Recombinant adeno-associated virus vector for direct myocardial reprogramming

Abstract

Provided is a recombinant adeno-associated virus vector which includes: a polynucleotide comprising at least one sequence selected from the group consisting of a periostin promoter sequence, a collagen 1a2 promoter sequence, and a cartilage oligomer matrix protein (COMP) promoter sequence; and an AAV-DJ capsid protein. The collagen 1a2 promoter sequence is any one of (2-1) to (2-4) described in the description. The cartilage oligomer matrix protein (COMP) promoter sequence is any one of (3-1) to (3-4) described in the description.

Description

Recombinant adeno-associated virus vectors for direct myocardial reprogramming

[0001] The present invention relates to a recombinant adeno-associated virus vector for direct myocardial reprogramming.

[0002] Cardiovascular disease is the leading cause of death worldwide. It is known that cardiac fibroblasts are activated and proliferate after myocardial infarction, a type of cardiovascular disease, leading to myocardial fibrosis and cardiac dysfunction. However, adult cardiomyocytes are terminally differentiated cells with little regenerative capacity, limiting treatment options for heart disease.

[0003] Previously, methods for inducing cardiomyocytes from cardiac fibroblasts using myocardial reprogramming factors (e.g., GATA4, MEF2C, and TBX5) have been discovered (Patent Document 1, Non-Patent Documents 1-3). It has been disclosed that in vivo cardiac reprogramming using retroviral vectors, Sendai virus vectors, etc., can reprogram cardiac fibroblasts into induced cardiomyocytes and repair the hearts of infarcted mice (Non-Patent Document 2). It has also been disclosed that cardiac reprogramming improves myocardial contractility and suppresses fibrosis through its regenerative and anti-fibrotic effects in a mouse model of chronic myocardial infarction (Non-Patent Document 3).

[0004] International Publication No. 2011 / 139688

[0005] Cell 2010, 142, 375-386Cell Stem Cell 2018, 22, 91-103Circulation 2023, 147, 223-238.

[0006] The methods disclosed in the above literature use retroviral vectors, Sendai virus vectors, etc., but each has the potential to cause insertional mutagenesis, immune reactions, etc. Therefore, there is a need for the development of a safe and efficient gene transfer system. An objective of the present invention is to provide a novel vector that can perform direct myocardial reprogramming in vivo.

[0007] To solve the above problems, the inventors conducted extensive research and found that cardiac fibroblasts can be induced to develop into cardiomyocytes in vivo by using a recombinant adeno-associated virus vector containing a nucleic acid molecule containing a specific promoter sequence and an adeno-associated virus DJ capsid protein. The present invention was completed based on these findings and includes the following broad aspects of the invention.

[0008] [Item 1] A recombinant adeno-associated virus vector comprising an AAV-DJ capsid protein and a polynucleotide comprising at least one selected from the group consisting of a periostin promoter sequence, a collagen 1a2 promoter sequence, and a cartilage oligomeric matrix protein (COMP) promoter sequence, wherein the collagen 1a2 promoter sequence is any of the following (2-1) to (2-4), and the cartilage oligomeric matrix protein (COMP) promoter sequence is any of the following (3-1) to (3-4): (2-1) a nucleotide sequence represented by SEQ ID NO: 9; (2-2) a nucleotide sequence in which one or several nucleotides have been substituted, added, or deleted in the nucleotide sequence represented by SEQ ID NO: 9; (2-3) a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 9; (2-4) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 9; (3-1) a nucleotide sequence represented by SEQ ID NO: 10; (3-2) A nucleotide sequence in which one or several nucleotides have been substituted, added, or deleted in the nucleotide sequence represented by SEQ ID NO: 10, (3-3) A nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 10, (3-4) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 10. [Item 2] The recombinant adeno-associated viral vector according to Item 1, wherein the recombinant adeno-associated viral vector comprises a polynucleotide encoding at least one reprogramming factor selected from the group consisting of Gata4, Hand2, Tbx5, and Mef2c. [Item 3] A pharmaceutical composition comprising the recombinant adeno-associated viral vector according to Item 1 or 2. [Item 4] An agent for improving cardiac function, comprising the recombinant adeno-associated viral vector according to Item 2. [Item 5] A method for expressing a gene in activated cardiac fibroblasts, comprising administering the recombinant adeno-associated viral vector according to Item 1 or 2. [Item 6] The gene expression method according to Item 5, wherein the activated cardiac fibroblasts include cardiac fibroblasts after myocardial infarction. [Item 7] A method for inducing cardiomyocytes from cardiac fibroblasts, comprising administering the recombinant adeno-associated virus vector according to Item 1 or 2.

[0009] According to the present invention, a novel vector capable of direct myocardial reprogramming in vivo can be provided.

[0010] Schematic diagram of in vitro and in vivo screening of multiple AAV capsids (AAV1-10 and AAV-DJ). The AAV vector contained a CMV promoter-driven GFP (AAV-CMV-GFP). In vitro screening of AAV capsids. After 1 week, a multiplicity of infection of 2.5 × 10 5 Quantitative data from FACS analysis of GFP expression in cardiac fibroblasts transduced with the AAV-CMV-GFP vector in VG / cell (n = 3). Mean ± SD. *p < 0.05, **p < 0.01. In vitro screening of AAV capsids. After 1 week, a multiplicity of infection of 2.5 × 10 5 Quantitative data from FACS analysis of GFP expression in cardiomyocytes transduced with the AAV-CMV-GFP vector in VG / cell (n = 3). Mean ± SD. *p < 0.05, **p < 0.01. In vitro screening of AAV capsid cellular tropism. After 1 week, a multiplicity of infection of 2.5x10 5 Representative immunofluorescence GFP expression in cardiomyocytes (top) and cardiac fibroblasts (bottom) transduced with the AAV-CMV-GFP vector at VG / cell. Scale bars represent 50 μm. In vivo screening of AAV capsids. AAV-CMV-GFP vector (1 × 10 11GFP-positive cells (VG / mouse) were directly injected into the hearts of mice 1 week after myocardial infarction. Most GFP+ cells were α-actinin+ cardiomyocytes. Quantitative data for GFP expression in non-myocytes (left) and cardiomyocytes (right) are shown (n = 5). Mean ± SD. *p < 0.05, **p < 0.01. AAV-DJ-Postn vectors were constructed by packaging AAV6, 9, and DJ capsids into an AAV expression cassette containing the Postn promoter-driven Cre recombinase (AAV-Postn-Cre). A schematic diagram of the analysis of R26-tomato mice in which the AAV-Postn-Cre vector was directly injected into the heart after myocardial infarction is shown. Immunohistochemical staining for Tomato, cTnT, and DAPI was performed in the hearts of Tomato mice 2 weeks after myocardial infarction injected with AAV6-Postn-Cre, AAV9-Postn-Cre, or AAV-DJ-Postn-Cre. Higher magnification images (bottom) show the border area. The scale bar represents 50 μm. Quantitative data for Tomato+ cells following injection of AAV6-Postn-Cre, AAV9-Postn-Cre, and AAV-DJ-Postn-Cre are shown (n = 5). Mean ± SD. **p < 0.01. Immunohistochemical staining for Tomato, cTnT, COL1, SMMHC, CD31, and DAPI was performed in the hearts of Tomato mice injected with AAV-DJ-Postn-Cre 2 weeks after myocardial infarction. Most Tomato+ cells were immunopositive for COL1. The ratios of cTnT+ cardiomyocytes (CMs), COL1+ cardiac fibroblasts (CFs), SMMHC+ smooth muscle cells (SMCs), and CD31+ endothelial cells (ECs) to Tomato+ cells are shown (n = 5). Mean ± SD. **p < 0.01. Experimental scheme for injection of AAV-DJ-Postn-GFP into the hearts of ICR mice after myocardial infarction is shown. Immunohistochemical staining for GFP, cTnT, and DAPI was performed on the hearts of AAV-DJ-Postn-GFP-injected mice 1, 2, and 4 weeks after myocardial infarction. Quantitative data for GFP+ cells in the border / infarct area are shown (n = 5). Mean ± SD. ** p < 0.01. Schematic diagram showing direct injection of the AAV-DJ-Postn-GFP vector into the heart of an ICR mouse after myocardial infarction.Immunohistochemical staining for GFP and DAPI was performed in the liver, lung, and skeletal muscle one week after direct injection of the AAV-DJ-Postn-GFP vector into the heart following myocardial infarction. Quantitative analysis of GFP+ / DAPI+ cells is shown (n = 5). Quantitation was performed at the cardiac border / infarct area. Mean ± SD. **p < 0.01. FACS analysis of GFP+ cells was performed in the heart (all ventricles), liver, lung, and skeletal muscle of ICR mice after direct injection of the AAV-DJ-Postn-GFP vector into the heart following myocardial infarction. Quantitative analysis of GFP+ cells is shown (n = 5). Quantitation was performed in the entire ventricle. Mean ± SD. **p < 0.01. Schematic diagram showing systemic administration of the AAV-DJ-Postn-Cre vector into healthy Tomato mice via tail vein injection. Immunohistochemical staining was performed in the heart, liver, lung, and skeletal muscle one week after systemic administration of the AAV-DJ-Postn-Cre vector into healthy Tomato mice. Quantitative analysis of Tomato+ / DAPI+ cells is shown (n = 5). Mean ± SD. Schematic diagram showing systemic administration of the AAV-DJ-Postn-Cre vector into Tomato mice with myocardial infarction via tail vein injection. AAV-DJ-Postn-Cre was injected immediately after coronary artery ligation. Immunohistochemical staining of Tomato was performed in the heart, liver, lung, and skeletal muscle after myocardial infarction 1 week after systemic AAV-DJ-Postn-Cre administration. Quantitative analysis of Tomato+ / DAPI+ cells is shown (n = 5 independent biological experiments). Mean ± SD. **p < 0.01. The structures of MEF2C and MEF2C-TAD are shown. AAV-DJ-Postn vectors encoding GATA4, HAND2, TBX5, and MEF2C, and AAV-DJ-Postn vectors encoding GATA4, HAND2, TBX5, and MEF2C-TAD (M-TAD) were constructed. A schematic diagram of the generation of Postn MCM / Tomato mice is shown below. After myocardial infarction, Postn MCM / Tomato mice were directly injected with PBS (Ctrl), AAV-DJ-Postn-GHT / M, or AAV-DJ-Postn-GHT / M-TAD into the heart. Subsequently, they were administered tamoxifen-containing food pellets (TAM food) for two consecutive weeks.Immunohistochemical staining for Tomato, α-actinin, and DAPI in the hearts of Postn MCM / Tomato mice 4 weeks after myocardial infarction is shown. The high-magnification inset shows sarcomere organization. The scale bar represents 50 μm. Quantitative analysis of α-actinin+ / Tomato+ cells in the border zone is shown (n = 5). A total of 36,000-60,000 cells were counted for each mouse, and five mice were analyzed per group. Mean ± SD. * p < 0.05; ** p < 0.01. A schematic diagram of the generation of Tcf21 iCre / Tomato mice is shown. Tcf21 iCre / Tomato mice were intraperitoneally injected with tamoxifen for 5 days, followed by myocardial infarction and AAV gene transfer 1 week later. Four weeks after myocardial infarction, hearts were removed and immunohistochemically stained. Immunohistochemical staining for Tomato, α-actinin, and DAPI was performed on sections from Tcf21 iCre / Tomato mouse hearts 4 weeks after myocardial infarction. Scale bar represents 50 μm. Quantitative analysis of α-actinin+ / Tomato+ cells in the border zone is shown (n = 5). A total of 36,000-60,000 cells were counted for each mouse, with five mice analyzed per group. Mean ± SD. *p < 0.05; **p < 0.01. Gene set enrichment analysis of genes with increased and decreased expression in the hearts of GHT / M-TAD-injected mice compared to Ctrl mice is shown. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured by echocardiography in Ctrl, AAV-DJ-Postn-GFP, and AAV-DJ-Postn-GHT / M-TAD-injected mice 4 weeks after myocardial infarction (n = 15). Mean ± SD. *p < 0.05. Fibrosis was compared between Ctrl, AAV-DJ-Postn-GFP, and AAV-DJ-Postn-GHT / M-TAD hearts 4 weeks after myocardial infarction. Fibrosis was assessed at the base, mid, and apex levels using Masson's trichrome staining. Quantitative analysis of fibrotic area at each level is shown (n = 5). Mean ± SD. *p < 0.05; **p < 0.01.1 shows a schematic diagram of the AAV-DJ-Col1a2-GFP vector and the AAV-DJ-COMP-GFP vector. A: Quantitative data of GFP expression in mouse embryonic fibroblasts. B: Quantitative data of GFP expression in cardiac fibroblasts. A: Results of quantitative analysis of GFP+ / cTNT+ cells in cardiomyocytes. B: Results of quantitative analysis of GFP+ / cTNT+ cells in non-cardiomyocytes. An outline of the experiment in Example 9 is shown. Results of immunohistochemical staining of cardiac fibroblasts in vivo in ICR mice with myocardial infarction are shown.

