1
|
Bengtsson NE, Tasfaout H, Chamberlain JS. The road toward AAV-mediated gene therapy of Duchenne muscular dystrophy. Mol Ther 2025; 33:2035-2051. [PMID: 40181545 DOI: 10.1016/j.ymthe.2025.03.065] [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: 03/10/2025] [Revised: 03/31/2025] [Accepted: 03/31/2025] [Indexed: 04/05/2025] Open
Abstract
Forty years after the dystrophin gene was cloned, significant progress has been made in developing gene therapy approaches for Duchenne muscular dystrophy (DMD). The disorder has presented numerous challenges, including the enormous size of the gene (2.2 MB), the need to target muscles body wide, and immunogenic issues against both vectors and dystrophin. Among human genetic disorders, DMD is relatively common, and the genetics are complicated since one-third of all cases arise from a spontaneous new mutation, resulting in thousands of independent lesions throughout the locus. Many approaches have been pursued in the goal of finding an effective therapy, including exon skipping, nonsense codon suppression, upregulation of surrogate genes, gene replacement, and gene editing. Here, we focus specifically on methods using AAV vectors, as these approaches have been tested in numerous clinical trials and are able to target muscles systemically. We discuss early advances to understand the structure of dystrophin, which are crucial for the design of effective DMD gene therapies. Included is a summary of efforts to deliver micro-, mini-, and full-length dystrophins to muscles. Finally, we review current approaches to adapt gene editing to the enormous DMD gene with prospects for improved therapies using all these methods.
Collapse
Affiliation(s)
- Niclas E Bengtsson
- Department of Neurology, University of Washington School of Medicine, Seattle, WA 98109, USA; Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA 98109, USA.
| | - Hichem Tasfaout
- Department of Neurology, University of Washington School of Medicine, Seattle, WA 98109, USA; Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA 98109, USA.
| | - Jeffrey S Chamberlain
- Department of Neurology, University of Washington School of Medicine, Seattle, WA 98109, USA; Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA 98109, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA 98109, USA; Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98109, USA.
| |
Collapse
|
2
|
Chang J, Yang X, Zhang T, Sun H, Cheng H, Jia Z, Li Y, Ma S, Sun T, Cao J. High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System. Molecules 2025; 30:1811. [PMID: 40333840 PMCID: PMC12029788 DOI: 10.3390/molecules30081811] [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: 02/26/2025] [Revised: 04/08/2025] [Accepted: 04/12/2025] [Indexed: 05/09/2025] Open
Abstract
Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.
Collapse
Affiliation(s)
- Jiasong Chang
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Xiulong Yang
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Tong Zhang
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Hao Sun
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Hongying Cheng
- Department of Preschool Education, Lvliang Teachers College, Lvliang 033001, China;
| | - Zhangrong Jia
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Yiying Li
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Sanyuan Ma
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Teng Sun
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Jimin Cao
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| |
Collapse
|
3
|
Haque US, Yokota T. Gene Editing for Duchenne Muscular Dystrophy: From Experimental Models to Emerging Therapies. Degener Neurol Neuromuscul Dis 2025; 15:17-40. [PMID: 40241992 PMCID: PMC12002074 DOI: 10.2147/dnnd.s495536] [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: 01/23/2025] [Accepted: 04/08/2025] [Indexed: 04/18/2025] Open
Abstract
The CRISPR system has emerged as a ground-breaking gene-editing tool, offering promising therapeutic potential for Duchenne muscular dystrophy (DMD), a severe genetic disorder affecting approximately 1 in 5000 male births globally. DMD is caused by mutations in the dystrophin gene, which encodes a critical membrane-associated protein essential for maintaining muscle structure, function and repair. Patients with DMD experience progressive muscle degeneration, loss of ambulation, respiratory insufficiency, and cardiac failure, with most succumbing to the disease by their third decade of life. Despite the well-characterized genetic basis of DMD, curative treatments- such as exon skipping therapies, micro-dystrophin, and steroids- remain elusive. Recent preclinical studies have demonstrated the promise of CRISPR-based approaches in restoring dystrophin expression across various models, including human cells, murine systems, and large animal models. These advancements highlight the potential of gene editing to fundamentally alter the trajectory of the disease. However, significant challenges persist, including immunogenicity, off-target effects, and limited editing efficiency, which hinder clinical translation. This review provides a comprehensive analysis of the latest developments in CRISPR-based therapeutic strategies for DMD. It emphasizes the need for further innovation in gene-editing technologies, delivery systems, and rigorous safety evaluations to overcome current barriers and harness the full potential of CRISPR/Cas as a durable and effective treatment for DMD.
Collapse
Affiliation(s)
- Umme Sabrina Haque
- Department of Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
- Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
| | - Toshifumi Yokota
- Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
- The Friends of Garrett Cumming Research & Muscular Dystrophy Canada HM Toupin Neurological Science Research Chair, Edmonton, AB, T6G 2H7, Canada
| |
Collapse
|
4
|
He Q, Wang Y, Tan Z, Zhang X, Yu C, Jiang X. Mapping the therapeutic landscape of CRISPR-Cas9 for combating age-related diseases. Front Genome Ed 2025; 7:1558432. [PMID: 40255230 PMCID: PMC12006052 DOI: 10.3389/fgeed.2025.1558432] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2025] [Accepted: 03/19/2025] [Indexed: 04/22/2025] Open
Abstract
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) has emerged as a transformative genome-editing tool with significant therapeutic potential for age-related diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disorders, and osteoporosis. This study presents a bibliometric analysis of CRISPR-Cas9 research in age-related diseases, identifying key contributors, major research hotspots, and critical technological advancements. While promising applications have been demonstrated in gene repair, functional regulation, and molecular interventions, significant barriers persist, including off-target effects, low delivery efficiency, and limited editing in non-dividing cells. Ethical concerns over germline editing and gaps in long-term safety data further complicate clinical translation. Future directions emphasize the development of high-precision Cas9 variants, homology-directed repair-independent tools, and efficient delivery systems, alongside the establishment of international regulatory frameworks and multicenter clinical trials. These efforts are essential to fully realize the potential of CRISPR-Cas9 in addressing the global health challenges of aging.
Collapse
Affiliation(s)
- Qiyu He
- Department of Urology, West China Hospital of Sichuan University, Chengdu, Sichuan, China
| | - Yida Wang
- Key Laboratory of BioResource and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan, China
| | - Zhimin Tan
- Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, Sichuan, China
| | - Xian Zhang
- Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, Sichuan, China
| | - Chao Yu
- Department of Anesthesiology, West China Second Hospital of Sichuan University, Chengdu, Sichuan, China
- Key Laboratory of Birth Defects and Related Diseases of Women and Children, Sichuan University, Ministry of Education, Chengdu, China
| | - Xiaoqin Jiang
- Department of Anesthesiology, West China Second Hospital of Sichuan University, Chengdu, Sichuan, China
- Key Laboratory of Birth Defects and Related Diseases of Women and Children, Sichuan University, Ministry of Education, Chengdu, China
- Department of Anesthesiology, Chengdu Hi-Tech Zone Hospital for Women and Children, Chengdu, China
| |
Collapse
|
5
|
Wang AYL, Aviña AE, Liu YY, Kao HK. Pluripotent Stem Cells: Recent Advances and Emerging Trends. Biomedicines 2025; 13:765. [PMID: 40299329 PMCID: PMC12025069 DOI: 10.3390/biomedicines13040765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2025] [Accepted: 03/19/2025] [Indexed: 04/30/2025] Open
Abstract
The field of induced pluripotent stem cells (iPSCs) continues to evolve, offering unprecedented potential for regenerative medicine, disease modeling, and therapeutic applications [...].
Collapse
Affiliation(s)
- Aline Yen Ling Wang
- Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (A.E.A.); (Y.-Y.L.)
| | - Ana Elena Aviña
- Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (A.E.A.); (Y.-Y.L.)
- International PhD Program in Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
| | - Yen-Yu Liu
- Center for Vascularized Composite Allotransplantation, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; (A.E.A.); (Y.-Y.L.)
| | - Huang-Kai Kao
- Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan;
- College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
| |
Collapse
|
6
|
Padmaswari MH, Agrawal S, Nelson CE. Preclinical development of genome editing to treat Duchenne muscular dystrophy by exon skipping. J Neuromuscul Dis 2025:22143602251326993. [PMID: 40105473 DOI: 10.1177/22143602251326993] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/20/2025]
Abstract
Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations to the gene encoding dystrophin. Restoring the reading frame of dystrophin by removing internal out-of-frame exons may address symptoms of DMD. Therefore, the principle of exon skipping has been at the center stage in drug development for Duchenne muscular dystrophy (DMD) over the past two decades. Antisense oligonucleotides (AONs) have proven effective in modulating splicing sites for exon skipping, resulting in the FDA approval of several drugs using this technique in recent years. However, due to the temporary nature of AON, researchers are actively exploring genome editing as a potential long-term, single-administration treatment. The advancements in genome-editing technology over the last decade have boosted this transition. While no clinical trials for exon skipping in DMD via genome editing have been conducted as of this writing, preclinical studies show encouraging results. This review describes the preclinical landscape of genome editing for exon skipping in DMD treatment. Along with highlighting the adaptability of genome editing in exon skipping, this review also describes delivery challenges and outlines future research directions that could set a new stage for enhanced therapeutic development in DMD.
