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Matuszek Z, Brown BL, Yrigollen CM, Keiser MS, Davidson BL. Current trends in gene therapy to treat inherited disorders of the brain. Mol Ther 2025; 33:1988-2014. [PMID: 40181540 DOI: 10.1016/j.ymthe.2025.03.057] [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: 03/17/2025] [Revised: 03/28/2025] [Accepted: 03/28/2025] [Indexed: 04/05/2025] Open
Abstract
Gene therapy development, re-engineering, and application to patients hold promise to revolutionize medicine, including therapies for disorders of the brain. Advances in delivery modalities, expression regulation, and improving safety profiles are of critical importance. Additionally, each inherited disorder has its own unique characteristics as to regions and cell types impacted and the temporal dynamics of that impact that are essential for the design of therapeutic design strategies. Here, we review the current state of the art in gene therapies for inherited brain disorders, summarizing key considerations for vector delivery, gene addition, gene silencing, gene editing, and epigenetic editing. We provide examples from animal models, human cell lines, and, where possible, clinical trials. This review also highlights the various tools available to researchers for basic research questions and discusses our views on the current limitations in the field.
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Affiliation(s)
- Zaneta Matuszek
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
| | - Brandon L Brown
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Center for Epilepsy and Neurodevelopmental Disorders (ENDD), Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Carolyn M Yrigollen
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Megan S Keiser
- Department of Neurological Surgery, The Ohio State Wexner Medical Center, Columbus, OH 43210, USA
| | - Beverly L Davidson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Center for Epilepsy and Neurodevelopmental Disorders (ENDD), Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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Korchanová Z, Milovanov A, Švec M, Doležel J, Bartoš J, Valárik M. Progress and innovations of gene cloning in wheat and its close relatives. TAG. THEORETICAL AND APPLIED GENETICS. THEORETISCHE UND ANGEWANDTE GENETIK 2025; 138:106. [PMID: 40295316 PMCID: PMC12037653 DOI: 10.1007/s00122-025-04897-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/12/2024] [Accepted: 04/02/2025] [Indexed: 04/30/2025]
Abstract
KEY MESSAGE Wheat and its close relatives have large and complex genomes, making gene cloning difficult. Nevertheless, developments in genomics over the past decade have made it more feasible. The large and complex genomes of cereals, especially bread wheat, have always been a challenge for gene mapping and cloning. Nevertheless, recent advances in genomics have led to significant progress in this field. Currently, high-quality reference sequences are available for major wheat species and their relatives. New high-throughput genotyping platforms and next-generation sequencing technologies combined with genome complexity reduction techniques and mutagenesis have opened new avenues for gene cloning. In this review, we provide a comprehensive overview of the genes cloned in wheat so far and discuss the strategies used for cloning these genes. We highlight the advantages and drawbacks of individual approaches and show how particular genomic progress contributed to wheat gene cloning. A wide range of new resources and approaches have led to a significant increase in the number of successful cloning projects over the past decade, demonstrating that it is now feasible to perform rapid gene cloning of agronomically important genes, even in a genome as large and complex as that of wheat.
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Affiliation(s)
- Zuzana Korchanová
- Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany of the Czech Academy of Sciences, 77900, Olomouc, Czech Republic
- Department of Cell Biology and Genetics, Faculty of Science, Palacký University, 77900, Olomouc, Czech Republic
| | - Alexander Milovanov
- Department of Botany, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, 84104, Slovakia
| | - Miroslav Švec
- Department of Botany, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, 84104, Slovakia
| | - Jaroslav Doležel
- Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany of the Czech Academy of Sciences, 77900, Olomouc, Czech Republic
| | - Jan Bartoš
- Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany of the Czech Academy of Sciences, 77900, Olomouc, Czech Republic
| | - Miroslav Valárik
- Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany of the Czech Academy of Sciences, 77900, Olomouc, Czech Republic.
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Mohanta R, Maiti P, Sharangi AB, Roy S, Hazra S, Chakraborty S, Ghorai S. Directed mutagenesis in fruit crops. 3 Biotech 2025; 15:104. [PMID: 40177007 PMCID: PMC11958931 DOI: 10.1007/s13205-025-04268-8] [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: 01/02/2025] [Accepted: 03/07/2025] [Indexed: 04/05/2025] Open
Abstract
Fruit crops are rich source of important vitamins, minerals, and dietary fibres. They are essential for global agriculture with respect to nutritional security. Globally, there is a rapid decline in the genetic base of fruit crops warranting breeding strategies to overcome the challenge. Applied mutagenesis has emerged as a viable approach for the focused enhancement of fruit crops utilizing precise genetic alterations to increase a variety of desirable characteristics. However, traditional mutagenesis using physical and chemical mutagens are majorly random in nature. Directed mutagenesis with advancements in genetic engineering and molecular technology allows precise manipulation of genes, which facilitates the efficient and precise knockout of target genes and the targeted insertion or modification of specific DNA sequences within the genome via homologous recombination (HR)-mediated gene replacement. This review presents an in-depth exploration of several directed mutagenesis techniques including CRISPR-Cas9, TILLING, TALEN, MutMap, and MutMap + emphasizing their transformative applications in fruit crops. It also discusses about space mutagenesis. These advanced techniques empower researchers to precisely introduce specific mutations into the genome, skilfully altering gene expression and reshaping protein function with remarkable precision. This review highlights successful examples of directed mutagenesis in a variety of fruit crops such as apples, grapes, citrus, and strawberries and elucidates the impact of directed mutagenesis on traits such as fruit size, colour, flavour, shelf-life, and resistance to diseases and environmental stresses.
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Affiliation(s)
- Rajdeep Mohanta
- Department of Agriculture, Brainware University, Barasat, Kolkata, 700125 West Bengal India
| | - Payal Maiti
- Department of Post-Harvest Management, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, 741252 West Bengal India
| | - Amit Baran Sharangi
- Department of Plantation Spices Medicinal & Aromatic Crops, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, 741252 West Bengal India
| | - Sourav Roy
- Department of Agriculture, Brainware University, Barasat, Kolkata, 700125 West Bengal India
| | - Soham Hazra
- Department of Agriculture, Brainware University, Barasat, Kolkata, 700125 West Bengal India
| | - Souvik Chakraborty
- Department of Post-Harvest Management, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, 741252 West Bengal India
| | - Subhadwip Ghorai
- Department of Agriculture, Brainware University, Barasat, Kolkata, 700125 West Bengal India
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Ashmore-Harris C, Ayabe H, Yoshizawa E, Arisawa T, Takada Y, Takebe T, Fruhwirth GO. Gene editing enables non-invasive in vivo PET imaging of human induced pluripotent stem cell-derived liver bud organoids. Mol Ther Methods Clin Dev 2025; 33:101406. [PMID: 39927149 PMCID: PMC11803834 DOI: 10.1016/j.omtm.2025.101406] [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: 03/07/2024] [Accepted: 01/06/2025] [Indexed: 02/11/2025]
Abstract
Human induced pluripotent stem cell (hiPSC)-derived liver cell therapies such as hepatocyte-like cells and liver organoids could provide unlimited therapeutic cells for clinical transplantation, but an inadequate understanding of their in vivo fate impedes translation. Whole body in vivo imaging could enable monitoring of transplanted cell survival and/or expansion non-invasively over time, permitting robust comparisons between emerging therapies to identify those most effective. The human sodium iodide symporter (hNIS) is a radionuclide reporter gene facilitating whole body in vivo cell tracking by positron emission tomography (PET). We gene-edited a clinical Good Manufacturing Practice-compliant hiPSC line at the AAVS1 safe harbor locus enabling constitutive expression of a hNIS-monomeric(m)GFP fusion reporter in hiPSCs and their differentiated progeny. We confirmed reporter integration did not impact pluripotency or differentiation capacity, and radiotracer uptake capacity was retained post-differentiation. In vivo trackable liver bud (LB) organoids were generated from traceable hNIS fused to monomeric GFP (hNIS-mGFP)-hiPSCs and transplanted into healthy and liver-injured mice. LB were imaged quantitatively by 18FBF4 --PET with imaging results confirmed histologically. We report, for the first time, hNIS-mGFP-hiPSC progeny retain differentiated function and PET trackability in vivo using LB. In vivo monitoring could accelerate regenerative cell therapy development by identifying efficacious candidate cells, successful engraftment/survival strategies and addressing safety concerns.
