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Jabri A, Taftafa B, Mhannayeh A, Alsharif M, Abbad T, Ahmed S, Alshehri EA, Elsalti A, Khan J, Mir TA, Yaqinuddin A. Cardiac Tissue Engineering for Translational Cardiology: From In Vitro Models to Regenerative Therapies. Bioengineering (Basel) 2025; 12:518. [PMID: 40428138 PMCID: PMC12109445 DOI: 10.3390/bioengineering12050518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2025] [Revised: 05/07/2025] [Accepted: 05/09/2025] [Indexed: 05/29/2025] Open
Abstract
Cardiovascular diseases (CVD) are the primary cause of death and disability around the world. Over the past decades, several conventional model systems based on two-dimensional (3D) monolayer cultures or experimental animals have been adopted to dissect and understand heart diseases in order to develop treatment modalities. However, traditional models exhibit several limitations in recapitulating human-specific key physiological and pathological characteristics, which highlights the necessity of developing physiologically relevant models. In recent years, tissue engineering approaches have been extensively employed to generate revolutionary three-dimensional (3D) cardiac models. In particular, the combined use of various bioengineering strategies and cellular reprogramming approaches has facilitated the development of various models. This review presents an overview of different approaches (bioprinting, scaffolding, and electrospinning) for creating bioengineered cardiac tissue models. Next, a broad survey of recent research related to the modeling of various cardiac diseases is presented. Finally, current challenges and future directions are proposed to foster further developments in the field of cardiac tissue engineering.
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Affiliation(s)
- Abdullah Jabri
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Bader Taftafa
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Abdulaziz Mhannayeh
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Mohamed Alsharif
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Tasnim Abbad
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Sana Ahmed
- Tissue/Organ Bioengineering & BioMEMS Laboratory, Organ Transplant Centre of Excellence (TR&I-Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia; (S.A.); (E.A.A.)
| | - Eman A. Alshehri
- Tissue/Organ Bioengineering & BioMEMS Laboratory, Organ Transplant Centre of Excellence (TR&I-Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia; (S.A.); (E.A.A.)
| | - Abdulrahman Elsalti
- International School of Medicine, Istanbul Medipol University, Istanbul 34810, Turkey;
| | - Jibran Khan
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
| | - Tanveer Ahmad Mir
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
- Tissue/Organ Bioengineering & BioMEMS Laboratory, Organ Transplant Centre of Excellence (TR&I-Dpt), King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia; (S.A.); (E.A.A.)
| | - Ahmed Yaqinuddin
- College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia; (A.J.); (B.T.); (A.M.); (M.A.); (T.A.); (J.K.)
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Okuno S, Higo S, Kondo T, Shiba M, Kameda S, Inoue H, Tabata T, Ogawa S, Morishita Y, Sun C, Ishino S, Honda T, Miyagawa S, Sakata Y. SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes. Sci Rep 2023; 13:21397. [PMID: 38049441 PMCID: PMC10696029 DOI: 10.1038/s41598-023-48084-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2023] [Accepted: 11/22/2023] [Indexed: 12/06/2023] Open
Abstract
Although an increased risk of myocarditis has been observed after vaccination with mRNA encoding severe acute respiratory syndrome coronavirus 2 spike protein, its underlying mechanism has not been elucidated. This study investigated the direct effects of spike receptor-binding domain (S-RBD) on human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs). Immunostaining experiments using ACE2 wild-type (WT) and knockout (KO) iPSC-CMs treated with purified S-RBD demonstrated that S-RBD was bound to ACE2 and internalized into the subcellular space in the iPSC-CMs, depending on ACE2. Immunostaining combined with live cell imaging using a recombinant S-RBD fused to the superfolder GFP (S-RBD-sfGFP) demonstrated that S-RBD was bound to the cell membrane, co-localized with RAB5A, and then delivered from the endosomes to the lysosomes in iPSC-CMs. Quantitative PCR array analysis followed by single cell RNA sequence analysis clarified that S-RBD-sfGFP treatment significantly upregulated the NF-kβ pathway-related gene (CXCL1) in the differentiated non-cardiomyocytes, while upregulated interferon (IFN)-responsive genes (IFI6, ISG15, and IFITM3) in the matured cardiomyocytes. S-RBD-sfGFP treatment promoted protein ISGylation, an ISG15-mediated post-translational modification in ACE2-WT-iPSC-CMs, which was suppressed in ACE2-KO-iPSC-CMs. Our experimental study demonstrates that S-RBD is internalized through the endolysosomal pathway, which upregulates IFN-responsive genes and promotes ISGylation in the iPSC-CMs.
