disease, disorder, or condition and / or its symptoms.

[0061] The terms "individual", "subject", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to humans and non-human primates (e.g., apes); mammalian sport animals (e.g., horses); mammalian farm animals (such as sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0062] When used with the compositions of the present invention, "administering" refers both to direct administration (administering to a subject by a medical professional or by the subject himself / herself) and / or to indirect administration (prescribing the composition to a patient). Generally, an effective amount is administered, and this amount can be determined by those skilled in the art and any method of administration can be used. Administration to a subject can be by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade coronary artery injection, retrograde injection, transendocardial myocardial injection, or molecular cardiac surgery combined with recirculating delivery techniques (MCARD). Intravenous administration is preferred.

[0063] Terms such as "effective amount" in relation to the amount of a composition refer to an amount sufficient to induce a desired physiological outcome (e.g., cell reprogramming or treatment of a disease). The effective amount can be administered in one or more than one administration, application, or dose. Such release depends on many variables, including the time of using a single dose unit, the bioavailability of the composition, the route of administration, etc. However, it should be understood that the specific amount of the composition (e.g., rAAV virions) for any particular subject depends on a variety of factors, including the activity of the specific drug used, the age, weight, general health, gender, and diet of the subject, the time of administration, the excretion rate, the combination of compositions, the severity of the particular disease being treated, and the form of administration.

[0064] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms within the scope of reasonable medical judgment that are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and having a reasonable benefit / risk ratio.

[0065] The term "heart cell" refers to any cell present in the heart that provides heart function, such as heart contraction or blood supply, or is otherwise used to maintain the structure of the heart. Heart cells as used herein include cells present in the epicardium, myocardium, or endocardium of the heart. Heart cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiovascular system, such as cells of the coronary artery or coronary vein. Other non-limiting examples of heart cells include epithelial cells, endothelial cells, fibroblasts, heart stem cells or heart progenitor cells, cardiac conduction cells, and cardiac pacemaker cells, which make up the myocardium, blood vessels, and heart cell support structures. Heart cells can be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0066] The term "CNS" or central nervous system refers to the anatomical and functional core of the nervous system, including various interconnected organs, which as used herein can include the brain, midbrain, cerebellum, brainstem, hypothalamus, pituitary gland, optic nerve, and spinal cord. These organs together or individually form the central processing unit for sensory input integration, motor control, and higher cognitive functions within the body. The term "cardiomyocyte" refers to a striated muscle cell containing sarcomeres that is naturally present in the mammalian heart, as opposed to skeletal muscle cells. Cardiomyocytes are characterized by the expression of specialized molecules, such as proteins like myosin heavy chain, myosin light chain, and cardiac α-actinin. The term "cardiomyocyte" as used herein is an umbrella term that includes any subset or subtype of cardiomyocytes, such as atrial cardiomyocytes, ventricular cardiomyocytes, and pacemaker cardiomyocytes.

[0067] The terms "heart pathology" or "heart dysfunction" are used interchangeably and refer to any impairment of the heart's pumping function. This includes, for example, systolic impairment, diastolic capacity impairment (sometimes referred to as diastolic dysfunction), abnormal or improper heart valve function, myocardial diseases (sometimes referred to as cardiomyopathies), diseases such as angina, myocardial ischemia, and / or infarct diseases characterized by insufficient myocardial blood supply, such as infiltrative diseases like amyloidosis and hemochromatosis, global or local hypertrophy (such as may occur in certain types of cardiomyopathies or systemic hypertension), and abnormal connections between heart chambers.

[0068] The term "cardiomyopathy" refers to any disease or dysfunction that directly affects the myocardium. The etiology of the disease or disorder can be, for example, genetic, inflammatory, metabolic, toxic, infiltrative, fibrotic, hematological, or of unknown origin. The two recognized basic forms are (1) primary, consisting of myocardial diseases of unknown cause; and (2) secondary, consisting of myocardial diseases of known cause or myocardial diseases associated with diseases involving other organ systems. "Specific cardiomyopathy" refers to heart diseases associated with certain systemic diseases or heart diseases; examples include hypertensive cardiomyopathy and metabolic cardiomyopathy. Cardiomyopathy includes dilated cardiomyopathy (DCM), for example, a disease in which the systolic pump function of the left ventricle and / or right ventricle is impaired due to gene mutations encoding the following: titin (TTN), myosin (MHY 6; MYH7 and MYBPC3), or actin (ACTC1 and ACTC2), or tropomyosin (TPM1), as well as other sarcomere genes, or nuclear proteins (such as LMNA), ion channel proteins (such as SCNA5), cytoskeletal proteins (such as DES), or other proteins (such as BAG3 or RBM20), which lead to progressive cardiac enlargement; hypertrophic cardiomyopathy (HCM), characterized, for example, by left ventricular hypertrophy due to gene mutations encoding sarcomere proteins (such as, MYH7, MYBPC3, TNNT2, TNNI3, TPM1, ACTC1, MYL2, MYL3, CSRP3), or other genes, such as FHL1, MYOZ2, PLN, TCAP, TRIM63, or TTN, etc.; and restrictive cardiomyopathy, characterized by abnormal diastolic function and overly rigid ventricular walls that impede ventricular filling. Cardiomyopathy also includes left ventricular noncompaction, arrhythmogenic right ventricular cardiomyopathy (ARVC), and arrhythmogenic right ventricular dysplasia, which are due to gene mutations encoding, for example, PKP2, DSP, DSG2, DSC2, JUP, or TMEM43, etc.

[0069] "Heart failure" refers to a pathological state in which an abnormality in cardiac function results in the heart being unable to pump blood at a rate commensurate with the requirements of tissue metabolism and / or only allows the heart to pump blood from an abnormally elevated diastolic volume. Heart failure includes systolic failure and diastolic failure. Patients with heart failure are classified into patients with low cardiac output (usually secondary to ischemic heart disease, arterial and pulmonary hypertension, dilated cardiomyopathy, and / or valvular or pericardial diseases or congenital heart diseases) and patients with elevated cardiac output (usually due to hyperthyroidism, anemia, pregnancy, arteriovenous fistula, beriberi, and Paget's disease).

[0070] Unless otherwise indicated, all medical terms are given their ordinary meaning as used by medical professionals, e.g., in Harrison's Principles of Internal Medicine, 15th edition, particularly in the chapters on heart or cardiovascular diseases, disorders, conditions, and dysfunctions.

[0071] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of tissue culture, immunology, molecular biology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1995) PCR: A Practical Approach; Herzenberg et al., eds. (1996) Weir’s Handbook of Experimental Immunology.

[0072] In some embodiments, the invention relates to a recombinant AAV-derived vector comprising a nucleic acid construct of a gene of interest, wherein the vector comprises a nucleic acid molecule encoding a capsid protein variant comprising the following consensus sequence (SEQ ID NO: 201):

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

[0074] The letters refer to single-letter amino acid codes, and the letters within the brackets are interchangeable for their respective positions. In the case where there is no amino acid at a certain position, a gap is allowed and represented by "-".

[0075] This consensus sequence was obtained from the sequences of the present invention as shown in Figure 4 by multiple sequence alignment (Clustal Omega algorithm, using Geneious prime v2023.2.1 software).

[0076] In a preferred embodiment, the present invention relates to a recombinant AAV-derived vector comprising a nucleic acid construct for a gene of interest, wherein the vector comprises a nucleic acid molecule encoding a capsid protein variant, and the capsid protein variant comprises any sequence of the present invention as shown in Figure 4.

[0077] To engineer the vector to obtain a cardiotropic capsid variant, the applicant applied two previously described methods and their combination: DNA shuffling and peptide display (Wang et al., Nat. Rev. Drug Discov. 2019, 18, 358 - 378; Becker et al., Pathogens 2022, 11, 756; Grimm et al., J. Virol. 2008, 82, 5887 - 5911; Hermann et al., Acs. Synth. Biol. 2019, 8, 194 - 206). Reference Figure 2 。

[0078] DNA shuffling

[0079] The parental capsid sequences are from wild-type AAV, and their sequences are shown in Figure 3.

[0080] These parental nucleotide sequences 1 to parental nucleotide sequence 8 mentioned in Figure 3 were PCR amplified and subjected to partial DNase I digestion to generate fragments smaller than 1 kb. Due to the high homology (average > 90%) between the parental sequences, these fragments self-annealed in primerless PCR to generate full-length cap sequences ( Figure 2A). To increase the homology between parental sequences and thus improve the recombination rate and library yield, the sequence homology between parental capsid sequences is increased by local codon optimization, as described by Cabanes-Creus et al., Mol. Ther–Methods Clin. Dev., 2019, 12, 71-84. The process of DNA shuffling is random, and thus, it is impossible to predict the final capsid sequence.