Collapse
Affiliation(s)
- Made Harumi Padmaswari
- Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR, USA
| | - Shilpi Agrawal
- Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA
| | - Christopher E Nelson
- Biomedical Engineering, University of Arkansas, Fayetteville, AR, USA
- Cell and Molecular Biology, University of Arkansas, Fayetteville, AR, USA
| |
Collapse
|
7
|
Kakizaki M, Hashimoto R, Nagata N, Yamamoto T, Okura T, Katoh H, Kitai Y, Akahori Y, Shirato K, Ryo A, Takayama K, Takeda M. The respective roles of TMPRSS2 and cathepsins for SARS-CoV-2 infection in human respiratory organoids. J Virol 2025; 99:e0185324. [PMID: 39601592 PMCID: PMC11784140 DOI: 10.1128/jvi.01853-24] [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: 10/19/2024] [Accepted: 10/31/2024] [Indexed: 11/29/2024] Open
Abstract
A critical aspect of the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the protease-mediated activation of the viral spike (S) protein. The type II transmembrane serine protease TMPRSS2 is crucial for SARS-CoV-2 infection in lung epithelial Calu-3 cells and murine airways. However, the importance of TMPRSS2 needs to be re-examined because the ability to utilize TMPRSS2 is significantly reduced in the Omicron variants that spread globally. For this purpose, replication profiles of SARS-CoV-2 were analyzed in human respiratory organoids. All tested viruses, including Omicron variants, replicated efficiently in these organoids. Notably, all SARS-CoV-2 strains retained replication ability in TMPRSS2-gene knockout (KO) respiratory organoids, suggesting that TMPRSS2 is not essential for SARS-CoV-2 infection in human respiratory tissues. However, TMPRSS2-gene knockout significantly reduces the inhibitory effect of nafamostat, indicating the advantage of TMPRSS2-utilizing ability for the SARS-CoV-2 infection in these organoids. Interestingly, Omicron variants regained the TMPRSS2-utilizing ability in recent subvariants. The basal infectivity would be supported mainly by cathepsins because the cathepsin inhibitor, EST, showed a significant inhibitory effect on infection with any SARS-CoV-2 strains, mainly when used with nafamostat. A supplementary contribution of other serine proteases was also suggested because the infection of the Delta variant was still inhibited partially by nafamostat in TMPRSS2 KO organoids. Thus, various proteases, including TMPRSS2, other serine proteases, and cathepsins, co-operatively contribute to SARS-CoV-2 infection significantly in the respiratory organoids. Thus, SARS-CoV-2 infection in the human respiratory tissues would be more complex than observed in cell lines or mice. IMPORTANCE We explored how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects human respiratory organoids, which are a cultured cell model made to mimic the physiological conditions of the human airways. We focused on understanding the role of different proteases of host cells in activating the virus spike proteins. Specifically, we looked at TMPRSS2, a transmembrane serine protease, and cathepsin L, a lysosomal enzyme, which helps the virus enter cells by cutting the viral spike protein. We discovered that while TMPRSS2 is crucial for the virus in certain cells and animal models, other proteases, including cathepsins and various serine proteases, also play significant roles in the SARS-CoV-2 infection of human respiratory organoids. We suggest that SARS-CoV-2 uses a more complex mechanism involving multiple proteases to infect human airways, differing from what we see in conventional cell lines or animal models. This complexity might help explain how different variants can spread and infect people effectively.
Collapse
Affiliation(s)
- Masatoshi Kakizaki
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Rina Hashimoto
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Noriyo Nagata
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takuya Yamamoto
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
- Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan
| | - Takashi Okura
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hiroshi Katoh
- Department of Microbiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yuki Kitai
- Department of Microbiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yukiko Akahori
- Department of Microbiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
| | - Kazuya Shirato
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Akihide Ryo
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuo Takayama
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Makoto Takeda
- Department of Microbiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
- Pandemic Preparedness, Infection and Advanced Research Center, The University of Tokyo, Tokyo, Japan
| |
Collapse
|
8
|
Mondéjar-Parreño G, Moreno-Manuel AI, Ruiz-Robles JM, Jalife J. Ion channel traffic jams: the significance of trafficking deficiency in long QT syndrome. Cell Discov 2025; 11:3. [PMID: 39788950 PMCID: PMC11717978 DOI: 10.1038/s41421-024-00738-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Accepted: 09/10/2024] [Indexed: 01/12/2025] Open
Abstract
A well-balanced ion channel trafficking machinery is paramount for the normal electromechanical function of the heart. Ion channel variants and many drugs can alter the cardiac action potential and lead to arrhythmias by interfering with mechanisms like ion channel synthesis, trafficking, gating, permeation, and recycling. A case in point is the Long QT syndrome (LQTS), a highly arrhythmogenic disease characterized by an abnormally prolonged QT interval on ECG produced by variants and drugs that interfere with the action potential. Disruption of ion channel trafficking is one of the main sources of LQTS. We review some molecular pathways and mechanisms involved in cardiac ion channel trafficking. We highlight the importance of channelosomes and other macromolecular complexes in helping to maintain normal cardiac electrical function, and the defects that prolong the QT interval as a consequence of variants or the effect of drugs. We examine the concept of "interactome mapping" and illustrate by example the multiple protein-protein interactions an ion channel may undergo throughout its lifetime. We also comment on how mapping the interactomes of the different cardiac ion channels may help advance research into LQTS and other cardiac diseases. Finally, we discuss how using human induced pluripotent stem cell technology to model ion channel trafficking and its defects may help accelerate drug discovery toward preventing life-threatening arrhythmias. Advancements in understanding ion channel trafficking and channelosome complexities are needed to find novel therapeutic targets, predict drug interactions, and enhance the overall management and treatment of LQTS patients.
Collapse
Affiliation(s)
| | | | | | - José Jalife
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
- CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain.
- Departments of Medicine and Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.
| |
Collapse
|
9
|
Mondéjar-Parreño G, Sánchez-Pérez P, Cruz FM, Jalife J. Promising tools for future drug discovery and development in antiarrhythmic therapy. Pharmacol Rev 2025; 77:100013. [PMID: 39952687 DOI: 10.1124/pharmrev.124.001297] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 08/30/2024] [Accepted: 10/04/2024] [Indexed: 01/22/2025] Open
Abstract
Arrhythmia refers to irregularities in the rate and rhythm of the heart, with symptoms spanning from mild palpitations to life-threatening arrhythmias and sudden cardiac death. The complex molecular nature of arrhythmias complicates the selection of appropriate treatment. Current therapies involve the use of antiarrhythmic drugs (class I-IV) with limited efficacy and dangerous side effects and implantable pacemakers and cardioverter-defibrillators with hardware-related complications and inappropriate shocks. The number of novel antiarrhythmic drugs in the development pipeline has decreased substantially during the last decade and underscores uncertainties regarding future developments in this field. Consequently, arrhythmia treatment poses significant challenges, prompting the need for alternative approaches. Remarkably, innovative drug discovery and development technologies show promise in helping advance antiarrhythmic therapies. In this article, we review unique characteristics and the transformative potential of emerging technologies that offer unprecedented opportunities for transitioning from traditional antiarrhythmics to next-generation therapies. We assess stem cell technology, emphasizing the utility of innovative cell profiling using multiomics, high-throughput screening, and advanced computational modeling in developing treatments tailored precisely to individual genetic and physiological profiles. We offer insights into gene therapy, peptide, and peptibody approaches for drug delivery. We finally discuss potential strengths and weaknesses of such techniques in reducing adverse effects and enhancing overall treatment outcomes, leading to more effective, specific, and safer therapies. Altogether, this comprehensive overview introduces innovative avenues for personalized rhythm therapy, with particular emphasis on drug discovery, aiming to advance the arrhythmia treatment landscape and the prevention of sudden cardiac death. SIGNIFICANCE STATEMENT: Arrhythmias and sudden cardiac death account for 15%-20% of deaths worldwide. However, current antiarrhythmic therapies are ineffective and have dangerous side effects. Here, we review the field of arrhythmia treatment underscoring the slow progress in advancing the cardiac rhythm therapy pipeline and the uncertainties regarding evolution of this field. We provide information on how emerging technological and experimental tools can help accelerate progress and address the limitations of antiarrhythmic drug discovery.
Collapse
Affiliation(s)
| | | | | | - José Jalife
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; Department of Medicine, University of Michigan, Ann Arbor, Michigan; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.
| |
Collapse
|
10
|
Azeez SS, Hamad RS, Hamad BK, Shekha MS, Bergsten P. Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine. Front Genome Ed 2024; 6:1509924. [PMID: 39726634 PMCID: PMC11669675 DOI: 10.3389/fgeed.2024.1509924] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 11/28/2024] [Indexed: 12/28/2024] Open
Abstract
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.