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Affiliation(s)
- Candice Ashmore-Harris
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK
- Imaging Therapies and Cancer Group, Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, King’s College London, Guy’s Cancer Centre, London SE1 1UL, UK
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
| | - Hiroaki Ayabe
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
- Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Emi Yoshizawa
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
| | - Tetsu Arisawa
- Department of Physiology, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
| | - Yuuki Takada
- Department of Physiology, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
| | - Takanori Takebe
- Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
- Center for Stem Cell & Organoid Medicine (CuSTOM), Division of Gastroenterology, Hepatology and Nutrition & Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
- Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), and Division of Stem Cell and Organoid Medicine, Osaka University, Suita, Osaka 565-0871, Japan
| | - Gilbert O. Fruhwirth
- Imaging Therapies and Cancer Group, Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, King’s College London, Guy’s Cancer Centre, London SE1 1UL, UK
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Busquets O, Li H, Syed KM, Jerez PA, Dunnack J, Bu RL, Verma Y, Pangilinan GR, Martin A, Straub J, Du Y, Simon VM, Poser S, Bush Z, Diaz J, Sahagun A, Gao J, Hong S, Hernandez DG, Levine KS, Booth EO, Blanchette M, Bateup HS, Rio DC, Blauwendraat C, Hockemeyer D, Soldner F. iSCORE-PD: an isogenic stem cell collection to research Parkinson's Disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.02.12.579917. [PMID: 38405931 PMCID: PMC10888955 DOI: 10.1101/2024.02.12.579917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/27/2024]
Abstract
Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of 65 human stem cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G/FS, SYNJ1 R258Q/FS, VPS13C A444P/FS, VPS13C W395C/FS, GBA1 IVS2+1/FS). All mutations were introduced into a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using different genome editing techniques. To ensure the genetic integrity of these cell lines, we implemented rigorous quality controls, including whole-genome sequencing of each line. Our analysis of the genetic variation in this cell line collection revealed that while genome editing, particularly using CRISPR/Cas9, can introduce rare off-target mutations, the predominant source of genetic variants arises from routine cell culture and are fixed in cell lines during clonal isolation. The observed genetic variation was minimal compared to that typically found in patient-derived iPSC experiments and predominantly affected non-coding regions of the genome. Importantly, our analysis outlines strategies for effectively managing genetic variation through stringent quality control measures and careful experimental design. This systematic approach ensures the high quality of our stem cell collection, highlights advantages of prime editing over conventional CRISPR/Cas9 methods and provides a roadmap for the generation of gene-edited hPSC collections at scale in an academic setting. Our iSCORE-PD collection represents an easily accessible and valuable platform to study PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.
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Affiliation(s)
- Oriol Busquets
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA
- Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- These authors contributed equally
| | - Hanqin Li
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA
- These authors contributed equally
| | - Khaja Mohieddin Syed
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- These authors contributed equally
| | - Pilar Alvarez Jerez
- Center for Alzheimer’s and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK
- These authors contributed equally
| | - Jesse Dunnack
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- These authors contributed equally
| | - Riana Lo Bu
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
| | - Yogendra Verma
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Gabriella R. Pangilinan
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Annika Martin
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Jannes Straub
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - YuXin Du
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Vivien M. Simon
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Steven Poser
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
| | - Zipporiah Bush
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Genetics, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA
| | - Jessica Diaz
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
| | - Atehsa Sahagun
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- Department of Neuroscience, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Jianpu Gao
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Samantha Hong
- Center for Alzheimer’s and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Dena G. Hernandez
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Kristin S. Levine
- Center for Alzheimer’s and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Ezgi O. Booth
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | | | - Helen S. Bateup
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- Department of Neuroscience, University of California, Berkeley, Berkeley, CA 94720, USA
- Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA
| | - Donald C. Rio
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Cornelis Blauwendraat
- Center for Alzheimer’s and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, 20892, USA
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Dirk Hockemeyer
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
- Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA
- Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA
| | - Frank Soldner
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA
- Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA
- Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- Department of Genetics, Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA
- Lead contact
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Ahmadikhah A, Zarabizadeh H, Nayeri S, Abbasi MS. Advancements in genome editing tools for genetic studies and crop improvement. FRONTIERS IN PLANT SCIENCE 2025; 15:1370675. [PMID: 39963359 PMCID: PMC11830681 DOI: 10.3389/fpls.2024.1370675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 12/31/2024] [Indexed: 02/20/2025]
Abstract
The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.
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Affiliation(s)
- Asadollah Ahmadikhah
- Department of Cellular and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
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Allisha J, Das J, Dunnigan T, Sharfstein ST, Datta P. Stipulations of cell and gene therapy and the ties to biomanufacturing. Biotechnol Prog 2025:e3521. [PMID: 39846483 DOI: 10.1002/btpr.3521] [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: 08/22/2024] [Revised: 11/15/2024] [Accepted: 11/20/2024] [Indexed: 01/24/2025]
Abstract
Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.
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Affiliation(s)
- Justin Allisha
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Juthika Das
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Thomas Dunnigan
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Susan T Sharfstein
- Department of Nanoscale Science and Engineering and The RNA Institute, University at Albany, State University of New York, Albany, New York, USA
| | - Payel Datta
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
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8
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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.
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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.
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Xu J, Gong W, Mo C, Hou X, Ou M. Global Knowledge Map and Emerging Research Trends in Induced Pluripotent Stem Cells and Hereditary Diseases: A CiteSpace-based Visualization and Analysis. Stem Cell Rev Rep 2025; 21:126-146. [PMID: 39377988 DOI: 10.1007/s12015-024-10799-z] [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] [Accepted: 10/01/2024] [Indexed: 01/26/2025]
Abstract
The rise of induced pluripotent stem cells (iPSCs) technology has ushered in a landmark shift in the study of hereditary diseases. However, there is a scarcity of reports that offer a comprehensive and objective overview of the current state of research at the intersection of iPSCs and hereditary diseases. Therefore, this study endeavors to categorize and synthesize the publications in this field over the past decade through bibliometric methods and visual knowledge mapping, aiming to visually analyze their research focus and clinical trends. The English language literature on iPSCs and hereditary diseases, published from 2014 to 2023 in the Web of Science Core Collection (WoSCC), was examined. The CiteSpace (version 6.3.R1) software was utilized to visualize and analyze country/region, institution, scholar, co-cited authors, and co-cited journals. Additionally, the co-occurrence, clustering, and bursting of co-cited references were displayed. Analysis of 347 articles that met the inclusion criteria revealed a steady increase in the number of published articles and citation frequency in the field over the past decade. With regard to the countries/regions, institutions, scholars, and journals where the articles were published, the highest numbers were found in the USA, the University of California System, Suren M. Zakian, and Stem Cell Research, respectively. The current research is focused on the construction of disease models, both before and after correction, as well as drug target testing for single-gene hereditary diseases. Chromosome transplantation genomic therapy for hereditary diseases with abnormal chromosome structures may emerge as a future research hotspot in this field.
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Affiliation(s)
- Jiajun Xu
- Laboratory Center, Guangxi Key Laboratory of Metabolic Reprogramming and In- telligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
- Laboratory Center, Guangxi Health Commission Key Laboratory of Glucose and Lipid Metabolism Disorders, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
| | - Weiwei Gong
- Laboratory Center, Guangxi Key Laboratory of Metabolic Reprogramming and In- telligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
- Laboratory Center, Guangxi Health Commission Key Laboratory of Glucose and Lipid Metabolism Disorders, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
| | - Chune Mo
- Laboratory Center, Guangxi Key Laboratory of Metabolic Reprogramming and In- telligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
- Laboratory Center, Guangxi Health Commission Key Laboratory of Glucose and Lipid Metabolism Disorders, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
| | - Xianliang Hou
- Laboratory Center, Guangxi Key Laboratory of Metabolic Reprogramming and In- telligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
- Laboratory Center, Guangxi Health Commission Key Laboratory of Glucose and Lipid Metabolism Disorders, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China
| | - Minglin Ou
- Laboratory Center, Guangxi Key Laboratory of Metabolic Reprogramming and In- telligent Medical Engineering for Chronic Diseases, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China.
- Laboratory Center, Guangxi Health Commission Key Laboratory of Glucose and Lipid Metabolism Disorders, The Second Affiliated Hospital of Guilin Medical University, Guilin, 541199, China.
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10
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Tsuji‐Hosokawa A, Tsuchiya I, Shimizu K, Terao M, Yasuhara M, Miyamoto N, Kikuchi S, Ogawa Y, Nakamura K, Matsubara Y, Takada S. Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs. J Inherit Metab Dis 2025; 48:e12803. [PMID: 39380247 PMCID: PMC11729594 DOI: 10.1002/jimd.12803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2024] [Revised: 09/13/2024] [Accepted: 09/17/2024] [Indexed: 10/10/2024]
Abstract
Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model PahhR111X mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in PahhR111X mice. PahhR111X mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of PahhR111X mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.
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Affiliation(s)
- Atsumi Tsuji‐Hosokawa
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
- Division of Diversity ResearchNational Research Institute for Child Health and DevelopmentTokyoJapan
- Department of Pediatrics and Developmental Biology, Graduate School of Medical and Dental SciencesTokyo Medical and Dental University (TMDU)TokyoJapan
| | - Iku Tsuchiya
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
- Department of NCCHD, Graduate School of Medical and Dental SciencesTokyo Medical and Dental University (TMDU)TokyoJapan
| | - Kie Shimizu
- Department of PharmacologyNational Research Institute for Child Health and DevelopmentTokyoJapan
- Division of Life Science, Graduate School of Science and EngineeringSaitama UniversitySaitamaJapan
| | - Miho Terao
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
| | - Mito Yasuhara
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
- Department of NCCHD, Graduate School of Medical and Dental SciencesTokyo Medical and Dental University (TMDU)TokyoJapan
| | - Natsuho Miyamoto
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
| | - Saki Kikuchi
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
| | - Yuya Ogawa
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
- Department of NCCHD, Graduate School of Medical and Dental SciencesTokyo Medical and Dental University (TMDU)TokyoJapan
| | - Kazuaki Nakamura
- Department of PharmacologyNational Research Institute for Child Health and DevelopmentTokyoJapan
- Division of Life Science, Graduate School of Science and EngineeringSaitama UniversitySaitamaJapan
| | - Yoichi Matsubara
- National Center for Child Health and DevelopmentSetagayaTokyoJapan
| | - Shuji Takada
- Department of Systems BioMedicineNational Research Institute for Child Health and DevelopmentTokyoJapan
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11
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Wu Y, Zhong A, Sidharta M, Kim TW, Ramirez B, Persily B, Studer L, Zhou T. Robust and inducible genome editing via an all-in-one prime editor in human pluripotent stem cells. Nat Commun 2024; 15:10824. [PMID: 39737975 PMCID: PMC11685797 DOI: 10.1038/s41467-024-55104-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2024] [Accepted: 11/27/2024] [Indexed: 01/01/2025] Open
Abstract
Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small insertions or deletions at a specific genomic locus. Here, we systematically compare a panel of prime editing conditions in hPSCs and generate a potent prime editor, "PE-Plus", through co-inhibition of mismatch repair and p53-mediated cellular stress responses. We further establish an inducible prime editing platform in hPSCs by incorporating the PE-Plus into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.