[0011] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that also encompasses "consist essentially of" and "consist of."

[0012] As used herein, the term "polynucleotide" can be alternatively referred to as a "nucleic acid" or a "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, the term "base sequence" can be alternatively referred to as a "nucleic acid sequence" or a "nucleotide sequence."

[0013] The "viral genome" and "polynucleotide" of the present invention may each exist in the form of DNA (e.g., cDNA or genomic DNA), but may also be in the form of RNA (e.g., mRNA). The viral genome and polynucleotide used herein may each be double-stranded or single-stranded DNA. In the case of single-stranded DNA or RNA, it may be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand). Unless otherwise specified, when describing the genetic arrangement of a promoter, gene of interest, polyadenylation signal, etc. encoded by the rAAV genome, the strand itself will be described if the rAAV genome is the sense strand, and the complementary strand will be described if the rAAV genome is the antisense strand.

[0014] As used herein, the term "protein" can also be referred to as "polypeptide."

[0015] As used herein, "packaging" refers to events including preparation of a single-stranded viral genome, assembly of a capsid, and encapsidation of the viral genome, etc. When an appropriate plasmid vector (usually multiple plasmids) is introduced into a packaging-capable cell line under appropriate conditions, recombinant viral particles (i.e., viral virions, viral vectors) are assembled and secreted into the culture.

[0016] As used herein, "operably linked" refers to a functional linkage between nucleic acids that confers a desired function, such as transcription, translation, etc. For example, it includes a functional linkage between a nucleic acid expression control sequence, such as a promoter or signal sequence, and a polynucleotide sequence encoding a protein of interest. The expression control sequence affects the transcription and / or translation of the polynucleotide sequence encoding the protein of interest.

[0017] As used herein, "activated cardiac fibroblasts" refers to cardiac fibroblasts that are not in a quiescent state. When cardiac fibroblasts are exposed to stress, such as myocardial infarction, cardiac injury, or chronic hypertension, their migration activity increases, they begin to proliferate, and they are transformed into myofibroblasts. Myofibroblasts are characterized by enhanced motility, expression of smooth muscle actin, and increased synthesis of collagen and other ECM components. As used herein, such non-quiescent cardiac fibroblasts are collectively referred to as "activated cardiac fibroblasts."

[0018] 1. Recombinant Adeno-Associated Virus Vector The present invention provides a recombinant adeno-associated virus vector (rAAV vector). The rAAV vector of the present invention comprises a polynucleotide containing at least one promoter sequence selected from the group consisting of a periostin promoter sequence, a collagen 1a2 promoter sequence, and a cartilage oligomeric matrix protein (COMP) promoter sequence, and an AAV-DJ capsid protein. By using the rAAV vector of the present invention for gene expression, it is possible to selectively express a protein of interest in activated cardiac fibroblasts (e.g., cardiac fibroblasts after myocardial infarction).

[0019] (Adeno-associated virus) Natural adeno-associated virus (AAV) is nonpathogenic and has low immunogenicity. In wild-type AAV, the viral genome, a single-stranded DNA packaged in a capsid, contains the rep gene and the cap gene, flanked by inverted terminal repeat (ITR) sequences at both ends. The Rep proteins (rep78, rep68, rep52, and rep40) produced by the rep gene are required for viral genome replication, transcriptional regulation, and chromosomal integration. The cap gene encodes three capsid proteins (VP1, VP2, and VP3).

[0020] (Capsid Protein) The rAAV vectors of the present invention comprise an AAV-DJ capsid protein. AAV-DJ forms hybrid capsids derived from eight different natural serotypes through DNA family shuffling (Grimm D., et al., J. Virol., 82(12), 5887-5911 (2008)). In one embodiment, the AAV-DJ capsid protein comprised in the rAAV vectors of the present invention includes a protein that has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 and is capable of forming a capsid. Furthermore, examples of proteins that can form capsids include those that contain an amino acid sequence of SEQ ID NO: 1 in which, for example, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue has been deleted, substituted, inserted, and / or added. A combination of two or more of these deletions, substitutions, insertions, and additions may also be present. In one embodiment, the capsid protein used in the present invention has the function of forming a capsid. Furthermore, a polynucleotide containing a gene of interest (e.g., a sequence encoding a reprogramming factor) is packaged within the capsid.

[0021] The polynucleotide encoding the AAV-DJ capsid protein used in the present invention encodes a protein functionally equivalent to the capsid protein capable of forming the rAAV vector of the present invention. Examples of such polynucleotides include: (i) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2; (ii) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 in which one or more nucleotides have been substituted, added, or deleted; (iii) a polynucleotide comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO: 2; and (iv) a polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2.

[0022] In (i) above, a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 and capable of forming a capsid in the rAAV vector of the present invention is included.

[0023] In (ii) above, the number of one or several bases to be substituted, deleted, added, or inserted is not particularly limited, as long as it is an integer of 1 or greater. For example, it can be from 1 to several tens of bases, preferably from 1 to 20, more preferably from 1 to 15, even more preferably from 1 to 10, and particularly preferably from 1 to 5. Polynucleotides containing a base sequence in which one or several bases have been substituted, added, or deleted in the base sequence represented by SEQ ID NO: 2 are encompassed, and are capable of forming capsids in the rAAV vectors of the present invention.

[0024] In one aspect, the number of one or more bases to be substituted, added, or deleted can be about 1 to 220 so that the base sequence has 90% or more identity with the base sequence of SEQ ID NO: 2, preferably about 1 to 110 so that the identity is 95% or more, more preferably about 1 to 66 so that the identity is 97% or more, even more preferably about 1 to 44 so that the identity is 98% or more, and particularly preferably about 1 to 22 so that the identity is 99% or more.