Collapse
Affiliation(s)
- Sarkar Sardar Azeez
- Department of Medical Laboratory Technology, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Rahin Shareef Hamad
- Nursing Department, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Bahra Kakamin Hamad
- Department of Medical Laboratory Technology, Erbil Health and Medical Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Mudhir Sabir Shekha
- Department of Biology, College of Science, Salahaddin University, Erbil, Kurdistan Region, Iraq
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Peter Bergsten
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| |
Collapse
|
11
|
Uno N, Miyamoto H, Yamazaki K, Egawa M, Kobayashi H, Kazuki K, Osaki M, Suzuki T, Hamamichi S, Oshimura M, Tomizuka K, Kazuki Y. Microcell-mediated chromosome transfer between non-identical human iPSCs. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102382. [PMID: 39634136 PMCID: PMC11616053 DOI: 10.1016/j.omtn.2024.102382] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/26/2023] [Accepted: 10/31/2024] [Indexed: 12/07/2024]
Abstract
Microcell-mediated chromosome transfer (MMCT) is anticipated as a unique strategy to manipulate numbers of chromosomes, including the generation of hyperaneuploidy syndrome models with human induced pluripotent stem cells (hiPSCs). Mouse A9/Chinese hamster ovary (CHO) cell libraries of human monochromosomal hybrids as chromosome donor cells frequently exhibit chromosomal rearrangement in the components. The generation of a new A9/CHO library is time-consuming and laborious. Here, we developed an MMCT method using hiPSCs as chromosome donor and recipient cells, through micronucleation using paclitaxel and reversine. Membrane fusion during the MMCT was mediated through interactions between the ecotropic viral envelope transiently expressed in chromosome donor cells and mCAT-1 in chromosome recipient cells. This approach involved tagging Chr21 and ChrY by CRISPR-Cas9 and transferring human/mouse artificial chromosomes, Chr21, ChrX, and ChrY, wherein there are no previous reports demonstrating a full-length introduction. Thus, a strategy that combing CRISPR-Cas9-mediated chromosome tagging and MMCT from hiPSCs as chromosome donor cells to hiPSCs as recipient cells systematically produced isogenic disease model hiPSCs with hyperaneuploidy. This approach allows the study of rare diseases and promises to provide new insights into early developmental mechanisms by introducing a comprehensive set of influential chromosomes/regions into hiPSCs.
Collapse
Affiliation(s)
- Narumi Uno
- Laboratory of Bioengineering, Faculty of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
- Department of Chromosome Biomedical Engineering, Integrated Medical Sciences, Graduate School of Medical Sciences, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Hitomaru Miyamoto
- Department of Chromosome Biomedical Engineering, Integrated Medical Sciences, Graduate School of Medical Sciences, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Kyotaro Yamazaki
- Department of Chromosome Biomedical Engineering, Integrated Medical Sciences, Graduate School of Medical Sciences, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
- Chromosome Engineering Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan
- Homeostatic Regulation, National Institute for Physiological Sciences, National Institutes of Natural Sciences (NINS), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan
| | - Masaya Egawa
- Laboratory of Bioengineering, Faculty of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
| | - Hiroaki Kobayashi
- Department of Chromosome Biomedical Engineering, Integrated Medical Sciences, Graduate School of Medical Sciences, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Kanako Kazuki
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Mitsuhiko Osaki
- Division of Experimental Pathology, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Teruhiko Suzuki
- Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan
| | - Shusei Hamamichi
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Mitsuo Oshimura
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| | - Kazuma Tomizuka
- Laboratory of Bioengineering, Faculty of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
| | - Yasuhiro Kazuki
- Department of Chromosome Biomedical Engineering, Integrated Medical Sciences, Graduate School of Medical Sciences, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
- Chromosome Engineering Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan
- Chromosome Engineering Research Center, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
| |
Collapse
|
12
|
Aslan C, Zolbanin NM, Faraji F, Jafari R. Exosomes for CRISPR-Cas9 Delivery: The Cutting Edge in Genome Editing. Mol Biotechnol 2024; 66:3092-3116. [PMID: 38012525 DOI: 10.1007/s12033-023-00932-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Accepted: 10/02/2023] [Indexed: 11/29/2023]
Abstract
Gene mutation correction was challenging until the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). CRISPR is a new era for genome modification, and this technology has bypassed the limitations of previous methods such as zinc-finger nuclease and transcription activator-like effector nuclease. Currently, this method is becoming the method of choice for gene-editing purposes, especially therapeutic gene editing in diseases such as cardiovascular, neurological, renal, genetic, optical, and stem cell, as well as blood disorders and muscular degeneration. However, finding the optimum delivery system capable of carrying this large complex persists as the main challenge of this technology. Therefore, it would be ideal if the delivery vehicle could direct the introduction of editing functions to specific cells in a multicellular organism. Exosomes are membrane-bound vesicles with high biocompatibility and low immunogenicity; they offer the best and most reliable way to fill the CRISPR/Cas9 system delivery gap. This review presents the current evidence on the molecular mechanisms and challenges of CRISPR/Cas9-mediated genome modification. Also, the role of CRISPR/Cas9 in the development of treatment and diagnosis of numerous disorders, from malignancies to viral infections, has been discussed. Lastly, the focus is on new advances in exosome-delivery technologies that may play a role in CRISPR/Cas9 delivery for future clinical settings.
Collapse
Affiliation(s)
- Cynthia Aslan
- Research Center for Integrative Medicine in Aging, Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Naime Majidi Zolbanin
- Experimental and Applied Pharmaceutical Sciences Research Center, Urmia University of Medical Sciences, Urmia, Iran
- Department of Pharmacology and Toxicology, School of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran
| | - Fatemeh Faraji
- Hazrat-e Rasool General Hospital, Antimicrobial Resistance Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Floor 3, Building No. 3, Niyayesh St, Sattar Khan St, Tehran, 1445613131, Iran.
| | - Reza Jafari
- Cellular and Molecular Research Center, Cellular and Molecular Medicine Research Institute, Clinical Research Institute, Urmia University of Medical Sciences, Shafa St., Ershad Blvd., P.O. Box: 1138, Urmia, 57147, Iran.
- Department of Immunology and Genetics, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
| |
Collapse
|
13
|
Lauerer AM, Caravia XM, Maier LS, Chemello F, Lebek S. Gene editing in common cardiovascular diseases. Pharmacol Ther 2024; 263:108720. [PMID: 39284367 DOI: 10.1016/j.pharmthera.2024.108720] [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: 05/20/2024] [Revised: 07/29/2024] [Accepted: 09/01/2024] [Indexed: 09/22/2024]
Abstract
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, highlighting the high socioeconomic impact. Current treatment strategies like compound-based drugs or surgeries are often limited. On the one hand, systemic administration of substances is frequently associated with adverse side effects; on the other hand, they typically provide only short-time effects requiring daily intake. Thus, new therapeutic approaches and concepts are urgently needed. The advent of CRISPR-Cas9 genome editing offers great promise for the correction of disease-causing hereditary mutations. As such mutations are often very rare, gene editing strategies to correct them are not broadly applicable to many patients. Notably, there is recent evidence that gene editing technology can also be deployed to disrupt common pathogenic signaling cascades in a targeted, specific, and efficient manner, which offers a more generalizable approach. However, several challenges remain to be addressed ranging from the optimization of the editing strategy itself to a suitable delivery strategy up to potential immune responses to the editing components. This review article discusses important CRISPR-Cas9-based gene editing approaches with their advantages and drawbacks and outlines opportunities in their application for treatment of cardiovascular diseases.
Collapse
Affiliation(s)
- Anna-Maria Lauerer
- Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany
| | - Xurde M Caravia
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Lars S Maier
- Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany
| | - Francesco Chemello
- Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
| | - Simon Lebek
- Department of Internal Medicine II, University Hospital Regensburg, Regensburg, Germany.
| |
Collapse
|
14
|
Chaudhary R, Rehman M, Agarwal V, Kumar A, Kaushik AS, Srivastava S, Srivastava S, Verma R, Rajinikanth PS, Mishra V. Terra incognita of glial cell dynamics in the etiology of leukodystrophies: Broadening disease and therapeutic perspectives. Life Sci 2024; 354:122953. [PMID: 39122110 DOI: 10.1016/j.lfs.2024.122953] [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: 02/05/2024] [Revised: 07/09/2024] [Accepted: 08/05/2024] [Indexed: 08/12/2024]
Abstract
Neuroglial cells, also known as glia, are primarily characterized as auxiliary cells within the central nervous system (CNS). The recent findings have shed light on their significance in numerous physiological processes and their involvement in various neurological disorders. Leukodystrophies encompass an array of rare and hereditary neurodegenerative conditions that were initially characterized by the deficiency, aberration, or degradation of myelin sheath within CNS. The primary cellular populations that experience significant alterations are astrocytes, oligodendrocytes and microglia. These glial cells are either structurally or metabolically impaired due to inherent cellular dysfunction. Alternatively, they may fall victim to the accumulation of harmful by-products resulting from metabolic disturbances. In either situation, the possible replacement of glial cells through the utilization of implanted tissue or stem cell-derived human neural or glial progenitor cells hold great promise as a therapeutic strategy for both the restoration of structural integrity through remyelination and the amelioration of metabolic deficiencies. Various emerging treatment strategies like stem cell therapy, ex-vivo gene therapy, infusion of adeno-associated virus vectors, emerging RNA-based therapies as well as long-term therapies have demonstrated success in pre-clinical studies and show promise for rapid clinical translation. Here, we addressed various leukodystrophies in a comprehensive and detailed manner as well as provide prospective therapeutic interventions that are being considered for clinical trials. Further, we aim to emphasize the crucial role of different glial cells in the pathogenesis of leukodystrophies. By doing so, we hope to advance our understanding of the disease, elucidate underlying mechanisms, and facilitate the development of potential treatment interventions.
Collapse
Affiliation(s)
- Rishabh Chaudhary
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Mujeeba Rehman
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Vipul Agarwal
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Anand Kumar
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Arjun Singh Kaushik
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Siddhi Srivastava
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Sukriti Srivastava
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Rajkumar Verma
- University of Connecticut School of Medicine, 200 Academic Way, Farmington, CT 06032, USA
| | - P S Rajinikanth
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India
| | - Vikas Mishra
- Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, U.P., India.
| |
Collapse
|
15
|
Zhao M, Taniguchi Y, Shimono C, Jonouchi T, Cheng Y, Shimizu Y, Nalbandian M, Yamamoto T, Nakagawa M, Sekiguchi K, Sakurai H. Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2308306. [PMID: 38685581 PMCID: PMC11234437 DOI: 10.1002/advs.202308306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 03/26/2024] [Indexed: 05/02/2024]
Abstract
Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.