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Affiliation(s)
- Youjun Wu
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Aaron Zhong
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Mega Sidharta
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Tae Wan Kim
- The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Bernny Ramirez
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Benjamin Persily
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA
| | - Lorenz Studer
- The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA.
| | - Ting Zhou
- The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY, USA.
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12
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Ma H. Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1. Bio Protoc 2024; 14:e5108. [PMID: 39600977 PMCID: PMC11588425 DOI: 10.21769/bioprotoc.5108] [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: 06/27/2024] [Revised: 09/06/2024] [Accepted: 09/09/2024] [Indexed: 11/29/2024] Open
Abstract
Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array, efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase, permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences, no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects. Key features • A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells • Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment • Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types.
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Affiliation(s)
- Haiting Ma
- Department of Cell and Developmental Biology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
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13
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Hu X, Zhang X, Sun W, Liu C, Deng P, Cao Y, Zhang C, Xu N, Zhang T, Zhang Y, Liu JJ, Wang H. Systematic discovery of DNA-binding tandem repeat proteins. Nucleic Acids Res 2024; 52:10464-10489. [PMID: 39189466 PMCID: PMC11417379 DOI: 10.1093/nar/gkae710] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 07/30/2024] [Accepted: 08/07/2024] [Indexed: 08/28/2024] Open
Abstract
Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.
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Affiliation(s)
- Xiaoxuan Hu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Xuechun Zhang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Wen Sun
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Chunhong Liu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Pujuan Deng
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China
| | - Yuanwei Cao
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Chenze Zhang
- National Key Laboratory of Efficacy and Mechanism on Chinese Medicine for Metabolic Diseases, Beijing University of Chinese Medicine, Beijing 100029, China
| | - Ning Xu
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Tongtong Zhang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
| | - Yong E Zhang
- University of Chinese Academy of Sciences, Beijing 100049, China
- Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jun-Jie Gogo Liu
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China
| | - Haoyi Wang
- Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
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14
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Lam I, Ndayisaba A, Lewis AJ, Fu Y, Sagredo GT, Kuzkina A, Zaccagnini L, Celikag M, Sandoe J, Sanz RL, Vahdatshoar A, Martin TD, Morshed N, Ichihashi T, Tripathi A, Ramalingam N, Oettgen-Suazo C, Bartels T, Boussouf M, Schäbinger M, Hallacli E, Jiang X, Verma A, Tea C, Wang Z, Hakozaki H, Yu X, Hyles K, Park C, Wang X, Theunissen TW, Wang H, Jaenisch R, Lindquist S, Stevens B, Stefanova N, Wenning G, van de Berg WDJ, Luk KC, Sanchez-Pernaute R, Gómez-Esteban JC, Felsky D, Kiyota Y, Sahni N, Yi SS, Chung CY, Stahlberg H, Ferrer I, Schöneberg J, Elledge SJ, Dettmer U, Halliday GM, Bartels T, Khurana V. Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions. Neuron 2024; 112:2886-2909.e16. [PMID: 39079530 PMCID: PMC11377155 DOI: 10.1016/j.neuron.2024.06.002] [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: 11/29/2022] [Revised: 10/26/2023] [Accepted: 06/03/2024] [Indexed: 09/07/2024]
Abstract
The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.
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Affiliation(s)
- Isabel Lam
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Alain Ndayisaba
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Division of Neurobiology, Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria
| | - Amanda J Lewis
- École Polytechnique Fédérale de Lausanne and University of Lausanne, Lausanne, Switzerland
| | - YuHong Fu
- The University of Sydney Brain and Mind Centre and Faculty of Medicine and Health School of Medical Science, Sydney, NSW, Australia
| | - Giselle T Sagredo
- The University of Sydney Brain and Mind Centre and Faculty of Medicine and Health School of Medical Science, Sydney, NSW, Australia
| | - Anastasia Kuzkina
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | | | - Meral Celikag
- Dementia Research Institute, University College London, London, UK
| | - Jackson Sandoe
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
| | - Ricardo L Sanz
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Aazam Vahdatshoar
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Timothy D Martin
- Harvard Medical School, Boston, MA, USA; Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Nader Morshed
- Harvard Medical School, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA; Boston Children's Hospital, Boston, MA, USA; The Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | | | - Arati Tripathi
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Nagendran Ramalingam
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Charlotte Oettgen-Suazo
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Theresa Bartels
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
| | - Manel Boussouf
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Max Schäbinger
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Erinc Hallacli
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Xin Jiang
- Yumanity Therapeutics, Cambridge, MA, USA
| | - Amrita Verma
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Challana Tea
- University of California, San Diego, San Diego, CA, USA
| | - Zichen Wang
- University of California, San Diego, San Diego, CA, USA
| | | | - Xiao Yu
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Kelly Hyles
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Chansaem Park
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA
| | - Xinyuan Wang
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | | | - Haoyi Wang
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
| | - Rudolf Jaenisch
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
| | - Susan Lindquist
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Beth Stevens
- Harvard Medical School, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA; Boston Children's Hospital, Boston, MA, USA; The Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Nadia Stefanova
- Division of Neurobiology, Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria
| | - Gregor Wenning
- Division of Neurobiology, Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria
| | | | - Kelvin C Luk
- University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | - Rosario Sanchez-Pernaute
- BioBizkaia Health Research Institute, Barakaldo, Spain; Ikerbasque, Basque Foundation for Science, Bilbao, Spain
| | | | - Daniel Felsky
- Centre for Addiction and Mental Health, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada
| | | | - Nidhi Sahni
- The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Baylor College of Medicine, Houston, TX, USA
| | - S Stephen Yi
- The University of Texas at Austin, Austin, TX, USA
| | | | - Henning Stahlberg
- École Polytechnique Fédérale de Lausanne and University of Lausanne, Lausanne, Switzerland
| | - Isidro Ferrer
- The University of Barcelona, Institut d'Investigacio Biomedica de Bellvitge IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
| | | | - Stephen J Elledge
- Harvard Medical School, Boston, MA, USA; Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Ulf Dettmer
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA
| | - Glenda M Halliday
- The University of Sydney Brain and Mind Centre and Faculty of Medicine and Health School of Medical Science, Sydney, NSW, Australia; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA
| | - Tim Bartels
- Dementia Research Institute, University College London, London, UK
| | - Vikram Khurana
- Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Division of Movement Disorders, American Parkinson Disease Association (APDA) Center for Advanced Research and MSA Center of Excellence, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA; Harvard Stem Cell Institute, Cambridge, MA, USA.
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15
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Biscaia-Caleiras M, Fonseca NA, Lourenço AS, Moreira JN, Simões S. Rational formulation and industrial manufacturing of lipid-based complex injectables: Landmarks and trends. J Control Release 2024; 373:617-639. [PMID: 39002799 DOI: 10.1016/j.jconrel.2024.07.021] [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: 04/05/2024] [Revised: 07/02/2024] [Accepted: 07/08/2024] [Indexed: 07/15/2024]
Abstract
Lipid-based complex injectables are renowned for their effectiveness in delivering drugs, with many approved products. While significant strides have been made in formulating nanosystems for small molecular weight drugs, a pivotal breakthrough emerged with the recognition of lipid nanoparticles as a promising platform for delivering nucleic acids. This finding has paved the way for tackling long-standing challenges in molecular and delivery aspects (e.g., mRNA stability, intracellular delivery) that have impeded the clinical translation of gene therapy, especially in the realm of immunotherapy. Nonetheless, developing and implementing new lipid-based delivery systems pose significant challenges, as industrial manufacturing of these formulations often involves complex, multi-batch processes, giving rise to issues related to scalability, stability, sterility, and regulatory compliance. To overcome these obstacles, embracing the principles of quality-by-design (QbD) is imperative. Furthermore, adopting cutting-edge manufacturing and process analytical tools (PAT) that facilitate the transition from batch to continuous production is essential. Herein, the key milestones and insights derived from the development of currently approved lipid- nanosystems will be explored. Additionally, a comprehensive and critical overview of the latest technologies and regulatory guidelines that underpin the creation of more efficient, scalable, and flexible manufacturing processes for complex lipid-based nanoformulations will be provided.
