Pluripotent stem cells derived from patients with arrhythmogenic cardiomyopathy, their use, and pharmaceuticals for treating arrhythmogenic cardiomyopathy

Abstract

The present invention provides a drug for treating arrhythmogenic cardiomyopathy that has, as an active ingredient, a gene therapy drug used for delivering a normal gene corresponding to a gene having a gene mutation to myocardial cells of a patient with arrhythmogenic cardiomyopathy caused by a gene mutation, such that a normal protein is caused to be expressed. The present invention also provides pluripotent stem cells established from cells of a patient with arrhythmogenic cardiomyopathy caused by a mutation of a plakophilin-2 gene or a desmoglein-2 gene.

Description

[Technical Field]

[0001] The present invention relates to pluripotent stem cells derived from patients with arrhythmogenic cardiomyopathy, the use thereof, and pharmaceuticals for treating arrhythmogenic cardiomyopathy. [Background technology]

[0002] Arrhythmogenic cardiomyopathy (AC) is a group of diseases primarily caused by genetic mutations, resulting in ventricular arrhythmias and impaired cardiac contractility. Previously, it was recognized as arrhythmogenic right ventricular cardiomyopathy (ARVC), a rare and intractable disease characterized by thinning of the right ventricular myocardium, ventricular dilation, and potentially fatal arrhythmias such as ventricular tachycardia and ventricular fibrillation. However, due to the increasing number of reported cases of left ventricular dysfunction, it has recently come to be collectively recognized as AC (Non-Patent Document 1). The primary cause of AC is known to be genetic abnormalities in the intercalated disc, and plakophilin-2 (PKP2) is the gene with the most commonly identified mutations responsible for AC. PKP2 gene knockout mice develop abnormal myocardial structure and ventricular wall rupture during the fetal stage, indicating that PKP2 is an important factor in heart formation (Non-Patent Document 2).

[0003] Disease-specific iPSCs (iPSCs) have been established from AC patients with PKP2 mutations. Cardiomyocytes differentiated from iPSCs derived from AC patients and those derived from iPSCs derived from healthy individuals have been shown to exhibit reduced intercalated disc-associated proteins, abnormal lipid droplet deposition, prolonged action potential rise time, and elevated PPARγ expression (Non-Patent Documents 3-5). However, these studies have only compared cardiomyocytes differentiated from iPSCs derived from healthy individuals with different genetic backgrounds. Instead, they have not used human disease model cells in which only the PKP2 gene mutation has been modified in the same genetic background, allowing for accurate evaluation of the pathology caused by PKP2 gene abnormalities. Furthermore, to reproduce the pathology of AC and develop treatments, a pathological model that mimics beating myocardial tissue, rather than a single myocardium, is needed.

[0004] Desmosomes, on the other hand, are localized in intercalated discs in many organs, including the heart, and play a role in maintaining cellular structure. The desmosomal cadherin genes that make up desmosomes include four desmogleins (DSG1-4) and three desmocollins (DSC1-3). DSG2 and DSC2 are specifically expressed in human and mouse cardiac tissue, and genetic mutations in these molecules primarily cause hereditary cardiomyopathies, including AC. Mice lacking DSG2 are embryonic lethal, while cardiac-specific knockout of DSG2 has been reported to exhibit ventricular dilation, cardiomyocyte death, and interstitial fibrosis, resulting in a dilated cardiomyopathy-like phenotype (Non-Patent Documents 6 and 7). Many heterozygous DSG2 mutations have been identified in human cases of AC, and compound heterozygous DSG2 mutations have been reported to cause severe heart failure (Non-Patent Document 8). However, no homozygous DSG2 mutations have been reported to date. Furthermore, there have been no reports demonstrating the reproducibility of cardiomyopathy caused by DSG2 gene mutations using human iPS-differentiated myocardium, or the therapeutic concept of introducing DSG2 molecules into human iPS-differentiated myocardium. [Prior art documents] [Non-patent literature]

[0005]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

[0006] An objective of the present invention is to provide a pharmaceutical for treating arrhythmogenic cardiomyopathy caused by a genetic mutation. Another objective of the present invention is to provide pluripotent stem cells established from patients with arrhythmogenic cardiomyopathy caused by a genetic mutation, pluripotent stem cells that have the same genetic background as the pluripotent stem cells but have a mating type mutation different from that of the patient, and pluripotent stem cells that have the same genetic background as the pluripotent stem cells but in which the mutated gene has been corrected to a normal gene. A further objective of the present invention is to provide a method for screening for a therapeutic drug for arrhythmogenic cardiomyopathy using the pluripotent stem cells or cardiomyocytes differentiated from the pluripotent stem cells. [Means for solving the problem]

[0007] The present invention includes the following inventions to solve the above problems. [1] A pharmaceutical for treating arrhythmogenic cardiomyopathy, the active ingredient of which is a gene therapy drug for delivering a normal gene corresponding to a gene having a genetic mutation to the cardiomyocytes of a patient with arrhythmogenic cardiomyopathy caused by the genetic mutation, thereby causing the expression of a normal protein. [2] The pharmaceutical composition according to [1] above, wherein the genetic mutation is a mutation in the plakophilin 2 gene or a mutation in the desmoglein 2 gene. [3] Pluripotent stem cells established from cells of a patient with arrhythmogenic cardiomyopathy caused by mutations in the plakophilin 2 gene or desmoglein 2 gene. [4] The pluripotent stem cell described in [3] above, wherein the mutation in the plakophilin 2 gene is a heterozygous frameshift mutation in which the guanine at position 1228 in the base sequence of the human plakophilin 2 gene shown in sequence number 2 is duplicated. [5] Pluripotent stem cells that have the same genetic background as the pluripotent stem cells described in [4] above and that have a homozygous mutation in the human plakophilin 2 gene that causes arrhythmogenic cardiomyopathy. [6] Pluripotent stem cells having the same genetic background as the pluripotent stem cells described in [4] above, in which the human plakophilin 2 gene has been corrected to a normal gene. [7] The pluripotent stem cell described in [3] above, wherein the mutation in the desmoglein 2 gene is a homozygous stop-gain mutation in which cytosine at position 355 in the base sequence of the human desmoglein 2 gene shown in sequence number 4 is replaced with thymine. [8] Pluripotent stem cells having the same genetic background as the pluripotent stem cells described in [7] above, and having a heterozygous mutation in the human desmoglein 2 gene that causes arrhythmogenic cardiomyopathy. [9] Pluripotent stem cells having the same genetic background as the pluripotent stem cells described in [7] above, in which the human desmoglein 2 gene has been corrected to a normal gene.

[10] A pluripotent stem cell set comprising two or three types of pluripotent stem cells selected from the group consisting of the pluripotent stem cells described in [4] above, the pluripotent stem cells described in [5] above, and the pluripotent stem cells described in [6] above.

[11] A pluripotent stem cell set comprising two or three types of pluripotent stem cells selected from the group consisting of the pluripotent stem cells described in [7] above, the pluripotent stem cells described in [8] above, and the pluripotent stem cells described in [9] above.

[12] A pluripotent stem cell for fluorescent imaging of endogenous desmoglein 2, in which a fluorescent protein gene has been inserted downstream of the desmoglein 2 gene of the pluripotent stem cell described in [4] above.

[13] A pluripotent stem cell for fluorescent imaging of endogenous desmoglein 2, in which a fluorescent protein gene has been inserted downstream of the desmoglein 2 gene of the pluripotent stem cell described in [5] above.

[14] A pluripotent stem cell for fluorescent imaging of endogenous desmoglein 2, in which a fluorescent protein gene has been inserted downstream of the desmoglein 2 gene of the pluripotent stem cell described in [6] above.

[15] A pluripotent stem cell set comprising two or three types of pluripotent stem cells selected from the group consisting of the pluripotent stem cells described in

[12] above, the pluripotent stem cells described in

[13] above, and the pluripotent stem cells described in

[14] above.

[16] A kit for researching arrhythmogenic cardiomyopathy, comprising the pluripotent stem cell set according to

[10] ,

[11] or

[15] above.

[17] A kit for screening therapeutic agents for arrhythmogenic cardiomyopathy, comprising the pluripotent stem cell set according to

[10] ,

[11] or

[15] above.

[18] A screening method for a therapeutic agent for arrhythmogenic cardiomyopathy using the pluripotent stem cells described in [5], [7] or

[13] above, or cardiomyocytes differentiated from the pluripotent stem cells, or cells in the process of inducing differentiation of the pluripotent stem cells into cardiomyocytes, comprising the steps of contacting or introducing a test substance into the cells, evaluating a phenotype in the cells that reflects arrhythmogenic cardiomyopathy, and comparing the evaluation result of the phenotype in the cells that have not been contacted or introduced with the test substance to select a test substance that improves arrhythmogenic cardiomyopathy. [Effects of the Invention]

[0008] The present invention can provide a pharmaceutical for treating arrhythmogenic cardiomyopathy caused by a genetic mutation. The present invention can also provide pluripotent stem cells established from patients with arrhythmogenic cardiomyopathy caused by a genetic mutation, pluripotent stem cells that have the same genetic background as the pluripotent stem cells but have a mating type mutation different from that of the patient, and pluripotent stem cells that have the same genetic background as the pluripotent stem cells but in which the mutated gene has been corrected to a normal gene. Furthermore, the present invention can provide a method for screening for a therapeutic drug for arrhythmogenic cardiomyopathy using the pluripotent stem cells or cardiomyocytes differentiated from the pluripotent stem cells. [Brief explanation of the drawings]