[0081] Peptide display

[0082] Two sites in the cap gene, position 453 and position 588 (based on the amino acid sequence positions), have previously been described as allowing the expression of short peptide motifs, which can alter tissue tropism (Büning et al., Mol. Ther.–Methods Clin. Dev. 2019, 12, 248-265). See Figure 2 B. The applicant uses these two sites (and their combinations) to incorporate random tetramers, pentamers, or hexamers (random peptide libraries), or hexamers with peptide motifs that have been described in the prior art (e.g., Ying et al., Gene Ther. 2010, 17, 980-990; US6303573B1).

[0083] Table 1 summarizes examples of peptide motifs and insertion positions used to obtain the sequences of the present invention.

[0084] Table 1

[0085]

[0086]

[0087]

[0088] DNA shuffling and peptide display are also used in combination ( Figure 2 C), and 18 sublibraries with different parental capsid sequences and diversification strategies were established from 8 parental cap gene sequences from Figures 3(a) to 3(h). The total diversity of the initial identification screening library for injection exceeded 2·10 8 .

[0089] In vivo screening

[0090] The pooled validation screening library was injected into three mice, three non-human primates (NHPs), and three pigs. Three weeks after injection, AAV capsid sequences were recovered from the left ventricular myocardium by PCR amplification, and a secondary validation screening library was established using these amplicons. In this step, unique molecular identifiers (UMIs) were introduced to facilitate subsequent high-throughput sequencing (Davidsson et al., Sci Rep-uk, 2016, 6, 37563; Davidsson et al., PNAS 2019, 116, 27053-27062). The secondary validation screening library was injected into four mice or two NHPs or two pigs, and three weeks after injection, organs were isolated for subsequent DNA isolation. Using the previously introduced UMIs, high-throughput short-read sequencing was applied to generate biodistribution maps, and cap gene sequences were obtained by matching the identified UMIs with the cap gene sequence information obtained from long-read sequencing of the plasmids produced from the secondary library.

[0091] Screening of cardiotropic AAV capsid variants

[0092] The UMI short-read data from the validation screening were quality-filtered, and the UMIs were identified and counted. The analysis followed the algorithm proposed by Weinmann et al. (Nat. Commun., 2020, 11, 5432), and the key formulas are described in Table 2. Briefly, the raw sequencing read counts for each UMI were normalized by the total sequencing read counts within the sample and subsequently by the frequencies within the input samples. Total AAV quantification for each tissue sample obtained by dPCR was used for tissue normalization before calculating tissue enrichment and specificity.

[0093] Novel cardiac capsid sequences

[0094] For each animal, UMIs with a heart tissue (left ventricle, right ventricle, and septum) enrichment factor higher than the enrichment factor of any other tissue were considered cardiophilic. The UMI sequences were mapped to the long-read sequencing data used for cap gene sequence retrieval, and a total of cardiophilic AAV capsid variants from the mouse validation screening (SEQ ID NO:78 to SEQ ID NO:87), from the non-human primate (NHP) screening (SEQ ID NO:90 to SEQ ID NO:108), and from the pig screening (SEQ ID NO:109 to SEQ ID NO:130) were identified. Refer to Figures 4(a) to 4(zzz).

[0095] Figures 7 to 57 Cardiac enrichment of the novel capsid sequences was shown to be superior to hepatic enrichment. Figures 7 to 57demonstrates the ability of the present invention to increase cardiac targeting while de-targeting the liver and CNS. In addition, Figures 7 to 57 shows that the left ventricular enrichment of the capsid variant exceeds that in off-target tissues such as the liver, adrenal gland, kidney, pancreas, spleen, lymph node, testis, fat, bone marrow, aorta, CNS (brain), lung, stomach, colon, rectum, etc. In addition, Figures 18 to 36 shows that the left ventricular enrichment of the capsid variant exceeds that in off-target tissues in non-human primates (NHP), Figures 37 to 57 shows that the left ventricular enrichment of each capsid variant exceeds that in off-target tissues in pigs.

[0096] In vivo validation screening

[0097] The variants identified in the identification screen and the recombinant variants (SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 131 to SEQ ID NO: 154) generated from these variants identified in the identification screen were generated individually for an additional validation screen library to compensate for potential biases caused by over-representation or under-representation of individual variants in the initial screen library. Each AAV in the validation screen library carried a unique DNA barcode in its genome for tracking by NGS. This enabled the creation of a validation library in which all AAVs were represented at equimolar ratios. The validation screen library included all variants identified in the identification screens in mice and NHP and was used for the evaluation and validation of the biodistribution profiles obtained in the identification screens in all species by the UMI method. For this purpose, the validation screen library was injected into five mice, three pigs, and three NHPs, and two weeks after injection, the organs were isolated for subsequent DNA isolation. Figures 58 to 7 Figure 4 shows the data obtained from the validation screen. Heart or heart tissue refers to the left ventricle, right ventricle, and septum.

[0098] Polynucleotides, vectors and uses for the production of AAV vectors

[0099] Another aspect of the present invention is a polynucleotide encoding a recombinantly expressed form of a cardiotropic capsid variant protein. The polynucleotide can be DNA, RNA, or a synthetic or semi-synthetic nucleic acid.

[0100] The present invention also includes sequence variants and recombinants of the polynucleotides of the present invention, which comprise a deletion and / or insertion of one or more nucleotides, in particular an insertion or deletion of one or more codons, mainly at the ends (3' or 5') of the oligonucleotide, but also showing at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the polynucleotide sequences described in the present invention and recombination. The polynucleotide sequences according to the present invention are similar to the sequences shown in Figure 4 and can be characterized and isolated according to any technique known in the art.

[0101] The present invention also provides functional fragments of the above-mentioned nucleotide sequences of the present invention, which are AAV-derived capsid proteins and exhibit enhanced cardiac tropism but reduced hepatic tropism. Those skilled in the art can easily determine which nucleic acid sequences are related to the nucleotide sequence of Figure 4(a) and are fragments thereof and still have the same function as the full-length sequence by using standard assays.

[0102] The present invention also provides polynucleotide sequences which are redundant compared to any of the nucleotide sequences given above due to the degeneracy of the genetic code, in particular comprising any one of SEQ ID NO:1 to SEQ ID NO:77. Thus, these variant polynucleotide sequences will encode the same amino acid sequence as the polynucleotide from which they are derived, in particular an amino acid sequence selected from SEQ ID NO:78 to SEQ ID NO:154.

[0103] In some embodiments, the polynucleotide encodes an AAV capsid variant comprising or consisting of a sequence selected from SEQ ID NO:90 to SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96 to SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:128, SEQ ID NO:133 to SEQ ID NO:137 and SEQ ID NO:149 to SEQ ID NO:150. In certain embodiments, the polynucleotide is selected from SEQ ID NO:13 to SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:19 to SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:56 to 60 and SEQ ID NO:72 to SEQ ID NO:73.

[0104] In some embodiments, the AAV capsid variants described herein:

[0105] - are cardiotropic;

[0106] - exhibit higher transduction efficiency in cardiac cells than in liver cells and CNS cells;

[0107] - show an increased selectivity for cardiac cells relative to liver cells and an increased selectivity for cardiac cells relative to CNS cells compared to native AAV serotypes, such as AAV1, AAV2, AAV6, AAV8, AAV9, particularly AAV9.

[0108] The cardiotropism of the AAV capsid for cardiac cells, such as cardiomyocytes or tissues or organs, such as the heart, can be determined by measuring the ability of AAV vector particles to infect or transduce a specific type of cardiac cell or tissue using standard assays well known in the art, such as those disclosed in the examples of this application, or according to the evaluations described in Examples 1 and 4.

[0109] The AAV capsid variants can be evaluated in vivo, preferably in a mouse or a relevant large animal model, such as a pig or a primate, particularly an NHP.

[0110] The AAV capsid variants have an increased tropism for the heart and are at least de-targeted from the liver. Preferably, the AAV capsid variants have an increased tropism for the heart and do not target the liver and CNS. In particular, the AAV capsid variants have an improved tropism for the heart and are de-targeted from the liver, CNS, and at least one other organ selected from the group consisting of atrium, diaphragm, quadriceps femoris, cerebellum, lung, stomach, pancreas, colon, kidney, adrenal gland, spleen, cervical lymph node, mesenteric lymph node, bone marrow, salivary gland, and testis.

[0111] "Increased tropism for the heart" means that the cardiac enrichment of the AAV capsid variant exceeds off-target organ enrichment, at least in the liver and brain. This increased cardiac enrichment of the AAV capsid variant will likely result in a reduction in the required vector dose, along with a reduction in the dose-dependent vector-related liver toxicity and immunogenicity risks.