[0025] In the above (iii), the identity of the nucleotide sequence can be 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The homology or identity of the nucleotide sequence can be less than 100%. The homology and identity between nucleotide sequences can be determined using known algorithms such as BLAST. Polynucleotides containing a nucleotide sequence that is 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 2 and that can form a capsid in the rAAV vector of the present invention are also included.

[0026] In (iv) above, "stringent conditions" refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Examples of stringent conditions include hybridization at 42°C in 1x SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6x SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA 2Na, pH 7.4), followed by washing with 0.5x SSC at 42°C. These conditions are not limited to these. Examples of such conditions are described, for example, in M.R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012). Polynucleotides containing a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2 and are capable of forming capsids in the rAAV vectors of the present invention.

[0027] (Polynucleotide (rAAV Genome)) In the polynucleotide (rAAV genome) packaged in the rAAV vector of the present invention, a polynucleotide sequence encoding a protein of interest (e.g., a reprogramming factor) is operably combined with a promoter sequence to express the protein. In the present invention, at least one promoter sequence selected from the group consisting of a periostin promoter sequence, a collagen 1a2 promoter sequence, and a cartilage oligomeric matrix protein (COMP) promoter sequence is used. Periostin is an extracellular matrix protein expressed only in activated cardiac fibroblasts. Cardiac fibroblasts are known to be activated in myocardial tissue subjected to stress such as myocardial infarction or pressure overload. Use of the periostin promoter sequence allows the protein of interest to be expressed selectively in activated cardiac fibroblasts (e.g., cardiac fibroblasts after myocardial infarction). Use of the periostin promoter sequence allows the protein of interest to be expressed in activated cardiac fibroblasts in the acute phase of myocardial infarction (within 10 days after the onset of myocardial infarction). Collagen 1a2 is a protein expressed in all cardiac fibroblasts, regardless of whether myocardial infarction is present or not. Using the collagen 1a2 promoter sequence, it is possible to selectively express a target protein in cardiac fibroblasts. Therefore, it is possible to express a target protein in cardiac fibroblasts in both the acute and chronic phases of myocardial infarction. Cartilage oligomeric matrix protein (COMP) is a protein whose expression level increases in the chronic phase of myocardial infarction (after 10 days after the onset of myocardial infarction). Using the cartilage oligomeric matrix protein (COMP) promoter sequence, it is possible to selectively express a target protein in cardiac fibroblasts in the chronic phase of myocardial infarction.

[0028] The periostin promoter sequence of the present invention is preferably any one of the following (1-1) to (1-4): (1-1) A nucleotide sequence represented by SEQ ID NO: 3. (1-2) A nucleotide sequence in which one or several nucleotides have been substituted, added, or deleted in the nucleotide sequence represented by SEQ ID NO: 3. (1-3) A nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 3. (1-4) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3.

[0029] In the above (1-1), the base sequence represented by SEQ ID NO: 3, which has the function of a periostin promoter, is included. SEQ ID NO: 3 is a sequence in which a mouse periostin promoter sequence is fused to a mouse periostin enhancer sequence (Table 1).

[0030] In (1-2) above, the number of one or several bases to be substituted, deleted, added, or inserted is not particularly limited, as long as it is an integer of 1 or more. For example, it can be about 1 to several tens of bases, preferably about 1 to 20, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. Base sequences in which one or several bases have been substituted, added, or deleted in the base sequence of SEQ ID NO: 3 and which have the function of a periostin promoter are included.

[0031] In one aspect, the number of one or more bases to be substituted, added, or deleted can be about 1 to 140 so that the base sequence has 90% or more identity with the base sequence of SEQ ID NO: 3, preferably about 1 to 69 so that the identity is 95% or more, more preferably about 1 to 42 so that the identity is 97% or more, even more preferably about 1 to 28 so that the identity is 98% or more, and particularly preferably about 1 to 14 so that the identity is 99% or more.

[0032] In the above (1-3), the identity of the nucleotide sequences can be 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The homology or identity of the nucleotide sequences can be less than 100%. The homology and identity between nucleotide sequences can be determined using known algorithms such as BLAST. A nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 3 and having the function of a periostin promoter is included.

[0033] In the above (1-4), "stringent conditions" refer to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Examples of stringent conditions include, but are not limited to, hybridization in 1xSSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6xSSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA 2Na, pH 7.4) at 42°C, followed by washing with 0.5xSSC at 42°C. Such conditions are described, for example, in M.R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012). A nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and has the function of a periostin promoter is also included.

[0034] The collagen 1a2 promoter sequence of the present invention is any one of the following (2-1) to (2-4): (2-1) A base sequence represented by SEQ ID NO: 9. (2-2) A base sequence in which one or more bases have been substituted, added, or deleted in the base sequence represented by SEQ ID NO: 9. (2-3) A base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 9. (2-4) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO: 9.

[0035]

[0036] In the above (2-2), the number of one or several bases to be substituted, deleted, added, or inserted can be preferably about 1 to 20, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. This includes a base sequence in which one or several bases have been substituted, added, or deleted in the base sequence represented by SEQ ID NO: 9, and which has the function of a collagen 1a2 promoter.

[0037] In one aspect, the number of one or more bases to be substituted, added, or deleted can be about 1 to 195 so that the base sequence has 90% or more identity with the base sequence of SEQ ID NO: 9, preferably about 1 to 97 so that the identity is 95% or more, more preferably about 1 to 58 so that the identity is 97% or more, even more preferably about 1 to 39 so that the identity is 98% or more, and particularly preferably about 1 to 19 so that the identity is 99% or more.

[0038] In the above (2-3), the identity of the base sequence can be 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The homology or identity of the base sequence can be less than 100%. The homology and identity between base sequences can be determined using known algorithms such as BLAST. Base sequences that have 90% or more identity with the base sequence represented by SEQ ID NO: 9 and have collagen 1a2 promoter function are included.

[0039] In the above (2-4), "stringent conditions" refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Examples of stringent conditions include hybridization at 42°C in 1xSSC (0.9M NaCl, 0.09M trisodium citrate) or 6xSSPE (3M NaCl, 0.2M NaH2PO4, 20mM EDTA 2Na, pH 7.4), followed by washing with 0.5xSSC at 42°C. These conditions are not limited to these. Examples of such conditions are described, for example, in M.R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012). Examples of stringent conditions include a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:9 and has collagen 1a2 promoter function.

[0040] The cartilage oligomeric matrix protein (COMP) promoter sequence of the present invention is any one of the following (3-1) to (3-4): (3-1) A nucleotide sequence represented by SEQ ID NO: 10. (3-2) A nucleotide sequence in which one or more nucleotides have been substituted, added, or deleted in the nucleotide sequence represented by SEQ ID NO: 10. (3-3) A nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 10. (3-4) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 10.

[0041]

[0042] In the above (3-2), the number of one or several bases to be substituted, deleted, added, or inserted is not particularly limited, as long as it is an integer of 1 or more. For example, it can be about 1 to several tens of bases, preferably about 1 to 20, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. This includes base sequences in which one or several bases have been substituted, added, or deleted in the base sequence represented by SEQ ID NO: 10, and which have the function of a cartilage oligomeric matrix protein (COMP) promoter.

[0043] In one aspect, the number of one or more bases to be substituted, added, or deleted can be about 1 to 170 so that the base sequence has 90% or more identity with the base sequence of SEQ ID NO: 10, preferably about 1 to 85 so that the identity is 95% or more, more preferably about 1 to 51 so that the identity is 97% or more, even more preferably about 1 to 34 so that the identity is 98% or more, and particularly preferably about 1 to 17 so that the identity is 99% or more.

[0044] In the above (3-3), the identity of the nucleotide sequence can be 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The homology or identity of the nucleotide sequence can be less than 100%. The homology and identity between nucleotide sequences can be determined using known algorithms such as BLAST. The nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 10 and having the function of a cartilage oligomeric matrix protein (COMP) promoter is also included.

[0045] In the above (3-4), "stringent conditions" refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Examples of stringent conditions include hybridization in 1x SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6x SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA 2Na, pH 7.4) at 42°C, followed by washing with 0.5x SSC at 42°C. These conditions are not limited to these. Examples of such conditions are described, for example, in M.R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012). Examples of stringent conditions include a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 10 and has the function of a cartilage oligomeric matrix protein (COMP) promoter.

[0046] The polynucleotide packaged in the rAAV vector of the present invention incorporates a polynucleotide encoding a protein of interest. The protein may be one or more types, but the total length of the polynucleotide packaged in the rAAV vector, including the polynucleotide encoding the protein of interest, is preferably comparable to that of the wild-type genome. That is, the total length is preferably comparable to the wild-type total length of 5 kb, for example, about 2 to 6 kb, preferably about 4 to 6 kb. Furthermore, the polynucleotide encoding the protein of interest is preferably, but not limited to, about 0.01 to 3.7 kb in length, more preferably about 0.01 to 2.5 kb, and even more preferably about 0.01 to 2 kb in length.