[0009] [Figure 1] (A) is the patient's (arrow) pedigree; squares indicate males and circles indicate females. Patients with ventricular arrhythmias are indicated by black circles or black squares. (B) is an echocardiogram of the patient; the left is a parasternal left ventricular short-axis view, and the right is a parasternal four-chamber view. LV: left ventricle; RV: right ventricle; EF: ejection fraction; LVDd / s: left ventricular diastolic / systolic diameter. The scale bar indicates 20 mm. [Figure 2]This figure shows that direct Sanger sequencing was performed on genomic DNA extracted from the patient's peripheral blood to screen for genetic mutations associated with hereditary cardiovascular disease, and a heterozygous frameshift mutation (1228 dupG) in the PKP2 gene was found. [Figure 3] FIG. 1 shows the results of immunostaining patient-derived iPS cells (hereinafter referred to as "iPSCs") with antibodies against pluripotency marker proteins (SSEA4, TRA-1-60, OCT4, NANOG) (scale bar: 50 μm). [Figure 4] FIG. 1 shows the results of karyotype analysis of patient-derived iPSCs. [Figure 5] This figure shows the results of direct Sanger sequencing analysis using genomic DNA from patient-derived iPSCs and cDNA obtained by reverse transcription from total RNA. (A) shows the results for genomic DNA, and (B) shows the results for cDNA. [Figure 6] FIG. 1 shows the results of Western blotting to detect the expression levels of PKP2 protein in patient-derived iPSCs and iPSCs prepared from healthy control subjects. [Figure 7] This figure shows a common ddPCR probe that detects transcripts of both the wild-type allele and the mutant allele (1228 dupG) in droplet digital PCR (ddPCR), a probe that detects the transcript of the wild-type allele, and a probe that detects the transcript of the mutant allele (1228 dupG). [Figure 8] This figure shows the results of droplet digital PCR performed on cDNA samples obtained from patient-derived iPSCs and cardiomyocytes differentiated from those iPSCs. Representative positive droplet signals from PCR probes targeting each PKP2 transcript are shown. [Figure 9] This figure shows the results of comparing the PKP2 transcript concentration (copies / μL) of each sample calculated from droplet digital PCR analysis (*: p<0.01, n=3, means ± SD). Relative transcript concentrations were calculated as a ratio normalized by the concentration of the wild-type transcript. [Figure 10] FIG. 1 shows the results of measuring the percentage of troponin T-positive cells in cardiomyocytes (iPSC-CMs) differentiated from patient-derived iPSCs by FACS analysis (n=3, mean±SD). [Figure 11] Figure 1 shows the relative copy number of each PKP2 transcript in iPSCs or iPSC-CMs (*: p < 0.01, #: p = 0.06, n = 3, mean ± SD). The relative copy number is normalized by the value of the wild-type transcript in iPSCs. [Figure 12] This figure shows four gRNAs (#1, #2, #3, #4) designed for genome editing of the flanking region of a mutation (1228 dupG) in exon 5 of the human PKP2 gene. [Figure 13] FIG. 1 shows the results of evaluating the cleavage activity of each gRNA by single-strand annealing assay, where (A) shows the cleavage results for the wild-type allele and (B) shows the cleavage results for the mutant allele. [Figure 14] FIG. 1 shows the results of evaluating the cleavage activity of gRNA#1 and gRNA#4 targeting the endogenous PKP2 locus in HEK293T cells by Cel-I assay. [Figure 15] FIG. 1 shows the results of sequence analysis of the area surrounding the mutation site in the PKP2 gene in genomic DNA obtained from each of the generated isogenic iPSC clones (HDR, hetero, NHEJ). [Figure 16] Fig. 1 shows the predicted protein lengths of PKP2 from the generated isogenic iPSC clones (HDR, hetero, NHEJ). [Figure 17] FIG. 1 shows the homology-directed repair (HDR) template structure with a 1035 bp 5′ homology arm and a 497 bp 3′ homology arm corresponding to the genomic sequence around exon 5 of the PKP2 gene. [Figure 18]This figure shows the results of quantitative real-time PCR analysis of cDNA obtained from each isogenic iPSC clone (HDR, hetero, and NHEJ) using a common probe targeting both wild-type and mutant transcripts. The control is a sample obtained from iPSCs generated from a healthy human (*: p<0.01 vs. Het, n=3, mean±SD). [Figure 19] Figure 1 shows the results of droplet digital PCR (ddPCR) analysis using cDNA obtained from each isogenic iPSC clone (HDR, heterozygous, NHEJ) (*: p<0.01 vs. HDR or NHEJ, #: p<0.01 vs. heterozygous, n=3, means±SD). [Figure 20] Figure 1 shows the results of Western blotting analysis using cell lysates from each isogenic iPSC clone (HDR, hetero, NHEJ) (*: p<0.05 vs. hetero; #: p<0.01 vs. hetero; n=3, means±SD). [Figure 21] FIG. 1 shows a scheme for an experiment to induce differentiation of iPSCs into cardiomyocytes. [Figure 22] Figure 1 shows the results of evaluating the differentiation induction efficiency of cardiomyocytes (hetero-iPSC-CM, HDR-iPSC-CM, NHEJ-iPSC-CM) induced in monolayer from each isogenic iPSC (hetero-iPSC-CM, HDR-iPSC-CM, NHEJ-iPSC-CM) by FACS using an anti-troponin T antibody. [Figure 23] This figure shows the results of continuous observation of monolayer HDR-iPSC-CMs and monolayer NHEJ-iPSC-CMs 8 to 10 days after the start of differentiation induction (scale bar: 1 mm). [Figure 24] FIG. 11 shows torn cardiac muscle fibers observed in NHEJ-iPSC-CMs 10 days after the start of differentiation induction. [Figure 25]Monolayer iPSC-CMs were continuously observed using cell motility analysis. Bright-field images of HDR-iPSC-CMs and NHEJ-iPSC-CMs at fixed positions at specific coordinates were shown on days 14, 18, and 28 after the start of differentiation induction (scale bar: 200 μm). The white box indicates the area enlarged in Figure 27. [Figure 26] Figure 1 shows (A) contraction velocity (CV) and (B) average deformation distance (ADD) of HDR-iPSC-CMs and NHEJ-iPSC-CMs on days 14 and 28, as calculated by motion vector analysis. [Figure 27] FIG. 26 shows an enlarged image of the area enclosed in the white frame in the bright-field image of HDR-iPSC-CM in FIG. 25. [Figure 28] This figure shows the results of excitation propagation in NHEJ-iPSC-CMs on days 21 and 28 after the start of differentiation induction, as determined by label-free detection using motion vectors (scale bar: 200 μm). [Figure 29] This figure shows the results of continuous observation of excitation propagation through the oriented fiber structure of NHEJ-iPSC-CMs on days 14 and 19 after the start of differentiation induction. [Figure 30] FIG. 1 shows a scheme for an experiment to induce differentiation of iPSCs into cardiomyocytes. [Figure 31] HDR-iPSC-CMs and NHEJ-iPSC-CMs were replated onto a 96-well plate on day 10 after the start of differentiation induction. The results were then immunostained with anti-troponin T antibody on day 16 (scale bar 50 μm) (*: p<0.01, n=3, means±SD). [Figure 32] This figure shows the results of quantitative real-time PCR performed on HDR-iPSC-CM and NHEJ-iPSC-CM 10 days after the start of differentiation induction (*: p<0.001 vs. HDR, n=3, mean±SD). [Figure 33]FIG. 11 shows the results of Western blotting using NHEJ-iPSC-CM and HDR-iPSC-CM samples on days 14 and 28 after the start of differentiation induction. [Figure 34] This figure shows the results of immunostaining with anti-plakoglobin antibody on day 16 of HDR-iPSC-CMs and NHEJ-iPSC-CMs replated onto a 96-well plate on day 10 after the start of differentiation induction (scale bar 50 μm). Enlarged images of the white boxes in the second row are shown in the bottom two rows. [Figure 35] This figure shows the results of immunostaining with anti-plakophilin 2 antibody on day 16 of HDR-iPSC-CMs and NHEJ-iPSC-CMs replated onto a 96-well plate on day 10 after the start of differentiation induction (scale bar 50 μm). Enlarged images of the white boxes in the second row are shown in the bottom two rows. [Figure 36] HDR-iPSC-CMs and NHEJ-iPSC-CMs were replated onto a 96-well plate on day 10 after the start of differentiation induction, and immunostained with anti-desmoglein 2 or anti-desmocollin 2 antibodies on day 16 (scale bar 50 μm). Enlarged images of the white boxes in the second row are shown in the bottom two rows. [Figure 37] This figure shows the results of immunostaining with anti-N-cadherin antibody on day 16 of HDR-iPSC-CMs and NHEJ-iPSC-CMs replated onto a 96-well plate on day 10 after the start of differentiation induction (scale bar 50 μm). The bottom two rows show enlarged images of the white boxes in the second row. [Figure 38] This figure shows the results of observing Hetero-iPSC-CMs and HDR-iPSC-CMs replated onto a 24-well plate on day 14 after the start of differentiation induction, followed by observation on day 28 (scale bar, 500 μm). [Figure 39] FIG. 1 shows a scheme of a PKP2 gene transfer experiment. [Figure 40]NHEJ-iPSC-CMs were replated onto a 96-well plate on day 10 after the start of differentiation induction, and the PKP2 gene was transduced via AAV2 on day 11. The results of immunostaining with anti-FLAG and anti-troponin T antibodies were obtained on day 16 (scale bar: 50 μm). The image on the right is an enlarged image of the area within the white box on the left. [Figure 41] NHEJ-iPSC-CMs were replated onto a 96-well plate on day 10 after the start of differentiation induction. On day 11, the PKP2 gene or EGFP gene was transduced via AAV2. On day 16, the NHEJ-iPSC-CMs were immunostained with anti-desmoglein 2, anti-desmocollin 2, or anti-N-cadherin antibodies (scale bar: 50 μm). [Figure 42] This figure shows the results of quantifying the expression level of each protein using high-content imaging for images obtained from the immunostained specimens in Figure 40 (*: p<0.001, n=3, means±SD, 6 images in each experiment). The viral genome amounts used were 1.04×104 viral genomes (vg) / cell for AAV2-EGFP and 1.04×104 or 2.08×104 vg / cell for AAV2-PKP2. [Figure 43] The cells were replated onto a 96-well plate on day 10 after the start of differentiation induction, and on day 11, the PKP2 gene or EGFP gene was introduced via AAV2. On day 16, the cells were immunostained with an anti-troponin T antibody, and the area of ​​troponin T-positive cardiomyocytes was measured using high-content imaging (*: p<0.01, n=3, means±SD). [Figure 44] NHEJ-iPSC-CMs were transduced with the EGFP gene via AAV2 on day 10 after the start of differentiation induction, and were observed under a fluorescent microscope (left) and in bright field (right) on day 24. [Figure 45] This figure shows the results of Western blotting performed on NHEJ-iPSC-CMs on day 10 after the start of differentiation induction, after AAV2-mediated introduction of the PKP2 gene or EGFP gene, and on day 24, using these cells as samples. [Figure 46] NHEJ-iPSC-CMs were transfected with the PKP2 gene or EGFP gene via AAV2 on day 10 after the start of differentiation induction. The following figures show images of the cells on day 24 (scale bar: 200 μm), an image in which excitation propagation was converted into a color map using motion vector analysis, and contraction velocity (CV) and deformation distance (DD) calculated by motion vector analysis. [Figure 47] (A) shows the domain structure of desmoglein 2 (DSG2), with arginine 119 located in EC1 (extracellular cadherin domain 1). (B) shows the pedigree of the patient (arrow). Squares represent males, circles represent females, and cases with ventricular arrhythmias are indicated by black boxes. (C) shows the results of Sanger sequencing of genomic DNA from the patient (II-1) and his parents (I-1 and I-2). [Figure 48] Left ventricular myocardial tissue from a patient with a homozygous C355T mutation (II-1) and a patient with dilated cardiomyopathy (DCM control) was immunostained with anti-desmoglein 2 antibody (scale bar 50 μm). The lower panel shows an enlarged image of the box in the upper panel. [Figure 49] FIG. 1 shows the results of transmission electron microscopic observation of left ventricular myocardial tissue from a patient with homozygous C355T mutation (II-1) and a patient with dilated cardiomyopathy (DCM control) (scale bar: 500 nm). [Figure 50] (A) shows the results of RT-PCR analysis of DSG2 mRNA levels in RNA extracted from patient-derived iPSCs and iPSCs generated from healthy control subjects (ACTB was used as an endogenous control). (B) shows the results of immunostaining of fixed patient-derived iPSCs and iPSCs generated from healthy control subjects with anti-desmoglein 2 antibody (nuclei were stained with Hoechst) (scale bar 100 μm). [Figure 51]Figure 1 shows the genomic DNA sequences and translated amino acid sequences of patient-derived iPSCs (R119X-iPSCs) and isogenic iPSCs (HDR-iPSCs) in which the heterozygous C355T mutation in the patient-derived iPSCs was modified to a normal sequence by genome editing. [Figure 52] (A) R119X-iPSCs and HDR-iPSCs were fixed and immunostained with anti-Desmoglein 2 antibody (nuclei were stained with Hoechst) (scale bar, 100 μm). (B) Protein samples were collected from R119X-iPSCs and HDR-iPSCs, and Desmoglein 2 expression was detected by Western blotting. [Figure 53] Three-dimensional self-assembled rings were fabricated using cardiomyocytes induced to differentiate from R119X-iPSCs (R119X-iPSC-CM) and cardiomyocytes induced to differentiate from HDR-iPSCs (HDR-iPSC-CM). The results show that myocardial contractile force was measured using a compressive strength measurement device. (A) shows a representative result for R119X-iPSC-CM, (B) shows a representative result for HDR-iPSC-CM, and (C) shows a comparison of the average values ​​for both (*: p<0.05, R119X-iPSC-CM n=4, HDR-iPSC-CM n=3, mean±SD). [Figure 54] Figure 1 shows the myocardial structure of R119X-iPSC-CMs and HDR-iPSC-CMs on day 30 after the start of differentiation induction (day 16 after reseeding) (scale bar 100 μm). (A) The upper row is a bright-field image, the middle row is an image of fixed cells immunostained with anti-troponin T antibody, and the lower row is an image of cells immunostained with anti-troponin T antibody (nuclei stained with Hoechst). (B) shows the relative ratio of troponin T-positive areas in R119X-iPSC-CMs to HDR-iPSC-CMs (*: p<0.001, n=4, mean±SD). [Figure 55]Fig. 10 shows the results of Western blotting analysis of desmoglein 2 and desmocollin 2 expression in protein samples collected from R119X-iPSC-CM and HDR-iPSC-CM on day 14 after the start of differentiation induction. [Figure 56] R119X-iPSC-CM and HDR-iPSC-CM were replated onto a 96-well plate 14 days after the start of differentiation induction. Seven days later, the cells were fixed and immunostained with anti-desmoglein 2 and anti-desmocollin 2 antibodies (scale bar, 50 μm). [Figure 57] R119X-iPSC-CM and HDR-iPSC-CM were replated onto a 96-well plate 14 days after the start of differentiation induction. The cells were then fixed and observed under a transmission electron microscope 30 days later (scale bar, 1 μm). [Figure 58] R119X-iPSC-CM were replated onto a 96-well plate on day 14 after the start of differentiation induction. Four days after replated, they were infected with AAV2-DSG2-cHA to introduce the DSG2 gene. Seven days after infection, the cells were fixed and immunostained with an anti-desmoglein 2 antibody (scale bar 50 μm). The top row shows R119X-iPSC-CM without the DSG2 gene. [Figure 59] This figure shows the results of Western blotting analysis of desmoglein 2 expression in R119X-iPSC-CMs on day 21 after the start of differentiation induction, which were infected with 2.0 × 105 vg / cell or 6.0 × 105 vg / cell of AAV2-DSG2-cHA for gene transfer. Protein samples were collected from cardiomyocytes 7 days after infection. [Figure 60] This figure shows the results of infecting R119X-iPSC-CM with AV2-DSG2-cHA on day 9 after the start of differentiation induction, re-seeding on day 14 after the start of differentiation induction, and observing them under bright field conditions on day 30 after the start of differentiation induction. [Figure 61]The top panel shows images of hetero-iPSC-CMs and HDR-iPSC-CMs immunostained with anti-desmoglein 2 antibody on day 14 after the start of differentiation induction, where desmoglein 2-positive areas were detected using high-content imaging. The bottom panel shows the results of quantification of desmoglein 2-positive areas using high-content imaging (*: p<0.0001, n=32 for each). [Figure 62] FIG. 1 shows the positions of guide RNAs targeting downstream of the stop codon of the human DSG2 gene and the positions of primers used in PCR. [Figure 63] FIG. 63 shows the results of PCR performed using the primers shown in FIG. 62 as templates for the genomic DNA of DSG2-tdT-Hetero-iPSCs, DSG2-tdT-HDR-iPSCs, and DSG2-tdT-NHEJ-iPSCs, in which tdTomato was inserted at the 3' end of the DSG2 gene by genome editing, as well as the genomic DNA of Hetero-iPSCs, HDR-iPSCs, and NHEJ-iPSCs that had not been subjected to genome editing. [Figure 64] This figure shows the results of Sanger sequencing analysis of the genome of DSG2-tdT-NHEJ-iPSCs. The upper panel shows the results of direct sequencing of PCR products derived from the wild-type allele, and the lower panel shows the results of direct sequencing of PCR products derived from the tdTomato knock-in allele. [Figure 65] FIG. 1 shows the established sets of PKP2 gene mutant isogenic iPSCs that produce wild-type DSG2 and DSG2-tdTomato fusion transcripts, respectively. [Figure 66] This figure shows the results of karyotype analysis of DSG2-tdT-Hetero-iPSCs, DSG2-tdT-HDR-iPSCs, and DSG2-tdT-NHEJ-iPSCs, as well as the results of immunostaining with antibodies against pluripotency marker proteins (OCT4, SSEA4, and NANOG) (scale bar: 50 μm). [Figure 67](A) Western blotting results for lysates from DSG2-tdT-Hetero-iPSCs, DSG2-tdT-HDR-iPSCs, and DSG2-tdT-NHEJ-iPSCs, using anti-desmoglein 2, anti-red fluorescent protein (RFP), anti-plakoglobin, anti-plakophilin 2, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibodies, respectively. (B) Hoechst staining of nuclei from fixed DSG2-tdT-Hetero-iPSCs, DSG2-tdT-HDR-iPSCs, and DSG2-tdT-NHEJ-iPSCs (scale bar 50 μm). [Figure 68] This figure shows the results of time-lapse imaging using DSG2-tdT-HDR-iPSCs and DSG2-tdT-HDR-iPSC-CMs on day 14 after the start of differentiation induction. The upper row shows bright-field (BF) images, and the lower row shows fluorescent images. [Figure 69] This figure shows the results of immunostaining of DSG2-tdT-Hetero-iPSC-CM, DSG2-tdT-HDR-iPSC-CM, and DSG2-tdT-NHEJ-iPSC-CM cells fixed 14 days after the start of differentiation induction (scale bar 50 μm). The upper row shows fluorescent images of DSG2-tdTomato, and the lower row shows images immunostained with anti-troponin T antibody (nuclei stained with Hoechst). [Figure 70] (A) Bright-field (BF) images (left, scale bar 50 μm) of DSG2-tdT-Hetero-iPSC-CMs and DSG2-tdT-HDR-iPSC-CMs on day 14 after the start of differentiation induction, a tdTomato fluorescent image (center), and a magnified image of the box in the center image (right). (B) Quantification of desmoglein 2-positive areas using high-content imaging for the live imaging of (A). [Figure 71]Figure 1 shows the results of time-lapse imaging of DSG2-tdT-NHEJ-iPSC-CMs infected with an adeno-associated virus encoding full-length human PKP2 (AAV2-PKP2, see Example 1) on day 14 after the start of differentiation induction for 4 days. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Medicine for treating arrhythmogenic cardiomyopathy] The present invention provides a pharmaceutical for treating arrhythmogenic cardiomyopathy (AC) (hereinafter referred to as the "pharmaceutical of the present invention"). The pharmaceutical of the present invention may contain, as an active ingredient, a gene therapy drug for delivering a normal gene corresponding to a gene having a mutation to the cardiomyocytes of a patient with AC caused by a genetic mutation, thereby expressing a normal protein. Examples of genetic mutations that cause AC include the plakophilin 2 (PKP2) gene, desmoglein 2 (DSG2) gene, plakoglobin (JUP) gene, desmoplakin (DSP) gene, desmocollin 2 (DSC2) gene, TMEM43 gene, lamin A / C (LMNA) gene, desmin (DES) gene, catenin alpha 3 (CTNNA3) gene, phospholamban (PLN) gene, TGFB3 gene, titin (TTN) gene, SCN5A gene, and N-cadherin (CDH2) gene (see Non-Patent Document 1).