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Affiliation(s)
- Shota Okuno
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shuichiro Higo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
| | - Takumi Kondo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Mikio Shiba
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Satoshi Kameda
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Hiroyuki Inoue
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Tomoka Tabata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shou Ogawa
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yu Morishita
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Congcong Sun
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Saki Ishino
- CoMIT Omics Center, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Tomoyuki Honda
- Department of Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-Ku, Okayama, 700-8558, Japan
- Department of Virology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Kita-Ku, Okayama, 700-8558, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yasushi Sakata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
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Petit C, Bonnet C, Safieddine S. Deafness: from genetic architecture to gene therapy. Nat Rev Genet 2023; 24:665-686. [PMID: 37173518 DOI: 10.1038/s41576-023-00597-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/27/2023] [Indexed: 05/15/2023]
Abstract
Progress in deciphering the genetic architecture of human sensorineural hearing impairment (SNHI) or loss, and multidisciplinary studies of mouse models, have led to the elucidation of the molecular mechanisms underlying auditory system function, primarily in the cochlea, the mammalian hearing organ. These studies have provided unparalleled insights into the pathophysiological processes involved in SNHI, paving the way for the development of inner-ear gene therapy based on gene replacement, gene augmentation or gene editing. The application of these approaches in preclinical studies over the past decade has highlighted key translational opportunities and challenges for achieving effective, safe and sustained inner-ear gene therapy to prevent or cure monogenic forms of SNHI and associated balance disorders.
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Affiliation(s)
- Christine Petit
- Institut Pasteur, Université Paris Cité, Inserm, Institut de l'Audition, F-75012, Paris, France.
- Collège de France, F-75005, Paris, France.
| | - Crystel Bonnet
- Institut Pasteur, Université Paris Cité, Inserm, Institut de l'Audition, F-75012, Paris, France
| | - Saaïd Safieddine
- Institut Pasteur, Université Paris Cité, Inserm, Institut de l'Audition, F-75012, Paris, France
- Centre National de la Recherche Scientifique, F-75016, Paris, France
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Tejero M, Duzenli OF, Caine C, Kuoch H, Aslanidi G. Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors. Genes (Basel) 2023; 14:1866. [PMID: 37895215 PMCID: PMC10606878 DOI: 10.3390/genes14101866] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Revised: 09/18/2023] [Accepted: 09/21/2023] [Indexed: 10/29/2023] Open
Abstract
The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.
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Affiliation(s)
- Marcos Tejero
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Ozgun F. Duzenli
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Colin Caine
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - Hisae Kuoch
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
| | - George Aslanidi
- Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55455, USA; (M.T.)
- Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
- Institute Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA
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Kar A, Gupta S, Matilal A, Kumar D, Sarkar S. Nanotherapeutics for the Myocardium: A Potential Alternative for Treating Cardiac Diseases. J Cardiovasc Pharmacol 2023; 82:180-188. [PMID: 37341530 DOI: 10.1097/fjc.0000000000001444] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Accepted: 06/03/2023] [Indexed: 06/22/2023]
Abstract
ABSTRACT Cardiovascular diseases (CVDs) are the foremost cause of morbidity and mortality worldwide. Current clinical interventions include invasive approaches for progressed conditions and pharmacological assistance for initial stages, which has systemic side effects. Preventive, curative, diagnostic, and theranostic (therapeutic + diagnostic) approaches till date are not very useful in combating the ongoing CVD epidemic, which demands a promising efficient alternative approach. To combat the growing CVD outbreak globally, the ideal strategy is to make the therapeutic intervention least invasive and direct to the heart to reduce the bystander effects on other organs and increase the bioavailability of the therapeutics to the myocardium. The application of nanoscience and nanoparticle-mediated approaches have gained a lot of momentum because of their efficient passive and active myocardium targeting capability owing to their improved specificity and controlled release. This review provides extensive insight into the various types of nanoparticles available for CVDs, their mechanisms of targeting (eg, direct or indirect), and the utmost need for further development of bench-to-bedside cardiac tissue-based nanomedicines. Furthermore, the review aims to summarize the different ideas and methods of nanoparticle-mediated therapeutic approaches to the myocardium till date with present clinical trials and future perspectives. This review also reflects the potential of such nanoparticle-mediated tissue-targeted therapies to contribute to the sustainable development goals of good health and well-being.