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Affiliation(s)
- Mariana Biscaia-Caleiras
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, 3004-504 Coimbra, Portugal; Bluepharma-Indústria Farmacêutica, S.A., São Martinho do Bispo, 3045-016 Coimbra, Portugal; Univ Coimbra-University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
| | - Nuno A Fonseca
- Bluepharma-Indústria Farmacêutica, S.A., São Martinho do Bispo, 3045-016 Coimbra, Portugal
| | - Ana Sofia Lourenço
- Bluepharma-Indústria Farmacêutica, S.A., São Martinho do Bispo, 3045-016 Coimbra, Portugal
| | - João Nuno Moreira
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, 3004-504 Coimbra, Portugal; Univ Coimbra-University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
| | - Sérgio Simões
- CNC - Center for Neurosciences and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, 3004-504 Coimbra, Portugal; Bluepharma-Indústria Farmacêutica, S.A., São Martinho do Bispo, 3045-016 Coimbra, Portugal; Univ Coimbra-University of Coimbra, CIBB, Faculty of Pharmacy, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
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16
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Ghavi Hossein-Zadeh N. An overview of recent technological developments in bovine genomics. Vet Anim Sci 2024; 25:100382. [PMID: 39166173 PMCID: PMC11334705 DOI: 10.1016/j.vas.2024.100382] [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: 08/22/2024] Open
Abstract
Cattle are regarded as highly valuable animals because of their milk, beef, dung, fur, and ability to draft. The scientific community has tried a number of strategies to improve the genetic makeup of bovine germplasm. To ensure higher returns for the dairy and beef industries, researchers face their greatest challenge in improving commercially important traits. One of the biggest developments in the last few decades in the creation of instruments for cattle genetic improvement is the discovery of the genome. Breeding livestock is being revolutionized by genomic selection made possible by the availability of medium- and high-density single nucleotide polymorphism (SNP) arrays coupled with sophisticated statistical techniques. It is becoming easier to access high-dimensional genomic data in cattle. Continuously declining genotyping costs and an increase in services that use genomic data to increase return on investment have both made a significant contribution to this. The field of genomics has come a long way thanks to groundbreaking discoveries such as radiation-hybrid mapping, in situ hybridization, synteny analysis, somatic cell genetics, cytogenetic maps, molecular markers, association studies for quantitative trait loci, high-throughput SNP genotyping, whole-genome shotgun sequencing to whole-genome mapping, and genome editing. These advancements have had a significant positive impact on the field of cattle genomics. This manuscript aimed to review recent advances in genomic technologies for cattle breeding and future prospects in this field.
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Affiliation(s)
- Navid Ghavi Hossein-Zadeh
- Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, 41635-1314, Iran
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17
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Chen X, Yang G, Ji P, Liu G, Zhang L. Identification of Site in the UTY Gene as Safe Harbor Locus on the Y Chromosome of Pig. Genes (Basel) 2024; 15:1005. [PMID: 39202365 PMCID: PMC11353466 DOI: 10.3390/genes15081005] [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: 07/02/2024] [Revised: 07/23/2024] [Accepted: 07/24/2024] [Indexed: 09/03/2024] Open
Abstract
Genomic Safe Harbors (GSH) are loci used for the insertion of exogenous genetic elements, enabling exogenous gene expressing predictably without alterations of the host genome. These sites are becoming increasingly important as the gene editing technologies advance rapidly. Currently, only a few GSHs have been identified in the pig genome. In this study, a novel strategy was demonstrated for the efficient insertion of exogenous genetic material into the third exon of the UTY gene on the Y chromosome using CRISPR/Cas9-mediated homology arm-mediated end joining. The safety of the locus was verified according to the proper expression of the inserted EGFP gene without altering the expression of UTY. This approach enables the integration and expression of the exogenous gene at this locus, indicating that the UTY locus serves as a genomic safe harbor site for gene editing in the pig genome. Located on the Y chromosome, this site can be utilized for sex-biased pig breeding and developing biomedical models.
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Affiliation(s)
- Xiaomei Chen
- State Key Laboratory of Farm Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; (X.C.); (G.Y.); (P.J.); (G.L.)
- College of Animal Science and Technology, Sanya Institute of China Agricultural University, Sanya 572025, China
| | - Guang Yang
- State Key Laboratory of Farm Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; (X.C.); (G.Y.); (P.J.); (G.L.)
- College of Animal Science and Technology, Sanya Institute of China Agricultural University, Sanya 572025, China
| | - Pengyun Ji
- State Key Laboratory of Farm Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; (X.C.); (G.Y.); (P.J.); (G.L.)
| | - Guoshi Liu
- State Key Laboratory of Farm Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; (X.C.); (G.Y.); (P.J.); (G.L.)
- College of Animal Science and Technology, Sanya Institute of China Agricultural University, Sanya 572025, China
| | - Lu Zhang
- State Key Laboratory of Farm Animal Biotech Breeding, Frontiers Science Center for Molecular Design Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; (X.C.); (G.Y.); (P.J.); (G.L.)
- College of Animal Science and Technology, Sanya Institute of China Agricultural University, Sanya 572025, China
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18
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Teles D, Fine BM. Using induced pluripotent stem cells for drug discovery in arrhythmias. Expert Opin Drug Discov 2024; 19:827-840. [PMID: 38825838 PMCID: PMC11227103 DOI: 10.1080/17460441.2024.2360420] [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: 03/18/2024] [Accepted: 05/23/2024] [Indexed: 06/04/2024]
Abstract
INTRODUCTION Arrhythmias are disturbances in the normal rhythm of the heart and account for significant cardiovascular morbidity and mortality worldwide. Historically, preclinical research has been anchored in animal models, though physiological differences between these models and humans have limited their clinical translation. The discovery of human induced pluripotent stem cells (iPSC) and subsequent differentiation into cardiomyocyte has led to the development of new in vitro models of arrhythmias with the hope of a new pathway for both exploration of pathogenic variants and novel therapeutic discovery. AREAS COVERED The authors describe the latest two-dimensional in vitro models of arrhythmias, several examples of the use of these models in drug development, and the role of gene editing when modeling diseases. They conclude by discussing the use of three-dimensional models in the study of arrythmias and the integration of computational technologies and machine learning with experimental technologies. EXPERT OPINION Human iPSC-derived cardiomyocytes models have significant potential to augment disease modeling, drug discovery, and toxicity studies in preclinical development. While there is initial success with modeling arrhythmias, the field is still in its nascency and requires advances in maturation, cellular diversity, and readouts to emulate arrhythmias more accurately.
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Affiliation(s)
- Diogo Teles
- Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA
| | - Barry M. Fine
- Department of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USA
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19
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Sirois CL, Guo Y, Li M, Wolkoff NE, Korabelnikov T, Sandoval S, Lee J, Shen M, Contractor A, Sousa AMM, Bhattacharyya A, Zhao X. CGG repeats in the human FMR1 gene regulate mRNA localization and cellular stress in developing neurons. Cell Rep 2024; 43:114330. [PMID: 38865241 PMCID: PMC11240841 DOI: 10.1016/j.celrep.2024.114330] [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: 11/18/2023] [Revised: 04/18/2024] [Accepted: 05/22/2024] [Indexed: 06/14/2024] Open
Abstract
The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.
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Affiliation(s)
- Carissa L Sirois
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Yu Guo
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Meng Li
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Natalie E Wolkoff
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Tomer Korabelnikov
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Soraya Sandoval
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Jiyoun Lee
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Minjie Shen
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Amaya Contractor
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Andre M M Sousa
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Anita Bhattacharyya
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Xinyu Zhao
- Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA.
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20
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Yin X, Li Q, Shu Y, Wang H, Thomas B, Maxwell JT, Zhang Y. Exploiting urine-derived induced pluripotent stem cells for advancing precision medicine in cell therapy, disease modeling, and drug testing. J Biomed Sci 2024; 31:47. [PMID: 38724973 PMCID: PMC11084032 DOI: 10.1186/s12929-024-01035-4] [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: 01/26/2024] [Accepted: 04/30/2024] [Indexed: 05/12/2024] Open
Abstract
The field of regenerative medicine has witnessed remarkable advancements with the emergence of induced pluripotent stem cells (iPSCs) derived from a variety of sources. Among these, urine-derived induced pluripotent stem cells (u-iPSCs) have garnered substantial attention due to their non-invasive and patient-friendly acquisition method. This review manuscript delves into the potential and application of u-iPSCs in advancing precision medicine, particularly in the realms of drug testing, disease modeling, and cell therapy. U-iPSCs are generated through the reprogramming of somatic cells found in urine samples, offering a unique and renewable source of patient-specific pluripotent cells. Their utility in drug testing has revolutionized the pharmaceutical industry by providing personalized platforms for drug screening, toxicity assessment, and efficacy evaluation. The availability of u-iPSCs with diverse genetic backgrounds facilitates the development of tailored therapeutic approaches, minimizing adverse effects and optimizing treatment outcomes. Furthermore, u-iPSCs have demonstrated remarkable efficacy in disease modeling, allowing researchers to recapitulate patient-specific pathologies in vitro. This not only enhances our understanding of disease mechanisms but also serves as a valuable tool for drug discovery and development. In addition, u-iPSC-based disease models offer a platform for studying rare and genetically complex diseases, often underserved by traditional research methods. The versatility of u-iPSCs extends to cell therapy applications, where they hold immense promise for regenerative medicine. Their potential to differentiate into various cell types, including neurons, cardiomyocytes, and hepatocytes, enables the development of patient-specific cell replacement therapies. This personalized approach can revolutionize the treatment of degenerative diseases, organ failure, and tissue damage by minimizing immune rejection and optimizing therapeutic outcomes. However, several challenges and considerations, such as standardization of reprogramming protocols, genomic stability, and scalability, must be addressed to fully exploit u-iPSCs' potential in precision medicine. In conclusion, this review underscores the transformative impact of u-iPSCs on advancing precision medicine and highlights the future prospects and challenges in harnessing this innovative technology for improved healthcare outcomes.