[0112] In addition, AAV capsid variants are de-targeted from the CNS (brain). For the development of gene therapy drug products for cardiovascular diseases, achieving very precise targeting of the heart is an absolute requirement and an indispensable factor. While the concept of simultaneously targeting multiple organs such as the central nervous system and the cardiovascular system may seem attractive for certain conditions such as Friedreich's ataxia, in which both the central nervous system and the heart are affected, it is important to note that severe toxicity has been observed in the central nervous system, particularly within the dorsal root ganglia, when high systemic doses of AAV vectors are administered (Hordeaux, J. et al.; Hum Gene Ther 2020, 31, 808–818; Hinderer, C. et al.; Hum Gene Ther 2018, 29, 285–298). In addition, due to differences in transduction efficiency, promoter activity, and promoter tissue specificity, the technical complexity and near-impossibility of achieving therapeutic expression levels using a single AAV vector targeting two organs pose a significant challenge. Specifically, the expression levels achieved by a single AAV vector in two organs may prove insufficient to be effective in one organ while being too high and thus toxic in the other organ. In summary, the pursuit of multi-organ targeting via a single AAV vector represents a technically challenging endeavor with inherent risks. Therefore, it is prudent to use highly specific vectors when developing gene therapy drug products.

[0113] Accordingly, in some embodiments, the AAV capsid is de-targeted from at least one additional organ. In other embodiments, the de-targeted organ is associated with the risk of dose-dependent vector-related toxicity or other adverse events. For example, the AAV capsid variants described herein are capable of de-targeting organs selected from the group consisting of the liver, adrenal gland, kidney, pancreas, spleen, lymph nodes, testis, fat, bone marrow, aorta, CNS, lung, stomach, colon, rectum, and the like.

[0114] For example, with respect to the testis, low transduction in the reproductive organs is required for therapeutic administration to patients of reproductive age.

[0115] In mice, pigs, and NHPs of different species, the AAV capsid variants described herein showed increased cardiac enrichment relative to the liver and brain, respectively. This indicates that the cardiac transduction ability is conserved across species.

[0116] The AAV capsid variants according to the present invention showed a higher cardiac tissue enrichment factor than the tissue enrichment factors in any other tissue. The tissue enrichment factor can be determined by the method of Example 1 and calculated by the formula in Table 2.

[0117] The AAV-derived capsid proteins according to the present invention achieved significant cardiac enrichment superior to that of the liver and brain tissues. Figures 19 to 57It is shown that the number of vector readings in the heart of pigs or NHPs is about 5 to about 100 times that in the liver.

[0118] In addition, compared to at least one natural (wild-type) AAV serotype, preferably compared to serotypes AAV2, AAV8, and / or AAV9, the AAV-capsid variant exhibits increased tropism for the heart and is at least de-targeted from the liver and brain.

[0119] For example, according to the method in Example 1 and the formula in Table 2, or according to the method in Example 4 and the formula in Table 3, the enrichment and de-targeting of the AAV capsid variant can be determined and calculated.

[0120] Enrichment means that the count of the AAV capsid variant in the heart exceeds the count of the AAV capsid variant in the off-target organ. Enrichment means that compared to another AAV capsid protein, such as natural AAV, like AAV2, it is enriched in the heart by at least about 1.5-fold, 2-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 30-fold, 50-fold, 100-fold, or more than 100-fold.

[0121] De-targeting refers to the count of the AAV capsid variant in the off-target organ compared to another AAV, such as natural AAV, like AAV2 or AAV9. De-targeting means that compared to another AAV capsid protein, such as natural AAV, like AAV2 or AAV9, it is reduced to at most about 1 / 1.5, 1 / 2, 1 / 4, 1 / 5, 1 / 8, 1 / 10, 1 / 15, 1 / 30, 1 / 50, 1 / 100, or less than 1 / 100 in the off-target organ. For example, Figure 60 shows that in pigs, the liver AAV load of the AAV capsid variant is reduced to at most 1 / 4 compared to AAV9, especially 1 / 5 to 1 / 300, and in NHPs, the liver AAV load is reduced to at most 1 / 5 compared to AAV9, especially 1 / 10 to 1 / 340.

[0122] In pigs and / or NHPs, the heart enrichment of the AAV-capsid variant relative to the liver is higher than that of AAV9, and the heart enrichment relative to the brain is higher than that of AAV9.

[0123] In some embodiments, in pigs and / or NHPs, the heart enrichment of the AAV capsid variant relative to the liver is higher than that of AAV9, and the heart enrichment relative to the brain is higher than that of AAV9. An embodiment of such an AAV capsid variant is shown in Table 4.

[0124] In additional embodiments, the AAV capsid variants exhibit improved cardiac enrichment relative to the liver and brain in both pigs and NHPs, and are selected from SEQ ID NO:80, SEQ ID NO:84 to SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:94 to SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99 to SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:109 to SEQ ID NO:111, SEQ ID NO:114 to SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:124 to SEQ ID NO:126, SEQ ID NO:130 to SEQ ID NO:131, SEQ ID NO:133 to SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144 to SEQ ID NO:146, SEQ ID NO:149, and SEQ ID NO:153 to SEQ ID NO:154.

[0125] In certain embodiments, the AAV capsid variants exhibit improved cardiac enrichment and off-target organ detargeting in both porcine and NHP species, and are selected from SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:99 to SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:133 to SEQ ID NO:135, SEQ ID NO:137, and SEQ ID NO:149. Off-target organs include the liver, CNS, adrenal glands, kidneys, pancreas, spleen, lymph nodes, testes, fat, bone marrow, and aorta.

[0126] In another embodiment, the AAV capsid variant exhibits improved cardiac enrichment in pigs and / or off-target organ de-targeting in pigs and NHPs and is selected from SEQ ID NO:90 to SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96 to SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:128, SEQ ID NO:133 to SEQ ID NO:137, and SEQ ID NO:149 to SEQ ID NO:150, where the off-target organs also include the adrenal gland, kidney, pancreas, spleen, lymph node, testis, fat, bone marrow, and aorta (Figures 61 to 71).

[0127] The polynucleotide is advantageously inserted into a recombinant vector that, in a non-limiting manner, comprises a linear or circular DNA or RNA molecule composed of chromosomal, extrachromosomal, synthetic, or semi-synthetic nucleic acids, such as, in particular, viral vectors, plasmids, or RNA vectors.

[0128] To introduce and maintain a nucleic acid molecule of interest in a eukaryotic host cell, many vectors into which the nucleic acid molecule of interest can be inserted are known per se. The choice of the appropriate vector depends on the intended use of the vector (e.g., replication of the sequence of interest, expression of the sequence, maintenance of the sequence in an extrachromosomal form, or integration into the chromosomal material of the host), and also on the nature of the host cell.

[0129] The recombinant vector for use in the present invention is an expression vector that contains suitable means for expressing the cardiotropic capsid variant protein and possibly also the AAV Rep protein. Generally, each coding sequence (AAV Cap variant and AAV Rep) is inserted into separate expression cassettes in the same vector or different vectors. Each expression cassette contains a coding sequence (open reading frame or ORF) functionally linked to regulatory sequences that permit the expression of the corresponding protein in AAV-producing cells, such as, in particular, a promoter, promoter / enhancer, start codon (ATG), stop codon, transcription termination signal. Alternatively, using an internal ribosome entry site (IRES) inserted between the two coding sequences, hybrid AAV Cap and AAV Rep proteins can be expressed from a unique expression cassette. In addition, the codon sequences encoding the cardiotropic capsid variant protein and AAV Rep (if present) are advantageously optimized for expression in AAV-producing cells, particularly human-producing cells.

[0130] The vector, preferably a recombinant plasmid, can be used to produce hybrid AAV vectors containing the cardiotropic capsid variant protein of the present invention using standard AAV production methods well known in the art (see Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102:1045-1054).

[0131] After co-transfection, the cells are incubated for a time sufficient to produce AAV vector particles, then the cells are harvested, lysed, and the AAV vector particles are purified by standard purification methods such as affinity chromatography or cesium chloride density gradient ultracentrifugation.

[0132] AAV particles, pharmaceutical compositions and therapeutic uses

[0133] Another aspect of the present invention is an AAV particle comprising a nucleotide sequence encoding the variant cap protein of the present invention.

[0134] Preferably, the AAV particle is an AAV vector particle. The genome of the AAV vector can be a single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003, Dec., 10(26), 2112-2118). Self-complementary vectors are generated by deleting the terminal resolution site (trs) from one AAV terminal repeat. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome, have a tendency to package DNA dimers. The AAV genome is flanked by ITRs. In certain embodiments, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are from different serotypes of AAV.