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Affiliation(s)
- Abhik Kar
- Department of Zoology, University of Calcutta, Kolkata, West Bengal, India
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Higo S. Disease modeling of desmosome-related cardiomyopathy using induced pluripotent stem cell-derived cardiomyocytes. World J Stem Cells 2023; 15:71-82. [PMID: 37007457 PMCID: PMC10052339 DOI: 10.4252/wjsc.v15.i3.71] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 02/10/2023] [Accepted: 03/17/2023] [Indexed: 03/23/2023] Open
Abstract
Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation. Although optimized medical therapies have been developed for heart failure during the last few decades, some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies. Desmosome, which is a dynamic cell-to-cell junctional component, maintains the structural integrity of heart tissues. Genetic mutations in desmosomal genes cause arrhythmogenic cardiomyopathy (AC), a rare inheritable disease, and predispose patients to sudden cardiac death and heart failure. Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies. Among desmosomal genes, mutations in PKP2 (which encodes PKP2) are most frequently identified in patients with AC. PKP2 deficiency causes various pathological cardiac phenotypes. Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs) in combination with genome editing, which allows the precise arrangement of the targeted genome, are powerful experimental tools for studying disease. This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency.
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Affiliation(s)
- Shuichiro Higo
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita 565-0871, Japan
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Tabata T, Masumura Y, Higo S, Kunimatsu S, Kameda S, Inoue H, Okuno S, Ogawa S, Takashima S, Watanabe M, Miyagawa S, Hikoso S, Sakata Y. Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption. Biochem Biophys Res Commun 2022; 637:40-49. [PMID: 36375249 DOI: 10.1016/j.bbrc.2022.10.088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Accepted: 10/25/2022] [Indexed: 11/06/2022]
Abstract
Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.
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Affiliation(s)
- Tomoka Tabata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yuki Masumura
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shuichiro Higo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan; Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
| | - Suzuka Kunimatsu
- Department of Clinical Laboratory and Biomedical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Satoshi Kameda
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Hiroyuki Inoue
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shota Okuno
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shou Ogawa
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Seiji Takashima
- Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Mikio Watanabe
- Department of Clinical Laboratory and Biomedical Sciences, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shungo Hikoso
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yasushi Sakata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
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Nishiga M, Liu C, Qi LS, Wu JC. The use of new CRISPR tools in cardiovascular research and medicine. Nat Rev Cardiol 2022; 19:505-521. [PMID: 35145236 PMCID: PMC10283450 DOI: 10.1038/s41569-021-00669-3] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 12/21/2021] [Indexed: 02/07/2023]
Abstract
Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.
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Affiliation(s)
- Masataka Nishiga
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
| | - Chun Liu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA
| | - Lei S Qi
- Department of Bioengineering, Stanford University, Stanford, CA, USA
- Department of Chemical & Systems Biology, Stanford University, Stanford, CA, USA
- ChEM-H Institute, Stanford University, Stanford, CA, USA
| | - Joseph C Wu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
- Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
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Tsuji S, Stephens CJ, Bortolussi G, Zhang F, Baj G, Jang H, de Alencastro G, Muro AF, Pekrun K, Kay MA. Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice. Nat Biotechnol 2022; 40:1285-1294. [PMID: 35393561 PMCID: PMC11648996 DOI: 10.1038/s41587-022-01240-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Accepted: 01/28/2022] [Indexed: 12/12/2022]
Abstract
Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.