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Affiliation(s)
- Xiya Yin
- Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, China
| | - Qingfeng Li
- Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
| | - Yan Shu
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Hongbing Wang
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Biju Thomas
- Keck School of Medicine, Roski Eye Institute, University of Southern California, Los Angeles, CA, 90033, USA
| | - Joshua T Maxwell
- Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA
| | - Yuanyuan Zhang
- Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA.
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21
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Lindenhofer D, Haendeler S, Esk C, Littleboy JB, Brunet Avalos C, Naas J, Pflug FG, van de Ven EGP, Reumann D, Baffet AD, von Haeseler A, Knoblich JA. Cerebral organoids display dynamic clonal growth and tunable tissue replenishment. Nat Cell Biol 2024; 26:710-718. [PMID: 38714853 PMCID: PMC11098754 DOI: 10.1038/s41556-024-01412-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 03/28/2024] [Indexed: 05/18/2024]
Abstract
During brain development, neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells, resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids, to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids.
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Affiliation(s)
- Dominik Lindenhofer
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria
- Vienna Biocenter PhD Program, University of Vienna and the Medical University of Vienna, Vienna, Austria
- Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Simon Haendeler
- Vienna Biocenter PhD Program, University of Vienna and the Medical University of Vienna, Vienna, Austria
- Center of Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna and Medical University of Vienna, Vienna BioCenter, Vienna, Austria
| | - Christopher Esk
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria.
- Institute of Molecular Biology, University of Innsbruck, Innsbruck, Austria.
| | - Jamie B Littleboy
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria
- Vienna Biocenter PhD Program, University of Vienna and the Medical University of Vienna, Vienna, Austria
| | | | - Julia Naas
- Vienna Biocenter PhD Program, University of Vienna and the Medical University of Vienna, Vienna, Austria
- Center of Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna and Medical University of Vienna, Vienna BioCenter, Vienna, Austria
| | - Florian G Pflug
- Center of Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna and Medical University of Vienna, Vienna BioCenter, Vienna, Austria
- Biological Complexity Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan
| | - Eline G P van de Ven
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria
| | - Daniel Reumann
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria
| | - Alexandre D Baffet
- Institut Curie, PSL Research University, CNRS UMR144, Paris, France
- Institut national de la santé et de la recherche médicale, Paris, France
| | - Arndt von Haeseler
- Vienna Biocenter PhD Program, University of Vienna and the Medical University of Vienna, Vienna, Austria
- Faculty of Computer Science, Bioinformatics and Computational Biology, University of Vienna, Vienna, Austria
| | - Jürgen A Knoblich
- Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna BioCenter, Vienna, Austria.
- Department of Neurology, Medical University of Vienna, Vienna, Austria.
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22
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Chang CH, Liu F, Militi S, Hester S, Nibhani R, Deng S, Dunford J, Rendek A, Soonawalla Z, Fischer R, Oppermann U, Pauklin S. The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms. Nat Commun 2024; 15:3580. [PMID: 38678032 PMCID: PMC11055877 DOI: 10.1038/s41467-024-47680-z] [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: 12/30/2022] [Accepted: 04/09/2024] [Indexed: 04/29/2024] Open
Abstract
The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.
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Affiliation(s)
- Chao-Hui Chang
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Feng Liu
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Stefania Militi
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Svenja Hester
- Target Discovery Institute, Nuffield Department of Medicine, Old Road, University of Oxford, Oxford, OX3 7FZ, UK
| | - Reshma Nibhani
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Siwei Deng
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - James Dunford
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Aniko Rendek
- Department of Histopathology, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
| | - Zahir Soonawalla
- Department of Hepatobiliary and Pancreatic Surgery, Oxford University Hospitals NHS, Oxford, UK
| | - Roman Fischer
- Target Discovery Institute, Nuffield Department of Medicine, Old Road, University of Oxford, Oxford, OX3 7FZ, UK
| | - Udo Oppermann
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK
| | - Siim Pauklin
- Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Old Road, Oxford, OX3 7LD, UK.
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23
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Davis JC, Ryaboshapkina M, Kenty JH, Eser PÖ, Menon S, Tyrberg B, Melton DA. IAPP Marks Mono-hormonal Stem-cell Derived β Cells that Maintain Stable Insulin Production in vitro and in vivo. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.10.587726. [PMID: 38645166 PMCID: PMC11030367 DOI: 10.1101/2024.04.10.587726] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/23/2024]
Abstract
Islet transplantation for treatment of diabetes is limited by availability of donor islets and requirements for immunosuppression. Stem cell-derived islets might circumvent these issues. SC-islets effectively control glucose metabolism post transplantation, but do not yet achieve full function in vitro with current published differentiation protocols. We aimed to identify markers of mature subpopulations of SC-β cells by studying transcriptional changes associated with in vivo maturation of SC-β cells using RNA-seq and co-expression network analysis. The β cell-specific hormone islet amyloid polypeptide (IAPP) emerged as the top candidate to be such a marker. IAPP+ cells had more mature β cell gene expression and higher cellular insulin content than IAPP- cells in vitro. IAPP+ INS+ cells were more stable in long-term culture than IAPP- INS+ cells and retained insulin expression after transplantation into mice. Finally, we conducted a small molecule screen to identify compounds that enhance IAPP expression. Aconitine up-regulated IAPP and could help to optimize differentiation protocols.
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Affiliation(s)
- Jeffrey C. Davis
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Boston MA 02138, United States of America
| | - Maria Ryaboshapkina
- Translational Science and Experimental Medicine, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Jennifer H. Kenty
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Boston MA 02138, United States of America
| | | | - Suraj Menon
- RDI Operations, Granta Park, AstraZeneca, Cambridge CB21 6GP, UK
| | - Björn Tyrberg
- Global Insights, Analytics & Commercial Excellence, BioPharmaceuticals Business Unit, AstraZeneca, Gothenburg, Sweden
| | - Douglas A. Melton
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Boston MA 02138, United States of America
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24
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Zheng Y, Li Y, Zhou K, Li T, VanDusen NJ, Hua Y. Precise genome-editing in human diseases: mechanisms, strategies and applications. Signal Transduct Target Ther 2024; 9:47. [PMID: 38409199 PMCID: PMC10897424 DOI: 10.1038/s41392-024-01750-2] [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: 05/17/2023] [Revised: 01/15/2024] [Accepted: 01/17/2024] [Indexed: 02/28/2024] Open
Abstract
Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these tools has led to a revolution in the life sciences, which promises to deliver novel therapies for genetic disease. Precise genome-editing can be traced back to the 1950s with the discovery of DNA's double-helix and, after 70 years of development, has evolved from crude in vitro applications to a wide range of sophisticated capabilities, including in vivo applications. Nonetheless, precise genome-editing faces constraints such as modest efficiency, delivery challenges, and off-target effects. In this review, we explore precise genome-editing, with a focus on introduction of the landmark events in its history, various platforms, delivery systems, and applications. First, we discuss the landmark events in the history of precise genome-editing. Second, we describe the current state of precise genome-editing strategies and explain how these techniques offer unprecedented precision and versatility for modifying the human genome. Third, we introduce the current delivery systems used to deploy precise genome-editing components through DNA, RNA, and RNPs. Finally, we summarize the current applications of precise genome-editing in labeling endogenous genes, screening genetic variants, molecular recording, generating disease models, and gene therapy, including ex vivo therapy and in vivo therapy, and discuss potential future advances.
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Affiliation(s)
- Yanjiang Zheng
- Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
| | - Yifei Li
- Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
| | - Kaiyu Zhou
- Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
| | - Tiange Li
- Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China
| | - Nathan J VanDusen
- Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
| | - Yimin Hua
- Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, 610041, China.
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25
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Li S, Wang Y, van der Stoel M, Zhou X, Madhusudan S, Kanerva K, Nguyen VD, Eskici N, Olkkonen VM, Zhou Y, Raivio T, Ikonen E. HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells. Genome Biol 2024; 25:58. [PMID: 38409044 PMCID: PMC10895734 DOI: 10.1186/s13059-024-03187-w] [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: 05/30/2023] [Accepted: 02/14/2024] [Indexed: 02/28/2024] Open
Abstract
Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.
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Affiliation(s)
- Shiqian Li
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland.
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland.
| | - Yafei Wang
- Stem Cells and Metabolism Research Program, Research Programs Unit, and Department of Physiology, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
| | - Miesje van der Stoel
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland
| | - Xin Zhou
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland
| | - Shrinidhi Madhusudan
- Stem Cells and Metabolism Research Program, Research Programs Unit, and Department of Physiology, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
| | - Kristiina Kanerva
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland
| | - Van Dien Nguyen
- Systems Immunity Research Institute, Cardiff University, Cardiff, CF14 4XN, UK
- Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK
| | - Nazli Eskici
- Stem Cells and Metabolism Research Program, Research Programs Unit, and Department of Physiology, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
| | - Vesa M Olkkonen
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland
| | - You Zhou
- Systems Immunity Research Institute, Cardiff University, Cardiff, CF14 4XN, UK
- Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK
| | - Taneli Raivio
- Stem Cells and Metabolism Research Program, Research Programs Unit, and Department of Physiology, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland
- New Children's Hospital, Pediatric Research Center, Helsinki University Hospital, 00290, Helsinki, Finland
| | - Elina Ikonen
- Department of Anatomy and Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland.