[0011] The normal protein is not limited to a protein consisting of a wild-type amino acid sequence as long as it exhibits normal functions, but is preferably a protein consisting of a wild-type amino acid sequence. The nucleotide sequence of the normal gene (wild-type gene) corresponding to a gene having a genetic mutation that causes AC and the amino acid sequence of the normal protein (wild-type protein) encoded by it can be obtained from publicly known databases (such as NCBI).

[0012] The pharmaceutical of the present invention may be a pharmaceutical for treating AC, containing as an active ingredient a gene therapy drug for expressing normal PKP2, which is intended for administration to AC patients having a mutation in the PKP2 gene. The amino acid sequence of normal human PKP2 (wild-type human PKP2) (NCBI Reference Sequence: NP_001005242.2) is shown in SEQ ID NO: 1. The nucleotide sequence of DNA for expressing normal PKP2 is not limited as long as it encodes the amino acid sequence shown in SEQ ID NO: 1. The nucleotide sequence of DNA for expressing normal PKP2 may be, for example, the nucleotide sequence shown in SEQ ID NO: 2 (positions 47 to 2560 in the nucleotide sequence of NCBI Reference Sequence: NM_001005242.3).

[0013] The pharmaceutical of the present invention may be a pharmaceutical for treating AC, the active ingredient of which is a gene therapy drug for expressing normal DSG2, and which is intended for administration to AC patients with a mutation in the DSG2 gene. The amino acid sequence of normal human DSG2 (wild-type human DSG2) (NCBI Reference Sequence: NP_001934.2) is shown in SEQ ID NO: 3. The nucleotide sequence of DNA for expressing normal DSG2 is not limited as long as it encodes the amino acid sequence shown in SEQ ID NO: 3. The nucleotide sequence of DNA for expressing normal DSG2 may be, for example, the nucleotide sequence shown in SEQ ID NO: 4 (positions 76 to 3432 in the nucleotide sequence of NCBI Reference Sequence: NM_001943.5).

[0014] Gene therapy drugs for delivering a normal gene corresponding to a gene mutation to express a normal protein can be administered in the form of a non-viral or viral vector. When administered in the form of a non-viral vector, methods for introducing DNA using liposomes (such as the liposome method, HVJ-liposome method, cationic liposome method, lipofection method, and lipofectamine method), microinjection, methods for transferring DNA to cells together with a carrier (metal particles) using a gene gun, and the method described in Nature Biotechnology, Volume 31, 898-907 (2013) can be used. When administered in the form of a viral vector, the target DNA can be introduced into a DNA or RNA virus such as a detoxified retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, or SV40, and then the gene can be introduced into cardiomyocytes by infecting the cardiomyocytes with this recombinant virus. An adeno-associated viral vector is preferred.

[0015] When the pharmaceutical of the present invention is an adeno-associated virus vector into which DNA encoding a target normal protein has been introduced, the dosage is appropriately determined taking into consideration the age, weight, severity of the disease, etc. of the patient. For example, 10 DRP(DNase-resistant particles)~1×10 14 The adeno-associated viral vector of the DRP may be administered as a single dose into the patient's coronary artery using a catheter.

[0016] [Pluripotent stem cells derived from AC patients] The present invention provides pluripotent stem cells established from cells of patients with AC caused by mutations in the PKP2 gene or DSG2 gene. Pluripotent stem cells are stem cells that have the pluripotency to differentiate into all cells present in the body and also have the ability to proliferate. Examples of pluripotent stem cells include induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). iPS cells (iPSCs) are preferred.

[0017] iPSCs are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of nucleic acids (DNA or RNA) or proteins. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO 2007 / 069666).

[0018] Somatic cells refer to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0019] The reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products or non-coding RNAs, genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNAs, or low-molecular-weight compounds. Examples of genes included in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, W O2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO 2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al.(2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0020] In a preferred embodiment, Oct3 / 4, Sox2, and Klf4 (OSK) can be used as reprogramming factors. More preferably, in addition to the three factors, a Myc family member (M) selected from L-Myc, N-Myc, and c-Myc (including T58A mutant) can be used. Furthermore, since Lin28 promotes the formation of TRA-1-60-positive cells and inhibits reversion to TRA-1-60-negative cells, it is also preferred to use Lin28 as a reprogramming factor in addition to the three factors (OSK) or four factors (OSKM).

[0021] Examples of the reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, and the like, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), and the like)], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., BIX-01294, small molecule inhibitors such as those listed above, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl and G9a, L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt signaling inhibitors (e.g., soluble Also included are factors used to improve establishment efficiency, such as Wnt3a, neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and DMRTBl. In this specification, factors used to improve establishment efficiency are not particularly distinguished from reprogramming factors.

[0022] When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), or microinjection.

[0023] When the reprogramming factor is in the form of DNA, it can be introduced into somatic cells by, for example, vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, microinjection, etc. Viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010 / 008054), etc. Artificial chromosome vectors include, for example, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. The plasmid may be a mammalian cell plasmid (Science, 322:949-953, 2008). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome-binding sequence, terminator, and polyadenylation site to enable expression of the nuclear reprogramming substance. Furthermore, if necessary, the vector may contain a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG. Furthermore, the vector may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise both the promoter and the gene after introduction into somatic cells.

[0024] When the reprogramming factor is in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0025] Examples of culture media for iPSC induction include DMEM, DMEM / F12, or DME culture media containing 10-15% FBS (these culture media can further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate), as well as commercially available culture media such as culture media for mouse ES cells (TX-WES culture medium, Thrombo-X), culture media for primate ES cells (culture medium for primate ES / iPS cells, ReproCell), and serum-free pluripotent stem cell maintenance media (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

[0026] As an example of a culture method, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM / F12 culture medium containing 10% FBS at 37°C in the presence of 5% CO2 and cultured for approximately 4 to 7 days, and then the cells are plated on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) and cultured in a bFGF-containing culture medium for primate ES cell culture from approximately 10 days after contacting the somatic cells with the reprogramming factors, and iPS-like colonies can be generated approximately 30 to 45 days or more after the contact.

[0027] Alternatively, ES-like colonies can be generated after approximately 25 to 30 days or more of culture on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) at 37°C in the presence of 5% CO2. Desirably, somatic cells to be reprogrammed themselves can be used instead of feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010 / 137746), or extracellular matrix (e.g., Laminin-5 (WO2009 / 123349) and Matrigel (BD)).

[0028] Another example is a method of culturing iPSCs using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci U.S.A. 106:15720-15725). Furthermore, to increase establishment efficiency, iPSCs may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or WO2010 / 013845).

[0029] During the above culture, the culture medium is replaced with fresh medium once a day from the second day onwards. The number of somatic cells used for nuclear reprogramming is not limited, but it is recommended to use a 100cm culture dish. 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0030] iPSCs can be selected based on the morphology of the colonies they form. Alternatively, if a drug-resistance gene that is expressed in conjunction with a gene expressed during somatic cell reprogramming (e.g., Oct3 / 4 or Nanog) is introduced as a marker gene, established iPSCs can be selected by culturing them in a culture medium containing the corresponding drug (selective culture medium). Furthermore, if the marker gene is a fluorescent protein gene, iPSCs can be selected by observing them under a fluorescent microscope; if the marker gene is a luciferase gene, by adding a luminescent substrate; or if the marker gene is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0031] The pluripotent stem cells of the present invention may be iPSCs established from peripheral blood mononuclear cells of an AC patient (diagnosed as ARVC) with a heterozygous frameshift mutation resulting in a duplication of guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2. These iPSCs have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074) and have been assigned HPS numbers HPS4885, HPS4886, and HPS4887. These iPSCs can be obtained from the RIKEN BRC.