[0135] The AAV particle further comprises a gene of interest. As described above, the gene of interest is any nucleic acid sequence capable of modifying a target gene or target cell pathway, particularly in (cardiac) muscle cells. For example, the gene can modify the expression, sequence, or regulation of a target gene or cell pathway. In some embodiments, the gene of interest is a functional version of a gene or a fragment thereof. The functional version of the gene includes the wild-type gene, variant genes such as variants belonging to the same family and other families, or truncated versions that at least partially retain the function of the encoded protein. The functional form of the gene can be used for replacement or addition gene therapy to replace defective or non-functional genes in a patient. In other embodiments, the gene of interest is a gene that inactivates a dominant allele causing an autosomal dominant genetic disease. The gene fragment can be used as a recombination template in combination with a genome editing enzyme. Alternatively, the gene of interest can encode a protein of interest (e.g., an antibody or antibody fragment, a genome editing enzyme) or RNA for a particular application. In some embodiments, the protein is a therapeutic protein, including a therapeutic antibody or antibody fragment, or a genome editing enzyme. In some embodiments, the RNA is a therapeutic RNA. The gene of interest is a functional gene capable of producing the encoded protein, peptide, or RNA in the target cells of the disease, particularly muscle cells. The AAV viral vector comprises the gene of interest in an expressible form in muscle cells including cardiomyocytes and skeletal muscle cells. In particular, the gene of interest is operably linked to a ubiquitous, tissue-specific, or inducible promoter that is functional in muscle cells. The gene of interest can be inserted into an expression cassette that also contains a polyA sequence.

[0136] The RNA is advantageously complementary to a target DNA or RNA sequence or binds to a target protein. For example, the RNA is interfering RNA such as shRNA, microRNA, guide RNA (gRNA) used in combination with a Cas enzyme or a similar enzyme for genome editing, antisense RNA capable of skipping exons such as modified small nuclear RNA (snRNA) or long non-coding RNA. The interfering RNA or microRNA can be used to regulate the expression of a target gene involved in (cardiac) muscle diseases. The guide RNA complexed with a Cas enzyme or a similar enzyme for genome editing can be used to modify the sequence of a target gene, particularly to correct the sequence of a mutant / defective gene or to modify the expression of a target gene associated with a disease, particularly a neuromuscular disease. The antisense RNA capable of skipping exons is particularly used to correct the reading frame and restore the expression of a defective gene with a disrupted reading frame. In some embodiments, the RNA is a therapeutic RNA.

[0137] The genome editing enzymes according to the present invention are any enzymes or enzyme complexes capable of modifying a target gene or a target cellular pathway, particularly in muscle cells. For example, the genome editing enzymes can modify the expression, sequence or regulation of a target gene or cellular pathway. Advantageously, the genome editing enzymes are engineered nucleases, such as but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), Cas enzymes from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, and the like. Genome editing enzymes, particularly engineered nucleases such as Cas enzymes and the like, can be functional nucleases that generate double-strand breaks (DSBs) at target genomic loci and are used in site-specific genome editing applications, including but not limited to: gene correction, gene replacement, gene knock-in, gene knockout, mutagenesis, chromosomal translocation, chromosomal deletion, etc. For site-specific genome editing applications, the genome editing enzymes, particularly engineered nucleases such as Cas enzymes and the like, can be used in combination with a homologous recombination (HR) substrate or template (also referred to as a DNA donor template), which modifies the target genomic locus by homologous recombination induced by the double-strand break (DSB). Specifically, the HR template can introduce a transgenic of interest into the target genomic locus or repair a mutation in the target genomic locus, preferably repairing a mutation in an abnormal or defective gene that causes a neuromuscular disease. Alternatively, the genome editing enzymes, such as Cas enzymes and the like, can be engineered to be nuclease-deficient and used as DNA-binding proteins for various genome engineering applications, such as but not limited to: transcriptional activation, transcriptional repression, epigenomic modification, genome imaging, DNA or RNA co-precipitation, etc.

[0138] Another aspect of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of AAV particles, said particles comprising the recombinant variant AAV capsid protein of the present invention, preferably AAV vector particles packaging a therapeutically relevant gene of interest. In some embodiments of the present invention, the pharmaceutical composition of the present invention is used as a medicament, particularly in gene therapy. The present invention includes the use of the pharmaceutical composition of the present invention as a medicament, particularly for treating diseases by gene therapy.

[0139] Gene therapy can be carried out by gene transfer, gene editing, exon skipping, RNA interference, trans-splicing or any other genetic modification of any coding or regulatory sequence in a cell, said cell including sequences contained in the nucleus, mitochondria or as symbiotic nucleic acids, such as but not limited to viral sequences contained in a cell.

[0140] The three main types of gene therapy are as follows:

[0141] Therapies aimed at providing a functional replacement gene for a defective / abnormal gene or silencing it: These are replacement or addition gene therapies and gene silencing therapies;

[0142] Therapy for gene or genome editing: In this case, the aim is to provide the cell with the necessary tools to correct the sequence or modify the expression or regulation of defective / abnormal genes in order to express functional genes or to inhibit (inactivate) abnormal genes: This is gene editing therapy.

[0143] In addition gene therapy, the therapeutic gene can be a functional version of a gene that is defective or mutated in the patient, for example in genetic diseases. In this case, the therapeutic gene will restore the expression of the functional gene. In gene silencing, the expression of a malfunctioning or unwarranted gene causing disease is inhibited by a suitable interfering nucleic acid that prevents the expression of the harmful gene product.

[0144] Gene or genome editing uses one or more therapeutic genes, for example:

[0145] (i) Genes encoding therapeutic RNAs as defined above, such as interfering RNAs such as shRNAs or microRNAs, guide RNAs (gRNAs) used in combination with Cas enzymes or similar enzymes, or antisense RNAs such as modified small nuclear RNAs (snRNAs) capable of exon skipping; and

[0146] (ii) Genes encoding genome editing enzymes as defined above, such as engineered nucleases, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), Cas enzymes or similar enzymes; or combinations of these genes, which can also be fragments of the functional version of the gene used as a recombination template, as described above.

[0147] According to the invention, gene therapy is used for treating diseases affecting myocardial tissue.

[0148] This includes:

[0149] · Treatment of cardiovascular or cardiopulmonary indications, regardless of origin (acquired or hereditary; e.g., ischemic cardiomyopathy / heart failure after myocardial infarction, hypertensive heart disease, cor pulmonale, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, atrial cardiomyopathy, left ventricular non-compaction cardiomyopathy, heart failure with reduced ejection fraction / diastolic heart failure or Takotsubo syndrome). The treatment may include editing or silencing mutations or abnormal expressions or restoring the expression of healthy gene copies, and the genes encode but are not limited to: cardiac troponin T; cardiac sarcomeric proteins; β-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac α-actin; α-tropomyosin; cardiac troponin I; cardiac myosin binding protein C; four and a half LIM domain protein 1; titin; 5'-AMP-activated protein kinase subunit γ-2; troponin I type 3, myosin light chain 2, cardiac actin α1; cardiac LIM protein; caveolin 3 (CAV3); alpha-galactosidase A (GLA); lysosome-associated membrane protein 2 (LAMP2); mitochondrial tRNA for glycine (MTTG); mitochondrial tRNA for isoleucine (MTTI); mitochondrial tRNA for lysine (MTTK); mitochondrial tRNA for glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoplasmic / endoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal cell-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); β-ARKct (β-adrenergic receptor kinase C-terminus); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia-inducible growth factor; thymosin β4 (TMSB4X); nitric oxide synthase-3 (NOS3); apolipoprotein-E (ApoE), superoxide dismutase (SOD), RNA binding motif protein 20 (RMB20) and S100A1, titin (TTN), myosin (MHY 6;MYH7), myosin-binding protein 3 (MYBPC3), actin (ACTC1 and ACTC2), tropomyosin (TPM1), (lamin A and C) LMNA, sodium channel A5 (SCNA5), desmin (DES), BAG3 or RBM20, troponin T (TNNT2), troponin I (TNNI3), TPM1, MYL2, MYL3, CSRP3, FHL1, MYOZ2, PLN, TCAP, TRIM63 or TTN, S100A1, S100A6, S100A4, S100B, SERCA2a, AC6, inhibitor-1, VEGF-A isoforms, SCF, PKP2, DSP, DSG2, DSC2, JUP or TMEM43, ERBB2-4, NRG1, CDK, YAP, FGF isoforms, HGF, miR-195, miR15a, miR-15b, miR-16 or miR-497, miR-323-3p, miR-187, miR-124, miR-31a-5p, miR-378, lncRNA Sarrah or UCA1 or FTX, circular RNA SNRK or CircFndc3b, HIF-1a, Bcl-2 and Bcl-xl, GATA4, MEF2C, TBX5, HAND2, MESP1, NKX2.5, MYOCD, ETV2, GMT, TRPV4, relaxin receptor, MRTF-A, TRPC isoforms, LRP6, BRG1, Nrf2 / HO-1, HO1, GSTP1, NQO1, ZBTB20, SIRT3, SOD1 / 2, LEF1 and / or IL-10.;

[0150] · Treatment of any syndrome disease involving the cardiovascular or cardiopulmonary system, such as Friedreich's ataxia, Danon disease or Duchenne muscular dystrophy, Down syndrome, Turner syndrome, 22q11.1 deletion syndrome, Williams syndrome, Noonan syndrome, Kabuki syndrome, Alagille syndrome.