- Minerva Foundation Institute for Medical Research, 00290, Helsinki, Finland.
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BORA J, MALIK S, KISHORE S, RUSTAGI S, AHMAD F, FAGOONEE S, PELLICANO R, HAQUE S. Therapeutic applications of CRISPR-Cas9 in diabetes mellitus: a perspective review. MINERVA BIOTECHNOLOGY AND BIOMOLECULAR RESEARCH 2024; 35. [DOI: 10.23736/s2724-542x.23.02996-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/09/2025]
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27
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Volodina OV, Fabrichnikova AR, Anuchina AA, Mishina OS, Lavrov AV, Smirnikhina SA. Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases. Curr Gene Ther 2024; 25:46-61. [PMID: 38623982 DOI: 10.2174/0115665232295117240405070809] [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: 01/04/2024] [Revised: 03/19/2024] [Accepted: 03/20/2024] [Indexed: 04/17/2024]
Abstract
The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.
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Affiliation(s)
- Olga V Volodina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | | | - Arina A Anuchina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Olesya S Mishina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Alexander V Lavrov
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Svetlana A Smirnikhina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
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28
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Zeng S, Lei S, Qu C, Wang Y, Teng S, Huang P. CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia. Hum Genet 2023; 142:1677-1703. [PMID: 37878144 DOI: 10.1007/s00439-023-02610-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Accepted: 10/10/2023] [Indexed: 10/26/2023]
Abstract
Beta-thalassemia (β-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of β-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for β-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including β-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent β-thalassemia (TDT). In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for β-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of β-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating β-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.
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Affiliation(s)
- Shujun Zeng
- The Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, People's Republic of China
| | - Shuangyin Lei
- The Second Norman Bethune Clinical College of Jilin University, Changchun, Jilin, People's Republic of China
| | - Chao Qu
- The First Norman Bethune Clinical College of Jilin University, Changchun, Jilin, People's Republic of China
| | - Yue Wang
- The Second Norman Bethune Clinical College of Jilin University, Changchun, Jilin, People's Republic of China
| | - Shuzhi Teng
- The Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, People's Republic of China.
| | - Ping Huang
- The Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, People's Republic of China.
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29
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Bora J, Dey A, Lyngdoh AR, Dhasmana A, Ranjan A, Kishore S, Rustagi S, Tuli HS, Chauhan A, Rath P, Malik S. A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus. NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY 2023; 396:3459-3481. [PMID: 37522916 DOI: 10.1007/s00210-023-02631-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Accepted: 07/14/2023] [Indexed: 08/01/2023]
Abstract
Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.
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Affiliation(s)
- Jutishna Bora
- Amity Institute of Biotechnology, Amity University Jharkhand, Ranchi, 834001, India
| | - Ankita Dey
- Department of Biochemistry, North Eastern Hill University, Shillong, Meghalaya, 793022, India
| | - Antonia R Lyngdoh
- Department of Biochemistry, North Eastern Hill University, Shillong, Meghalaya, 793022, India
| | - Archna Dhasmana
- Himalayan School of Biosciences, Swami Rama Himalayan University, Jolly Grant, Dehradun, Uttarakhand, India
| | - Anuj Ranjan
- Academy of Biology and Biotechnology, Southern Federal University, Stachki 194/1, Rostov-On-Don, 344090, Russia
| | - Shristi Kishore
- Amity Institute of Biotechnology, Amity University Jharkhand, Ranchi, 834001, India
| | - Sarvesh Rustagi
- School of Applied and Life Sciences, Uttaranchal University, 22 Dehradun, Uttarakhand, India
| | - Hardeep Singh Tuli
- Department of Biotechnology, Maharishi Markandeshwar Engineering College, Maharishi Markandeshwar (Deemed to Be University), Mullana-Ambala, 133207, India
| | - Abhishek Chauhan
- Amity Institute of Environmental Toxicology Safety and Management, Amity University, Sector 125, Noida, Uttar Pradesh, India
| | - Prangya Rath
- Amity Institute of Environmental Sciences, Amity University, Noida, Uttar Pradesh, 201303, India
| | - Sumira Malik
- Amity Institute of Biotechnology, Amity University Jharkhand, Ranchi, 834001, India.
- School of Applied and Life Sciences, Uttaranchal University, 22 Dehradun, Uttarakhand, India.
- Guru Nanak College of Pharmaceutical Sciences, Dehradun, Uttarakhand, India.
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30
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Elizalde MJ, Gorelick DA. Mechanistic toxicology in light of genetic compensation. Toxicol Sci 2023; 197:kfad113. [PMID: 37941503 PMCID: PMC10823772 DOI: 10.1093/toxsci/kfad113] [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] [Indexed: 11/10/2023] Open
Abstract
Mechanistic toxicology seeks to identify the molecular and cellular mechanisms by which toxicants exert their deleterious effects. One powerful approach is to generate mutations in genes that respond to a particular toxicant, and then test how such mutations change the effects of the toxicant. CRISPR is a rapid and versatile approach to generate mutations in cultured cells and in animal models. Many studies use CRISPR to generate short insertions or deletions in a target gene and then assume that the resulting mutation, such as a premature termination codon, causes a loss of functional protein. However, recent studies demonstrate that this assumption is flawed. Cells can compensate for short insertion and deletion mutations, leading toxicologists to draw erroneous conclusions from mutant studies. In this review, we will discuss mechanisms by which a mutation in one gene may be rescued by compensatory activity. We will discuss how CRISPR insertion and deletion mutations are susceptible to compensation by transcriptional adaptation, alternative splicing, and rescue by maternally derived gene products. We will review evidence that measuring levels of messenger RNA transcribed from a mutated gene is an unreliable indicator of the severity of the mutation. Finally, we provide guidelines for using CRISPR to generate mutations that avoid compensation.
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Affiliation(s)
- Mary Jane Elizalde
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
| | - Daniel A Gorelick
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
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Militi S, Nibhani R, Jalali M, Pauklin S. RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling. Cell Rep 2023; 42:113146. [PMID: 37725511 DOI: 10.1016/j.celrep.2023.113146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Revised: 05/30/2023] [Accepted: 08/31/2023] [Indexed: 09/21/2023] Open
Abstract
The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/β-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.
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Affiliation(s)
- Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK
| | - Morteza Jalali
- Anne McLaren Laboratory for Regenerative Medicine, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Old Road, Headington, Oxford OX3 7LD, UK.
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Balmas E, Sozza F, Bottini S, Ratto ML, Savorè G, Becca S, Snijders KE, Bertero A. Manipulating and studying gene function in human pluripotent stem cell models. FEBS Lett 2023; 597:2250-2287. [PMID: 37519013 DOI: 10.1002/1873-3468.14709] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 07/04/2023] [Accepted: 07/05/2023] [Indexed: 08/01/2023]
Abstract
Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss-, gain-, and change-of-function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single-cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.
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Affiliation(s)
- Elisa Balmas
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Federica Sozza
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Sveva Bottini
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Maria Luisa Ratto
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Giulia Savorè
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Silvia Becca
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Kirsten Esmee Snijders
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Alessandro Bertero
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
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33
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Kizub IV. Induced pluripotent stem cells for cardiovascular therapeutics: Progress and perspectives. REGULATORY MECHANISMS IN BIOSYSTEMS 2023; 14:451-468. [DOI: 10.15421/10.15421/022366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2025] Open
Abstract
The discovery of methods for reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) opens up prospects of developing personalized cell-based therapy options for a variety of human diseases as well as disease modeling and new drug discovery. Like embryonic stem cells, iPSCs can give rise to various cell types of the human body and are amenable to genetic correction. This allows usage of iPSCs in the development of modern therapies for many virtually incurable human diseases. The review summarizes progress in iPSC research in the context of application in the cardiovascular field including modeling cardiovascular disease, drug study, tissue engineering, and perspectives for personalized cardiovascular medicine.
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34
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Wang JH, Wu SJ, Li Y, Zhao Y, Liu ZM, Deng SL, Lian ZX. Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase. Cells 2023; 12:1818. [PMID: 37508483 PMCID: PMC10378061 DOI: 10.3390/cells12141818] [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: 05/02/2023] [Revised: 07/02/2023] [Accepted: 07/04/2023] [Indexed: 07/30/2023] Open
Abstract
The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.
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Affiliation(s)
- Jia-Hao Wang
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
- Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing 100871, China
| | - Su-Jun Wu
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Yan Li
- Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing 100071, China;
| | - Yue Zhao
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Zhi-Mei Liu
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Shou-Long Deng
- NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100021, China
| | - Zheng-Xing Lian
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
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35
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Moore ST, Nakamura T, Nie J, Solivais AJ, Aristizábal-Ramírez I, Ueda Y, Manikandan M, Reddy VS, Romano DR, Hoffman JR, Perrin BJ, Nelson RF, Frolenkov GI, Chuva de Sousa Lopes SM, Hashino E. Generating high-fidelity cochlear organoids from human pluripotent stem cells. Cell Stem Cell 2023; 30:950-961.e7. [PMID: 37419105 PMCID: PMC10695300 DOI: 10.1016/j.stem.2023.06.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 05/15/2023] [Accepted: 06/14/2023] [Indexed: 07/09/2023]
Abstract
Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.