[0032] The pluripotent stem cells of the present invention may be iPSCs established from peripheral blood mononuclear cells of an AC patient (diagnosed as dilated cardiomyopathy) with a homozygous stop-gain mutation in which cytosine at position 355 in the base sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine. These iPSCs have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074) and have been assigned HPS numbers HPS4879, HPS4880, and HPS4881. These iPSCs can be obtained from the RIKEN BRC.

[0033] [Pluripotent stem cells with the same genetic background as pluripotent stem cells derived from AC patients] The present invention provides isogenic genetically modified pluripotent stem cells that have the same genetic background as pluripotent stem cells established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated. Specifically, the present invention provides iPSCs that have the same genetic background as the iPSCs established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated and that have a homozygous mutation in the human PKP2 gene that causes AC. Furthermore, the present invention provides iPSCs that have the same genetic background as the iPSCs established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated and that have no mutations in both alleles of the human PKP2 gene, in which the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene.

[0034] iPSCs with the same genetic background as the above-mentioned iPSCs established from an AC patient with a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, and with a homozygous mutation in the human PKP2 gene that causes AC, have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074) and assigned HPS numbers HPS4889 and HPS4890. These iPSCs can be obtained from the RIKEN BRC. Furthermore, iPSCs with the same genetic background as the above-mentioned iPSCs established from an AC patient with a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, and in which the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene, have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture 305-0074) and assigned the HPS number HPS4888. These iPSCs can be obtained from the RIKEN BRC.

[0035] The present invention also provides isogenic genetically modified pluripotent stem cells that have the same genetic background as pluripotent stem cells established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine. Specifically, the present invention provides iPSCs that have the same genetic background as the iPSCs established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine and that have a heterozygous mutation in the human DSG2 gene that causes AC. Furthermore, the present invention provides iPSCs that have the same genetic background as the iPSCs established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine and that do not have a mutation in the human DSG2 gene, in which both alleles of the homozygous stop-gain mutation in the human DSG2 gene have been corrected to normal.

[0036] iPSCs with the same genetic background as the iPSCs established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the base sequence of the human DSG2 gene (shown in SEQ ID NO: 4) was replaced with thymine, and carrying a heterozygous mutation that causes AC in the human DSG2 gene (iPSCs in which the heterozygous C355T mutation in the patient-derived iPSCs was modified to a normal sequence) have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki 305-0074) and assigned the HPS number HPS4882. These iPSCs are available from the RIKEN BRC.

[0037] Genetically modified pluripotent stem cells with the same genetic background as pluripotent stem cells established from AC patients (isogenic) can be produced using known genetic recombination techniques, such as homologous recombination using a targeting vector, or genome editing techniques such as CRISPR / Cas9, TALEN, and ZFN.

[0038] The iPSCs established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, and the homozygous mutation in the human PKP2 gene that causes AC has the same genetic background as the iPSCs established from the AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated. The mutation in the human PKP2 gene in the iPSCs that causes AC may be the same as the mutation carried by AC patients, i.e., the frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, or may be a mutation different from the mutation carried by AC patients. The mutation different from the mutation carried by AC patients is not particularly limited as long as it is a mutation that causes AC, but a mutation similar to the mutation carried by AC patients is preferred. The frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, resulting in a stop codon due to the frameshift, resulting in a mutant PKP2 protein of 424 amino acids in length (the wild-type PKP2 protein is 837 amino acids in length). Therefore, the mutation different from that carried by AC patients is preferably a mutation that results in a mutant PKP2 protein having an amino acid length similar to that of 424. The similar amino acid length may be, for example, ±20 amino acids, ±15 amino acids, or ±10 amino acids.

[0039] The iPSCs established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is replaced with thymine have the same genetic background as the iPSCs established from the AC patient with a heterozygous mutation in the human DSG2 gene that causes AC. The mutation in the human DSG2 gene in the iPSCs may be the same as the mutation carried by AC patients, i.e., the stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is replaced with thymine, or it may be a mutation different from the mutation carried by AC patients. If the mutation is different from the mutation carried by AC patients, it is not particularly limited as long as it is a mutation that causes AC, but a mutation similar to the mutation carried by AC patients is preferred. A homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is replaced with thymine results in a mutant DSG2 protein with a full length of 118 amino acids due to the resulting stop codon (wild-type DSG2 protein is 1118 amino acids in length). Therefore, the mutation different from that carried by AC patients is preferably a mutation that results in a mutant DSG2 protein having an amino acid length similar to that of 118 amino acids. The similar amino acid length may be, for example, ±20 amino acids, ±15 amino acids, or ±10 amino acids.

[0040] [AC patient-derived pluripotent stem cells for fluorescent imaging of endogenous desmoglein 2] The present invention provides pluripotent stem cells established from an AC patient carrying a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated; pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) and carrying a homozygous mutation in the human PKP2 gene that causes AC; and pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) in which the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene, and which have been modified so that the endogenous DSG2 protein in each of these pluripotent stem cells is expressed as a fusion protein with a fluorescent protein.

[0041] Pluripotent stem cells established from an AC patient carrying a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 may be any of the iPSCs assigned the above HPS numbers HPS4885, HPS4886, and HPS4887. Pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) and carrying a homozygous mutation in the human PKP2 gene that causes AC may be any of the iPSCs assigned the above HPS numbers HPS4889 and HPS4890. Pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) and in which the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene may be any of the iPSCs assigned the above HPS number HPS4888.

[0042] The pluripotent stem cells for endogenous DSG2 fluorescent imaging of the present invention can be prepared by inserting a fluorescent protein gene downstream of the DSG2 gene of each of the above pluripotent stem cells so that a fusion protein of DSG2 and a fluorescent protein is expressed. The above pluripotent stem cells expressing a fusion protein of DSG2 and a fluorescent protein can be prepared using known genetic recombination techniques. For example, they can be prepared using homologous recombination techniques using a targeting vector, or genome editing techniques such as CRISPR / Cas9, TALEN, and ZFN.

[0043] The fluorescent protein used is not particularly limited, and can be appropriately selected from known fluorescent proteins that can be used for biological cell imaging. Examples of known fluorescent proteins include tdTomato (red fluorescent protein), GFP (green fluorescent protein), and fluorescent proteins containing derivatives thereof. Amino acid sequence information of known fluorescent proteins and nucleotide sequence information of the genes encoding them can be obtained from known databases (NCBI, etc.).

[0044] iPSCs for endogenous DSG2 fluorescence imaging, in which the tdTomato gene was inserted downstream of the DSG2 gene of iPSCs established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, have been deposited at the RIKEN BioResource Research Center (RIKEN BRC, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, 305-0074) and assigned the HPS number HPS5039. iPSCs for endogenous DSG2 fluorescence imaging, in which the tdTomato gene was inserted downstream of the DSG2 gene of iPSCs with a homozygous mutation in the human PKP2 gene that causes AC and has the same genetic background as the iPSCs established from an AC patient with a heterozygous frameshift mutation in which a guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, have been deposited at the RIKEN BioResource Research Center and assigned the HPS number HPS5041. iPSCs for endogenous DSG2 fluorescence imaging, which have the same genetic background as iPSCs established from an AC patient with a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated, and the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene, have been modified to have the tdTomato gene inserted downstream of the DSG2 gene, and have been deposited at the RIKEN BioResource Research Center and assigned the HPS number HPS5040. These iPSCs can be obtained from the RIKEN BioResource Research Center.

[0045] [Isogenic pluripotent stem cell set] The present invention provides a pluripotent stem cell set comprising three types of pluripotent stem cells: pluripotent stem cells established from an AC patient carrying a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated; pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) and carrying a homozygous mutation in the human PKP2 gene that causes AC; and pluripotent stem cells having the same genetic background as the pluripotent stem cells (isogenic) in which the heterozygous frameshift mutation in the human PKP2 gene has been corrected to a normal gene.

[0046] The pluripotent stem cell set of the present invention may be a set of two of the above three types of pluripotent stem cells. A set of two types of pluripotent stem cells may be a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells, a set of pluripotent stem cells having a heterozygous mutation and normal pluripotent stem cells, or a set of pluripotent stem cells having a homozygous mutation and pluripotent stem cells having a heterozygous mutation, although a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells is preferred.

[0047] The present invention provides a pluripotent stem cell set comprising three types of pluripotent stem cells: pluripotent stem cells established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the base sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine; pluripotent stem cells with the same genetic background as the pluripotent stem cells (isogenic) and a heterozygous mutation in the human DSG2 gene that causes AC; and pluripotent stem cells with the same genetic background as the pluripotent stem cells (isogenic) in which both alleles of the homozygous stop-gain mutation in the human DSG2 gene have been corrected to normal genes.

[0048] The pluripotent stem cell set of the present invention may be a set of two of the above three types of pluripotent stem cells. A set of two types of pluripotent stem cells may be a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells, a set of pluripotent stem cells having a heterozygous mutation and normal pluripotent stem cells, or a set of pluripotent stem cells having a homozygous mutation and pluripotent stem cells having a heterozygous mutation, although a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells is preferred.

[0049] The present invention provides a pluripotent stem cell set containing three types of pluripotent stem cells: pluripotent stem cells for endogenous DSG2 fluorescent imaging, in which the tdTomato gene has been inserted downstream of the DSG2 gene of pluripotent stem cells established from an AC patient with a heterozygous frameshift mutation in which guanine at position 1228 in the base sequence of the human PKP2 gene shown in SEQ ID NO: 2 has been duplicated; pluripotent stem cells for endogenous DSG2 fluorescent imaging, in which the tdTomato gene has been inserted downstream of the DSG2 gene of pluripotent stem cells that have the same genetic background as the pluripotent stem cells (isogenic) and have a homozygous mutation in the human PKP2 gene that causes AC; and pluripotent stem cells for endogenous DSG2 fluorescent imaging, in which the tdTomato gene has been inserted downstream of the DSG2 gene of pluripotent stem cells that have the same genetic background as the pluripotent stem cells (isogenic) and have had the heterozygous frameshift mutation in the human PKP2 gene corrected to a normal gene.

[0050] The pluripotent stem cell set of the present invention may be a set of two of the above three types of pluripotent stem cells. A set of two types of pluripotent stem cells may be a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells, a set of pluripotent stem cells having a heterozygous mutation and normal pluripotent stem cells, or a set of pluripotent stem cells having a homozygous mutation and pluripotent stem cells having a heterozygous mutation, although a set of pluripotent stem cells having a homozygous mutation and normal pluripotent stem cells is preferred.

[0051] The pluripotent stem cells of the set of isogenic pluripotent stem cells of the present invention may be iPSCs.

[0052] [AC research kit] The present invention provides a kit for AC research. The AC research kit of the present invention may include the pluripotent stem cell set of the present invention. The other components of the kit are not particularly limited, and may include reagents, equipment such as trays and tubes, and instruction manuals, as required depending on the research objectives. By using the kit of the present invention, highly accurate AC research can be carried out simply and quickly.

[0053] [AC therapeutic drug screening kit] The present invention provides a screening kit for AC therapeutic agents. The screening kit of the present invention may be any kit that includes the pluripotent stem cell set of the present invention. The other components of the kit are not particularly limited, and may include necessary reagents, tools such as trays and tubes, and instruction manuals. Use of the kit of the present invention allows for the simple and rapid implementation of the screening method for AC therapeutic agents described below.

[0054] [Method for screening AC therapeutic drugs] The present invention provides a screening method for a therapeutic agent for AC. The screening method of the present invention may use pluripotent stem cells that have the same genetic background as pluripotent stem cells established from an AC patient with a heterozygous frameshift mutation in which guanine at position 1228 in the nucleotide sequence of the human PKP2 gene shown in SEQ ID NO: 2 is duplicated and that have a homozygous mutation in the human PKP2 gene that causes AC, or pluripotent stem cells for endogenous DSG2 fluorescence imaging in which the tdTomato gene has been inserted downstream of the DSG2 gene of the pluripotent stem cells, or pluripotent stem cells established from an AC patient with a homozygous stop-gain mutation in which cytosine at position 355 in the nucleotide sequence of the human DSG2 gene shown in SEQ ID NO: 4 is substituted with thymine (hereinafter, these pluripotent stem cells are referred to as "homozygous mutant cells").