Collapse
Affiliation(s)
- Mingming Zhao
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
- Center for Medical EpigeneticsSchool of Basic Medical SciencesChongqing Medical University1 Yixueyuan Road, Yuzhong DistrictChongqing400016China
| | - Yukimasa Taniguchi
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Chisei Shimono
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Tatsuya Jonouchi
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Yushen Cheng
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Yasuhiro Shimizu
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Minas Nalbandian
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Takuya Yamamoto
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Masato Nakagawa
- Department of Life Science FrontiersCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| | - Kiyotoshi Sekiguchi
- Division of Matrixome Research and ApplicationInstitute for Protein ResearchOsaka University3‐2 Yamadaoka, SuitaOsaka565‐0871Japan
| | - Hidetoshi Sakurai
- Department of Clinical ApplicationCenter for iPS Cell Research and Application (CiRA)Kyoto University53 Shogoin‐Kawahara‐cho, Sakyo‐kuKyoto606‐8507Japan
| |
Collapse
|
16
|
Dhoke NR, Kim H, Azzag K, Crist SB, Kiley J, Perlingeiro RCR. A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs. Cells 2024; 13:972. [PMID: 38891104 PMCID: PMC11171783 DOI: 10.3390/cells13110972] [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: 05/15/2024] [Revised: 05/25/2024] [Accepted: 05/30/2024] [Indexed: 06/21/2024] Open
Abstract
Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.
Collapse
Affiliation(s)
- Neha R. Dhoke
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
| | - Hyunkee Kim
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
| | - Karim Azzag
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
| | - Sarah B. Crist
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
| | - James Kiley
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
| | - Rita C. R. Perlingeiro
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA; (N.R.D.); (H.K.); (K.A.); (S.B.C.); (J.K.)
- Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| |
Collapse
|
17
|
Laurent M, Geoffroy M, Pavani G, Guiraud S. CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments. Cells 2024; 13:800. [PMID: 38786024 PMCID: PMC11119143 DOI: 10.3390/cells13100800] [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: 03/26/2024] [Revised: 05/03/2024] [Accepted: 05/05/2024] [Indexed: 05/25/2024] Open
Abstract
In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.
Collapse
Affiliation(s)
- Marine Laurent
- INTEGRARE, UMR_S951, Genethon, Inserm, Univ Evry, Université Paris-Saclay, 91190 Evry, France
| | | | - Giulia Pavani
- Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Simon Guiraud
- SQY Therapeutics, 78180 Montigny-le-Bretonneux, France
| |
Collapse
|
18
|
Alizadeh F, Abraghan YJ, Farrokhi S, Yousefi Y, Mirahmadi Y, Eslahi A, Mojarrad M. Production of Duchenne muscular dystrophy cellular model using CRISPR-Cas9 exon deletion strategy. Mol Cell Biochem 2024; 479:1027-1040. [PMID: 37289342 DOI: 10.1007/s11010-023-04759-3] [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: 03/24/2023] [Accepted: 05/03/2023] [Indexed: 06/09/2023]
Abstract
Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disorder caused by loss-of-function mutations in the dystrophin gene. Although the search for a definitive cure has failed to date, extensive efforts have been made to introduce effective therapeutic strategies. Gene editing technology is a great revolution in biology, having an immediate application in the generation of research models. DMD muscle cell lines are reliable sources to evaluate and optimize therapeutic strategies, in-depth study of DMD pathology, and screening the effective drugs. However, only a few immortalized muscle cell lines with DMD mutations are available. In addition, obtaining muscle cells from patients also requires an invasive muscle biopsy. Mostly DMD variants are rare, making it challenging to identify a patient with a particular mutation for a muscle biopsy. To overcome these challenges and generate myoblast cultures, we optimized a CRISPR/Cas9 gene editing approach to model the most common DMD mutations that include approximately 28.2% of patients. GAP-PCR and sequencing results show the ability of the CRISPR-Cas9 system to efficient deletion of mentioned exons. We showed producing truncated transcript due to the targeted deletion by RT-PCR and sequencing. Finally, mutation-induced disruption of dystrophin protein expression was confirmed by western blotting. All together, we successfully created four immortalized DMD muscle cell lines and showed the efficacy of the CRISPR-Cas9 system for the generation of immortalized DMD cell models with the targeted deletions.
Collapse
Affiliation(s)
- Farzaneh Alizadeh
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Yousef Jafari Abraghan
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Shima Farrokhi
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Yasamin Yousefi
- Department of Biochemistry, Mashhad University of Ferdowsi, Mashhad, Iran
| | - Yeganeh Mirahmadi
- Department of Biochemistry, Genetics and Molecular Biology, Islamic Azad University, Mashhad, Iran
| | - Atieh Eslahi
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| | - Majid Mojarrad
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
- Genetic Center of Khorasan Razavi, Mashhad, Iran.
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
| |
Collapse
|
19
|
Wang Y, Zhai Y, Zhang M, Song C, Zhang Y, Zhang G. Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications. Cell Mol Biol Lett 2024; 29:48. [PMID: 38589794 PMCID: PMC11003099 DOI: 10.1186/s11658-024-00565-x] [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: 10/01/2023] [Accepted: 03/21/2024] [Indexed: 04/10/2024] Open
Abstract
Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.
Collapse
Affiliation(s)
- Ying Wang
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
- School of Public Health, Qingdao University, Qingdao, China
| | - Yujing Zhai
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
- School of Public Health, Qingdao University, Qingdao, China
| | - Mingzhe Zhang
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Chunlin Song
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Yuqing Zhang
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Gang Zhang
- The Cancer Institute, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China.
| |
Collapse
|
20
|
Lotfi M, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Abbaszadegan MR. Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells. Mol Biotechnol 2024; 66:517-530. [PMID: 37266832 DOI: 10.1007/s12033-023-00739-6] [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: 01/16/2023] [Accepted: 03/27/2023] [Indexed: 06/03/2023]
Abstract
Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.
Collapse
Affiliation(s)
- Malihe Lotfi
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atefeh Ashouri
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Majid Mojarrad
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| | - Mohammad Reza Abbaszadegan
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| |
Collapse
|
21
|
Saad FA, Saad JF, Siciliano G, Merlini L, Angelini C. Duchenne Muscular Dystrophy Gene Therapy. Curr Gene Ther 2024; 24:17-28. [PMID: 36411557 DOI: 10.2174/1566523223666221118160932] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Revised: 09/27/2022] [Accepted: 10/11/2022] [Indexed: 11/23/2022]
Abstract
Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that could address the genetic cause of the disease at its root are crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.
Collapse
Affiliation(s)
- Fawzy A Saad
- Department of Biology, Padua University School of Medicine, Via Trieste 75, Padova 35121, Italy
- Department of Gene Therapy, Saad Pharmaceuticals, Tornimäe 7-26, Tallinn, 10145, Estonia
| | - Jasen F Saad
- Department of Gene Therapy, Saad Pharmaceuticals, Tornimäe 7-26, Tallinn, 10145, Estonia
| | - Gabriele Siciliano
- Department of Clinical and Experimental Medicine, Pisa University School of Medicine, Pisa, Italy
| | - Luciano Merlini
- Department of Biomedical and Neuromotor Sciences, Bologna University School of Medicine, 40126 Bologna, Italy
| | - Corrado Angelini
- Department Neurosciences, Padova University School of Medicine, Padova, Italy
| |
Collapse
|
22
|
Szwec S, Kapłucha Z, Chamberlain JS, Konieczny P. Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review. BioDrugs 2024; 38:95-119. [PMID: 37917377 PMCID: PMC10789850 DOI: 10.1007/s40259-023-00632-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/10/2023] [Indexed: 11/04/2023]
Abstract
Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic treatment, decades of research have resulted in first therapeutics able to restore the affected reading frame of dystrophin transcripts or induce synthesis of a truncated dystrophin protein from a vector, with other strategies based on gene therapy and cell signaling in preclinical or clinical development. Nevertheless, recent reports show that potentially therapeutic dystrophins can be immunogenic in patients. This raises the question of whether a dystrophin paralog, utrophin, could be a more suitable therapeutic protein. Here, we compare dystrophin and utrophin amino acid sequences and structures, combining published data with our extended in silico analyses. We then discuss these results in the context of therapeutic approaches for Duchenne muscular dystrophy. Specifically, we focus on strategies based on delivery of micro-dystrophin and micro-utrophin genes with recombinant adeno-associated viral vectors, exon skipping of the mutated dystrophin pre-mRNAs, reading through termination codons with small molecules that mask premature stop codons, dystrophin gene repair by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genetic engineering, and increasing utrophin levels. Our analyses highlight the importance of various dystrophin and utrophin domains in Duchenne muscular dystrophy treatment, providing insights into designing novel therapeutic compounds with improved efficacy and decreased immunoreactivity. While the necessary actin and β-dystroglycan binding sites are present in both proteins, important functional distinctions can be identified in these domains and some other parts of truncated dystrophins might need redesigning due to their potentially immunogenic qualities. Alternatively, therapies based on utrophins might provide a safer and more effective approach.