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Affiliation(s)
- Stephen T Moore
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Takashi Nakamura
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Otolaryngology-Head & Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Jing Nie
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Alexander J Solivais
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | | | - Yoshitomo Ueda
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Mayakannan Manikandan
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - V Shweta Reddy
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Daniel R Romano
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - John R Hoffman
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Benjamin J Perrin
- Department of Biology, Purdue School of Science, Indianapolis, IN 46202, USA
| | - Rick F Nelson
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | | | | | - Eri Hashino
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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36
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Cho G, Hyun K, Choi J, Shin E, Kim B, Kim H, Kim J, Han YM. Arginine 65 methylation of Neurogenin 3 by PRMT1 is required for pancreatic endocrine development of hESCs. Exp Mol Med 2023; 55:1506-1519. [PMID: 37394590 PMCID: PMC10393949 DOI: 10.1038/s12276-023-01035-8] [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: 10/18/2022] [Revised: 04/07/2023] [Accepted: 04/17/2023] [Indexed: 07/04/2023] Open
Abstract
Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs) in the developing pancreas. Previous studies have shown that the stability and activity of NGN3 are regulated by phosphorylation. However, the role of NGN3 methylation is poorly understood. Here, we report that protein arginine methyltransferase-1 (PRMT1)-mediated arginine 65 methylation of NGN3 is required for the pancreatic endocrine development of human embryonic stem cells (hESCs) in vitro. We found that inducible PRMT1-knockout (P-iKO) hESCs did not differentiate from EPs into endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused NGN3 accumulation in the cytoplasm of EPs and decreased the transcriptional activity of NGN3. We found that PRMT1 specifically methylates NGN3 arginine 65 and that this modification is a prerequisite for ubiquitin-mediated degradation. Our findings demonstrate that arginine 65 methylation of NGN3 is a key molecular switch in hESCs permitting their differentiation into pancreatic ECs.
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Affiliation(s)
- Gahyang Cho
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Kwangbeom Hyun
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Jieun Choi
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Eunji Shin
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Bumsoo Kim
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Hail Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Jaehoon Kim
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.
| | - Yong-Mahn Han
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.
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37
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Perez-Bermejo JA, Judge LM, Jensen CL, Wu K, Watry HL, Truong A, Ho JJ, Carter M, Runyon WV, Kaake RM, Pulido EH, Mandegar MA, Swaney DL, So PL, Krogan NJ, Conklin BR. Functional analysis of a common BAG3 allele associated with protection from heart failure. NATURE CARDIOVASCULAR RESEARCH 2023; 2:615-628. [PMID: 39195919 DOI: 10.1038/s44161-023-00288-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Accepted: 05/18/2023] [Indexed: 08/29/2024]
Abstract
Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism remains elusive. In this study, we used induced pluripotent stem cells to test if the only coding variant in this allele block, BAG3C151R, alters protein and cellular function in human cardiomyocytes. Quantitative protein interaction analysis identified changes in BAG3C151R protein partners specific to cardiomyocytes. Knockdown of genes encoding for BAG3-interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in a dose-dependent manner. This study suggests that BAG3C151R could be responsible for the cardioprotective effect of the haplotype block, by increasing cardiomyocyte protection from stress. Preferential binding partners of BAG3C151R may reveal potential targets for cardioprotective therapies.
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Affiliation(s)
| | - Luke M Judge
- Gladstone Institutes, San Francisco, CA, USA
- Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA
| | | | - Kenneth Wu
- Gladstone Institutes, San Francisco, CA, USA
| | | | | | - Jaclyn J Ho
- Tenaya Therapeutics, South San Francisco, CA, USA
| | | | | | - Robyn M Kaake
- Gladstone Institutes, San Francisco, CA, USA
- Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA
| | | | | | - Danielle L Swaney
- Gladstone Institutes, San Francisco, CA, USA
- Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA
| | - Po-Lin So
- Gladstone Institutes, San Francisco, CA, USA
| | - Nevan J Krogan
- Gladstone Institutes, San Francisco, CA, USA
- Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA
| | - Bruce R Conklin
- Gladstone Institutes, San Francisco, CA, USA.
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
- Innovative Genomics Institute, Berkeley, CA, USA.
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38
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Ueda Y, Nakamura T, Nie J, Solivais AJ, Hoffman JR, Daye BJ, Hashino E. Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution. Development 2023; 150:dev201071. [PMID: 37381908 PMCID: PMC10323240 DOI: 10.1242/dev.201071] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Accepted: 05/24/2023] [Indexed: 06/29/2023]
Abstract
The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.
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Affiliation(s)
- Yoshitomo Ueda
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Takashi Nakamura
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
- Department of Otolaryngology-Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | - Jing Nie
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Alexander J. Solivais
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - John R. Hoffman
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Becca J. Daye
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Eri Hashino
- Department of Otolaryngology-Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
- Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
- Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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39
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Eisen B, Binah O. Modeling Duchenne Muscular Dystrophy Cardiomyopathy with Patients' Induced Pluripotent Stem-Cell-Derived Cardiomyocytes. Int J Mol Sci 2023; 24:ijms24108657. [PMID: 37240001 DOI: 10.3390/ijms24108657] [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: 04/20/2023] [Revised: 05/05/2023] [Accepted: 05/10/2023] [Indexed: 05/28/2023] Open
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene, resulting in death by the end of the third decade of life at the latest. A key aspect of the DMD clinical phenotype is dilated cardiomyopathy, affecting virtually all patients by the end of the second decade of life. Furthermore, despite respiratory complications still being the leading cause of death, with advancements in medical care in recent years, cardiac involvement has become an increasing cause of mortality. Over the years, extensive research has been conducted using different DMD animal models, including the mdx mouse. While these models present certain important similarities to human DMD patients, they also have some differences which pose a challenge to researchers. The development of somatic cell reprograming technology has enabled generation of human induced pluripotent stem cells (hiPSCs) which can be differentiated into different cell types. This technology provides a potentially endless pool of human cells for research. Furthermore, hiPSCs can be generated from patients, thus providing patient-specific cells and enabling research tailored to different mutations. DMD cardiac involvement has been shown in animal models to include changes in gene expression of different proteins, abnormal cellular Ca2+ handling, and other aberrations. To gain a better understanding of the disease mechanisms, it is imperative to validate these findings in human cells. Furthermore, with the recent advancements in gene-editing technology, hiPSCs provide a valuable platform for research and development of new therapies including the possibility of regenerative medicine. In this article, we review the DMD cardiac-related research performed so far using human hiPSCs-derived cardiomyocytes (hiPSC-CMs) carrying DMD mutations.
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Affiliation(s)
- Binyamin Eisen
- Cardiac Research Laboratory, Department of Physiology, Biophysics and Systems Biology, Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 3200003, Israel
| | - Ofer Binah
- Cardiac Research Laboratory, Department of Physiology, Biophysics and Systems Biology, Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 3200003, Israel
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40
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Li D, Liu Q, Yang M, Xu H, Zhu M, Zhang Y, Xu J, Tian C, Yao J, Wang L, Liang Y. Nanomaterials for mRNA-based therapeutics: Challenges and opportunities. Bioeng Transl Med 2023; 8:e10492. [PMID: 37206219 PMCID: PMC10189457 DOI: 10.1002/btm2.10492] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2022] [Revised: 01/03/2023] [Accepted: 01/04/2023] [Indexed: 01/31/2023] Open
Abstract
Messenger RNA (mRNA) holds great potential in developing immunotherapy, protein replacement, and genome editing. In general, mRNA does not have the risk of being incorporated into the host genome and does not need to enter the nucleus for transfection, and it can be expressed even in nondividing cells. Therefore, mRNA-based therapeutics provide a promising strategy for clinical treatment. However, the efficient and safe delivery of mRNA remains a crucial constraint for the clinical application of mRNA therapeutics. Although the stability and tolerability of mRNA can be enhanced by directly retouching the mRNA structure, there is still an urgent need to improve the delivery of mRNA. Recently, significant progress has been made in nanobiotechnology, providing tools for developing mRNA nanocarriers. Nano-drug delivery system is directly used for loading, protecting, and releasing mRNA in the biological microenvironment and can be used to stimulate the translation of mRNA to develop effective intervention strategies. In the present review, we summarized the concept of emerging nanomaterials for mRNA delivery and the latest progress in enhancing the function of mRNA, primarily focusing on the role of exosomes in mRNA delivery. Moreover, we outlined its clinical applications so far. Finally, the key obstacles of mRNA nanocarriers are emphasized, and promising strategies to overcome these obstacles are proposed. Collectively, nano-design materials exert functions for specific mRNA applications, provide new perception for next-generation nanomaterials, and thus revolution of mRNA technology.