[0055] Furthermore, the screening method of the present invention may use cardiomyocytes induced to differentiate from the homozygous mutant cells, or may use cells in the process of inducing differentiation from the homozygous mutant cells to cardiomyocytes. The method for inducing differentiation from pluripotent stem cells to cardiomyocytes is not particularly limited, and known differentiation induction methods can be suitably used. The cells in the process of inducing differentiation from homozygous mutant cells to cardiomyocytes may be cells at any stage in the differentiation induction process. Hereinafter, the cells used in the screening method of the present invention will be referred to as "test cells."

[0056] The screening method of the present invention may be any method that includes the following steps (1) to (3). (1) contacting or introducing a test substance into the test cells; (2) assessing a phenotype reflective of AC in the cells; and (3) A step of selecting a test substance that improves AC by comparing with the evaluation result of the phenotype in the cells that have not been contacted or introduced with the test substance.

[0057] In step (1), a test substance is contacted with or introduced into the test cells. The test substance is not particularly limited and may be, for example, a nucleic acid, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, a cell culture supernatant, a plant extract, a mammalian tissue extract, or plasma. The test substance may be a novel substance or a known substance. The test substance may form a salt. As the salt of the test substance, a salt with a physiologically acceptable acid or base is used.

[0058] The method for contacting the test substance with the test cells is not particularly limited. For example, the test substance may be added to a medium for culturing the test cells. The method for introducing the test substance into the test cells is also not particularly limited. For example, lipofection, microinjection, a viral vector, a plasmid vector, etc. may be used. It is also preferable to provide a control group in which the test substance is not contacted or introduced.

[0059] In step (2), a phenotype reflecting AC is evaluated in the test cells contacted or transfected with the test substance in step (1). Evaluation items for phenotypes reflecting AC include, for example, (i) abnormalities in contraction velocity, expansion velocity, contraction / expansion distance, and propagation velocity in motion vector analysis, (ii) abnormalities in intracellular calcium concentration and calcium transients in calcium imaging, (iii) detection of abnormal electrical potentials using a microelectrode array, (iv) abnormalities in tissue strength and contractile force using tissue-reconstructed iPS-differentiated myocardium, (v) abnormal localization of desmosomal proteins by immunostaining or live imaging using fluorescently labeled proteins, and (vi) changes in desmosomal protein expression levels by Western blotting.

[0060] Motion vector analysis can be performed, for example, by the method described in "J Mol Cell Cardiol 77, 178-191, doi:10.1016 / j.yjmcc.2014.09.010 (2014)." Calcium imaging can be performed by appropriately selecting from known methods. For example, the method described in "Toxicol Sci 148, 503-516, doi:10.1093 / toxsci / kfv201 (2015)" may be used. Abnormal potentials can be detected using a microelectrode array, for example, by the method described in "Stem Cell Reports 9, 1546-1559, doi:10.1016 / j.stemcr.2017.09.007 (2017)." Functional analysis of tissue-constructed iPS-differentiated myocardium can be performed, for example, using the method described in "bioRxiv preprint first posted online Jul. 27, 2019; doi: http: / / dx.doi.org / 10.1101 / 717108." Immunostaining, live imaging using fluorescently labeled proteins, and Western blotting can be performed using known methods.

[0061] In step (3), a test substance that improves AC by contact or introduction is selected by comparing the phenotypic evaluation results with those of test cells not contacted or introduced with the test substance. For example, when evaluating the results of motion vector analysis, a test substance is selected that significantly improves abnormalities in contraction velocity, expansion velocity, contraction / expansion distance, and propagation velocity in AC compared to controls without the test substance. When evaluating by calcium imaging, a test substance is selected that significantly improves abnormalities in intracellular calcium concentration and calcium transients in AC compared to controls without the test substance. When detecting potentials using a microelectrode array, a test substance is selected that significantly reduces or eliminates abnormal potentials in AC compared to controls without the test substance. When evaluating tissue-reconstructed iPS-differentiated myocardium, a test substance is selected that significantly improves abnormalities in tissue strength and contractile force in AC compared to controls without the test substance. When evaluating by immunostaining or live imaging using fluorescently labeled proteins, a test substance is selected that significantly improves abnormal localization of desmosomal proteins in AC compared to controls without the test substance. When evaluation is performed by Western blotting, a test substance that significantly improves abnormal changes in the expression levels of desmosomal proteins in AC compared to a control to which the test substance is not added is selected.

[0062] In the screening method of the present invention, heterozygous mutant cells isogenic to the test cells and / or normal cells isogenic to the test cells may be used as control cells. When heterozygous mutant cells isogenic to the test cells and / or normal cells isogenic to the test cells are used as control cells, only the disease gene mutation is modified in the isogenic cells, which is useful for detecting abnormalities specific to the mutant gene and analyzing its function, compared to the use of conventional control cells established from healthy individuals. [Example]

[0063] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0064] Example 1: Establishment of iPSCs from an AC patient with a heterozygous frameshift mutation in PKP2 and generation of an isogenic iPSC set 〔material and method〕 (1) Human samples The use of samples obtained from patients and genomic analysis was approved by the Ethics Committee of Osaka University Hospital, and written informed consent was obtained.

[0065] (2) Antibodies and reagents The following antibodies and reagents were used: anti-Oct-3 / 4 antibody (C-10) (Santa Cruz Biotechnology, Dallas, Texas, USA, Cat# sc-5279, RRID: AB_628051), anti-TRA-1-60 antibody (Merck Millipore, Burlington, Massachusetts, USA, Cat# MAB4360, RRID: AB_2119183), anti-SSEA-4 antibody (Merck Millipore, Cat# MAB4304, RRID: AB_177629), anti-Nanog antibody (Abcam, Cambridge, MA, USA, Cat# ab80892, RRID: AB_2150114), and anti-DSG2 antibody (AH12).2)(Santa Cruz Biotechnology, Cat# sc-80663 RRID: AB_2093438), anti-PKP2 antibody (PROGEN, Germany, Cat#651167) (immunostaining), anti-PKP2 antibody (Abcam Cat# ab151402), anti-γ-Catenin antibody (Cell Signaling Technology, Tokyo, Japan, Cat# 2309, RRID: AB_823448), anti-Desmocollin-2 / 3 antibody (7G6) (Thermo Fisher Scientific, Waltham, Massachusetts, USA, Cat#32-6200, RRID: AB_2533090), anti-Connexin 43 antibody (Cell Signaling Technology, Cat# 3512, RRID: AB_2294590), anti-Troponin T antibody (Abcam Cat# ab64623, RRID: AB_1139590)(ウエスタンブロッティング), anti-GAPDH antibody (Santa Cruz Biotechnology Cat# sc-47724, RRID: AB_627678), anti-Sarcomeric Alpha Actinin antibody (EA-53) (Abcam, Cat# ab9465, RRID: AB_307264), anti-Vimentin antibody (Abcam Cat# ab24525 RRID: AB_778824), BV421 Mouse IgG1, κ Isotype Control (BD Bioscience, Tokyo, Japan, Cat# 562438, RRID: AB_2721018), BV421 Mouse Anti-Cardiac Troponin T (BD Bioscience, Cat# 565618, RRID: AB_2739306)(フローサイトメトリー), Puromycin dihydrochloride(SIGMA, cat# P9620-10ML).

[0066] (3)Analysis Genomic DNA was extracted from patients' peripheral blood using a QIAAmp DNA Mini Kit (QIAGEN). Genomic DNA libraries were prepared using the Ion AmpliSeq Library Kit and the Ion Ampliseq Cardiovascular Research Panel (10,430 PCR amplicons covering 404 genes known to harbor mutations affecting cardiovascular function), and sequencing was performed using an Ion PGM with a 318 chip. Sequencing data were analyzed using TorrentSuite (version 5.2.2, Life Technologies). Variants with a low quality score of <30 or a low read depth of <30 were excluded. Synonymous mutations without amino acid changes were excluded. Variants were classified as benign if they were present at an allele frequency of >1% in the Human Genetic Variation Database (HGMD) or ESP 6500 databases. The heterozygous frameshift mutation in PKP2 (c.1228 dupG, p.D410fs) had not been reported in any of the HGMD, ESP 6500, 1000 Genomes databases, or the ARVD / C genetic variation database.

[0067] (4) Cell culture and cardiomyocyte differentiation HEK293T cells were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and penicillin / streptomycin / glutamine (PSG, Gibco).

[0068] iPSCs were established from peripheral blood mononuclear cells (PBMCs) of a patient diagnosed with AC (arterial vascular venous cytopathy) with the 1228 dupG mutation (diagnosed as ARVC). PBMCs were isolated from peripheral whole blood using Ficoll-Paque (GE). Reprogramming was performed using a Sendai virus vector containing OCT3 / 4, SOX2, KLF4, and c-MYC (CytoTune-iPS 2.0 Sendai Reprogramming Kit, Life Technologies). iPSCs were cultured under feeder-free conditions on laminin-coated plates using StemFit AK02N (AJINOMOTO) (Nakagawa, M. et al., A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells, Sci Rep 4, 3594, doi:10.1038 / srep03594 (2014)). iPSCs were differentiated into cardiomyocytes according to the protocol of Burridge et al. (Burridge, PW et al., Chemically defined generation of human cardiomyocytes, Nat Methods 11, 855-860, doi:10.1038 / nmeth.2999 (2014)) and maintained in RPMI 1640 medium supplemented with human albumin and ascorbic acid. For serial observation experiments using cardiomyocytes differentiated from HDR, NHEJ, and hetero-iPSCs (iPSC-CMs), differentiated monolayer cardiomyocytes were cultured for 10-12 days and purified in medium containing 5 mM sodium DL-lactate (Sigma-Aldrich) for 2-4 days. On day 14, the medium was then replaced with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2 mM L-glutamine (PSG, Gibco, Thermo Fisher Scientific).For immunostaining experiments and subsequent high-content imaging, differentiated iPSC-CMs were trypsinized, replated onto 96-well clear plates (Greiner) precoated with gelatin (Nitta Gelatin), and incubated with serum-containing DMEM.

[0069] (5) Immunofluorescence staining iPSCs were seeded at 1,000 cells / well into 96-well clear plates (Greiner) and incubated at 37°C for colony formation. Differentiated iPSC-CMs were treated with 0.25% trypsin-EDTA, suspended in DMEM containing 10% FBS, PSG, and 10 μM Y-27632 (Wako), and filtered through a 100 μm cell strainer (Falcon). Cardiomyocytes were seeded at 10,000 cells / well into 96-well clear plates precoated with gelatin (Nitta Gelatin). For immunostaining, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, and blocked with 1% BSA for 30 minutes at room temperature or overnight at 4°C. Primary antibodies were diluted in 1% bovine serum albumin (BSA) and added to each well. The cells were incubated for 1 hour at room temperature or overnight at 4°C. Secondary antibodies conjugated with Alexa Fluor Dyes (Molecular Probes) including Hoechst 33342 for nuclear staining or Alexa 568-conjugated phalloidin (Thermo) were added and incubated for 30 min at room temperature. All images were acquired using an IN Cell Analyzer 6000 (GE Healthcare).

[0070] (6) Single-strand annealing assay The target genomic sequence was cloned into the multiple cloning site (MCS) located in the middle of the EGFP sequence of the pCAG-EGxxFP vector (Mashiko, D. et al., Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA, Sci Rep 3, 3355, doi:10.1038 / srep03355 (2013)). When the target genomic sequence is cleaved by the gRNA and CAS9 complex, the EGFP sequence is restored, causing the cells to emit green fluorescence. The pCAG-EGxxFP vector and the pX459 vector encoding SpCas9 and the designated sgRNA (Ran, FA et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308, doi:10.1038 / nprot.2013.143 (2013)) were cultured in a Greiner CELLSTAR 96-well plate (1 × 10 4 293T cells pre-seeded at 1000 x g (1000 x g, ...

[0071] (7) Cel-I assay HEK293T cells were seeded in 24-well plates (5 × 10 4Cells were transfected with the pX459 vector using Lipofectamine 3000 (Life Technologies) one day later. Two days after transfection, the medium was replaced with medium containing 1.0 μg / mL puromycin to select for cells expressing Cas9. After puromycin selection, genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN). PCR (KOD Fx Neo, TOYOBO) was performed to amplify the target region using the following conditions: 94°C for 2 minutes, followed by 33 cycles of 98°C for 30 seconds, annealing temperature depending on the primers for 30 seconds, and 68°C for 30 seconds. After purifying the PCR product using a QIAquick PCR Purification Kit (QIAGEN), the PCR fragments from both the untreated and treated alleles were hybridized to form heteroduplex DNA. The hybridized PCR heteroduplexes were then enzymatically digested with the mismatch-specific endonuclease Cel-I at 42°C for 60 min (SURVEYOR Mutation Detection Kit) and subjected to electrophoresis.