[0047] In one embodiment, the target protein is a reprogramming factor. Examples of reprogramming factors include Gata4, Hand2, Tbx5, Mef2c, Mef2c-TAD, MYOCD (myocardin), ASCL1 (Achaete-scute family bHLH transcription factor 1), miR-133 (microRNA), and the like. These may be used alone or in combination of two or more. The reprogramming factor encoded in the polynucleotide packaged in the rAAV vector of the present invention is preferably at least one selected from the group consisting of Gata4, Hand2, Tbx5, Mef2c, Mef2c-TAD, MYOCD, ASCL1, and miR-133, more preferably two or more, even more preferably three or more, and particularly preferably four or more. In one embodiment, the polynucleotide packaged within the rAAV vector of the present invention encodes Gata4, Hand2, and Tbx5, and preferably further encodes Mef2c or Mef2c-TAD, and more preferably encodes Gata4, Hand2, Tbx5, and Mef2c-TAD.

[0048] Gata4: Gata4 polypeptide is a member of the GATA family of zinc finger transcription factors that recognizes and binds to the GATA motif present in the promoter regions of many genes. Amino acid sequences for Gata4 polypeptides from various species and nucleotide sequences encoding Gata4 polypeptides are known in the art. Accession numbers for amino acid sequences for Gata4 polypeptides include, for example, NP_002043 (Homo sapiens), NP_032118 (Mus musculus), and NP_653331 (Rattus norvegicus). Accession numbers for nucleotide sequences encoding Gata4 polypeptides include, for example, NM_002052 (Homo sapiens), NM_008092 (Mus musculus), and NM_144730 (Rattus norvegicus).

[0049] In some embodiments, the amino acid sequence of the Gata4 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in one of the above GenBank entries.

[0050] In some embodiments, a nucleotide sequence encoding a Gata4 polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in one of the above GenBank entries.

[0051] In some embodiments, a polypeptide functionally equivalent to a Gata4 polypeptide (or a nucleotide sequence encoding such a functional equivalent) can be used. For example, in some embodiments, a Gata5 polypeptide (or a nucleotide sequence encoding a Gata5 polypeptide) is used. In other embodiments, a Gata6 polypeptide (or a nucleotide sequence encoding a Gata6 polypeptide) is used.

[0052] The amino acid sequence of Gata5 polypeptide and the nucleotide sequence encoding Gata5 polypeptide are known in the art. Accession numbers for the amino acid sequence of Gata5 polypeptide include, for example, NP_536721 (Homo sapiens), NP_032119 (Mus musculus), and NP_001019487 (Rattus norvegicus). Accession numbers for the nucleotide sequence encoding Gata5 polypeptide include, for example, NM_080473 (Homo sapiens), NM_008093 (Mus musculus), and NM_001024316 (Rattus norvegicus).

[0053] The amino acid sequence of Gata6 polypeptide and the nucleotide sequence encoding Gata6 polypeptide are known in the art. Accession numbers for the amino acid sequence of Gata6 polypeptide include, for example, NP_005248 (Homo sapiens), NP_034388 (Mus musculus), and NP_062058 (Rattus norvegicus). Accession numbers for the nucleotide sequence encoding Gata6 polypeptide include, for example, NM_005257 (Homo sapiens), NM_010258 (Mus musculus), and NM_019185 (Rattus norvegicus).

[0054] In some embodiments, a suitable functional equivalent of a Gata4 polypeptide is a polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of a Gata5 polypeptide or a Gata6 polypeptide.

[0055] In some embodiments, a nucleotide sequence encoding a functional equivalent of a Gata4 polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity to a nucleotide sequence encoding a Gata5 polypeptide or a Gata6 polypeptide.

[0056] Hand2 The Hand2 polypeptide belongs to the helix-loop-helix transcription factor family and is known to play an important role in cardiac development.

[0057] Amino acid sequences for Hand2 polypeptides from various species and nucleotide sequences encoding Hand2 polypeptides are known in the art. Accession numbers for amino acid sequences for Hand2 polypeptides include, for example, NP_068808.1 (Homo sapiens), NP_034532.3 (Mus musculus), and NP_073187.1 (Rattus orvegicus). Accession numbers for nucleotide sequences encoding Hand2 polypeptides include, for example, NM_021973.3 (Homo sapiens), NM_010402.4 (Mus musculus), and NM_022696.2 (Rattus orvegicus).

[0058] In some embodiments, the amino acid sequence of the Hand2 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in one of the above GenBank entries.

[0059] In some embodiments, the nucleotide sequence encoding the Hand2 polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in one of the above GenBank entries.

[0060] Tbx5 (T-box transcription factor 5) is a transcription factor that recognizes and binds to T-boxes in the promoter regions of some genes, activating the transcription of genes operably linked to the promoter. Amino acid sequences of Tbx5 polypeptides from various species and nucleotide sequences encoding Tbx5 polypeptides are known in the art. Accession numbers for amino acid sequences of Tbx5 polypeptides include, for example, CAA70592.1 (Homo sapiens), NP_000183 (Homo sapiens), NP_035667 (Mus musculus), and NP_001009964.1 (Rattus orvegicus). Accession numbers for nucleotide sequences encoding Tbx5 polypeptides include, for example, Y09445 (Homo sapiens), NM_000192 (Homo sapiens), NM_011537 (Mus musculus), and NM_001009964 (Rattus orvegicus).

[0061] In some embodiments, the amino acid sequence of the Tbx5 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in one of the above GenBank entries.

[0062] In some embodiments, the nucleotide sequence encoding the Tbx5 polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in one of the above GenBank entries.

[0063] Mef2c Mef2c (muscle cell-specific enhancer factor 2c) is a transcriptional activator that specifically binds to the MEF2 element present in the regulatory regions of many muscle-specific genes. Mef2c may be post-translationally modified, for example, by phosphorylation on Ser-59 and Ser-396; sumoylation on Lys-391; and acetylation on Lys-4. The amino acid sequences of Mef2c polypeptides from various species and the nucleotide sequences encoding them are known in the art. Examples of amino acid sequences for Mef2c polypeptides and accession numbers for the nucleotide sequences encoding them are shown below.

[0064] Accession numbers for amino acid sequences related to Mef2c polypeptides include, for example, NP_001124477 (Homo sapiens), NP_002388 (Homo sapiens), NP_079558.1 (Mus musculus), NP_001164008 (Mus musculus), XP_001056692 (Rattus norvegicus), etc. Accession numbers for nucleotide sequences encoding Mef2c polypeptides include, for example, NM_001131005 (Homo sapiens), NM_002397 (Homo sapiens), NM_025282 (Mus musculus), NM_001170537 (Mus musculus), XM_001056692 (Rattus norvegicus), etc.

[0065] In some embodiments, the amino acid sequence of the Mef2c polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in one of the above GenBank entries.

[0066] In some embodiments, the nucleotide sequence encoding the Mef2c polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in one of the above GenBank entries.

[0067] In some embodiments, a polypeptide functionally equivalent to a Mef2c polypeptide (or a nucleotide sequence encoding such a functional equivalent) is used. For example, in some embodiments, a Mef2a polypeptide (or a nucleotide sequence encoding a Mef2a polypeptide) is used. In other embodiments, a Mef2b polypeptide (or a nucleotide sequence encoding a Mef2b polypeptide) is used. In other embodiments, a Mef2d polypeptide (or a nucleotide sequence encoding a Mef2d polypeptide) is used.

[0068] The amino acid sequence of Mef2a polypeptide and the nucleotide sequence encoding Mef2a polypeptide are known in the art. Accession numbers for amino acid sequences of Mef2a polypeptide include, for example, NP_005578.2 (Homo sapiens), NP_001124398.1 (Homo sapiens), NP_001124399.1 (Homo sapiens), and NP_001124400.1 (Homo sapiens). Accession numbers for nucleotide sequences encoding Mef2a polypeptide include, for example, NM_005587 (Homo sapiens), NM_001130926 (Homo sapiens), NM_001130927 (Homo sapiens), and NM_001130928 (Homo sapiens).

[0069] The amino acid sequence of Mef2b polypeptide and the nucleotide sequence encoding Mef2b polypeptide are known in the art. Accession numbers for amino acid sequences of Mef2b polypeptide include, for example, NP_001139257.1 (Homo sapiens), NP_005910.1 (Homo sapiens), NP_032604.2 (Mus musculus), and NP_001038949.1 (Mus musculus). Accession numbers for nucleotide sequences encoding Mef2b polypeptide include, for example, NM_001145785 (Homo sapiens), NM_005919 (Homo sapiens), NM_008578 (Mus musculus), and NM_001045484 (Mus musculus).

[0070] The amino acid sequence of the Mef2d polypeptide and the nucleotide sequence encoding the Mef2d polypeptide are known in the art. Accession numbers for the amino acid sequence of the Mef2d polypeptide include, for example, NP_005911.1 (Homo sapiens) and NP_598426.1 (Mus musculus). Accession numbers for the nucleotide sequence encoding the Mef2d polypeptide include, for example, NM_005920 (Homo sapiens) and NM_133665 (Mus musculus).

[0071] In some embodiments, a suitable functional equivalent of a Mef2c polypeptide is a polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of a Mef2a polypeptide, a Mef2b polypeptide, or a Mef2d polypeptide.

[0072] In some embodiments, suitable nucleotide sequences encoding functional equivalents of Mef2c polypeptides include nucleotide sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity to a nucleotide sequence encoding a Mef2a polypeptide, a Mef2b polypeptide, or a Mef2d polypeptide.