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Affiliation(s)
- Shinnosuke Tsuji
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Calvin J Stephens
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Giulia Bortolussi
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
| | - Feijie Zhang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Gabriele Baj
- Light Microscopy Imaging Center, Department of Life Sciences, University of Trieste, Trieste, Italy
| | - Hagoon Jang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | | | - Andrés F Muro
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
| | - Katja Pekrun
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Mark A Kay
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA.
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Abstract
Cardiovascular disease remains the leading cause of morbidity and mortality in the developed world. In recent decades, extraordinary effort has been devoted to defining the molecular and pathophysiological characteristics of the diseased heart and vasculature. Mouse models have been especially powerful in illuminating the complex signaling pathways, genetic and epigenetic regulatory circuits, and multicellular interactions that underlie cardiovascular disease. The advent of CRISPR genome editing has ushered in a new era of cardiovascular research and possibilities for genetic correction of disease. Next-generation sequencing technologies have greatly accelerated the identification of disease-causing mutations, and advances in gene editing have enabled the rapid modeling of these mutations in mice and patient-derived induced pluripotent stem cells. The ability to correct the genetic drivers of cardiovascular disease through delivery of gene editing components in vivo, while still facing challenges, represents an exciting therapeutic frontier. In this review, we provide an overview of cardiovascular disease mechanisms and the potential applications of CRISPR genome editing for disease modeling and correction. We also discuss the extent to which mice can faithfully model cardiovascular disease and the opportunities and challenges that lie ahead.
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Affiliation(s)
- Ning Liu
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas
| | - Eric N Olson
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas
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11
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Affiliation(s)
- Yanjiang Zheng
- Department of Biochemistry, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, China
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Nathan J. VanDusen
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Catalina E. Butler
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Qing Ma
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Justin S. King
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
| | - William T. Pu
- Department of Cardiology, Boston Children’s Hospital, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Cambridge, MA 02138, USA
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12
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Bijlani S, Pang KM, Sivanandam V, Singh A, Chatterjee S. The Role of Recombinant AAV in Precise Genome Editing. Front Genome Ed 2022; 3:799722. [PMID: 35098210 PMCID: PMC8793687 DOI: 10.3389/fgeed.2021.799722] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Accepted: 12/14/2021] [Indexed: 12/14/2022] Open
Abstract
The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.
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13
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Inoue H, Nakamura S, Higo S, Shiba M, Kohama Y, Kondo T, Kameda S, Tabata T, Okuno S, Ikeda Y, Li J, Liu L, Yamazaki S, Takeda M, Ito E, Takashima S, Miyagawa S, Sawa Y, Hikoso S, Sakata Y. Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes. Stem Cell Reports 2022; 17:337-351. [PMID: 35063130 PMCID: PMC8828557 DOI: 10.1016/j.stemcr.2021.12.016] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 12/20/2021] [Accepted: 12/21/2021] [Indexed: 12/13/2022] Open
Abstract
Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.