[0072] (8) Plasmid A gRNA sequence targeting the genomic region surrounding the 1228 dupG mutation in PKP2 was designed using the CRISPR Design Tool and cloned into the pX459 vector. The DNA sequence of the 5' and 3' homology arms surrounding the 1228 dupG mutation in the PKP2 gene was amplified from wild-type genomic DNA and cloned into the pCR bluntII-TOPO vector (Thermo). The sequence encoding full-length human PKP2 was subcloned from an ORF clone (Dharmacon) into the pENTR / D-TOPO vector (Thermo). For expression in cultured cells, the PKP2 sequence was recombined into pcDNA3.1 / nV5-DEST (Thermo) using the Gateway system (Invitrogen). To generate an N-terminal FLAG-tagged protein, a FLAG epitope was inserted before the coding sequence by PCR-based mutagenesis. For AAV production, the N-terminal FLAG-tagged full-length PKP2 sequence was subcloned into the pAAV vector (TaKaRa).

[0073] (9) AAV production and purification To generate AAV2, HEK293T cells were transfected with pAAV vectors encoding N-terminally FLAG-tagged PKP2, pHelper vector, and pRC6 vector (AAVpro Helper-Free System, TaKaRa) using calcium phosphate transfection (CalPhos Mammalian Transfection Kit, TaKaRa). Seventy-two hours after transfection, HEK293T cells were detached by adding 1 / 80 volume of 0.5 M EDTA (pH 8.0) and pelleted by low-speed centrifugation (2000 × g, 10 min). The cell pellet was lysed in AAV Extraction Solution A and centrifuged (9000 × g, 10 min). The supernatant was collected, added to AAV Extraction Solution B, and stored at -80°C. AAV recovered from HEK293T cells was purified using the AAVpro Purification Kit (TaKaRa), and viral titers were measured using the AAV Titration Kit (TaKaRa).

[0074] (10) RNA extraction and quantitative real-time PCR Total RNA was extracted using the RNeasy mini kit (QIAGEN) and converted to cDNA using the high-capacity RNA-to-cDNA RT kit (Thermo). Quantitative real-time PCR was performed using Sybr Green or the probe method (THUNDERBIRD SYBR, probe qPCR mix, TOYOBO). All samples were run in duplicate. The level of each transcript was quantified by the threshold cycle (Ct) method using TBP or GAPDH as an internal control.

[0075] (11) Droplet digital PCR and quantitative real-time PCR Droplet digital PCR (ddPCR) was performed using a QX200 ddPCR system (BIORAD). To specifically detect transcripts from the wild-type or 1228 dupG allele of PKP2, HEX- or FAM-labeled probes were designed (see Figure 6) (Assay ID: dMDS329472318, BIORAD). To detect both the wild-type and 1228 dupG transcripts, a pre-made FAM-labeled probe for PKP2 was used (see Figure 6) (Assay ID: qHsaCIP0027871, BIORAD). Following PCR, the resulting droplets were detected and analyzed using a QX200 droplet reader (BIORAD). TBP was used as an internal control (Assay ID: dHsaCPE5058363, BIORAD).

[0076] (12) Transfection of plasmids into human iPSCs and selection of target clones The genome editing plasmid construct was transfected into iPSCs using a modified method described by Li et al. (Li, HL et al. Precise correction of the dystrophin gene in Duchenne muscular dystrophy patient-induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports 4, 143-154, doi:10.1016 / j.stemcr.2014.10.013 (2015)). Five micrograms of pX459 plasmid was transfected into 1 × 10 iPSCs using a NEPA21 electroporator. 5Cells were electroporated (poling pulse voltage: 125 V, pulse width: 5 ms, pulse number: 2, Nepagene). For HDR-mediated genome editing, 5 μg of repair template DNA plasmid (pCR bluntII-TOPO vector) was additionally transfected. Puromycin (0.3 μg / mL) was added within 48 hours after electroporation. Three days after transfection, iPSCs were passaged at a density of 200 cells into 35 mm dishes for clonal colony formation. Genomic DNA was extracted simultaneously, and genome editing results were evaluated by direct sequencing. After iPSC colony formation, individual colonies (at least 24 colonies) were picked and dispersed into single cells in a sterile tube. For both genotyping and cell expansion, the cell suspension was divided into two 96-well plates. Genomic DNA was extracted, and the target genomic region was amplified by PCR and cloned into the pCR bluntII-TOPO vector, followed by direct sequencing or sequence analysis. To obtain targeted single-clonal iPSCs, cells were repeatedly passaged into new dishes to form clonal colonies.

[0077] (13) Western blotting For Western blotting, cells were washed with cold PBS and directly lysed in SDS buffer (10% SDS, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA). Protein concentration was measured using a BCA Protein Assay Kit (Thermo). Cell lysates were mixed with 4x Laemmli sample buffer (BIORAD) containing 2.5% mercaptoethanol. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Antibodies were diluted in 3% nonfat milk. After blocking with 3% nonfat milk for 1 hour, the membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 30 minutes at room temperature. Membrane signals were detected by chemiluminescence using ECL or ECL Prime Reagent (GE). Protein expression levels were quantified using an ImageQuant TL (GE). The expression levels of each protein were normalized by the expression level of GAPDH.

[0078] (14)Cell movement analysis Cell movement profiles of cardiomyocytes (iPSC-CMs) differentiated from iPSCs were acquired using a Cell Motion Imaging System (SI8000, SONY). Videos were recorded with a 4x objective at a frame rate of 150 fps and a resolution of 1024 x 1024 pixels. Videos were acquired from at least three fields of three wells of each isogenic iPSC-CM cultured in a 6-well plate. For each image, motion parameters were calculated from nine regions of interest (ROIs) of 64 x 64 pixels. Data were acquired from three independent differentiation experiments. Fixed positions, defined as the X and Y axes, were continuously observed during the observation period. Maximum contraction velocity (CV), relaxation velocity (RV), and mean deformation distance (DD) during the contraction-relaxation process were calculated as the total area under the CV and RV peaks. CV and DD, calculated by the motion vector, represent contractile function or contractile force, respectively. Color mapping images visualized the motion propagation of contracting iPSC-CMs.

[0079] (15) Statistical analysis Unless otherwise stated, data are presented as the mean ± standard deviation from at least three independent experiments. Statistical analysis was performed using JMP (SAS, Cary, NC) by either Student's t-test or Wilcoxon test for nonparametric data. p values ​​<0.05 were considered statistically significant.

[0080] [Experiment 1: Establishment of iPSCs from an AC patient with a heterozygous frameshift mutation in PKP2] A 19-year-old female patient who was resuscitated after out-of-hospital cardiac arrest due to ventricular fibrillation had a family history of arrhythmias in her father and uncle (Figure 1(A)). This patient was diagnosed with AC (diagnosis: ARVC) based on three major criteria: repolarization abnormalities, arrhythmias, and a family history with a pathogenic mutation in the PKP2 gene. Echocardiography of the patient showed no obvious right ventricular enlargement, and the ejection fraction and left ventricular diameter were within normal limits (Figure 1(B)).

[0081] Genomic DNA was extracted from the patient's peripheral blood and subjected to direct Sanger sequencing to determine the presence of genetic mutations associated with hereditary cardiovascular disease. Using the Ion Ampliseq Cardiovascular Research Panel, a total of 404 genes associated with hereditary cardiovascular disease were screened, and a previously unreported heterozygous frameshift mutation (c.1228dupG, pD410fsX425) in the PKP2 gene was identified (Figure 2). The frameshift mutation of aspartic acid (D410fs) results in an amino acid stop at residue 425 (X425). No deleterious mutations were detected in other desmosomal genes (DSC2, DSG2, JUP, or DSP) reported as causative genes for AC. This heterozygous frameshift mutation (c.1228dupG) in the PKP2 gene was also present in the patient's father.

[0082] iPSCs were established from peripheral blood mononuclear leukocytes obtained from the patient. The established iPSCs were positive for the pluripotency marker proteins SSEA4, TRA-1-60, OCT4, and NANOG (Figure 3) and possessed a normal karyotype (Figure 4).

[0083] Direct Sanger sequencing was performed using cDNA obtained by reverse transcription from genomic DNA and total RNA from patient-derived iPSCs. The results are shown in Figure 5. The electropherogram of the genomic DNA showed dual signals representing the wild-type and mutant alleles, whereas the electropherogram of the cDNA showed only a signal representing the wild-type allele. This suggests that the transcript from the mutant allele was unstable and degraded.

[0084] Cell lysates were prepared from patient-derived iPSCs and iPSCs established from healthy subjects (controls), and the expression levels of PKP2 protein were detected by Western blotting. The results are shown in Figure 6. The expression level of PKP2 protein was shown to be reduced in patient-derived iPSCs.

[0085] To accurately assess the amount of transcripts produced by each allele in patient-derived iPSCs and cardiomyocytes differentiated from those iPSCs (iPSC-CMs), droplet digital PCR (ddPCR) analysis was performed. The results are shown in Figures 8 and 9. The transcript copy number of the mutant allele (1228 dupG) in the patient-derived iPSCs was shown to be reduced to 27% of the transcript copy number of the wild-type allele.

[0086] Patient-derived iPSCs were differentiated into cardiomyocytes, and the percentage of troponin T-positive cells was measured by FACS analysis. The results are shown in Figure 10. Ten days after differentiation induction, approximately 80-90% of the cells were troponin T-positive cardiomyocytes.

[0087] Figure 11 shows the relative copy numbers of transcripts generated by each allele in patient-derived iPSCs and cardiomyocytes differentiated from them (iPSC-CMs). The transcript copy number of the wild-type allele in iPSC-CMs on day 10 after differentiation was approximately 30-fold higher than that in undifferentiated iPSCs. The increase in the transcript of the mutant allele (1228 dupG) between undifferentiated iPSCs and iPSC-CMs on day 10 after differentiation remained low. In differentiated iPSC-CMs, the transcript copy number of the wild-type allele was approximately 10-fold higher than that of the mutant allele. This suggests that the transcript of the mutant allele (1228 dupG) is unstable and that the absolute difference between the transcripts of the wild-type and mutant alleles (1228 dupG) increases in differentiated cardiomyocytes, which require high expression of PKP2.

[0088] [Experiment 2: Creation of isogenic iPSC sets] We designed four gRNAs targeting the flanking regions of the PKP2 mutation (1228 dupG) in exon 5 of the PKP2 gene to correct the mutant frameshift allele by homology-directed repair (HDR) and introduce a homozygous frameshift mutation by non-homologous end joining (NHEJ) in patient-derived iPSCs (Figure 12). gRNA#1 used the mutant AGG sequence as a PAM sequence, and gRNA#2 was designed to contain a 20-bp sequence corresponding to the mutant sequence in its 5' region. gRNA#3 and #4 were designed to target both the wild-type and mutant alleles.

[0089] The cleavage activity of each gRNA was evaluated by single-strand annealing (SSA) assay using the pCAG-EGxxFP and pX459 vectors in HEK293T cells. The results are shown in Figure 13. (A) shows the cleavage results for the wild-type allele, and (B) shows the cleavage results for the mutant allele. gRNA#1 specifically cleaved the mutant sequence containing the duplicated G but did not cleave the wild-type sequence. The cleavage activity of gRNA#2 was lower than that of gRNA#1. The cleavage activity of gRNA#3 and #4 was higher than that of gRNA#4.

[0090] The cleavage activity of gRNAs #1 and #4 targeting the endogenous PKP2 locus in HEK293T cells was evaluated using a Cel-I assay. The results are shown in Figure 14. The target sequence in the endogenous PKP2 locus in HEK293T cells was cleaved by gRNA #4 but not by gRNA #1. Patient-derived iPSCs were transfected with a px459 vector encoding gRNA #1, and Sanger sequencing analysis revealed that the genomic break was specifically introduced at the mutation site containing the 1228 dupG sequence. Based on these results, gRNAs #1 and #4 were selected to replace the target genome of the PKP2 gene in patient-derived iPSCs.

[0091] To introduce a homozygous frameshift mutation mimicking the patient's mutated sequence into the PKP2 gene by nonhomologous end joining (NEHJ), we transfected the pX459 vector encoding gRNA#4 into patient-derived iPSCs. After several rounds of sib selection, we isolated an iPSC clone containing a homozygous frameshift allele and named it NEHJ (Figure 15). The NHEJ clone carried a homozygous 31-bp deletion in both alleles and was expected to produce a shortened PKP2 protein of 411 amino acids (Figure 16). Next, we generated a repair template DNA containing the wild-type sequence, with a 1035-bp 5' homology arm and a 497-bp 3' homology arm positioned before and after the 1228G in PKP2, to replace the mutated sequence by homology-directed repair (HDR) (Figure 17). The repair template vector and the pX459 vector encoding gRNA#1 were combined and transfected into patient-derived iPSCs. By repeated sib selection, we isolated iPSC clones containing homozygous wild-type alleles, designated "HDR." During the same sib selection procedure, we also isolated control iPSC clones carrying a heterozygous frameshift mutation in PKP2, designated "Hetero." These iPSC clones exhibited uniformly round colonies, expressed pluripotency markers, and displayed a normal karyotype.