[0073] Mef2c-TAD Mef2c-TAD is an artificial transcription factor that fuses Mef2c with the transactivation domain (TAD) of MYOD, and overexpresses genes downstream of Mef2c. Mef2c-TAD is described, for example, in Kojima et al. (2023). Stem Cell Reports 18, 1274-1283. Microarray data and RNA-seq data for Mef2c-TAD can be found under accession numbers GSE196293 and GSE222869.

[0074] In some embodiments, the amino acid sequence of the Mef2c-TAD polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0075] In some embodiments, the nucleotide sequence encoding the Mef2c-TAD polypeptide comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:5.

[0076] In another embodiment, the polynucleotide encoding the protein of interest may be a marker gene. An rAAV vector containing a polynucleotide incorporating a marker gene is administered to a test animal, and the number and distribution of cells into which the gene has been introduced can be determined by identifying cells expressing the marker gene. Any known marker gene can be used without particular limitation. Examples of such marker genes include the green fluorescent protein (GFP) gene, the LacZ gene, and luminescent protein genes (such as firefly luciferase).

[0077] Other examples of target proteins include, but are not limited to, Cre recombinase.

[0078] A method for obtaining a polynucleotide encoding a protein of interest can include a method using a nucleic acid amplification method such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and these primers are used to perform PCR or the like using genomic DNA or cDNA as a template to amplify the DNA region between the two primers. This allows for the large-scale production of DNA fragments containing the polynucleotide of the present invention.

[0079] The polynucleotide packaged in the rAAV vector of the present invention is preferably a polynucleotide in which the polynucleotide of the internal region located between the ITR sequences located on the 5' and 3' sides of the AAV genome (i.e., one or both of the rep gene and the cap gene) is replaced with a gene cassette containing a polynucleotide encoding a protein of interest (e.g., a reprogramming factor) and at least one promoter sequence selected from the group consisting of the periostin promoter sequence, the collagen 1a2 promoter sequence, and the cartilage oligomeric matrix protein (COMP) promoter sequence. More preferably, the polynucleotide has ITR sequences at its 5' and 3' ends, respectively.

[0080] The ITR of an adeno-associated virus is a region approximately 145 bases long, and functions as a replication origin, etc. In the present invention, the ITR sequence of the polynucleotide may be derived from any AAV serotype, as long as it has at least one of the functions of an original ITR, such as a function as a replication origin or gene insertion into a host cell, but is preferably derived from AAV2.

[0081] In one embodiment, the 5' ITR sequence comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6. In one embodiment, the 3' ITR sequence comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 7. Generally, the ITR portion readily adopts a sequence in which the complementary sequence is reversed, and therefore, the 5' and 3' orientations of the ITRs included in the polynucleotide packaged in the rAAV vectors of the invention may be reversed.

[0082] 2. Preparation of rAAV Vectors The rAAV vectors of the present invention can be prepared by known methods. Such methods include, for example, transfecting cultured cells with an AAV helper plasmid (plasmid 1) and an rAAV vector plasmid (plasmid 2). The method can further include transfecting the cultured cells with an adenovirus helper plasmid or infecting the cultured cells with adenovirus. The method can further include culturing the transfected cultured cells and collecting the recombinant adeno-associated virus vector from the culture supernatant.

[0083] The AAV helper plasmid (plasmid 1) contains a polynucleotide encoding the AAV-DJ capsid protein and a polynucleotide encoding the Rep protein. The AAV-DJ capsid protein and the Rep protein may be incorporated into one or more plasmids. Furthermore, one or more of these AAV-DJ capsid protein and Rep protein may be contained in the rAAV genome. In the present invention, it is preferred that the AAV-DJ capsid protein and the Rep protein are all encoded by a single polynucleotide and used as an AAV helper plasmid. Furthermore, it is preferred that the polynucleotide be operably linked to a known promoter sequence that is operable in cultured cells. Examples of such promoter sequences that can be used include the cytomegalovirus (CMV) promoter, EF-1α promoter, and SV40 promoter. Furthermore, the AAV helper plasmid may contain known enhancer sequences, Kozak sequences, poly(A) addition signal sequences, and the like.

[0084] The Rep protein used in the present invention may comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the wild-type AAV Rep protein, as long as it has known functions, such as recognizing ITR sequences and replicating the genome depending on those sequences, recruiting and packaging an rAAV genome (or wild-type AAV genome) into a viral vector, and forming the rAAV vector of the present invention, to the same extent as the wild-type AAV Rep protein. In one embodiment, the Rep protein may comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the AAV-DJ Rep protein (SEQ ID NO: 8). In the present invention, a Rep protein derived from a known AAV-DJ is preferably used.

[0085] The polynucleotide encoding the Rep protein used in the present invention may contain a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence encoding the wild-type AAV Rep protein, as long as it encodes a Rep protein that has known functions, such as recognizing ITR sequences and replicating the genome depending on those sequences, recruiting an rAAV genome (or a wild-type AAV genome) into a viral vector and packaging it, and forming the rAAV vector of the present invention. In one embodiment, the polynucleotide may contain a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence encoding the Rep protein of AAV-DJ. In the present invention, a Rep gene derived from a known AAV-DJ is preferably used.

[0086] The rAAV vector plasmid (plasmid 2) is a plasmid containing a nucleotide sequence encoding a protein of interest located between two ITR sequences. Plasmid 2 contains a polynucleotide sequence encoding the protein of interest in a position operable with at least one promoter selected from the group consisting of the periostin promoter, collagen 1a2 promoter, and cartilage oligomeric matrix protein (COMP) promoter. Plasmid 2 may further contain known enhancer sequences, Kozak sequences, poly(A) addition signal sequences, etc., as appropriate.

[0087] Adenovirus helper plasmid is a plasmid encoding adenovirus-derived factors.AAV is a helper-dependent virus, so in order to prepare the rAAV vector of the present invention, it is necessary to co-infect cultured cells with a helper virus such as adenovirus when infecting them.Without co-infection with a helper virus, AAV will insert its viral genome into host cell chromosomes, but will not produce an infectious AAV vector derived from the inserted viral genome.When a host with the inserted viral genome is infected with a helper virus, it can produce an infectious AAV vector derived from the integrated genome.

[0088] In preparing the rAAV vector of the present invention, an adenovirus helper plasmid can be introduced into cultured cells simultaneously with the above-mentioned Plasmid 1 and Plasmid 2. In the present invention, the adenovirus helper plasmid is preferably derived from the same virus species as the cultured cells. For example, when using human cultured cells 293T, an adenovirus helper plasmid derived from human AdV can be used. Commercially available adenovirus helper plasmids can be used.

[0089] In preparing the rAAV vectors of the present invention, various known methods can be used to transfect cultured cells with one or more of the above-mentioned plasmids, such as the calcium phosphate method, lipofection, electroporation, etc. Such methods are described, for example, in Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997.

[0090] 3. Pharmaceutical Compositions Comprising the rAAV Vectors of the Present Invention In another embodiment, a pharmaceutical composition comprising the rAAV vectors of the present invention is provided. In one embodiment, the rAAV vectors of the present invention comprise a polynucleotide encoding a reprogramming factor. Pharmaceutical compositions comprising such rAAV vectors are useful for treating various cardiac disorders (e.g., myocardial infarction, heart failure, arrhythmia, etc.). The pharmaceutical compositions can be used as agents for improving cardiac function. The reprogramming factors described above can be preferably used.

[0091] The content of the rAAV vector in the pharmaceutical composition of the present invention can be approximately 0.001 to 100 parts by mass per 100 parts by mass of the pharmaceutical composition. In other words, the rAAV vector itself may be used as the pharmaceutical composition of the present invention.

[0092] The pharmaceutical composition of the present invention may be blended with pharmaceutically acceptable carriers or additives used in the manufacture of pharmaceutical compositions. Such carriers or additives are well known, and specific examples thereof include any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring agent, sweetener, etc.

[0093] The pharmaceutical composition of the present invention can be made into any dosage form by appropriately combining the above-mentioned carriers or compounds.Specific dosage forms include injections such as intravenous injections, infusions, implant injections, and sustained-release injections; tablets such as plain tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets; capsules such as hard capsules and soft capsules; granules including effervescent granules, sustained-release granules, and enteric-coated granules; powders; oral liquids such as elixirs, suspensions, emulsions, and lemonades; syrups such as syrups; and oral jellies.

[0094] The target animal for use with the pharmaceutical composition of the present invention is not limited to humans, and any living animal may be used, including laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, monkeys, and chimpanzees; pet animals such as dogs and cats; and any other animal species requiring protection.

[0095] The method of administration of the pharmaceutical composition of the present invention is not particularly limited, and any known administration method may be adopted, taking into account the above-mentioned administration target, dosage form, etc., as appropriate. Specific examples include administration into cardiac tissue, intravenously, orally, intramuscularly, intraarterially, etc., with administration into cardiac tissue or intravenously being preferred, and direct administration into cardiac tissue being more preferred. In one embodiment, the method of administration of the pharmaceutical composition of the present invention involves contacting the pharmaceutical composition of the present invention with fibroblasts of an individual. "Contact" includes administering the pharmaceutical composition of the present invention to an individual at or near the treatment site (e.g., in or around the heart). Examples of administration methods include a method in which a catheter inserted into a distal artery is guided to the vicinity of the affected area of ​​the heart, and the pharmaceutical composition of the present invention is injected from the tip of the catheter into the fibrotic affected tissue to contact it.