Generation of isogenic iPSC-CMs with a precise dose of plakophilin-2 Modeling reduced contractility and impaired desmosome assembly using iPSC-CMs Generation of isogenic iPSC-CMs for desmosome-imaging Proof of concept of PKP2 replacement using isogenic plakophilin-2-deficient iPSC-CMs
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Affiliation(s)
- Hiroyuki Inoue
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | | | - Shuichiro Higo
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
| | - Mikio Shiba
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yasuaki Kohama
- Cardiovascular Division, National Hospital Organization, Osaka-Minami Medical Center, Kawachinagano, Osaka 586-8512, Japan
| | - Takumi Kondo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Satoshi Kameda
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Tomoka Tabata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shota Okuno
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yoshihiko Ikeda
- Department of Pathology, National Cerebral and Cardiovascular Center, Suita, Osaka 564-8565, Japan
| | - Junjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan; Department of Design for Tissue Regeneration, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Li Liu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan; Department of Design for Tissue Regeneration, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Satoru Yamazaki
- Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center, Suita, Osaka 564-8565, Japan
| | - Maki Takeda
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Emiko Ito
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Seiji Takashima
- Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yoshiki Sawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shungo Hikoso
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yasushi Sakata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
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14
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Pavani G, Amendola M. Targeted Gene Delivery: Where to Land. Front Genome Ed 2021; 2:609650. [PMID: 34713234 PMCID: PMC8525409 DOI: 10.3389/fgeed.2020.609650] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Accepted: 12/16/2020] [Indexed: 12/12/2022] Open
Abstract
Genome-editing technologies have the potential to correct most genetic defects involved in blood disorders. In contrast to mutation-specific editing, targeted gene insertion can correct most of the mutations affecting the same gene with a single therapeutic strategy (gene replacement) or provide novel functions to edited cells (gene addition). Targeting a selected genomic harbor can reduce insertional mutagenesis risk, while enabling the exploitation of endogenous promoters, or selected chromatin contexts, to achieve specific transgene expression levels/patterns and the modulation of disease-modifier genes. In this review, we will discuss targeted gene insertion and the advantages and limitations of different genomic harbors currently under investigation for various gene therapy applications.
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Affiliation(s)
- Giulia Pavani
- INTEGRARE, UMR_S951, Genethon, Inserm, Univ Evry, Univ Paris-Saclay, Evry, France
| | - Mario Amendola
- INTEGRARE, UMR_S951, Genethon, Inserm, Univ Evry, Univ Paris-Saclay, Evry, France
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15
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Kalidasan V, Ng WH, Ishola OA, Ravichantar N, Tan JJ, Das KT. A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system. Sci Rep 2021; 11:19265. [PMID: 34584147 PMCID: PMC8478948 DOI: 10.1038/s41598-021-98657-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Accepted: 09/09/2021] [Indexed: 02/08/2023] Open
Abstract
Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.
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Affiliation(s)
- V. Kalidasan
- grid.11875.3a0000 0001 2294 3534Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia
| | - Wai Hoe Ng
- grid.11875.3a0000 0001 2294 3534Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia
| | - Oluwaseun Ayodeji Ishola
- grid.11875.3a0000 0001 2294 3534Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia ,Helmholtz Research Zentrum, Munich, Germany
| | - Nithya Ravichantar
- grid.11875.3a0000 0001 2294 3534Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia
| | - Jun Jie Tan
- grid.11875.3a0000 0001 2294 3534Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia
| | - Kumitaa Theva Das
- grid.11875.3a0000 0001 2294 3534Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Malaysia
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16
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Chingale M, Zhu D, Cheng K, Huang K. Bioengineering Technologies for Cardiac Regenerative Medicine. Front Bioeng Biotechnol 2021; 9:681705. [PMID: 34150737 PMCID: PMC8209515 DOI: 10.3389/fbioe.2021.681705] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 04/12/2021] [Indexed: 12/11/2022] Open
Abstract
Cardiac regenerative medicine faces big challenges such as a lack of adult cardiac stem cells, low turnover of mature cardiomyocytes, and difficulty in therapeutic delivery to the injured heart. The interaction of bioengineering and cardiac regenerative medicine offers innovative solutions to this field. For example, cell reprogramming technology has been applied by both direct and indirect routes to generate patient-specific cardiomyocytes. Various viral and non-viral vectors have been utilized for gene editing to intervene gene expression patterns during the cardiac remodeling process. Cell-derived protein factors, exosomes, and miRNAs have been isolated and delivered through engineered particles to overcome many innate limitations of live cell therapy. Protein decoration, antibody modification, and platelet membranes have been used for targeting and precision medicine. Cardiac patches have been used for transferring therapeutics with better retention and integration. Other technologies such as 3D printing and 3D culture have been used to create replaceable cardiac tissue. In this review, we discuss recent advancements in bioengineering and biotechnologies for cardiac regenerative medicine.