Collapse
Affiliation(s)
- Sylwia Szwec
- Institute of Human Biology and Evolution, Faculty of Biology, Adam Mickiewicz University, ul. Uniwersytetu Poznańskiego 6, 61-614, Poznań, Poland
| | - Zuzanna Kapłucha
- Institute of Human Biology and Evolution, Faculty of Biology, Adam Mickiewicz University, ul. Uniwersytetu Poznańskiego 6, 61-614, Poznań, Poland
| | - Jeffrey S Chamberlain
- Department of Neurology, University of Washington School of Medicine, Seattle, WA, 98109-8055, USA
- Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Washington School of Medicine, Seattle, WA, 98109-8055, USA
- Department of Biochemistry, University of Washington School of Medicine, Seattle, WA, 98109-8055, USA
- Department of Medicine, University of Washington School of Medicine, Seattle, WA, 98109-8055, USA
| | - Patryk Konieczny
- Institute of Human Biology and Evolution, Faculty of Biology, Adam Mickiewicz University, ul. Uniwersytetu Poznańskiego 6, 61-614, Poznań, Poland.
| |
Collapse
|
23
|
Punetha M, Saini S, Chaudhary S, Yadav PS, Whitworth K, Green J, Kumar D, Kues WA. Induced Pluripotent Stem Cells in the Era of Precise Genome Editing. Curr Stem Cell Res Ther 2024; 19:307-315. [PMID: 36880183 DOI: 10.2174/1574888x18666230307115326] [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: 07/14/2022] [Revised: 11/22/2022] [Accepted: 12/06/2022] [Indexed: 03/08/2023]
Abstract
Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.
Collapse
Affiliation(s)
- Meeti Punetha
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Sheetal Saini
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Suman Chaudhary
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Prem Singh Yadav
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Kristin Whitworth
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA
| | - Jonathan Green
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA
| | - Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Wilfried A Kues
- Department of Biotechnology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Höltystr 10, 31535, Neustadt, Germany
| |
Collapse
|
24
|
Lotfi M, Morshedi Rad D, Mashhadi SS, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Farrokhi S, Hashemi M, Lotfi M, Ebrahimi Warkiani M, Abbaszadegan MR. Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells. Stem Cell Rev Rep 2023; 19:2576-2596. [PMID: 37723364 PMCID: PMC10661828 DOI: 10.1007/s12015-023-10585-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/30/2023] [Indexed: 09/20/2023]
Abstract
Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.
Collapse
Affiliation(s)
- Malihe Lotfi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Dorsa Morshedi Rad
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia
| | - Samaneh Sharif Mashhadi
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atefeh Ashouri
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Majid Mojarrad
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Shima Farrokhi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Maryam Hashemi
- Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Marzieh Lotfi
- Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran
| | - Majid Ebrahimi Warkiani
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia.
- Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, Australia.
| | - Mohammad Reza Abbaszadegan
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| |
Collapse
|
25
|
Hosokawa M, Mikawa R, Hagiwara A, Okuno Y, Awaya T, Yamamoto Y, Takahashi S, Yamaki H, Osawa M, Setoguchi Y, Saito MK, Abe S, Hirai T, Gotoh S, Hagiwara M. Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC. iScience 2023; 26:107731. [PMID: 37701577 PMCID: PMC10494175 DOI: 10.1016/j.isci.2023.107731] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 08/05/2023] [Accepted: 08/23/2023] [Indexed: 09/14/2023] Open
Abstract
Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.
Collapse
Affiliation(s)
- Motoyasu Hosokawa
- Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
- Department of Developmental Biology and Functional Genomics, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan
| | - Ryuta Mikawa
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
- Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Atsuko Hagiwara
- Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
| | - Yukiko Okuno
- Medical Research Support Center, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Tomonari Awaya
- Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yuki Yamamoto
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
- Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Senye Takahashi
- Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Haruka Yamaki
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Mitsujiro Osawa
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Yasuhiro Setoguchi
- Department of Respiratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8519, Japan
| | - Megumu K Saito
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Shinji Abe
- Department of Respiratory Medicine Tokyo, Medical University Hospital, Shinjuku-ku, Tokyo 160-0023, Japan
| | - Toyohiro Hirai
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
| | - Shimpei Gotoh
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
- Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan
| | - Masatoshi Hagiwara
- Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| |
Collapse
|
26
|
Molugu K, Khajanchi N, Lazzarotto CR, Tsai SQ, Saha K. Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells. CRISPR J 2023; 6:473-485. [PMID: 37676985 PMCID: PMC10611976 DOI: 10.1089/crispr.2023.0033] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2023] [Accepted: 07/31/2023] [Indexed: 09/09/2023] Open
Abstract
Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.
Collapse
Affiliation(s)
- Kaivalya Molugu
- Biophysics Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA; St Jude Children's Research Hospital, Memphis, Tennessee, USA
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA; St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Namita Khajanchi
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA; St Jude Children's Research Hospital, Memphis, Tennessee, USA
- Department of Biomedical and Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA; and St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Cicera R. Lazzarotto
- Department of Hematology, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Shengdar Q. Tsai
- Department of Hematology, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Krishanu Saha
- Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA; St Jude Children's Research Hospital, Memphis, Tennessee, USA
- Department of Biomedical and Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA; and St Jude Children's Research Hospital, Memphis, Tennessee, USA
| |
Collapse
|
27
|
Shintani T, Imamura C, Ueyama-Toba Y, Inui J, Watanabe A, Mizuguchi H. Establishment of UGT1A1-knockout human iPS-derived hepatic organoids for UGT1A1-specific kinetics and toxicity evaluation. Mol Ther Methods Clin Dev 2023; 30:429-442. [PMID: 37663646 PMCID: PMC10471830 DOI: 10.1016/j.omtm.2023.08.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Accepted: 08/08/2023] [Indexed: 09/05/2023]
Abstract
Uridine diphosphate glucuronosyltransferases (UGTs) are highly expressed in the liver and are involved in the metabolism of many drugs. In particular, UGT1A1 has a genetic polymorphism that causes decreased activity, leading to drug-induced hepatotoxicity. Therefore, an in vitro evaluation system that accurately predicts the kinetics of drugs involving UGT1A1 is required. However, there is no such evaluation system because of the absence of the UGT1A1-selective inhibitor. Here, using human induced pluripotent stem (iPS) cells, genome editing technology, and organoid technology, we generated UGT1A1-knockout human iPS hepatocyte-derived liver organoids (UGT1A1-KO i-HOs) as a model for UGT1A1-specific kinetics and toxicity evaluation. i-HOs showed higher gene expression of many drug-metabolizing enzymes including UGT1A1 than human iPS cell-derived hepatocyte-like cells (iPS-HLCs), suggesting that hepatic organoid technology improves liver functions. Wild-type (WT) i-HOs showed similar levels of UGT1A1 activity to primary human (cryopreserved) hepatocytes, while UGT1A1-KO i-HOs completely lost the activity. Additionally, to evaluate whether this model can be used to predict drug-induced hepatotoxicity, UGT1A1-KO i-HOs were exposed to SN-38, the active metabolite of irinotecan, an anticancer drug, and acetaminophen and confirmed that these cells could predict UGT1A1-mediated toxicity. Thus, we succeeded in generating model cells that enable evaluation of UGT1A1-specific kinetics and toxicity.
Collapse
Affiliation(s)
- Tomohiro Shintani
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Chiharu Imamura
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Yukiko Ueyama-Toba
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
- Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka 565-0871, Japan
| | - Jumpei Inui
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Akira Watanabe
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
- Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
- Laboratory of Functional Organoid for Drug Discovery, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka 565-0871, Japan
- Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan
- Center for Infectious Disease Education and Research, Osaka University, Osaka 565-0871, Japan
| |
Collapse
|
28
|
Kita Y, Okuzaki Y, Naoe Y, Lee J, Bang U, Okawa N, Ichiki A, Jonouchi T, Sakurai H, Kojima Y, Hotta A. Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs. Stem Cell Reports 2023; 18:1753-1765. [PMID: 37625413 PMCID: PMC10545483 DOI: 10.1016/j.stemcr.2023.07.007] [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: 10/03/2022] [Revised: 07/30/2023] [Accepted: 07/31/2023] [Indexed: 08/27/2023] Open
Abstract
To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.
Collapse
Affiliation(s)
- Yuto Kita
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Yuya Okuzaki
- Nagoya University Graduate School of Bioagricultural Sciences, Avian Bioscience Research Center, Furo-cho, Chikusa-ku, Nagoya, Aishi 464-8601, Japan
| | - Youichi Naoe
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Joseph Lee
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Uikyu Bang
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Natsumi Okawa
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Akane Ichiki
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Tatsuya Jonouchi
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Yusuke Kojima
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
| | - Akitsu Hotta
- Center for iPS Cell Research and Application, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa 251-8555, Japan.
| |
Collapse
|
29
|
Gapinske M, Winter J, Swami D, Gapinske L, Woods WS, Shirguppe S, Miskalis A, Busza A, Joulani D, Kao CJ, Kostan K, Bigot A, Bashir R, Perez-Pinera P. Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 33:572-586. [PMID: 37637209 PMCID: PMC10448430 DOI: 10.1016/j.omtn.2023.07.029] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Accepted: 07/25/2023] [Indexed: 08/29/2023]
Abstract
Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) characterized by progressive muscle weakness, leading to loss of ambulation and decreased life expectancy. Since the current standard of care for Duchenne muscular dystrophy is to merely treat symptoms, there is a dire need for treatment modalities that can correct the underlying genetic mutations. While several gene replacement therapies are being explored in clinical trials, one emerging approach that can directly correct mutations in genomic DNA is base editing. We have recently developed CRISPR-SKIP, a base editing strategy to induce permanent exon skipping by introducing C > T or A > G mutations at splice acceptors in genomic DNA, which can be used therapeutically to recover dystrophin expression when a genomic deletion leads to an out-of-frame DMD transcript. We now demonstrate that CRISPR-SKIP can be adapted to correct some forms of Duchenne muscular dystrophy by disrupting the splice acceptor in human DMD exon 45 with high efficiency, which enables open reading frame recovery and restoration of dystrophin expression. We also demonstrate that AAV-delivered split-intein base editors edit the splice acceptor of DMD exon 45 in cultured human cells and in vivo, highlighting the therapeutic potential of this strategy.