[0096] The dosage of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dosage can be selected depending on various conditions such as the type of disease, the age and symptoms of the patient, the route of administration, the purpose of treatment, and the presence or absence of concomitant drugs. The dosage of the pharmaceutical composition of the present invention is, for example, usually about 0.1 to 50 mg / kg for humans, and usually about 0.1 to 50 mg / kg for mice. When vg (vector genome) is used as the dosage unit, for example, 10 mg / kg per kg of body weight is used. 9 ~10 14 vg, preferably 10 10 ~10 13 vg, more preferably 10 10 ~10 12The dosage can be selected within the range of 0.1 mg / kg, but is not limited thereto. For other target animals, the dosage can be appropriately determined based on the dosages for humans and mice described above. The pharmaceutical composition of the present invention may be administered in the above amount once a day or in divided doses. Furthermore, the administration interval may be daily, every other day, weekly, every other week, every 2-3 weeks, monthly, every other month, or every 2-3 months, as long as a therapeutic effect is achieved.

[0097] 4. Gene Expression Using the rAAV Vector of the Present Invention In one embodiment, a gene expression method using the AAV vector of the present invention is provided. The method comprises administering the rAAV vector of the present invention. The subject of administration may be an individual, tissue, cell, or the like. The AAV vector of the present invention can express a gene of interest in activated cardiac fibroblasts. The activated cardiac fibroblasts are preferably cardiac fibroblasts following myocardial infarction. In one embodiment, the individual is a non-human mammal.

[0098] In one embodiment, the gene expression method comprises administering an rAAV vector containing a polynucleotide encoding a reprogramming factor. The polynucleotide encoding the reprogramming factor is preferably one described above. The gene expression method using such an rAAV vector induces cardiomyocytes from activated cardiac fibroblasts.

[0099] 5. Kits for Preparing rAAV Vectors of the Present Invention In another embodiment, the present invention provides kits for preparing the rAAV vectors of the present invention. Such kits may include, for example, the above-described Plasmid 1 and Plasmid 2. In addition, kits for preparing the rAAV vectors of the present invention may further include any of the components described herein (e.g., adenovirus helper plasmids, etc.).

[0100] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention. All experiments shown below were conducted with the approval of the Animal Experiment Ethics Committee of the University of Tsukuba.

[0101] Materials and Methods (AAV Vector Construction) AAV vectors were constructed according to the methods described in Kurosaki et al., 2017 and Mizukami et al., 2006. Recombinant AAV vector plasmids were constructed by PCR amplification of the coding regions of GFP, Cre, GATA4, HAND2, TBX5, MEF2C, and MEF2C-TAD. The amplified DNA was subcloned into AAV-CMV (Agilent Technologies) and AAV-Postn (periostin) (Piras et al., 2016). Furthermore, amplified GFP DNA was subcloned into AAV-COMP and AAV-Col1a2. AAV-DJ Rep-Cap was purchased from Cell Biolabs, Inc. Packaging plasmids (AAV1-10, DJ) and helper plasmids (pHelper for AAV1-10: Agilent Technologies, pHelper for AAV-DJ: Cell Biolabs Inc.) were co-transfected into HEK293 cells using calcium phosphate precipitation. Virus-containing medium was collected after 3 days, and cells were resuspended in PBS. AAV vectors were purified using polyethylene glycol precipitation and ultracentrifugation using cesium chloride. The titer (VG) of each vector was measured by real-time PCR (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.). The amino acid sequence of the AAV-DJ capsid protein used in this example is shown in SEQ ID NO: 1. The polynucleotide sequence encoding the AAV-DJ capsid protein is shown in SEQ ID NO: 2. The polynucleotide sequence of the periostin promoter is shown in SEQ ID NO: 3. The amino acid sequence of the Mef2c-TAD polypeptide is shown in SEQ ID NO: 4. The polynucleotide sequence encoding the Mef2c-TAD polypeptide is shown in SEQ ID NO: 5. The polynucleotide sequence of the cartilage oligomeric matrix protein (COMP) promoter is shown in SEQ ID NO: 9. The polynucleotide sequence of the collagen 1a2 promoter is shown in SEQ ID NO: 10.

[0102] (Mice) Genetically modified Tcf21 iCre / Tomato mice were obtained by crossing Tcf21 iCre mice with R26R-tdTomato mice. Genetically modified Postn MCM / Tomato mice were obtained by crossing Postn MCM mice with R26R-tdTomato mice. ICR (Jcl:ICR) mice were purchased from CLEA Japan, Inc. The transgenic mice showed no immunodeficiency or other health problems and were drug-free and experimentally naive until the start of this experiment. All animals were group-housed in a dedicated specific pathogen-free (SPF) facility with a 12-hour / 12-hour light / dark cycle, with free access to food and water, and monitored daily. Health status was checked regularly to maintain SPF status. Animals undergoing surgical procedures were transferred to the SPF facility under the same conditions.

[0103] (Mouse Myocardial Infarction Model) Experiments were performed on 8-week-old male and female mice, randomly assigned to each group. Isoflurane was used to induce and maintain anesthesia. A hot mat was used to prevent hypothermia. Oral tracheal intubation was performed, and a thoracic approach was made through an intercostal incision above the skin and heart. The heart was exposed, and the left coronary artery was ligated using 7-0 silk suture. Immediately after coronary artery ligation, 60 μL of AAV vector-containing solution (1 × 10 11 For systemic administration, AAV vector-containing solution (1 × 10 VG / mouse) was injected into the tail vein immediately after coronary artery ligation at three sites at the border between the infarct and border zone using a 29-gauge needle. 12 Each mouse was injected with 200 μL of PEG (VGA / mouse). The mouse operators were blinded to the study. The mortality rate after myocardial infarction was recorded as less than 10%. Echocardiography was performed on the second postoperative day, and mice with an ejection fraction (EF) of 45% or greater were excluded from the analysis.

[0104] (Primary culture of rat cardiomyocytes and cardiac fibroblasts) Cells derived from postnatal Sprague-Dawley rats (P1-3) were separated into cardiomyocytes and cardiac fibroblasts based on centrifugation using Percoll gradient centrifugation, and then allowed to adhere to plastic dishes to obtain cardiomyocytes (Haginiwa et al., 2019). Cells were resuspended in DMEM / Medium 199 supplemented with 10% FBS and cultured at 37°C and 5% CO2, with the medium changed every 2-3 days. Rat cardiomyocytes and cardiac fibroblasts were cultured the following day after inoculation with the above vectors (2.5 × 10 5 The cells were transduced with 1000 ng / cell and cultured for another week.

[0105] (Mouse Embryonic Fibroblasts (MEF)) Mouse embryos were collected between 12.5 and 13.5 days of gestation. The head, heart, and other internal organs were removed aseptically under a stereomicroscope. The remaining torsos of 3-5 individuals were collected and finely minced with scissors. The resulting tissue fragments were transferred to 15 mL of 0.25% trypsin / EDTA solution pre-warmed to 37°C and incubated at 37°C for 20 minutes. To stop the reaction, an equal volume of FBS (15 mL) was added, and the tissue was dissociated by pipetting several times. The dissociated suspension was centrifuged, and the resulting pellet was resuspended in 10 mL of MEF medium. The resuspended cells were seeded into a 100 mm dish and incubated in 5% CO ２ The cells were cultured under conditions of 100°C, 37°C, and 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% non-essential amino acids (NEAA), and 1% antibiotic-antimycotic mixture (PSA) in Dulbecco's Modified Eagle Medium (DMEM).

[0106] Tamoxifen Administration: Tcf21 iCre / Tomato mice were intraperitoneally administered tamoxifen citrate (2 mg / day; Millipore Sigma, T5648) for five consecutive days. Tamoxifen was dissolved in 90% peanut oil (Millipore Sigma, P2144) and 10% ethanol to a concentration of 50 mg / mL. MI surgery was performed 7 days after the last day of tamoxifen administration. Postn MCM / Tomato mice were continuously administered tamoxifen-containing food pellets for two weeks after myocardial infarction surgery. Food pellets were prepared by adding 0.4 g of tamoxifen citrate (T2510, Tokyo Chemical Industry Co., Ltd.) and 49.6 g of sucrose per kg of standard mouse chow (MF, Oriental Yeast Co., Ltd.).

[0107] (In vitro infection with AAV vectors) Primary cultured neonatal rat cardiomyocytes and cardiac fibroblasts were infected with the indicated AAV vectors (2.5 × 10 5 The cells were transduced with 1000 ng / cell (1000 ng / cell) and cultured for an additional week. The cells were analyzed by FACS and immunofluorescence to determine the cellular tropism of AAV.

[0108] In vivo AAV vector infection: Direct injection into the heart yields 1 x 10 11 The AAV vectors were injected into the hearts of mice after myocardial infarction or intact mice. For systemic administration, 1 × 10 12 The VG AAV vector was injected into the tail vein of mice.