[0151] Thus, by gene editing or gene replacement, the correct version of the gene is provided in the cardiomyocytes of affected patients, which may contribute to an effective treatment against the disease.

[0152] In some embodiments, the target gene for gene therapy (additive gene therapy or gene editing) is a gene responsible for one of myotubular myopathy (MTM1 gene), Pompe disease or glycogen storage disease type III (GSD3) (AGL gene).

[0153] Dystrophinopathies are a group of X-linked muscle diseases caused by pathogenic variants in the DMD gene, which encodes the protein dystrophin. Muscular dystrophies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-related dilated cardiomyopathy.

[0154] Pompe disease is a genetic disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Mutations in the GAA gene prevent acid alpha-glucosidase from effectively breaking down glycogen, which allows this sugar to accumulate to toxic levels in lysosomes. This buildup can damage organs and tissues throughout the body, especially muscles. Muscle weakness is usually mild in childhood but becomes more severe in adulthood; some patients develop cardiomyopathy.

[0155] In some embodiments, the pharmaceutical compositions of the present invention are used to treat muscle diseases (i.e., myopathies) or muscle injuries, particularly neuromuscular genetic diseases, such as: Beck / Duchenne MD, myotonic MD, distal MD, limb-girdle MD, congenital MD, Emory-Dreyfus MD, facioscapulohumeral MD or oculopharyngeal MD, malignant hyperthermia, metabolic myopathy, hereditary cardiomyopathy or congenital myasthenic syndrome, ischemic cardiomyopathy / heart failure after myocardial infarction, hypertensive heart disease, cor pulmonale, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, atrial cardiomyopathy, heart failure with reduced ejection fraction / diastolic heart failure, Takotsubo syndrome, left ventricular noncompaction cardiomyopathy.

[0156] Pharmaceutical compositions of the invention comprising AAV vector particles with reduced liver tropism can be administered to patients with concurrent liver degeneration such as congestive liver disease, fibrosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, viral or toxic hepatitis, or underlying genetic diseases that induce liver degeneration.

[0157] In the context of this invention, a therapeutically effective amount refers to a dose sufficient to reverse, alleviate or inhibit the progression of the disorder or condition to which the term applies, or to reverse, alleviate or inhibit the progression of one or more symptoms of the disorder or condition to which the term applies.

[0158] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0159] Preferably, the pharmaceutical composition comprises a carrier or vehicle which is pharmaceutically acceptable for a formulation capable of being injected. These can be in particular isotonic sterile saline solutions (sodium dihydrogen phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of these salts), or dry, in particular lyophilized, compositions which can be made into an injection solution by adding sterile water or physiological saline as the case may be. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or suspensions. The solution or suspension may contain additives which are compatible with the viral vector and do not prevent the viral vector particles from entering the target cells. In all cases, the form must be sterile and must be a liquid which is easy to inject. It must be stable under the production and storage conditions and must prevent the contaminating action of microorganisms such as bacteria and fungi. Examples of suitable solutions are buffers such as phosphate buffered saline (PBS) or Ringer's lactate solution.

[0160] The present invention also provides a method for treating a disease affecting cardiac tissue, which comprises: administering to a patient a therapeutically effective amount of the above-mentioned pharmaceutical composition. The present invention also provides a method for treating a disease by expressing a therapeutic gene in muscle tissue, which comprises: administering to a patient a therapeutically effective amount of the above-mentioned pharmaceutical composition.

[0161] The present invention will now be described by way of example with reference to the accompanying drawings, which show:

[0162] Figure 1 : Identifying AAV capsid variants with high cardiac tropism for cardiac gene transfer

[0163] (1) An initial screening library of novel recombinant AAVs was established based on the previously described wild-type AAV cap gene sequence. All AAV capsid variants in this library were generated by a random recombination process, resulting in new and previously undescribed capsid variants. (2) The initial screening library was injected into mice by tail vein injection via the peripheral vein. (3) A secondary library containing cardiac tropic AAV capsid variants was established from the AAV capsid sequences recovered from the heart. (4) The secondary screening library was injected into mice by tail vein injection via the peripheral vein. (5) AAV capsid sequences were recovered from all tissues, and next-generation sequencing (NGS) allowed biodistribution analysis and identification of those AAV capsid variants that were enriched in the heart and de-targeted in all other organs (off-target organs).

[0164] Figure 2: Overview of methods for screening library generation. (A) DNA shuffling uses parental cap genes and recombines them in a random process by combining partial DNase I digestion with self-annealing and primerless PCR for ligation. In this example, the capsid gene sequences of cap9, cap8, and cap7 from AAV9, AAV8, and AAV7 were used, and a library of novel recombinant capsids composed of fragments of these parental AAV capsids was generated. To establish the initial screening library for injection, all the parental capsid genes mentioned in FIGS. 3(a) to 3(h) were used for DNA shuffling. (B) Peptide display utilizes two known sites (453 and 588) within the capsid to allow insertion and expression of targeted or random short peptide sequences. The resulting library contains highly conserved capsid sequences that differ only at the peptide display sites. In this example, the capsid gene sequence of cap9 was used to insert peptide motifs at position 453, position 588, or both positions simultaneously. To establish the initial screening library for injection, peptides were inserted into the parental capsid genes as mentioned in FIG. 3. (C) DNA shuffling and peptide display were also combined to generate capsid sequences that simultaneously have different parental fragments and different inserted peptides. Overall, more than 2·10 8 sequence variants were generated and subjected to a selection process for capsid sequences for identifying cardiac tropism.

[0165] Figures 3A to 3H : Parental sequences 1 to parental sequence 8.

[0166] Figures 4A to 4YYY : Sequences of capsid variants according to the present invention.

[0167] Figure 5 Biodistribution of AAV2 in mouse tissues.

[0168] FIG. 6 Biodistribution of AAV9 in tissues of (A) pigs, (B) NHPs, and (C) mice.

[0169] Figure 7 Cardiac enrichment of capsid variant 1 exceeds off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0170] Figure 8 Cardiac enrichment of capsid variant 3 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0171] Figure 9Heart enrichment of capsid variant 7 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0172] Figure 10 Heart enrichment of capsid variant 11 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0173] Figure 11 Heart enrichment of capsid variant 13 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0174] Figure 12 Heart enrichment of capsid variant 14 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0175] Figure 13 Heart enrichment of capsid variant 16 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0176] Figure 14 Heart enrichment of capsid variant 18 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0177] Figure 15 Heart enrichment of capsid variant 19 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0178] Figure 16 Heart enrichment of capsid variant 22 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0179] Figure 17Heart enrichment of capsid variant 31 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0180] Figure 18 Heart enrichment of capsid variant 32 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0181] Figure 19 Heart enrichment of capsid variant 33 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0182] Figure 20 Heart enrichment of capsid variant 35 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0183] Figure 21 Heart enrichment of capsid variant 36 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0184] Figure 22 Heart enrichment of capsid variant 37 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0185] Figure 23 Heart enrichment of capsid variant 38 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0186] Figure 24 Heart enrichment of capsid variant 39 relative to off-target organ enrichment. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0187] Figure 25Heart enrichment of capsid variant 41 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0188] Figure 26 Heart enrichment of capsid variant 42 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0189] Figure 27 Heart enrichment of capsid variant 43 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0190] Figure 28 Heart enrichment of capsid variant 44 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0191] Figure 29 Heart enrichment of capsid variant 45 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0192] Figure 30 Heart enrichment of capsid variant 46 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0193] Figure 31 Heart enrichment of capsid variant 47 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0194] Figure 32 Heart enrichment of capsid variant 48 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0195] Figure 33Heart enrichment of capsid variant 49 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0196] Figure 34 Heart enrichment of capsid variant 50 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0197] Figure 35 Heart enrichment of capsid variant 51 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0198] Figure 36 Heart enrichment of capsid variant 52 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0199] Figure 37 Heart enrichment of capsid variant 53 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0200] Figure 38 Heart enrichment of capsid variant 54 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0201] Figure 39 Heart enrichment of capsid variant 55 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0202] Figure 40 Heart enrichment of capsid variant 56 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0203] Figure 41Cardiac enrichment of capsid variant 57 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0204] Figure 42 Cardiac enrichment of capsid variant 58 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0205] Figure 43 Cardiac enrichment of capsid variant 59 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues.