Collapse
Affiliation(s)
- Michael Gapinske
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Jackson Winter
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Devyani Swami
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Lauren Gapinske
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Nick J. Holonyak Micro and Nano Technology Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Wendy S. Woods
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Shraddha Shirguppe
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Angelo Miskalis
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Anna Busza
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Dana Joulani
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Collin J. Kao
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Kurt Kostan
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Anne Bigot
- Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France
| | - Rashid Bashir
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Nick J. Holonyak Micro and Nano Technology Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Carle Illinois College of Medicine, Champaign, IL 61820, USA
| | - Pablo Perez-Pinera
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Carle Illinois College of Medicine, Champaign, IL 61820, USA
- Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
- Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| |
Collapse
|
30
|
Bez Batti Angulski A, Hosny N, Cohen H, Martin AA, Hahn D, Bauer J, Metzger JM. Duchenne muscular dystrophy: disease mechanism and therapeutic strategies. Front Physiol 2023; 14:1183101. [PMID: 37435300 PMCID: PMC10330733 DOI: 10.3389/fphys.2023.1183101] [Citation(s) in RCA: 56] [Impact Index Per Article: 28.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Accepted: 05/24/2023] [Indexed: 07/13/2023] Open
Abstract
Duchenne muscular dystrophy (DMD) is a severe, progressive, and ultimately fatal disease of skeletal muscle wasting, respiratory insufficiency, and cardiomyopathy. The identification of the dystrophin gene as central to DMD pathogenesis has led to the understanding of the muscle membrane and the proteins involved in membrane stability as the focal point of the disease. The lessons learned from decades of research in human genetics, biochemistry, and physiology have culminated in establishing the myriad functionalities of dystrophin in striated muscle biology. Here, we review the pathophysiological basis of DMD and discuss recent progress toward the development of therapeutic strategies for DMD that are currently close to or are in human clinical trials. The first section of the review focuses on DMD and the mechanisms contributing to membrane instability, inflammation, and fibrosis. The second section discusses therapeutic strategies currently used to treat DMD. This includes a focus on outlining the strengths and limitations of approaches directed at correcting the genetic defect through dystrophin gene replacement, modification, repair, and/or a range of dystrophin-independent approaches. The final section highlights the different therapeutic strategies for DMD currently in clinical trials.
Collapse
Affiliation(s)
| | | | | | | | | | | | - Joseph M. Metzger
- Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, United States
| |
Collapse
|
31
|
Lučanský V, Holubeková V, Kolková Z, Halašová E, Samec M, Golubnitschaja O. Multi-faceted CRISPR/Cas technological innovation aspects in the framework of 3P medicine. EPMA J 2023; 14:201-217. [PMID: 37275547 PMCID: PMC10201107 DOI: 10.1007/s13167-023-00324-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Accepted: 05/10/2023] [Indexed: 06/07/2023]
Abstract
Since 2009, the European Association for Predictive, Preventive and Personalised Medicine (EPMA, Brussels) promotes the paradigm change from reactive approach to predictive, preventive, and personalized medicine (PPPM/3PM) to protect individuals in sub-optimal health conditions from the health-to-disease transition, to increase life-quality of the affected patient cohorts improving, therefore, ethical standards and cost-efficacy of healthcare to great benefits of the society at large. The gene-editing technology utilizing CRISPR/Cas gene-editing approach has demonstrated its enormous value as a powerful tool in a broad spectrum of bio/medical research areas. Further, CRISPR/Cas gene-editing system is considered applicable to primary and secondary healthcare, in order to prevent disease spread and to treat clinically manifested disorders, involving diagnostics of SARS-Cov-2 infection and experimental treatment of COVID-19. Although the principle of the proposed gene editing is simple and elegant, there are a lot of technological challenges and ethical considerations to be solved prior to its broadly scaled clinical implementation. This article highlights technological innovation beyond the state of the art, exemplifies current achievements, discusses unsolved technological and ethical problems, and provides clinically relevant outlook in the framework of 3PM.
Collapse
Affiliation(s)
- Vincent Lučanský
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Veronika Holubeková
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Zuzana Kolková
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Erika Halašová
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Marek Samec
- Department of Pathophysiology, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia
| | - Olga Golubnitschaja
- Predictive, Preventive, Personalised (3P) Medicine, Department of Radiation Oncology, University Hospital Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53127 Bonn, Germany
| |
Collapse
|
32
|
Eslahi A, Alizadeh F, Avan A, Ferns GA, Moghbeli M, Reza Abbaszadegan M, Mojarrad M. New advancements in CRISPR based gene therapy of Duchenne muscular dystrophy. Gene 2023; 867:147358. [PMID: 36914142 DOI: 10.1016/j.gene.2023.147358] [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: 11/25/2022] [Revised: 03/02/2023] [Accepted: 03/07/2023] [Indexed: 03/13/2023]
Abstract
Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.
Collapse
Affiliation(s)
- Atieh Eslahi
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Farzaneh Alizadeh
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Amir Avan
- Metabolic Syndrome Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Basic Sciences Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Gordon A Ferns
- Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex BN1 9PH, UK
| | - Meysam Moghbeli
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohammad Reza Abbaszadegan
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
| | - Majid Mojarrad
- Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Medical Genetics and Molecular Medicine, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Genetic Center of Khorasan Razavi, Mashhad, Iran.
| |
Collapse
|
33
|
Muto V, Benigni F, Magliocca V, Borghi R, Flex E, Pallottini V, Rosa A, Compagnucci C, Tartaglia M. CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes. Int J Mol Sci 2023; 24:ijms24097988. [PMID: 37175696 PMCID: PMC10178052 DOI: 10.3390/ijms24097988] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Revised: 04/20/2023] [Accepted: 04/21/2023] [Indexed: 05/15/2023] Open
Abstract
Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.
Collapse
Affiliation(s)
- Valentina Muto
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
| | - Federica Benigni
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
- Department of Science, University Roma Tre, 00146 Rome, Italy
| | - Valentina Magliocca
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
| | - Rossella Borghi
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
| | - Elisabetta Flex
- Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy
| | - Valentina Pallottini
- Department of Science, University Roma Tre, 00146 Rome, Italy
- Neuroendocrinology Metabolism and Neuropharmacology Unit, IRCSS Fondazione Santa Lucia, 00143 Rome, Italy
| | - Alessandro Rosa
- Department of Biology and Biotechnologies "Charles Darwin", Sapienza University of Rome, 00185 Rome, Italy
- Center for Life Nano- & Neuro-Science, Fondazione Istituto Italiano di Tecnologia (IIT), 00161 Rome, Italy
| | - Claudia Compagnucci
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
| | - Marco Tartaglia
- Molecular Genetics and Functional Genomics, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy
| |
Collapse
|
34
|
Wang P, Li H, Zhu M, Han RY, Guo S, Han R. Correction of DMD in human iPSC-derived cardiomyocytes by base-editing-induced exon skipping. Mol Ther Methods Clin Dev 2023; 28:40-50. [PMID: 36588820 PMCID: PMC9792405 DOI: 10.1016/j.omtm.2022.11.010] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Accepted: 11/29/2022] [Indexed: 12/03/2022]
Abstract
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Previously, we showed that adenine base editing (ABE) can efficiently correct a nonsense point mutation in a DMD mouse model. Here, we explored the feasibility of base-editing-mediated exon skipping as a therapeutic strategy for DMD using cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs). We first generated a DMD hiPSC line with a large deletion spanning exon 48 through 54 (ΔE48-54) using CRISPR-Cas9 gene editing. Dystrophin expression was disrupted in DMD hiPSC-derived cardiomyocytes (iCMs) as examined by RT-PCR, western blot, and immunofluorescence staining. Transfection of ABE and a guide RNA (gRNA) targeting the splice acceptor led to efficient conversion of AG to GG (35.9% ± 5.7%) and enabled exon 55 skipping. Complete AG to GG conversion in a single clone restored dystrophin expression (42.5% ± 11% of wild type [WT]) in DMD iCMs. Moreover, we designed gRNAs to target the splice sites of exons 6, 7, 8, 43, 44, 46, and 53 in the mutational hotspots and demonstrated their efficiency to induce exon skipping in iCMs. These results highlight the great promise of ABE-mediated exon skipping as a promising therapeutic approach for DMD.
Collapse
Affiliation(s)
- Peipei Wang
- Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| | - Haiwen Li
- Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| | - Mandi Zhu
- Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| | - Rena Y. Han
- Olentangy Liberty High School, Powell, OH 43065, USA
| | - Shuliang Guo
- Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| | - Renzhi Han
- Department of Surgery, Davis Heart and Lung Research Institute, Biomedical Sciences Graduate Program, Biophysics Graduate Program, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
| |
Collapse
|
35
|
Padmaswari MH, Agrawal S, Jia MS, Ivy A, Maxenberger DA, Burcham LA, Nelson CE. Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy. BIOPHYSICS REVIEWS 2023; 4:011307. [PMID: 36864908 PMCID: PMC9969352 DOI: 10.1063/5.0131452] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 01/19/2023] [Indexed: 06/18/2023]
Abstract
Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.