[0092] To evaluate the transcript levels in each iPSC clone, quantitative real-time PCR analysis was performed using a common probe targeting both wild-type and mutant transcripts (see Figure 7). The results are shown in Figure 18. The relative mRNA expression levels of the PKP2 gene in HDR clones were restored from the heterozygote clones and were close to those of control iPSCs. Next, droplet digital PCR (ddPCR) analysis was performed, and the results are shown in Figure 19. The mutant transcript from the 1228dupG allele was completely reduced in both HDR and NHEJ clones, while the transcript from the wild-type allele in HDR clones was restored by exactly two-fold from the heterozygote clones. Furthermore, Western blotting analysis was performed, and the results are shown in Figure 20. PKP2 protein expression levels were undetectable in NHEJ clones but were restored in HDR clones. Plakoglobin, encoded by the JUP gene, desmoglein 2, encoded by the DSG2 gene, and desmocollin 2, encoded by the DSC2 gene, are major components of the desmosome structure, and the expression levels of these proteins have been reported to be reduced in cardiomyocytes differentiated from iPSCs of AC patients harboring PKP2 mutations and in the myocardium of AC patients harboring PKP2 mutations (Caspi, O. et al., Circ Cardiovasc Genet 6, 557-568, doi:10.1161 / CIRCGENETICS.113.000188 (2013); Ma, D. et al., Eur Heart J 34, 1122-1133, doi:10.1093 / eurheartj / ehs226 (2013); Rasmussen, T.B. et al., Circ Cardiovasc Genet 7, 230-240, doi:10.1161 / CIRCGENETICS.113.000338 (2014)). However, in undifferentiated isogenic iPSCs (hetero, HDR, and NHEJ), the expression levels of all proteins were not affected (Figure 20). Furthermore, the cellular localization of all proteins was not affected.

[0093] [Experiment 3: Evaluation of cardiomyocytes (iPSC-CMs) differentiated from each isogenic iPSC (cell motility analysis)] The experimental scheme is shown in Figure 21. Spontaneous beating was observed in cardiomyocytes induced to differentiate in monolayers from each isogenic iPSC (hetero, HDR, NHEJ) 7-8 days after the start of differentiation induction. The differentiation efficiency of the cells 7-8 days after the start of differentiation induction was evaluated by FACS analysis using an anti-troponin T antibody, and the results are shown in Figure 22. Differentiation efficiency was comparable between these iPSC-CMs.

[0094] Figure 23 shows the results of observations of HDR-iPSC-CMs and NHEJ-iPSC-CMs on days 8–10 after the start of differentiation induction. Connected layer structures and coordinated dynamic contractions were observed in HDR-iPSC-CMs. In contrast, hole-like defects appeared in connecting cardiomyocytes in NHEJ-iPSC-CMs, gradually increasing from day 8 to day 10. Ripped cardiomyocytes were detected in NHEJ-iPSCs (Figure 24), suggesting that cell-cell adhesions were weakened under increased contractile tension.

[0095] Contraction velocity (CV) and displacement distance (DD) calculated by motion vector analysis represent contractile function or contractile force, respectively, enabling real-time evaluation of the motility characteristics of cultured iPSC-CMs. Therefore, we continuously observed the contractile movement of HDR-iPSC-CMs and NHEJ-iPSC-CMs at the same position at specific coordinates in a 6-well plate. The results are shown in Figures 25 and 26. The contractile activity of HDR-iPSC-CMs was maintained, and both CV and DD remained unchanged during the observation period from days 14 to 28. The HDR-iPSC-CMs exhibited oriented contraction of cardiomyocytes from days 14 to 28 (Figure 27). This suggests that mechanical stress promoted cardiomyocyte maturation. In contrast, the CV and DD of NHEJ-iPSC-CMs were reduced compared to HDR-iPSC-CMs at day 14. Furthermore, the CV and DD of NHEJ-iPSC-CMs themselves gradually decreased from days 14 to 28.

[0096] Motion vector analysis converts the amplitude of movement into a color map, enabling label-free detection of excitation propagation. Directional excitation propagation was first observed through the fiber structure in NHEJ-iPSC-CMs at day 14. However, under continuous contractile tension, directional propagation was gradually impaired from day 21 to day 28 (Figure 28). Occasionally, conduction block was observed in NHEJ-iPSC-CM myofibers (Figure 29). These data suggest that NHEJ-iPSC-CMs exhibited a distinct phenotype, including impaired cardiomyocyte maturation, reduced contractile properties, and progressive conduction defects within 4 weeks of differentiation.

[0097] [Experiment 4: Histological comparison of cardiomyocytes differentiated from HDR and NHEJ] The experimental scheme is shown in Figure 30. HDR-iPSC-CMs and NHEJ-iPSC-CMs were replated onto 96-well plates on day 10 after the start of differentiation induction. The results of immunostaining with anti-troponin T antibody on day 16 are shown in Figure 31. The area of ​​troponin T-positive cardiomyocytes in NHEJ-iPSC-CMs was significantly reduced compared to HDR-iPSC-CMs.

[0098] Figure 32 shows the results of quantitative real-time PCR performed on HDR-iPSC-CMs and NHEJ-iPSC-CMs 10 days after the start of differentiation induction. The mRNA expression levels of NPPB (natriuretic peptide precursor B), MYL2 (myosin light chain 2), and MYH7 (myosin heavy chain 7), which are marker genes for cardiomyocyte maturation, were significantly reduced in NHEJ-iPSC-CMs. However, the levels of TNNT2 (troponin T2) and TNNI3 (troponin I3) were comparable between NHEJ-iPSC-CMs and HDR-iPSC-CMs. These results suggest that cardiomyocyte maturation is significantly impaired in NHEJ-iPSC-CMs.

[0099] Desmosomes and focal adhesions connect cardiomyocytes, mediate the intermediate filament network between cardiomyocytes, and influence cardiomyocyte maturation through mechanotransduction. PKP2 is a scaffolding protein that integrates proteins constituting desmosomes and focal adhesions. Therefore, we evaluated the expression and cellular localization of desmosome and focal adhesion proteins by Western blotting using NHEJ-iPSC-CMs and HDR-iPSC-CMs at days 14 and 28 after the start of differentiation induction, and immunostaining using NHEJ-iPSC-CMs and HDR-iPSC-CMs at day 16 after the start of differentiation induction. Western blotting results are shown in Figure 33. Plakoglobin immunostaining results are shown in Figure 34. Plakophilin-2 (PKP2) immunostaining results are shown in Figure 35. The results of immunostaining for desmoglein-2 and desmocollin-2 are shown in Figure 36. The results of immunostaining for N-cadherin are shown in Figure 37.

[0100] Plakoglobin is an anchor protein that connects desmosomal cadherins to desmoplakin and is expressed in both desmosomes and contact zones. Figures 33, 34, and 35 show that plakophilin 2 expression was completely abolished in NHEJ-iPSC-CMs, but the expression level and localization of plakoglobin in NHEJ-iPSC-CMs were not significantly different from those in HDR-iPSC-CMs.

[0101] Desmoglein 2 and desmocollin 2 are desmosomal cadherins that form homopolymers and heteropolymers with their C-terminal tails located in the intercellular space and in the cytoplasm bound to plakophilin-2. Both desmoglein 2 and desmocollin 2 were normally expressed at the intercellular junctions of HDR-iPSC-CMs. In contrast, in NHEJ-iPSC-CMs, the expression levels of these desmosomal proteins were significantly reduced (Figure 33), completely shed from the cell periphery, and scattered throughout the cytoplasm (Figure 36).

[0102] Focal adhesion junctions extend from the extracellular space to cytoskeletal actin filaments, and the junction complex contains transmembrane proteins primarily composed of N-cadherin. At 28 days after the start of differentiation, N-cadherin expression was reduced in NHEJ-iPSC-CMs compared to HDR-iPSC-CMs (Figure 33). Immunostaining revealed that N-cadherin expression was detected at cell-cell junctions but was substantially reduced in NHEJ-iPSC-CMs (Figure 37). These results suggest that PKP2 deficiency severely affects the stability of desmosomal cadherin proteins in NHEJ-iPSC-CMs, partially affecting the composition of focal adhesions in NHEJ-iPSC-CMs and impairing cardiac cell maturation.

[0103] [Experiment 5: Histological comparison of cardiomyocytes differentiated from HDR and Hetero mice] In contrast to NHEJ-iPSC-CMs, which rapidly exhibited distinct phenotypes after differentiation (see Figures 23 and 24), Hetero-iPSC-CMs did not exhibit significant morphological differences compared to HDR-iPSC-CMs over the same time course up to day 28. On day 14 after the start of differentiation induction, HDR-iPSC-CMs and hetero-iPSC-CMs were detached by trypsinization and replated in 24-well plates. Observations were continued until day 28. Thinning of the myocardial structure was observed in hetero-iPSC-CMs on day 28, whereas no such morphological abnormalities were observed in HDR-iPSC-CMs (Figure 38).

[0104] [Experiment 6: PKP2 gene delivery to cardiomyocytes differentiated from NHEJ] The experimental scheme is shown in Figure 39. An adeno-associated virus (AAV2-PKP2) encoding full-length human PKP2 with an N-terminal FLAG tag driven by a CMV promoter was generated. The AAV2 serotype was selected for the adeno-associated virus to efficiently transduce NHEJ-iPSC-CMs. NHEJ iPSCs were replated onto 96-well plates on day 10 after the start of differentiation induction, and transduced with AAV2-PKP2 on day 11 (upper panel of Figure 39, after day 10). Immunostaining with anti-FLAG and anti-troponin T antibodies on day 16 is shown in Figure 40. The transduced FLAG-tagged PKP2 protein was clearly localized to the periphery of NHEJ-iPSC-CMs.

[0105] AAV2 encoding EGFP (AAV2-EGFP) was used as a control. Cells were replated onto 96-well plates on day 10 after the start of differentiation induction and transfected with AAV2-PKP2 or AAV2-EGFP on day 11. Figure 41 shows the results of immunostaining with anti-desmoglein 2, anti-desmocollin 2, or anti-N-cadherin antibodies on day 16. Figure 42 shows the results of quantifying the expression levels of each protein using high-content imaging on the images obtained from the immunostained specimens in Figure 41. Transfection of NHEJ-iPSC-CMs with human PKP2 dose-dependently restored the expression levels and localization of desmoglein 2, desmocollin 2, and N-cadherin. Figure 43 shows the results of immunostaining with anti-troponin T antibodies on day 16. Transfection of NHEJ-iPSC-CMs with human PKP2 also restored the cardiomyocyte area.

[0106] The experiment was carried out according to the scheme shown in the lower part of Figure 39 from day 10 onwards. On day 10 after the start of differentiation induction, cells (NHEJ-iPSC-CM, approximately 1 × 10 6 cell / well), approximately 2.0 × 10 3NHEJ-iPSC-CMs were infected with either AAV2-PKP2 or AAV2-EGFP at 1000 μg / cell for gene transfer. Figure 44 shows the results of fluorescent and bright-field observations of AAV2-EGFP-infected NHEJ-iPSC-CMs on day 24. EGFP expression was confirmed by fluorescent microscopy. Figure 45 shows the results of Western blotting performed on NHEJ-iPSC-CMs on day 24 infected with AAV2-PKP2 or AAV2-EGFP. Introduction of human PKP2 into NHEJ-iPSC-CMs increased the expression of PKP2 (plakophilin 2), desmoglein 2, and desmocollin 2.

[0107] Figure 46 shows images of day 24 NHEJ-iPSC-CMs infected with AAV2-PKP2 or AAV2-EGFP, images of excitation propagation converted into color maps by motion vector analysis, and contraction velocity (CV) and displacement distance (DD) calculated by motion vector analysis. PKP2-transfected NHEJ-iPSC-CMs suppressed the formation of hole-like structures in contracting cardiomyocytes. Furthermore, the reduction in contraction velocity (CV) and displacement distance (DD) was restored. These results demonstrate that gene therapy using a normal PKP2 gene is effective in patients with AC caused by PKP2 gene mutations. Furthermore, these results suggest that NHEJ-iPSC-CMs can be a useful human model for therapeutic development and can be used to screen AC therapeutics.