[0206] Figure 44 Cardiac enrichment of capsid variant 60 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0207] Figure 45 Cardiac enrichment of capsid variant 61 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0208] Figure 46 Cardiac enrichment of capsid variant 62 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0209] Figure 47 Cardiac enrichment of capsid variant 63 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0210] Figure 48 Cardiac enrichment of capsid variant 65 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0211] Figure 49 Cardiac enrichment of capsid variant 66 relative to off-target organ enrichment. Enrichment refers to the cardiac UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0212] Figure 50 Heart enrichment of capsid variant 67 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0213] Figure 51 Heart enrichment of capsid variant 68 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0214] Figure 52 Heart enrichment of capsid variant 69 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0215] Figure 53 Heart enrichment of capsid variant 71 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0216] Figure 54 Heart enrichment of capsid variant 73 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0217] Figure 55 Heart enrichment of capsid variant 74 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0218] Figure 56 Heart enrichment of capsid variant 76 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0219] Figure 57 Heart enrichment of capsid variant 77 enriched relative to off-target organs. Enrichment refers to the heart UMI frequency relative to the UMI frequency in other tissues. If no capsid variant is found in the corresponding off-target organ, "divide by zero ∞" is indicated.

[0220] Figure 58Generation of cardiotropic capsid variants: The "recovery ratio" was calculated by dividing the number of AAV vector genome copies in the eluate fraction by the number of AAV vector genome copies in the input fraction.

[0221] Figure 59A AAV genomes (AAV / ng) per ng of isolated genomic DNA in the (A) liver and (B) brain of mice for AV9, AAV8, mutant 3 (SEQ ID NO:34 of WO 2022 / 003211), and the capsid variants.

[0222] Figure 60 AAV genomes (AAV / ng) per ng of isolated genomic DNA for AAV9 and the capsid variants in (A) pigs and (B) NHPs.

[0223] Figure 61 Comparison of AAV capsid variants in the heart versus the liver in (A) NHPs and (B) pigs.

[0224] Figure 62 Comparison of AAV capsid variants with AAV9 in the liver; (A) NHPs and (B) pigs.

[0225] Figure 63 Comparison of AAV capsid variants with AAV9 in the adrenal gland; (A) NHPs and (B) pigs.

[0226] Figure 64 Comparison of AAV capsid variants with AAV9 in the kidney; (A) NHPs and (B) pigs.

[0227] Figure 65 Comparison of AAV capsid variants with AAV9 in the pancreas; (A) NHPs and (B) pigs.

[0228] Figure 66 Comparison of AAV capsid variants with AAV9 in the spleen; (A) NHPs and (B) pigs.

[0229] Figure 67 Comparison of AAV capsid variants with AAV9 in the lymph nodes; (A) NHPs and (B) pigs.

[0230] Figure 68 Comparison of AAV capsid variants with AAV9 in the testis of NHPs.

[0231] Figure 69 Comparison of AAV capsid variants with AAV9 in the fat of NHPs.

[0232] Figure 70 Comparison of AAV capsid variants with AAV9 in the bone marrow; (A) NHPs and (B) pigs.

[0233] Figure 71 Comparison of AAV capsid variants with AAV9 in the aorta; (A) NHPs and (B) pigs.

[0234] Figure 72Production efficiency of recombinant AAV vectors using novel capsid variants. Data from 3 independent experiments are shown, and error bars indicate standard deviation.

[0235] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0236] Example 1: Identification, screening and evaluation of heart enrichment

[0237] Based on 8 parental wild-type adeno-associated viruses, a recombinant library of new synthetic AAV candidate viruses was created for subsequent screening of variants with cardiac tropism and liver de-targeting. The library was engineered by DNA shuffling, peptide display, and their combination. Both DNA shuffling and peptide display have been described previously (Wang, D. et al. Nat Rev Drug Discov 2019, 18, 358–378; Becker et al., Pathogens 2022, 11, 756; Grimm et al., J Virol 2008, 82, 5887–5911; Herrmann et al., Acs Synth Biol 2019, 8, 194–206).

[0238] For DNA shuffling, local codon optimization (Cabanes-Creus, M. et al. Mol Ther-Methods Clin Dev 2019, 12, 71-84) was used to increase the homology of the parental capsid gene (cap) sequence to improve library yield. The capsid gene (cap) was PCR amplified and partially digested with DNase I to obtain fragments less than 1 kb. Due to the high homology between parental sequences (average >90%), these self-annealed and randomly recombined full-length cap sequence fragments were recovered in a primerless PCR reaction. The cap sequence was cloned into an AAV production plasmid for subsequent AAV library generation.

[0239] For peptide display, two sites for inserting peptide motifs 4 to 6 amino acids in length were introduced into the same homology-optimized parental cap sequence. Sites at positions 453 and 588 are known to allow the expression of short peptide motifs, which can alter tissue tropism (Büning, H. et al., Mol Ther-Methods Clin Dev 2019, 12, 248–265; K. et al., Mol Ther 2020, 28, 1016–1032; Kienle, E. et al., J Vis Exp 2012, 1–11). Both of these sites (and their combinations) were used to integrate random tetramers, pentamers, or hexamers (random peptide library), or hexamers with peptide motifs previously described in the literature as heart-specific (targeted peptide display).

[0240] DNA shuffling and peptide display were also used in combination to create 18 sub-libraries of the selected parental capsid sequences and diversification strategies from 8 parental cap gene sequences. The overall diversity of the injected library exceeded 2·10 2 .

[0241] The pooled library was injected into three mice. Three weeks after injection, the AAVcap sequences were recovered from the left ventricular myocardium by PCR. These amplicons were again cloned into AAV production plasmids for subsequent production of a secondary AAV library. During PCR recovery, unique molecular identifiers (UMIs) were introduced into the cap sequences as previously described (Davidsson et al., Sci Rep-uk, 2016, 6, 37563; Davidsson et al., PNAS 2019, 116, 27053-27062). Prior to AAV library production, each recovered AAV cap was labeled at the plasmid DNA level. This allowed the generation of biodistribution maps based on short-read next-generation sequencing after the next round of in vivo screening. At the same time, long-read high-throughput sequencing was used to create a lookup table from the plasmid DNA used for AAV production. This allowed the matching between the UMIs of interest identified in the biodistribution maps and their cap sequences.

[0242] The secondary library was injected into four mice, and three weeks after injection, the myocardium (heart), atrium, liver, diaphragm, quadriceps femoris, brain, cerebellum, lung, stomach, pancreas, colon, kidney, adrenal gland, spleen, cervical lymph nodes, mesenteric lymph nodes, bone marrow, salivary glands, and testes were isolated for gDNA extraction. The UMI amplicons were recovered by PCR and short-read sequenced using 150b paired-end sequencing to obtain a biodistribution map, which was created using the algorithm proposed by Weinmann et al. (Weinmann, J. et al., Nat Commun 2020, 11, 5432).

[0243] First, the UMI short-read data were quality-filtered, and the UMI sequences were extracted and counted. Second, the sequencing read counts R of all variants α in tissue β were normalized by the sum of all variants α in β to obtain a proportion P αβ (Table 2 - Sequencing read count normalization). Third, P αβ was normalized by the proportion of each variant α in the initial library L α (Table 2 - Input normalization). Fourth, P * αβ was normalized by the total AAV G β determined by dPCR αβIt is a measurement of the proportion of variant α in all tissue β, representing tissue specificity (Table 2 - Tissue specificity (tissue-normalized)). T * αβ It is an additional measurement of tissue specificity, but is independent of the normalization performed with total AAV G measured by dPCR β (Table 2 - Tissue specificity (non-tissue-normalized)). Thus, it represents the specificity of variant α for tissue β based on enrichment in tissue β relative to the input tissue and relative to all other tissues.

[0244] Table 2: Formulas for biodistribution calculations.

[0245]

[0246] For all UMIs, calculate the cardiac T αβ (T αβ cardiac), and select all UMIs for which T αβ cardiac is 0.7 or greater than 0.7 and the P αβ of cardiac abundance is 0.0009 or greater than 0.0009. A T αβ of 0.7 or greater than 0.7 can be interpreted as 70% or greater than 70% of the variant-normalized UMIs measured in the experiment being located in the heart. Thus, this variant is considered to be cardiac tropic. The threshold of 0.0009 for P αβ reflects the signal-to-noise ratio in the experiment and allows selection of the most abundant cardiac variants. Similarly, T αβ is used to identify variants that allow simultaneous targeting of the heart and brain, or the heart and skeletal muscle.