[0109] Fluorescence-activated cell sorting (FACS) analysis: Mice were euthanized by CO2 inhalation, and the hearts were immediately cannulated and perfused with chilled PBS (50 mL). After removal, the atria and valves were dissected to separate the ventricles. The ventricular myocardium was then cut into approximately 1 mm pieces on a sterile dish on ice. The pieces were transferred to a 10 mL tube containing 3 mL of enzyme solution and incubated in a 37°C water bath for 45 minutes, with pipetting every 15 minutes. The enzyme solution contained 2 mg / mL collagenase type IV (Worthington Biochemical, CLS-4) and 1.2 U / mL Dispase II (Sigma, 255-914-4). After the final pipetting, the cell suspension was filtered through a 40 μm cell strainer. After removing debris by centrifugation using Debris Removal Solution (Miltenyi Biotec, 130-109-398), red blood cells were removed using RBC lysis buffer (pluriSelect, 60-00050-11). Cells were then analyzed using a flow cytometer (Beckman Coulter, CytoFLEX S) and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA) to detect GFP expression.

[0110] Immunocytochemistry: Cells were fixed with 4% PFA for 15 minutes at 25°C, blocked with 5% normal goat serum blocking solution (Vector Laboratories, S-1000), permeabilized with 0.2% Triton X-100 (Sigma) for 10 minutes, and incubated with a primary antibody against GFP. This was followed by incubation with a secondary antibody conjugated with Alexa Fluor 488. Immunocytochemistry was performed using an all-in-one fluorescence microscope (Keyence, BZX810).

[0111] Immunohistochemistry: Mouse heart, liver, lung, and skeletal muscle were fixed overnight in 4% PFA, then replaced with 20% sucrose solution, embedded in OCT, and frozen in liquid nitrogen. Cryosections were cut at 7 μm thickness. Sections were stained with primary antibodies against α-actinin (Sigma, A7811), CD31 (Abcam, ab28364), CD45 (Thermo, 30-F11), COL1 (Millipore, Sigma, AB765P), COMP (PGI, 28369-1-AP), cTnT (Thermo, MS-295-P1), GFP (MBL Life Sciences, 598), and SMMHC (Biomedical Technologies, Madrid, Spain, BT562). After incubation with Alexa 546- or Alexa 488-conjugated secondary antibodies, cell nuclei were stained with DAPI (Thermo, D21490). Confocal microscopy was performed using an LSM800 microscope (Carl Zeiss), and Z-stack images were collected according to standard protocols. Measurements and calculations were performed in a blinded manner. Comp+ areas were measured using the analysis tool in Adobe Photoshop. The proportion of immunoreactive cells was determined in six randomly selected fields per section. Data for each mouse were calculated based on 30 to 50 sections, with five mice observed per group. A total of 36,000–60,000 cells were counted for each mouse.

[0112] Masson's trichrome staining was performed on paraffin-embedded sections according to standard procedures. To measure scar size, the scar area (blue) and healthy area (red) were measured on transverse sections across three levels (apex, mid-base) within the left ventricle using Adobe Photoshop. Three tissue sections were measured at each level (a total of nine sections), and five mice per group were examined. Measurements and calculations were performed blinded.

[0113] (Echocardiography) Cardiac function was evaluated using an echocardiogram (Vevo 2100, Visual Sonics). Echocardiography was performed under isoflurane anesthesia. The depth of anesthesia was adjusted to maintain a heart rate of approximately 500 beats per minute. Left ventricular short-axis images were obtained, and M-mode measurements were taken at the papillary muscle level to determine the left ventricular end-diastolic and end-systolic dimensions, and the ejection fraction (EF) was calculated. The ejection fraction (EF) and fractional shortening (FS) were calculated using the Teichholtz formula.

[0114] RNA sequencing analysis was performed at Tsukuba i-Laboratory LLP. Total RNA was extracted from the left ventricular border infarct area of ​​PBS- and GHT / M-TAD-injected mice 4 weeks after myocardial infarction using TRIzol (Invitrogen) according to the manufacturer's protocol (n = 5 per group). RNA sequencing was performed using NextSeq500 (Illumina). Reads were mapped to the mm10 mouse reference genome and quantified using CLC Genomics Workbench (CLC-GW, Version 10.1.1; Qiagen, Venlo, Netherlands). Normalization was performed using the quantile method, and after adding 1, the normalized values ​​were log2-transformed to generate heat maps. Heat maps were created using Morpheus software (https: / / software.broadinstitute.org / morpheus). Differential gene expression analysis was performed using the Empirical Analysis of DGE in CLC. Principal component analysis (PCA) was performed using BioJupies software (Torre et al., 2018). Relative expression was analyzed using normalized expression values ​​as a ratio to the Ctrl group.

[0115] (Statistics) Statistical analysis was performed using GraphPad Prism version 8.4.3 (GraphPad Software Inc.). Statistical significance was examined using unpaired t-tests or one-way analysis of variance, followed by Tukey's or Dunnett's post-hoc tests. Differences with p values ​​<0.05 were considered significant.

[0116] Comparative Example 1 In Vitro and In Vivo Screening Using the CMV Promoter (In Vitro) To examine the cardiac fibroblast tropism of AAV capsids, 10 native types (AAV1-10) and one chimeric type (AAV-DJ) were screened. Eleven AAV vector serotypes expressing GFP under the control of the constitutive CMV promoter (AAV-CMV-GFP) were transfected at a multiplicity of infection of 2.5 × 10 5 Primary rat CF and CM cells were transduced in vitro with vector genomes per cell (VG / cell). Transduction efficiency was determined 1 week later using fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) (Figure 1). FACS analysis revealed that AAV1, AVV6, and AVV-DJ showed the highest transduction efficiency in cardiac fibroblasts among 11 serotypes (Figure 2). AAV2 and AAV9, which are widely used in cardiomyocyte gene therapy, showed low transduction efficiency in cardiac fibroblasts in vitro. In contrast, most AAV vectors, including AAV-DJ, showed moderate transduction efficiency (~40%) in cardiomyocytes in vitro (Figure 3). Immunofluorescence staining revealed that AAV1, AAV6, and AAV-DJ showed strong GFP expression in cardiac fibroblasts, while AAV1 and AAV4 showed strong GFP expression in cultured cardiomyocytes (Figure 4).

[0117] (In vivo) AAV-CMV-GFP (1 × 10) was transfected into the hearts of ICR mice after myocardial infarction caused by coronary artery ligation. 11We determined the cellular tropism of AAV capsids in vivo by directly injecting AAV-DJ-CMV-GFP into guinea pigs (vectors) (VG / mice). GFP expression in cardiomyocytes and non-myocytes was analyzed 2 weeks later using immunohistochemical staining (Figure 1). Notably, immunohistochemical staining revealed that all AAV capsids, including AAV6 and AAV-DJ, preferentially expressed GFP in α-actinin + CMs rather than in non-myocytes (Figure 5). The in vivo transduction efficiencies of AAV-DJ-CMV-GFP into non-myocytes and cardiomyocytes were ~20% and ~80%, respectively (Figure 5). These results suggest that the tropism of AAV vectors may differ between in vitro and in vivo conditions, which is consistent with previous studies (Guan et al., 2015; Zincarelli et al., 2010).

[0118] Example 1: In vivo gene expression in cardiac fibroblasts using AAV-DJ-Postn vectors. AAV constructs containing periostin (Postn) promoter-driven Cre recombinase (AAV-Postn-Cre) were constructed and packaged into AAV6, 9, and DJ capsids (Figure 6). The AAV6-Postn-Cre vector, AAV9-Postn-Cre vector, and AAV-DJ-Postn-Cre vector (hereinafter sometimes referred to simply as AAV6-Postn-Cre, AAV9-Postn-Cre, or AAV-DJ-Postn-Cre) were directly injected into the hearts of Tomato reporter mice after myocardial infarction, and transgene expression was measured (Figure 7). With AAV6-Postn-Cre, AAV9-Postn-Cre, and AAV-DJ-Postn-Cre, the Tomato reporter was expressed primarily in the infarct and border zones, but not in distant regions (Figure 8). In particular, AAV-DJ-Postn-Cre showed the highest transduction efficiency among the three serotypes (Fig. 9). Co-immunostaining with multiple antibodies against cardiomyocytes (cTnT), cardiac fibroblasts (COL1), smooth muscle cells (SMCs; SMMHC), and endothelial cells (ECs; CD31) demonstrated that Tomato was primarily expressed in resident CFs, and the transduction efficiency of AAV-DJ-Postn-Cre injection reached approximately 90% (Fig. 10). Tomato+ cells persisted in the border and infarct areas for 4 weeks after myocardial infarction.

[0119] [Example 2] Gene expression in primary cultured human cardiac fibroblasts using the AAV-DJ-Postn vector To examine the efficiency of gene transfer in human cardiac fibroblasts, human cardiac fibroblasts were cultured, AAV-DJ-Postn-GFP was constructed, and gene transfer was performed. The multiplicity of infection was 5x10. 4 The transfection efficiency was determined by immunofluorescence (IF) analysis 1 week after transfection. The AAV-DJ-Postn vector also induced gene expression in human cardiac fibroblasts in vitro (data not shown).

[0120] Example 3: Confirmation of the time course of transgene expression under the control of the Postn promoter To examine the time course of transgene expression under the control of the Postn promoter, AAV-DJ-Postn-GFP was constructed and directly injected into the hearts of mice after myocardial infarction (Figure 11). In the hearts after myocardial infarction, GFP expression decreased 2 and 4 weeks after myocardial infarction compared to 1 week after myocardial infarction (Figure 12).