Collapse
Affiliation(s)
| | - Shilpi Agrawal
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Mary S. Jia
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Allie Ivy
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Daniel A. Maxenberger
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Landon A. Burcham
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | | |
Collapse
|
36
|
Abstract
Transcription activator-like effector (TALE) nuclease (TALEN) is the second-generation genome editing tool consisting of TALE protein containing customizable DNA-binding repeats and nuclease domain of FokI enzyme. Each DNA-binding repeat recognizes one base of double-strand DNA, and functional TALEN can be created by a simple modular assembly of these repeats. To easily and efficiently assemble the highly repetitive DNA-binding repeat arrays, various construction systems such as Golden Gate assembly, serial ligation, and ligation-independent cloning have been reported. In this chapter, we summarize the updated situation of these systems and publicly available reagents and protocols, enabling optimal selection of best suited systems for every researcher who wants to utilize TALENs in various research fields.
Collapse
Affiliation(s)
- Tetsushi Sakuma
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
| | - Takashi Yamamoto
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
| |
Collapse
|
37
|
Kawada R, Jonouchi T, Kagita A, Sato M, Hotta A, Sakurai H. Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs. Sci Rep 2023; 13:94. [PMID: 36631509 PMCID: PMC9834395 DOI: 10.1038/s41598-022-26614-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Accepted: 12/16/2022] [Indexed: 01/13/2023] Open
Abstract
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.
Collapse
Affiliation(s)
- Ryu Kawada
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan ,grid.419836.10000 0001 2162 3360Discovery Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama, 331-9530 Japan
| | - Tatsuya Jonouchi
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Akihiro Kagita
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Masae Sato
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Akitsu Hotta
- grid.258799.80000 0004 0372 2033Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507 Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.
| |
Collapse
|
38
|
Milanese JS, Marcotte R, Costain WJ, Kablar B, Drouin S. Roles of Skeletal Muscle in Development: A Bioinformatics and Systems Biology Overview. ADVANCES IN ANATOMY, EMBRYOLOGY, AND CELL BIOLOGY 2023; 236:21-55. [PMID: 37955770 DOI: 10.1007/978-3-031-38215-4_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/14/2023]
Abstract
The ability to assess various cellular events consequent to perturbations, such as genetic mutations, disease states and therapies, has been recently revolutionized by technological advances in multiple "omics" fields. The resulting deluge of information has enabled and necessitated the development of tools required to both process and interpret the data. While of tremendous value to basic researchers, the amount and complexity of the data has made it extremely difficult to manually draw inference and identify factors key to the study objectives. The challenges of data reduction and interpretation are being met by the development of increasingly complex tools that integrate disparate knowledge bases and synthesize coherent models based on current biological understanding. This chapter presents an example of how genomics data can be integrated with biological network analyses to gain further insight into the developmental consequences of genetic perturbations. State of the art methods for conducting similar studies are discussed along with modern methods used to analyze and interpret the data.
Collapse
Affiliation(s)
| | - Richard Marcotte
- Human Health Therapeutics, National Research Council of Canada , Montreal, QC, Canada
| | - Willard J Costain
- Human Health Therapeutics, National Research Council of Canada, Ottawa, ON, Canada
| | - Boris Kablar
- Department of Medical Neuroscience, Anatomy and Pathology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada
| | - Simon Drouin
- Human Health Therapeutics, National Research Council of Canada , Montreal, QC, Canada.
| |
Collapse
|
39
|
Aslan A, Yuka SA. Stem Cell-Based Therapeutic Approaches in Genetic Diseases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1436:19-53. [PMID: 36735185 DOI: 10.1007/5584_2023_761] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Stem cells, which can self-renew and differentiate into different cell types, have become the keystone of regenerative medicine due to these properties. With the achievement of superior clinical results in the therapeutic approaches of different diseases, the applications of these cells in the treatment of genetic diseases have also come to the fore. Foremost, conventional approaches of stem cells to genetic diseases are the first approaches in this manner, and they have brought safety issues due to immune reactions caused by allogeneic transplantation. To eliminate these safety issues and phenotypic abnormalities caused by genetic defects, firstly, basic genetic engineering practices such as vectors or RNA modulators were combined with stem cell-based therapeutic approaches. However, due to challenges such as immune reactions and inability to target cells effectively in these applications, advanced molecular methods have been adopted in ZFN, TALEN, and CRISPR/Cas genome editing nucleases, which allow modular designs in stem cell-based genetic diseases' therapeutic approaches. Current studies in genetic diseases are in the direction of creating permanent treatment regimens by genomic manipulation of stem cells with differentiation potential through genome editing tools. In this chapter, the stem cell-based therapeutic approaches of various vital genetic diseases were addressed wide range from conventional applications to genome editing tools.
Collapse
Affiliation(s)
- Ayça Aslan
- Department of Bioengineering, Yildiz Technical University, Istanbul, Turkey
| | - Selcen Arı Yuka
- Department of Bioengineering, Yildiz Technical University, Istanbul, Turkey.
- Health Biotechnology Joint Research and Application Center of Excellence, Istanbul, Turkey.
| |
Collapse
|
40
|
Caron L, Testa S, Magdinier F. Induced Pluripotent Stem Cells for Modeling Physiological and Pathological Striated Muscle Complexity. J Neuromuscul Dis 2023; 10:761-776. [PMID: 37522215 PMCID: PMC10578229 DOI: 10.3233/jnd-230076] [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] [Accepted: 07/13/2023] [Indexed: 08/01/2023]
Abstract
Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.
Collapse
Affiliation(s)
- Leslie Caron
- Aix-Marseille Univ-INSERM, MMG, Marseille, France
| | | | | |
Collapse
|
41
|
Nalbandian M, Zhao M, Sakurai H. Evaluation of hiPSC-Derived Muscle Progenitor Cell Transplantation in a Mouse Duchenne Muscular Dystrophy Model. Methods Mol Biol 2023; 2587:527-536. [PMID: 36401048 DOI: 10.1007/978-1-0716-2772-3_28] [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] [Indexed: 06/16/2023]
Abstract
For cell therapy toward Duchenne muscle dystrophy (DMD), muscle progenitor cells derived from human-induced pluripotent stem cell (hiPSC-MuPCs) are recognized as a good candidate, and currently, cell transplantation of hiPSC-MuPCs is being tested with several DMD animal models. In this article, we describe an efficient method to dissociate, purify by cell sorting, transplant, and evaluate the transplantation efficacy of hiPSC-MuPCs.
Collapse
Affiliation(s)
- Minas Nalbandian
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Mingming Zhao
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Hidetoshi Sakurai
- Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
| |
Collapse
|
42
|
Berling E, Nicolle R, Laforêt P, Ronzitti G. Gene therapy review: Duchenne muscular dystrophy case study. Rev Neurol (Paris) 2023; 179:90-105. [PMID: 36517287 DOI: 10.1016/j.neurol.2022.11.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 11/17/2022] [Indexed: 12/14/2022]
Abstract
Gene therapy, i.e., any therapeutic approach involving the use of genetic material as a drug and more largely altering the transcription or translation of one or more genes, covers a wide range of innovative methods for treating diseases, including neurological disorders. Although they share common principles, the numerous gene therapy approaches differ greatly in their mechanisms of action. They also differ in their maturity for some are already used in clinical practice while others have never been used in humans. The aim of this review is to present the whole range of gene therapy techniques through the example of Duchenne muscular dystrophy (DMD). DMD is a severe myopathy caused by mutations in the dystrophin gene leading to the lack of functional dystrophin protein. It is a disease known to all neurologists and in which almost all gene therapy methods were applied. Here we discuss the mechanisms of gene transfer techniques with or without viral vectors, DNA editing with or without matrix repair and those acting at the RNA level (RNA editing, exon skipping and STOP-codon readthrough). For each method, we present the results obtained in DMD with a particular focus on clinical data. This review aims also to outline the advantages, limitations and risks of gene therapy related to the approach used.
Collapse
Affiliation(s)
- E Berling
- Neurology department, Raymond Poincaré university hospital, AP-HP, Garches, France; Nord-Est-Île-de-France neuromuscular reference center, FHU PHENIX, Garches, France; U 1179 Inserm, université Paris-Saclay, Montigny-Le-Bretonneux, France.
| | - R Nicolle
- Université Paris Cité, Inserm UMR1163, Imagine Institute, Clinical Bioinformatics laboratory, 75015 Paris, France
| | - P Laforêt
- Neurology department, Raymond Poincaré university hospital, AP-HP, Garches, France; Nord-Est-Île-de-France neuromuscular reference center, FHU PHENIX, Garches, France; U 1179 Inserm, université Paris-Saclay, Montigny-Le-Bretonneux, France
| | - G Ronzitti
- Université Paris Cité, Inserm UMR1163, Imagine Institute, Clinical Bioinformatics laboratory, 75015 Paris, France; Genethon, Evry, France
| |
Collapse
|
43
|
Wani AK, Akhtar N, Singh R, Prakash A, Raza SHA, Cavalu S, Chopra C, Madkour M, Elolimy A, Hashem NM. Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals. Vet Res Commun 2023; 47:1-16. [PMID: 35781172 DOI: 10.1007/s11259-022-09967-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/24/2022] [Indexed: 01/27/2023]
Abstract
Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.