[0247] For all variants, calculate the T * αβ for all other organs (off-target organs), and divide T αβ cardiac by the corresponding off-target T * αβ . This ratio is a measure of the degree to which the corresponding variant is more specific for the heart and more enriched in the heart compared to off-targets. These values can be seen in Figures 7 to 5 9.

[0248] Using long-read sequencing information, match the UMIs to their respective cap sequences.

[0249] Example 2: Generation of Cardiac-Tropic Capsid Variants

[0250] AAV capsid variants (Figure 4; SEQ ID NOs: 79 to 156) were prepared individually and purified by iodixanol density gradient centrifugation. Each AAV carried a unique DNA barcode in its genome. The AAVs were uniformly combined into phosphate-buffered saline (PBS), and 1x10 11 AAV vector genomes were applied to "Poros TM Capture Select TM AAVX affinity resin" (Thermo fisher scientific A36739). After incubation with stirring at room temperature for 20 minutes, the beads were washed three times with PBS. AAV was eluted using 0.1 M citric acid. The eluate was neutralized with Tris buffer.

[0251] The amount of each AAV in the input and eluate fractions was determined by PCR and next-generation sequencing of the unique DNA barcodes. The recovery ratio ( Figure 58 ) was calculated by dividing the number of AAV vector genomes in the eluate fraction by the number of AAV vector genomes in the input fraction.

[0252] Example 3: Quantification of AAV in the liver and brain of mice compared to AAV8 and AAV9

[0253] The copy numbers of AAV capsid variants in the liver and brain of mice were compared to the copy numbers of AAV8, AAV9, and mutant 3 (hybrid AAV8, SEQ ID NO: 34 of WO2022 / 003211).

[0254] A mixture of the AAV capsid variants generated in Example 2 with AAV9 (SEQ ID NO: 191), AAV8 (SEQ ID NO: 189), and mutant 3 was injected into 5 mice by intravenous injection. Two weeks after injection, the liver and brain were isolated for genomic DNA isolation. The vector copy numbers of each AAV capsid variant, AAV9, AAV8, and mutant 3 in each organ were determined by PCR and next-generation sequencing and calculated as AAV genomes per ng of isolated genomic DNA (AAV / ng): the average AAV / ng value for 5 animals was calculated.

[0255] Figure 59(A) shows the reduction of some AAV capsid variants in the mouse liver. Compared to AAV8 and AAV9, all AAV capsid variants were significantly detargeted in the liver. Sequence_42 was below the detection threshold in the livers of 5 mice; sequence_43 was detected in the livers of only 2 mice, sequence_44 was detected in the liver of 1 mouse, sequence_102 was detected in the livers of only 2 mice, and sequence_107 was detected in the liver of 1 mouse.

[0256] Figure 59(B) shows the reduction of AAV capsid variants in the mouse brain. Compared with AAV9, all AAV capsid variants were significantly detargeted. Compared with AAV8, sequences _36, sequences _42 to sequence _43, sequence _59, sequence _102, and sequence _107 were also detargeted in the mouse brain.

[0257] Example 4: Biodistribution in pigs and NHPs compared with AAV9

[0258] In pigs and NHPs, compared with AAV9, the cardiac enrichment of AAV capsid variants (SEQ ID NOs: 78 to 154) exceeded the off-target organ enrichment. AAV9 is a vector with high cardiac and muscle tropism (Haijar & Ishikawa, 2017; supra), and it has been reported to exhibit the highest transgene expression in the mouse heart among AAV serotypes (Zincarelli, et al.; supra). In addition, AAV9-based gene therapy drugs have been approved for the treatment of spinal muscular atrophy. Figure 6 shows the biodistribution of AAV9 that transduces different organs at similar levels and patterns in (A) pigs, (B) NHPs, and (C) mice.

[0259] A mixture of the AAV capsid variants produced in Example 2 and AAV9 (SEQ ID NO: 191) was injected into three pigs and three NHPs by intravenous injection. Two weeks after injection, the organs were isolated for genomic DNA isolation. The vector copy number of each AAV capsid variant in each organ was determined by PCR and next-generation sequencing and calculated as AAV genomes per ng of isolated genomic DNA (AAV / ng):

[0260] For each AAV capsid variant and each organ, the average AAV / ng value of 3 animals of each species (pigs, NHPs) was calculated in the same way as the calculation of Bαβ in Example 1. Figure 60 shows the AAV genomes per ng of isolated genomic DNA (AAV / ng) of AAV9 and capsid variants in (A) pigs and (B) NHPs. Injection of AAV capsid variants resulted in a liver AAV load in pigs that was at most 1 / 4, especially 1 / 5 to 1 / 300, of that of AAV9, and a liver AAV load in NHPs that was at most 1 / 5, especially 1 / 10 to 1 / 340, of that of AAV9.

[0261] The cardiac-organ enrichment factor E CapX heart / organ was calculated using the average AAV / ng value for each capsid (CapX) and organ (e.g., liver, brain, adrenal gland, kidney, pancreas, spleen, lymph node, testis, fat, bone marrow, aorta, etc.).

[0262] Then, for each AAV (CapX) and organ, the enrichment factor was normalized by the enrichment factor of AAV9: E normalized Capx 心脏 / 器官 .

[0263] Compared to AAV9, E normalized 心脏 / 肝脏 > 1 And E normalized 心脏 / 脑 AAV capsid variants with E normalized > 1 are more enriched in the heart compared to the liver (E CapX 心脏 / 肝脏 ), and more enriched in the heart compared to the brain (E CapX 心脏 / 脑 ).

[0264] Compared to AAV9, the AAV capsid variants described herein have better heart / liver, heart / brain, heart / spleen, and heart / lymph node (LN). This indicates that, compared to AAV9, the AAV capsid variants target the heart more effectively than the major off-target organs liver, brain, spleen, and lymphoid tissues.

[0265] Table 3: Formulas for calculating enrichment factors

[0266]

[0267] Table 4 shows the E normalized 心脏 / 肝脏 and E normalized 心脏 / 脑 values of all selected capsid variants in pigs (left) and NHPs (right). Capsid variants with E normalized 心脏 / 肝脏 > 1 And E normalized 心脏 / 脑 > 1 are highlighted in bold.

[0268]

[0269]

[0270]

[0271] Thus, in pigs and / or NHPs, relative to AAV9, the AAV capsid variants (SEQ ID NO:78 to SEQ ID NO:154) are enriched in the heart compared to the liver and in the heart compared to the brain.

[0272] In addition, some AAV capsid variants have shown favorable detargeting in other off-target organs in NHPs and pigs. In addition to the brain and liver, at least sequences 31 to 32, sequence 36, sequences 38 to 39, sequences 41 to 45, sequence 49, sequence 52, sequence 61, sequence 65, sequence 72, sequence 74, sequences 80 to 84, sequence 102, and sequence 104 exhibit additional detargeting relative to AAV9 in at least the adrenal gland, kidney, pancreas, spleen, lymph nodes, testis, fat, bone marrow, and aorta. For treatment safety and to avoid toxic events, significant detargeting in these organs is urgently needed:

[0273] Figure 61 shows that AAV capsid variants are significantly enriched in the heart compared to the liver in (A) NHPs (about 5-fold to about 14-fold) and (B) pigs (about 2 to 9-fold).

[0274] Figure 62 shows a comparison of detargeting of AAV capsid variants with AAV9 in the liver; (A) NHPs and (B) pigs.

[0275] Figure 63 shows a comparison of detargeting of AAV capsid variants with AAV9 in the adrenal gland; (A) NHPs and (B) pigs.

[0276] Figure 64 shows a comparison of detargeting of AAV capsid variants with AAV9 in the kidney; (A) NHPs and (B) pigs.

[0277] Figure 65 shows a comparison of detargeting of AAV capsid variants with AAV9 in the pancreas; (A) NHPs and (B) pigs.

[0278] Figure 66 shows a comparison of detargeting of AAV capsid variants with AAV9 in the spleen; (A) NHPs and (B) pigs.

[0279] Figure 67 shows a comparison of detargeting of AAV capsid variants with AAV9 in the lymph nodes; (A) NHPs and (B) pigs.

[0280] Figure 68 A comparison of detargeting of AAV capsid variants with AAV9 in the testis of NHPs is shown. Pigs were castrated and could not be evaluated. Administration in the treatment of patients of reproductive age requires low transduction in the reproductive organs.

[0281] Figure 69 A comparison of detargeting of AAV capsid variants relative to AAV9 in the fat of NHPs is shown.

[0282] Figure 70 shows a comparison of detargeting of AAV capsid variants with AAV9 in the bone marrow; (A) NHPs and (B) pigs.