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Affiliation(s)
- Mira Chingale
- Department of Molecular Biomedical Sciences and Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States
| | - Dashuai Zhu
- Department of Molecular Biomedical Sciences and Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States
- Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, Raleigh, NC, United States
| | - Ke Cheng
- Department of Molecular Biomedical Sciences and Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States
- Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, Raleigh, NC, United States
| | - Ke Huang
- Department of Molecular Biomedical Sciences and Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States
- Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, Raleigh, NC, United States
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17
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Shiba M, Higo S, Kondo T, Li J, Liu L, Ikeda Y, Kohama Y, Kameda S, Tabata T, Inoue H, Nakamura S, Takeda M, Ito E, Takashima S, Miyagawa S, Sawa Y, Hikoso S, Sakata Y. Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent stem cell-derived cardiomyocytes. Hum Mol Genet 2021; 30:1384-1397. [PMID: 33949662 PMCID: PMC8283207 DOI: 10.1093/hmg/ddab127] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 04/09/2021] [Accepted: 04/28/2021] [Indexed: 01/09/2023] Open
Abstract
Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.
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Affiliation(s)
- Mikio Shiba
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shuichiro Higo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.,Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Takumi Kondo
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Junjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.,Department of Design for Tissue Regeneration, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Li Liu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.,Department of Design for Tissue Regeneration, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yoshihiko Ikeda
- Department of Pathology, National Cerebral and Cardiovascular Center, Suita, Osaka 564-8565, Japan
| | - Yasuaki Kohama
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Satoshi Kameda
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Tomoka Tabata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Hiroyuki Inoue
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Satoki Nakamura
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.,Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Maki Takeda
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Emiko Ito
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Seiji Takashima
- Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yoshiki Sawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Shungo Hikoso
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
| | - Yasushi Sakata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
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18
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Zhang X, Li T, Ou J, Huang J, Liang P. Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing. Protein Cell 2021; 13:316-335. [PMID: 33945139 PMCID: PMC9008090 DOI: 10.1007/s13238-021-00838-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Accepted: 03/16/2021] [Indexed: 12/26/2022] Open
Abstract
Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.
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Affiliation(s)
- Xiya Zhang
- Center for Reproductive Medicine, the Third Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, 510630, China
| | - Tao Li
- Center for Reproductive Medicine, the Third Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, 510630, China
| | - Jianping Ou
- Center for Reproductive Medicine, the Third Affiliated Hospital of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, 510630, China.
| | - Junjiu Huang
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. .,Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
| | - Puping Liang
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. .,Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
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19
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DGK and DZHK position paper on genome editing: basic science applications and future perspective. Basic Res Cardiol 2021; 116:2. [PMID: 33449167 PMCID: PMC7810637 DOI: 10.1007/s00395-020-00839-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Accepted: 12/09/2020] [Indexed: 12/18/2022]
Abstract
For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.
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20
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Li J, Hua Y, Miyagawa S, Zhang J, Li L, Liu L, Sawa Y. hiPSC-Derived Cardiac Tissue for Disease Modeling and Drug Discovery. Int J Mol Sci 2020; 21:E8893. [PMID: 33255277 PMCID: PMC7727666 DOI: 10.3390/ijms21238893] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2020] [Revised: 11/18/2020] [Accepted: 11/18/2020] [Indexed: 12/20/2022] Open
Abstract
Relevant, predictive normal, or disease model systems are of vital importance for drug development. The difference between nonhuman models and humans could contribute to clinical trial failures despite ideal nonhuman results. As a potential substitute for animal models, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) provide a powerful tool for drug toxicity screening, modeling cardiovascular diseases, and drug discovery. Here, we review recent hiPSC-CM disease models and discuss the features of hiPSC-CMs, including subtype and maturation and the tissue engineering technologies for drug assessment. Updates from the international multisite collaborators/administrations for development of novel drug discovery paradigms are also summarized.
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Affiliation(s)
- Junjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
- Department of Cell Design for Tissue Construction, Faculty of Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Ying Hua
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
| | - Jingbo Zhang
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
| | - Lingjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
| | - Li Liu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
- Department of Design for Tissue Regeneration, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Yoshiki Sawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (J.L.); (Y.H.); (S.M.); (J.Z.); (L.L.)
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