Collapse
Affiliation(s)
- Atif Khurshid Wani
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Nahid Akhtar
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Reena Singh
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Ajit Prakash
- Department of Biochemistry and Biophysics, University of North Carolina, 120 Mason Farm Road, CB# 7260, 3093 Genetic Medicine, Chapel Hill, NC, 27599-2760, USA
| | - Sayed Haidar Abbas Raza
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Simona Cavalu
- Faculty of Medicine and Pharmacy, University of Oradea, P -ta 1Decembrie 10, 410073, Oradea, Romania
| | - Chirag Chopra
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Mahmoud Madkour
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Ahmed Elolimy
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Nesrein M Hashem
- Department of Animal and Fish Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, 21545, Egypt.
| |
Collapse
|
44
|
Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation. Biomolecules 2022; 12:biom12121814. [PMID: 36551242 PMCID: PMC9775085 DOI: 10.3390/biom12121814] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2022] [Revised: 12/02/2022] [Accepted: 12/02/2022] [Indexed: 12/12/2022] Open
Abstract
Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.
Collapse
|
45
|
Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping. Methods Mol Biol 2022; 2587:427-453. [PMID: 36401042 DOI: 10.1007/978-1-0716-2772-3_22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The CRISPR-Cas9 system has quickly become the standard tool for genome editing. To deliver this system to target cells, adeno-associated virus (AAV) vectors are commonly used. In fact, AAV vectors have been utilized to deliver the CRISPR-Cas9 system to induce genomic exon skipping and restore the dystrophin protein in various Duchenne muscular dystrophy model animals. Despite the high transduction efficiency, AAV vector-mediated delivery has several limitations, such as the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the risk of integrating a part of the AAV genomic sequence into the host cell. To overcome these issues, we have recently engineered a transient delivery system utilizing VSV-G pseudotyped extracellular vesicles (EVs) termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). NanoMEDIC utilizes an HIV-derived Gag protein to package Cas9 protein and gRNA into EVs. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to recruit Cas9 protein into EVs. sgRNA is packaged into EVs through an HIV-derived RNA packaging signal and is subsequently released by two self-cleaving ribozymes. Utilizing these features, NanoMEDIC can achieve highly efficient packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo. Here, we describe a step-by-step protocol, including the gRNA-expressing vector construction and large-scale NanoMEDIC production, for in vivo genome editing.
Collapse
|
46
|
Dong M, Liu J, Liu C, Wang H, Sun W, Liu B. CRISPR/CAS9: A promising approach for the research and treatment of cardiovascular diseases. Pharmacol Res 2022; 185:106480. [PMID: 36191879 DOI: 10.1016/j.phrs.2022.106480] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2022] [Revised: 09/27/2022] [Accepted: 09/29/2022] [Indexed: 10/31/2022]
Abstract
The development of gene-editing technology has been one of the biggest advances in biomedicine over the past two decades. Not only can it be used as a research tool to build a variety of disease models for the exploration of disease pathogenesis at the genetic level, it can also be used for prevention and treatment. This is done by intervening with the expression of target genes and carrying out precise molecular targeted therapy for diseases. The simple and flexible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technology overcomes the limitations of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). For this reason, it has rapidly become a preferred method for gene editing. As a new gene intervention method, CRISPR/Cas9 has been widely used in the clinical treatment of tumours and rare diseases; however, its application in the field of cardiovascular diseases is currently limited. This article reviews the application of the CRISPR/Cas9 editing technology in cardiovascular disease research and treatment, and discusses the limitations and prospects of this technology.
Collapse
Affiliation(s)
- Mengying Dong
- Department of Cardiology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, China, 130041
| | - Jiangen Liu
- Department of Cardiology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, China, 130041
| | - Caixia Liu
- Department of Neurology, The Liaoning Province People's Hospital, 33 Wenyi Road, ShenYang, China, 110016
| | - He Wang
- Department of Cardiology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, China, 130041
| | - Wei Sun
- Department of Cardiology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, China, 130041.
| | - Bin Liu
- Department of Cardiology, The Second Hospital of Jilin University, 218 Ziqiang Road, Changchun, China, 130041.
| |
Collapse
|
47
|
CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives. Cells 2022; 11:cells11192964. [PMID: 36230926 PMCID: PMC9564082 DOI: 10.3390/cells11192964] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2022] [Revised: 09/06/2022] [Accepted: 09/09/2022] [Indexed: 11/19/2022] Open
Abstract
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.
Collapse
|
48
|
Nishitani-Isa M, Mukai K, Honda Y, Nihira H, Tanaka T, Shibata H, Kodama K, Hiejima E, Izawa K, Kawasaki Y, Osawa M, Katata Y, Onodera S, Watanabe T, Uchida T, Kure S, Takita J, Ohara O, Saito MK, Nishikomori R, Taguchi T, Sasahara Y, Yasumi T. Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation. J Exp Med 2022; 219:213184. [PMID: 35482294 PMCID: PMC9059393 DOI: 10.1084/jem.20211889] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Revised: 02/28/2022] [Accepted: 03/31/2022] [Indexed: 12/12/2022] Open
Abstract
Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β–blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation.
Collapse
Affiliation(s)
| | - Kojiro Mukai
- Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan
| | - Yoshitaka Honda
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan.,Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan.,Department of Immunology, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Hiroshi Nihira
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Takayuki Tanaka
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Hirofumi Shibata
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Kumi Kodama
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Eitaro Hiejima
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Kazushi Izawa
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Yuri Kawasaki
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Mitsujiro Osawa
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Yu Katata
- Department of Neonatology, Miyagi Children's Hospital, Sendai, Japan
| | - Sachiko Onodera
- Department of Neonatology, Miyagi Children's Hospital, Sendai, Japan
| | - Tatsuya Watanabe
- Department of Neonatology, Miyagi Children's Hospital, Sendai, Japan
| | - Takashi Uchida
- Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Shigeo Kure
- Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Junko Takita
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| | - Osamu Ohara
- Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Japan
| | - Megumu K Saito
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Ryuta Nishikomori
- Department of Pediatrics, Kurume University Graduate School of Medicine, Kurume, Japan
| | - Tomohiko Taguchi
- Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan
| | - Yoji Sasahara
- Department of Pediatrics, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Takahiro Yasumi
- Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan
| |
Collapse
|
49
|
Morimoto Y, Tokumitsu A, Sone T, Hirota Y, Tamura R, Sakamoto A, Nakajima K, Toda M, Kawakami Y, Okano H, Ohta S. TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF). Neurochem Res 2022; 47:2741-2756. [PMID: 35622214 DOI: 10.1007/s11064-022-03629-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 05/03/2022] [Accepted: 05/05/2022] [Indexed: 11/25/2022]
Abstract
One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.
Collapse
Affiliation(s)
- Yukina Morimoto
- Department of Neurosurgery, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Ayako Tokumitsu
- Division of Translational Research, Keio University Hospital Clinical and Translational Research Center, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Takefumi Sone
- Department of Physiology, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Yuki Hirota
- Department of Anatomy, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Ryota Tamura
- Department of Neurosurgery, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Ayuna Sakamoto
- Division of Translational Research, Keio University Hospital Clinical and Translational Research Center, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Kazunori Nakajima
- Department of Anatomy, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Masahiro Toda
- Department of Neurosurgery, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Yutaka Kawakami
- Cellular Signaling, Institute for Advanced Medical Research, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.,Department of Immunology, School of Medicine, International University of Health and Welfare 4-3, Kozunomori, Narita, Chiba, 286-8686, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Shigeki Ohta
- Cellular Signaling, Institute for Advanced Medical Research, Keio University of School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. .,Department of Immunology, School of Medicine, International University of Health and Welfare 4-3, Kozunomori, Narita, Chiba, 286-8686, Japan.
| |
Collapse
|
50
|
Long-Term Protective Effect of Human Dystrophin Expressing Chimeric (DEC) Cell Therapy on Amelioration of Function of Cardiac, Respiratory and Skeletal Muscles in Duchenne Muscular Dystrophy. Stem Cell Rev Rep 2022; 18:2872-2892. [PMID: 35590083 PMCID: PMC9622520 DOI: 10.1007/s12015-022-10384-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/26/2022] [Indexed: 12/12/2022]
Abstract
Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in dystrophin encoding gene, causing progressive degeneration of cardiac, respiratory, and skeletal muscles leading to premature death due to cardiac and respiratory failure. Currently, there is no cure for DMD. Therefore, novel therapeutic approaches are needed for DMD patients. We have previously reported functional improvements which correlated with increased dystrophin expression following administration of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD. In the current study, we confirmed dose-dependent protective effect of human DEC therapy created from myoblasts of normal and DMD-affected donors, on restoration of dystrophin expression and amelioration of cardiac, respiratory, and skeletal muscle function at 180 days after systemic-intraosseous DEC administration to mdx/scid mouse model of DMD. Functional improvements included maintenance of ejection fraction and fractional shortening levels on echocardiography, reduced enhanced pause and expiration time on plethysmography and improved grip strength and maximum stretch induced contraction of skeletal muscles. Improved function was associated with amelioration of mdx muscle pathology revealed by reduced muscle fibrosis, reduced inflammation and improved muscle morphology confirmed by reduced number of centrally nucleated fibers and normalization of muscle fiber diameters. Our findings confirm the long-term systemic effect of DEC therapy in the most severely affected by DMD organs including heart, diaphragm, and long skeletal muscles. These encouraging preclinical data introduces human DEC as a novel therapeutic modality of Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt the progression of DMD and enhance quality of life of DMD patients.
Collapse
|