[0283] Figure 71 shows a comparison of the detargeting of AAV capsid variants with AAV9 in the aorta; (A) NHP and (B) pig.

[0284] Example 5: Production efficiency of rAVV vectors

[0285] The production efficiency of recombinant AAV vectors using variant capsids was analyzed using a standard triple transfection protocol. AAVpro Hek293T cells (Takara Bio) were transfected with a production plasmid containing the capsid sequence, a GFP reporter transgene flanked by AAV2 ITRs, and a helper plasmid. The AAV vector genomes in the supernatant and cell pellet were quantified by digital PCR. Figure 72 The total yields of the supernatant and cell pellet are shown. Data from 3 independent experiments are shown, and error bars represent standard deviation. All capsid variant sequences were able to produce at least as well as AAV2 (SEQ ID NO: 202), which is used for the approved gene therapy Luxturna and is being tested as a vector in several ongoing trials. These results demonstrate that rAAV vectors with the AAV capsid variants of the present invention are effective as vectors for gene delivery.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid molecule encoding a capsid protein variant and a heterologous nucleic acid encoding the product of one or more genes of interest, wherein the capsid protein variant comprises a sequence selected from SEQ ID NO: 78 to SEQ ID NO:

154.

2. The rAAV vector according to claim 1, wherein the nucleic acid molecule encoding the capsid protein variant is selected from the sequences of SEQ ID NO: 1 to SEQ ID NO:

77.

3. An rAAV vector comprising a nucleic acid molecule encoding a capsid protein variant and a heterologous nucleic acid encoding the product of one or more genes of interest, wherein the capsid protein variant comprises a sequence selected from SEQ ID NO: 90 to SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 96 to SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 128, SEQ ID NO: 133 to SEQ ID NO: 137, and SEQ ID NO: 149 to SEQ ID NO:

150.

4. The rAAV vector according to claim 3, wherein the nucleic acid molecule encoding the capsid protein variant is selected from the sequences of SEQ ID NO: 13 to SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 19 to SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 51, SEQ ID NO: 56 to SEQ ID NO: 60, and SEQ ID NO: 72 to SEQ ID NO:

73.

5. The rAAV vector according to any one of claims 1 to 4, wherein the product of the one or more genes of interest comprises a protein selected from cardiac troponin T; cardiac sarcomeric protein; β-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac α-actin; α-tropomyosin; cardiac troponin I; cardiac myosin binding protein C; Four-and-a-half LIM protein 1; titin; 5'-AMP-activated protein kinase subunit gamma-2; troponin I type 3, myosin light chain 2, cardiac actin alpha 1; cardiac LIM protein; caveolin 3 (CAV3); alpha-galactosidase (GLA); lysosome-associated membrane protein 2 (LAMP2); mitochondrial transfer RNA for glycine (MTTG); mitochondrial transfer RNA for isoleucine (MTTI); mitochondrial transfer RNA for lysine (MTTK); mitochondrial transfer RNA for glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoplasmic / endoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal cell-derived factor-1 (SDF-1); adenylate cyclase-6 (AC6); b-ARKct (b-adrenergic receptor kinase C terminus); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia-inducible growth factor; thymosin beta4 (TMSB4X); nitric oxide synthase-3 (NOS3); apolipoprotein-E (ApoE), superoxide dismutase (SOD), RNA-binding motif 20 (RMB20) and S100A1, titin (TTN), myosin (MHY6, MYH7), myosin-binding protein 3 (MYBPC3), actin (ACTC1 and ACTC2), tropomyosin (TPM1), (lamin A and C) LMNA, sodium channel A5 (SCNA5), desmin (DES), BAG3 or RBM20, troponin T (TNNT2), troponin I (TNNI3), TPM1, MYL2, MYL3, CSRP3, FHL1, MYOZ2, PLN, TCAP, TRIM63 or TTN, S100A1, S100A6, S100A4, S100B, SERCA2a, AC6, inhibitor-1, VEGF-A isoform, SCF, PKP2, DSP, DSG2, DSC2, JUP or TMEM43, ERBB2-4, NRG1, CDK, YAP, FGF isoform, HGF, miR-195, miR15a, miR-15b, miR-16 or miR-497, miR-323-3p, miR-187, miR-124, miR-31a-5p, miR-378, lncRNA Sarrah or UCA1 or FTX, circular RNA SNRK or CircFndc3b, HIF-1a, Bcl-2 and Bcl-xl, GATA4, MEF2C, TBX5, HAND2, MESP1, NKX2.

5. Therapeutic polypeptides of MYOCD, ETV2, GMT, TRPV4, relaxin receptor, MRTF-A, TRPC isoforms, LRP6, BRG1, Nrf2 / HO-1, HO1, GSTP1, NQO1, ZBTB20, SIRT3, SOD1 / 2, LEF1 and / or IL-10.

6. The rAAV vector according to any one of claims 1 to 4, wherein the product of one or more genes of interest comprises a genome editing enzyme selected from meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and Cas enzymes.

7. The rAAV vector according to any one of claims 1 to 4, wherein one or more genes of interest comprise a gene silencing tool selected from siRNA, microRNA, circular RNA, and lncRNA.

8. An rAAV capsid protein comprising a sequence selected from SEQ ID NO: 78 to SEQ ID NO:

154.

9. An rAAV capsid protein comprising a sequence selected from SEQ ID NO:90 to SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96 to SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:128, SEQ ID NO:133 to SEQ ID NO:137, and SEQ ID NO:149 to SEQ ID NO:

150.

10. The rAAV capsid protein according to claim 8 or 9, wherein the capsid protein: (i) shows an increased selectivity for cardiac cells compared to liver cells and an increased selectivity for cardiac cells compared to CNS cells; (ii) shows an increased transduction efficiency for cardiac cells compared to liver cells and an increased transduction efficiency for cardiac cells compared to CNS cells.

11. The rAAV capsid protein according to claim 10, wherein (i) and (ii) are evaluated in primates.

12. A polynucleotide molecule encoding the rAAV capsid protein according to claim 8.

13. The polynucleotide molecule according to claim 12, comprising a sequence selected from SEQ ID NO:1 to SEQ ID NO:

77.

14. A pharmaceutical composition comprising the rAAV vector according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

15. A kit comprising the pharmaceutical composition according to claim 14 and instructions for use.

16. A method for transducing cardiac cells, comprising contacting the cardiac cells with the rAAV vector according to any one of claims 1 to 7.

17. A cardiac cell transduced with the rAAV vector according to any one of claims 1 to 7.

18. The cardiac cell according to claim 17, wherein the cell is a cardiomyocyte.

19. The rAAV vector according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 14 for treating a disease or defect of cardiomyocytes, or for treating a disease or defect of muscle myocytes or skeletal muscle cells.

20. The rAAV vector or pharmaceutical composition for the use according to claim 19, wherein the rAAV vector or pharmaceutical composition is administered by: intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade coronary artery injection, retrograde injection, transendocardial myocardial injection, or molecular cardiac surgery combined with recirculation delivery technology (MCARD).

21. The rAAV vector or pharmaceutical composition for use according to claim 19 or 20, wherein the disease or defect of cardiomyocytes is ischemic cardiomyopathy / heart failure after myocardial infarction, hypertensive heart disease, cor pulmonale, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, atrial cardiomyopathy, left ventricular noncompaction cardiomyopathy, heart failure with reduced ejection fraction / diastolic heart failure or Takotsubo syndrome.

22. The rAAV vector or pharmaceutical composition for use according to claim 19 or 20, wherein the disease or defect is a syndromic disease with cardiovascular or cardiopulmonary involvement, which is selected from Friedreich's ataxia, Danon's disease or Duchenne muscular dystrophy, Down syndrome, Turner syndrome, 22q11.1 deletion syndrome, Williams syndrome, Noonan syndrome, Kabuki syndrome, Alaje syndrome, myotubular myopathy (MTM1 gene), Pompe disease, or glycogen storage disease type III (GSD3) (AGL gene), Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), DMD-related dilated cardiomyopathy.

23. The rAAV vector or pharmaceutical composition for use according to claim 19 or 20, wherein the defect or disease is a muscle disease or muscle injury, in particular a neuromuscular genetic disorder selected from Beck MD / Duchenne MD, myotonic MD, distal MD, limb-girdle MD, congenital MD, Emory-Dreyfus MD, facioscapulohumeral MD or oculopharyngeal MD, malignant hyperthermia, metabolic myopathy, hereditary cardiomyopathy or congenital myasthenic syndrome.

24. A method of treating a disease or defect of cardiomyocytes in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the rAAV vector of any one of claims 1 to 7, wherein the rAAV vector transduces cardiac tissue.

25. The method of claim 24, wherein the rAAV vector is administered by intravenous injection.

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