[0121] Example 4: Specificity of the AAV-DJ-Postn Vector. Immunohistochemical staining and FACS analysis confirmed that GFP+ cells were detected in the heart after 1 week, but not in other tissues, such as the liver, lung, or skeletal muscle (Figures 13-15). When AAV-DJ-Postn-GFP was directly injected into the hearts of healthy, non-infarcted mice, low levels of GFP expression were detected only at the injection site. Furthermore, when AAV-DJ-Postn-Cre was systemically administered via the tail vein to healthy control mice (Figures 16 and 17) and infarcted Tomato reporter mice (Figures 18 and 19), Tomato expression was observed only in the infarcted area, but not in healthy myocardium or other tissues. These results suggest that the AAV-DJ-Postn vector preferentially targets activated CFs after myocardial injury, but not quiescent CFs or other organs.

[0122] Example 5: In vivo myocardial reprogramming of cardiac fibroblasts using AAV-DJ-Postn vectors (1) We investigated whether in vivo cardiac fibroblasts could be reprogrammed into induced cardiomyocytes by gene transfer using AAV-DJ-Postn vectors. First, we constructed AAV-DJ-Postn vectors expressing reprogramming factors, including GATA4, HAND2, TBX5, MEF2C, and MEF2C-TAD (M-TAD) (Figure 20). To test cardiac reprogramming in vivo, we directly injected any of the following vectors into the hearts of Postn MCM / Tomato mice immediately after myocardial infarction: (i) phosphate-buffered saline (PBS) (control, Ctrl); (ii) AAV-DJ-Postn-GATA4, AAV-DJ-Postn-HAND2, AAV-DJ-Postn-TBX5, and AAV-DJ-Postn-MEF2C suspended in PBS (AAV-DJ-Postn-GHT / M); or (iii) AAV-DJ-Postn-GATA4, AAV-DJ-Postn-HAND2, AAV-DJ-Postn-TBX5, and AAV-DJ-Postn-MEF2C-TAD suspended in PBS (AAV-DJ-Postn-GHT / M-TAD). The activated CF lineages were then traced (Figures 21 and 22). Mice were treated with tamoxifen for two weeks after myocardial infarction, and hearts were harvested two weeks later (Kanisicak et al., 2016). Immunohistochemical staining revealed that both AAV-DJ-Postn-GHT / M and AAV-DJ-Postn-GHT / M-TAD induced α-actinin expression in Tomato+ cells (Figure 23). α-Actinin+ / Tomato+ induced cardiomyocytes displayed distinct sarcomere structures, and three-dimensional analysis confirmed that the cells expressed both proteins. Importantly, AAV-DJ-Postn-GHT / M-TAD generated twice as many induced cardiomyocytes in the border zone as AAV-DJ-Postn-GHT / M, suggesting that activated MEF2C promotes cardiac reprogramming in vivo (Figure 24).

[0123] Example 6: In vivo cardiac reprogramming of cardiac fibroblasts using AAV-DJ-Postn vectors (2). Tcf21 iCre / Tomato mice were treated with tamoxifen before myocardial infarction to specifically label quiescent cardiac fibroblasts, enabling lineage tracking of cardiac fibroblasts without leaky expression in cardiomyocytes. Tcf21 iCre / Tomato mice were treated with tamoxifen for 5 days to label Tcf21-expressing cardiac fibroblasts before myocardial infarction. Coronary artery ligation was performed 7 days after the final tamoxifen treatment. Similar to Postn MCM / Tomato mice, numerous Tomato+ cardiac fibroblasts were observed at the border / infarct area 4 weeks after myocardial infarction in Tcf21 iCre / Tomato mice (Figures 25-27). PBS or AAV-DJ-Postn-GHT / M-TAD was directly injected into the hearts of Tcf21 iCre / Tomato mice immediately after myocardial infarction. Immunohistochemical staining revealed that approximately 1.8% of Tomato+ cells at the border in the AAV-DJ-Postn-GHT / M-TAD group expressed α-actinin, compared with 0.3% in the Ctrl group (Figure 28). The induced cardiomyocytes showed a distinct sarcomer structure. These results demonstrate that the AAV-DJ-Postn vector expressing activated MEF2C and GHT efficiently reprograms resident cardiac fibroblasts into induced cardiomyocytes after myocardial infarction.

[0124] Example 7: Confirmation of Fibrosis Suppression by Myocardial Reprogramming with AAV-DJ-Postn Vector To investigate the molecular changes induced by cardiac reprogramming with AAV-DJ-Postn-GHT / M-TAD, we performed RNA sequencing analysis on hearts from Ctrl- and AAV-DJ-Postn-GHT / M-TAD-injected mice 4 weeks after myocardial infarction. Gene set enrichment analysis revealed that upregulated genes in GHT / M-TAD hearts were enriched in clusters related to cardiac function, such as cardiac contractility or metabolic processes, whereas downregulated genes were enriched in fibroblast signatures or inflammatory responses (Figure 29). Next, cardiac function in Ctrl-, AAV-DJ-Postn-GFP-, and AAV-DJ-Postn-GHT / M-TAD-injected mice was assessed using echocardiography 2 days, 2 weeks, and 4 weeks after myocardial infarction. AAV-DJ-Postn-GHT / M-TAD administration significantly improved left ventricular ejection fraction and fractional shortening after 4 weeks (Figure 30). Histological analysis using Masson's trichrome staining was also performed to quantify scar size after 4 weeks. The fibrotic area in AAV-DJ-Postn-GHT / M-TAD-treated hearts was significantly reduced compared with Ctrl and AAV-DJ-Postn-GFP-treated hearts (Figure 31). Thus, in vivo cardiac reprogramming using AAV-DJ-Postn-GHT / M-TAD improved cardiac function and reduced fibrosis after myocardial infarction.

[0125] Example 8 Gene Expression in Mouse Fetal Fibroblasts and Cardiac Fibroblasts Using AAV-DJ-Col1a2 Vector and AAV-DJ-COMP Vector To examine mouse fetal fibroblasts and gene transfer efficiency, these cells were cultured, and AAV-DJ-Col1a2-GFP vector and AAV-DJ-COMP-GFP vector were constructed (Figure 32), followed by gene transfer. Multiplicity of infection: 5x10 4Gene transfer was performed at 1000 ng / cell, and transfection efficiency was determined by immunofluorescence (IF) 1 week after transfection. The AAV-DJ-Col1a2 vector demonstrated gene expression in mouse embryonic fibroblasts and cardiac fibroblasts in vitro, although the efficiency was lower than that of the AAV-DJ-CMV and AAV-DJ-Postn vectors (Figure 33). The AAV-DJ-COMP-GFP vector also demonstrated gene expression in mouse embryonic fibroblasts and cardiac fibroblasts in vitro, although the transfection efficiency was lower (Figure 33). Similarly, cardiomyocytes were examined; no expression was observed in cardiomyocytes with either vector (Figure 34), confirming selectivity for non-cardiomyocytes.

[0126] Example 9: In vivo gene expression in cardiac fibroblasts using the AAV-DJ-Col1a2 vector. The AAV-DJ-Col1a2-GFP vector was directly injected into the hearts of ICR mice after myocardial infarction, and transgene expression was examined by immunohistochemical staining at 1, 2, and 4 weeks (Figure 35). Significant gene expression was observed at all time points after myocardial infarction (Figure 36). The AAV-DJ-Col1a2 vector demonstrated broad gene expression throughout the acute and chronic phases of myocardial infarction.

Claims

1. A recombinant adeno-associated virus vector comprising an AAV-DJ capsid protein and a polynucleotide comprising at least one selected from the group consisting of a periostin promoter sequence, a collagen 1a2 promoter sequence, and a cartilage oligomeric matrix protein (COMP) promoter sequence, wherein the collagen 1a2 promoter sequence is any of the following (2-1) to (2-4), and the cartilage oligomeric matrix protein (COMP) promoter sequence is any of the following (3-1) to (3-4): (2-1) a nucleotide sequence represented by SEQ ID NO: 9; (2-2) a nucleotide sequence in which one or several nucleotides have been substituted, added, or deleted in the nucleotide sequence represented by SEQ ID NO: 9; (2-3) a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 9; (2-4) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 9; (3-1) a nucleotide sequence represented by SEQ ID NO: 10; (3-2) A base sequence in which one or more bases have been substituted, added, or deleted in the base sequence represented by SEQ ID NO: 10; (3-3) A base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 10; (3-4) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO:

10.

2. The recombinant adeno-associated viral vector of claim 1, wherein the recombinant adeno-associated viral vector comprises a polynucleotide encoding at least one reprogramming factor selected from the group consisting of Gata4, Hand2, Tbx5, and Mef2c.

3. A pharmaceutical composition comprising the recombinant adeno-associated virus vector of claim 1 or 2.

4. A cardiac function improving agent comprising the recombinant adeno-associated virus vector of claim 2.

5. A method for gene expression in activated cardiac fibroblasts, comprising administering the recombinant adeno-associated virus vector of claim 1 or 2.

6. The gene expression method according to claim 5, wherein the activated cardiac fibroblasts include cardiac fibroblasts after myocardial infarction.

7. A method for inducing cardiomyocytes from cardiac fibroblasts, comprising administering the recombinant adeno-associated virus vector of claim 1 or 2.

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