[0108] Example 2: Establishment of iPSCs from an AC patient with a homozygous stop-gain mutation in DSG2 and generation of an isogenic iPSC set The use of samples obtained from patients and genomic analysis was approved by the Ethics Committee of Osaka University Hospital, and written informed consent was obtained.

[0109] [Experiment 7: Establishment of iPSCs from an AC patient with a homozygous stop-gain mutation in DSG2 and generation of isogenic iPSCs with a heterozygous mutation] A homozygous stop-gain mutation (a C355T base substitution resulting in a change of arginine at position 119 to a stop codon (R119X)) in the DSG2 gene was identified in a male patient with early-onset severe heart failure (diagnosed as dilated cardiomyopathy) who presented with treatment-resistant arrhythmias and progressive heart failure (Figure 47(A)). No other individuals in this patient's family had cardiomyopathy (Figure 47(B)). Sanger sequencing analysis of the genomic DNA of this patient and his parents revealed that both parents had a heterozygous C355T mutation, while the patient had a homozygous C355T mutation (Figure 47(C)).

[0110] Immunostaining of left ventricular myocardial tissue from this patient and other patients (controls) with dilated cardiomyopathy using anti-desmoglein 2 antibodies revealed a complete lack of DSG2 molecules in the patient's myocardial tissue (Figure 48). Transmission electron microscopy also revealed disruption of desmosome structures (Figure 49).

[0111] iPSCs (R119X-iPSCs) were established from peripheral blood mononuclear leukocytes of a patient with a homozygous C355T mutation using the same method as in Example 1. Compared to control iPSCs established from a healthy individual, R119X-iPSCs with a homozygous C355T mutation showed significantly reduced DSG2 mRNA expression (Figure 50(A)), and DSG2 protein expression was completely abolished (Figure 50(B)).

[0112] We designed a guide RNA targeting the DSG2 genomic sequence and introduced a synonymous substitution sequence into one allele of the DSG2 gene in homozygous C355T mutant iPSCs (R119X-iPSCs) by genome editing to establish isogenic iPSCs (HDR-iPSCs) in which the C355T mutation was repaired to heterozygosity (Figure 51). DSG2 protein expression was restored in HDR-iPSCs in which the C355T mutation was repaired to heterozygosity (Figure 52(A) and (B)).

[0113] To recapitulate myocardial tissue structure, we seeded R119X-iPSC-CMs and HDR-iPSC-CMs 14 days after differentiation induction and generated three-dimensional self-organized tissue rings (SOTRs) using spontaneously circulating traveling wave stimulation (Li, J. et al., doi:10.1101 / 717108 (2019)). Thirty days after reseeding, the contractile force of the SOTRs was measured using a MicroTester G2 compressive strength analyzer (CellScale). Compared to SOTRs derived from R119X-iPSC-CMs, SOTRs derived from HDR-iPSC-CMs showed significant recovery of myocardial contractile force (Figure 53).

[0114] The tissue formation process after reseeding was observed over time, and the myocardial structure was confirmed by troponin immunostaining 30 days after the start of differentiation induction (16 days after reseeding). R119X-iPSC-CMs showed impaired formation of continuous myocardial tissue structure, exhibiting a reticular myocardial morphology, whereas HDR-iPSC-CMs did not show similar changes (Figure 54).

[0115] Protein samples were collected from R119X-iPSC-CM and HDR-iPSC-CM on day 14 after the start of differentiation induction, and the expression of desmoglein 2 and desmocollin 2 was analyzed by Western blotting. Desmoglein 2 was not detected in R119X-iPSC-CM, and the expression level of desmocollin 2 was significantly lower than that of HDR-iPSC-CM (Figure 55). Furthermore, R119X-iPSC-CM and HDR-iPSC-CM on day 14 after the start of differentiation induction were replated in 96-well plates, and 7 days later, the cells were fixed and immunostained with anti-desmoglein 2 and anti-desmocollin 2 antibodies. Consistent with the Western blotting results, desmoglein 2 was not expressed in R119X-iPSC-CM, and the expression level of desmocollin 2 was significantly lower than that of HDR-iPSC-CM (Figure 56).

[0116] On day 14 after the start of differentiation induction, R119X-iPSC-CMs and HDR-iPSC-CMs were replated onto 96-well plates. 30 days later, the cells were fixed and observed under a transmission electron microscope. Normal desmosomal structures were observed in HDR-iPSC-CMs, whereas R119X-iPSC-CMs showed disrupted desmosomal structures (Figure 57).

[0117] [Experiment 8: DSG2 gene delivery into R119X-iPSC-CM] To test whether exogenous gene transfer could ameliorate the observed phenotype, we cloned the full-length human DSG2 gene and generated an adeno-associated virus (AAV2-DSG2-cHA) encoding full-length human DSG2 with a C-terminal HA tag driven by the CMV promoter.

[0118] On day 14 after the start of differentiation induction, R119X-iPSC-CMs were replated onto a 96-well plate, and 4 days later, 2.0 × 10 6 Transfection was performed using AAV2-DSG2-cHA at 1000 μg / cell. Seven days after infection, cells were fixed and immunostained with anti-desmoglein 2 antibody. In R119X-iPSC-CMs transfected with the DSG2 gene, DSG2 was localized to intercellular desmosomes (Figure 58).

[0119] 2.0 × 10 cells were added to R119X-iPSC-CMs 21 days after the start of differentiation induction. 5 vg / cell or 6.0 × 10 5 Cardiomyocytes were transfected with AAV2-DSG2-cHA at 1000 μg / cell. Seven days after infection, protein samples were collected from the cardiomyocytes and analyzed for Desmoglein 2 expression by Western blotting. Cardiomyocytes induced to differentiate from iPSCs established from healthy donors (Ctrl-iPSC-CM) served as a control. DSG2-transfected R119X-iPSC-CM expressed Desmoglein 2 at levels comparable to those observed in Ctrl-iPSC-CM (Figure 59).

[0120] R119X-iPSC-CMs were infected with AV2-DSG2-cHA on day 9 after the start of differentiation induction, replated on day 14 after the start of differentiation induction, and observed over time up to day 30. Figure 60 shows brightfield images taken on day 30 after the start of differentiation induction. Abnormal reticular myocardial morphology was observed in R119X-iPSC-CMs not transfected with the DSG2 gene, but such abnormal morphology was not observed in R119X-iPSC-CMs transfected with the DSG2 gene, indicating that DSG2 gene transfection suppresses damage to myocardial structure.

[0121] Example 3: Generation of a PKP2 gene mutant isogenic iPSC set capable of imaging the dynamics of desmoglein 2 protein In NHEJ-iPSC-CMs differentiated from isogenic iPSCs (NEHJ) containing a homozygous frameshift allele in the PKP2 gene prepared in Example 1, desmoglein 2 protein expression levels were significantly reduced (see Figure 33) and completely shed from the cell membrane (see Figure 36). Therefore, it was predicted that desmoglein 2 (DSG2) molecules would be a useful molecular marker related to the phenotype of this model cell. Therefore, to evaluate the dynamics of endogenous DSG2 molecules in more detail, we used genome editing technology to insert tdTomato fluorescent protein into the 3' end of each DSG2 gene in the PKP2 gene mutant isogenic iPSC sets (hetero-iPSCs, HDR-iPSCs, and NHEJ-iPSCs) prepared in Example 1.

[0122] [Experiment 9: Analysis of Desmoglein 2 Expression in Cardiomyocytes Differentiated from the Isogenic iPSC Set Prepared in Example 1] Desmoglein 2 immunostaining was performed on cardiomyocytes (hetero-iPSCs and HDR-iPSCs) derived from isogenic iPSCs containing a heterozygous frameshift allele of the PKP2 gene (hetero-iPSCs) and isogenic iPSCs containing a homozygous wild-type allele (HDR-iPSCs) at day 14 after differentiation induction (hetero-iPSC-CMs and HDR-iPSC-CMs) (Figure 61, top). DSG2 was normally distributed on the cell membrane in both hetero-iPSC-CMs and HDR-iPSC-CMs. However, quantitative analysis of the dotted DSG2 distribution using a high-content image analyzer (IN Cell Analyzer 6000, GE) revealed that the area of ​​individual desmosomes stained with DSG2 was significantly greater in HDR-iPSC-CMs than in hetero-iPSC-CMs (Figure 61, bottom, p<0.0001, n=32 for each). This confirms the usefulness of endogenous DSG2 as a molecular marker.

[0123] [Experiment 10: Insertion of tdTomato fluorescent protein into the 3' end of the DSG2 gene] As shown in Figure 62, a guide RNA specific to the 3' side of the DSG2 gene stop codon was designed and introduced by electroporation along with the repair template DNA. Because a single nucleotide polymorphism (SNP: C / T, a synonymous substitution encoding valine) was detected upstream of the DSG2 stop codon, a T was inserted at the SNP site in the 5' arm of the repair template to distinguish the genome-edited transcript. A CC-to-AA mutation was introduced in the 3' arm to prevent re-cutting by Cas9. Following colony selection, PCR analysis (Figure 63) and Sanger sequencing (Figure 64) of genomic DNA were performed. Following colony selection, isogenic iPSC sets were established in which the allele containing SNP: C remained wild-type and the allele containing SNP: T was heterozygously inserted with tdTomato (DSG2-tdT-Hetero-iPSC, DSG2-tdT-HDR-iPSC, and DSG2-tdT-NHEJ-iPSC) (Figure 65). The established isogenic iPSC set had a normal karyotype, and immunostaining confirmed the expression of undifferentiated markers (OCT4, SSEA4, NANOG) (Figure 66).

[0124] [Experiment 11: Confirmation of expression of DSG2-tdTomato fusion protein] Western blotting analysis (Figure 67(A)) and fluorescent imaging of Hoechst-stained nuclei (Figure 67(B)) confirmed that the DSG2-tdTomato fusion protein was equally expressed in pre-differentiation DSG2-tdT-Hetero-iPSCs, DSG2-tdT-HDR-iPSCs, and DSG2-tdT-NHEJ-iPSCs. Time-lapse imaging of live DSG2-tdT-HDR-iPSCs and DSG2-tdT-HDR-iPSC-CMs at day 14 after differentiation induction revealed that the DSG2-tdTomato fusion protein, which was localized at the cell periphery in iPSCs, displayed a dotted distribution after cardiac differentiation (Figure 68). On day 14 after the start of differentiation, DSG2-tdT-Hetero-iPSC-CM, DSG2-tdT-HDR-iPSC-CM, and DSG2-tdT-NHEJ-iPSC-CM were fixed and immunostained. In DSG2-tdT-Hetero-iPSC-CM and DSG2-tdT-HDR-iPSC-CM, desmosome signals were observed as dots on the cell membrane. In DSG2-tdT-NHEJ-iPSC-CM, the tdTomato fluorescent signal on the membrane disappeared upon differentiation (Figure 69). Live imaging of DSG2-tdT-Hetero-iPSC-CM and DSG2-tdT-HDR-iPSC-CM on day 14 after the start of differentiation induction was performed using a high-content image analyzer (IN Cell Analyzer 6000, GE) to quantify the area of ​​the DSG2-tdTomato fluorescent image localized in dots on the membrane. A significant increase was observed in DSG2-tdT-HDR-iPSC-CM compared to DSG2-tdT-Hetero-iPSC-CM (Figure 70).

[0125] [Experiment 12: PKP2 gene delivery into DSG2-tdT-NHEJ-iPSC-CMs] On day 14 after the start of differentiation induction, DSG2-tdT-NHEJ-iPSC-CMs were infected with an adeno-associated virus encoding full-length human PKP2 (AAV2-PKP2, see Example 1) to transduce the PKP2 gene, and daily time-lapse imaging was performed. Four days after AAV2-PKP2 infection, punctate accumulation of Desmoglein 2-tdTomato fusion protein was observed in the cell membrane (Figure 71). Introducing human PKP2 into DSG2-tdT-NHEJ-iPSC-CMs restored the expression level and localization of Desmoglein 2.

[0126] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

[Claim 1] A pharmaceutical for treating arrhythmogenic cardiomyopathy, the active ingredient of which is a gene therapy drug for delivering a normal gene corresponding to a gene having a gene mutation to the cardiomyocytes of a patient with arrhythmogenic cardiomyopathy caused by the gene mutation, thereby expressing a normal protein, wherein the gene mutation is a mutation in the desmoglein 2 gene, and the gene therapy drug is an adeno-associated virus vector into which DNA encoding normal human desmoglein 2 has an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 3 or has two or fewer amino acid substitutions.

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