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Affiliation(s)
- De‐feng Li
- Department of GastroenterologyShenzhen People's Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology)ShenzhenGuangdongChina
| | - Qi‐song Liu
- National Clinical Research Center for Infectious DiseasesShenzhen Third People's Hospital, Southern University of Science and TechnologyShenzhenChina
| | - Mei‐feng Yang
- Department of HematologyYantian District People's HospitalShenzhenGuangdongChina
| | - Hao‐ming Xu
- Department of Gastroenterology and HepatologyGuangzhou Digestive Disease Center, Guangzhou First People's Hospital, School of Medicine, South China University of TechnologyGuangzhouChina
| | - Min‐zheng Zhu
- Department of Gastroenterology and Hepatologythe Second Affiliated Hospital, School of Medicine, South China University of TechnologyGuangzhouGuangdongChina
| | - Yuan Zhang
- Department of Medical AdministrationHuizhou Institute of Occupational Diseases Control and PreventionHuizhouGuangdongChina
| | - Jing Xu
- Department of Gastroenterology and HepatologyGuangzhou Digestive Disease Center, Guangzhou First People's Hospital, School of Medicine, South China University of TechnologyGuangzhouChina
| | - Cheng‐mei Tian
- Department of EmergencyShenzhen People's Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology)ShenzhenGuangdongChina
| | - Jun Yao
- Department of GastroenterologyShenzhen People's Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology)ShenzhenGuangdongChina
| | - Li‐sheng Wang
- Department of GastroenterologyShenzhen People's Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology)ShenzhenGuangdongChina
| | - Yu‐jie Liang
- Department of Child and Adolescent PsychiatryShenzhen Kangning Hospital, Shenzhen Mental Health CenterShenzhenChina
- Affiliated Hospital of Jining Medical University, Jining Medical UniversityJiningShandongChina
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41
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Chen H, Yang QL, Xu JX, Deng X, Zhang YJ, Liu T, Rots MG, Xu GL, Huang KY. Efficient methods for multiple types of precise gene-editing in Chlamydomonas. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2023. [PMID: 37310200 DOI: 10.1111/tpj.16265] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 04/20/2023] [Accepted: 04/26/2023] [Indexed: 06/14/2023]
Abstract
Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3'-UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.
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Affiliation(s)
- Hui Chen
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Qing-Lin Yang
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Jia-Xi Xu
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Xuan Deng
- Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
| | - Yun-Jie Zhang
- Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
| | - Ting Liu
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Marianne G Rots
- Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, 9713 GZ, Groningen, The Netherlands
| | - Guo-Liang Xu
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
- Shanghai Key Laboratory of Medical Epigenetics, Laboratory of Cancer Epigenetics, Institutes of Biomedical Sciences, Medical College of Fudan University, Chinese Academy of Medical Sciences (RU069), Shanghai, China
| | - Kai-Yao Huang
- Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
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42
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Mao M, Qian Y, Zhang W, Zhou S, Wang Z, Chen X, Yang Y. Controlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction. Nat Commun 2023; 14:2172. [PMID: 37061509 PMCID: PMC10105765 DOI: 10.1038/s41467-023-37830-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2021] [Accepted: 04/03/2023] [Indexed: 04/17/2023] Open
Abstract
Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.
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Affiliation(s)
- Miaowei Mao
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
- Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
- Bio-Med Big Data Center, CAS Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Yajie Qian
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
- Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
| | - Wenyao Zhang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
| | - Siyu Zhou
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
- Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China
| | - Zefeng Wang
- Bio-Med Big Data Center, CAS Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Xianjun Chen
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China.
- Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China.
| | - Yi Yang
- Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China.
- Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China.
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Sidhaye J, Trepte P, Sepke N, Novatchkova M, Schutzbier M, Dürnberger G, Mechtler K, Knoblich JA. Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids. eLife 2023; 12:e85135. [PMID: 36989136 PMCID: PMC10059687 DOI: 10.7554/elife.85135] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Accepted: 03/16/2023] [Indexed: 03/30/2023] Open
Abstract
During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the posttranscriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5'TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach proposes novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.
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Affiliation(s)
- Jaydeep Sidhaye
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
| | - Philipp Trepte
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
| | - Natalie Sepke
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
| | - Maria Novatchkova
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
| | | | | | - Karl Mechtler
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
| | - Jürgen A Knoblich
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC)ViennaAustria
- Department of Neurology, Medical University of ViennaViennaAustria
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Feng Y, Cai L, Pook M, Liu F, Chang CH, Mouti MA, Nibhani R, Wu S, Deng S, Militi S, Dunford J, Philpott M, Fan Y, Fan GC, Liu Q, Qi J, Sadayappan S, Jegga AG, Oppermann U, Wang Y, Huang W, Jiang L, Pauklin S. BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.02.530770. [PMID: 36909530 PMCID: PMC10002796 DOI: 10.1101/2023.03.02.530770] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFβ/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.
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Ganipineni VDP, Gutlapalli SD, Danda S, Garlapati SKP, Fabian D, Okorie I, Paramsothy J. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in Cardiovascular Disease: A Comprehensive Clinical Review on Dilated Cardiomyopathy. Cureus 2023; 15:e35774. [PMID: 37025725 PMCID: PMC10071452 DOI: 10.7759/cureus.35774] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/05/2023] [Indexed: 03/07/2023] Open
Abstract
Dilated cardiomyopathy (DCM) is one of the most important causes of heart failure in developed and developing countries. Currently, most medical interventions in the treatment of DCM are mainly focused on mitigating the progression of the disease and controlling the symptoms. The vast majority of patients who survive till the late stages of the disease require cardiac transplantation; this is exactly why we need novel therapeutic interventions and hopefully treatments that can reverse the clinical cardiac deterioration in patients with DCM. Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a novel therapeutic intervention with such capacity; it can help us edit the genome of patients with genetic etiology for DCM and potentially cure them permanently. This review provides an overview of studies investigating CRISPR-based gene editing in DCM, including the use of CRISPR in DCM disease models, phenotypic screening, and genotype-specific precision therapies. The review discusses the outcomes of these studies and highlights the potential benefits of CRISPR in developing novel genotype-agnostic therapeutic strategies for the genetic causes of DCM. The databases we used to extract relevant literature include PubMed, Google Scholar, and Cochrane Central. We used the Medical Subject Heading (MeSH) strategy for our literature search in PubMed and relevant search keywords for other databases. We screened all the relevant articles from inception till February 22, 2023. We retained 74 research articles after carefully reviewing each of them. We concluded that CRISPR gene editing has shown promise in developing precise and genotype-specific therapeutic strategies for DCM, but there are challenges and limitations, such as delivering CRISPR-Cas9 to human cardiomyocytes and the potential for unintended gene targeting. This study represents a turning point in our understanding of the mechanisms underlying DCM and paves the way for further investigation into the application of genomic editing for identifying novel therapeutic targets. This study can also act as a potential framework for novel therapeutic interventions in other genetic cardiovascular diseases.
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Affiliation(s)
- Vijaya Durga Pradeep Ganipineni
- Department of Internal Medicine, SRM Medical College Hospital and Research Centre, Chennai, IND
- Department of General Medicine, Andhra Medical College/King George Hospital, Visakhapatnam, IND
| | - Sai Dheeraj Gutlapalli
- Department of Internal Medicine, Richmond University Medical Center, Staten Island, USA
- Internal Medicine and Clinical Research, California Institute of Behavioral Neurosciences & Psychology, Fairfield, USA
| | - Sumanth Danda
- Department of Internal Medicine, Katuri Medical College & Hospital, Guntur, IND
| | | | - Daniel Fabian
- Department of Internal Medicine, Richmond University Medical Center, Staten Island, USA
| | - Ikpechukwu Okorie
- Department of Internal Medicine, Richmond University Medical Center, Staten Island, USA
| | - Jananthan Paramsothy
- Department of Internal Medicine, Richmond University Medical Center, Staten Island, USA
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Castiello MC, Ferrari S, Villa A. Correcting inborn errors of immunity: From viral mediated gene addition to gene editing. Semin Immunol 2023; 66:101731. [PMID: 36863140 PMCID: PMC10109147 DOI: 10.1016/j.smim.2023.101731] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 01/25/2023] [Accepted: 02/14/2023] [Indexed: 03/04/2023]
Abstract
Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.
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Affiliation(s)
- Maria Carmina Castiello
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (IRGB-CNR), Milan, Italy
| | - Samuele Ferrari
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy
| | - Anna Villa
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (IRGB-CNR), Milan, Italy.
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A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells. Nat Commun 2023; 14:939. [PMID: 36805596 PMCID: PMC9941109 DOI: 10.1038/s41467-023-36294-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2022] [Accepted: 01/25/2023] [Indexed: 02/22/2023] Open
Abstract
Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.
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Suwito BE, Adji AS, Widjaja JS, Angel SCS, Al Hajiri AZZ, Salamy NFW, Choirotussanijjah C. A Review of CRISPR Cas9 for SCA: Treatment Strategies and Could Target β-globin Gene and BCL11A Gene using CRISPR Cas9 Prevent the Patient from Sickle Cell Anemia? Open Access Maced J Med Sci 2023. [DOI: 10.3889/oamjms.2023.11435] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/29/2023] Open
Abstract
BACKGROUND: Sickle cell anemia is a hereditary globin chain condition that leads to hemolysis and persistent organ damage. Chronic hemolytic anemia, severe acute and chronic pain, and end-organ destruction occur throughout the lifespan of sickle cell anemia. SCD is associated with a higher risk of mortality. Genome editing with CRISPR-associated regularly interspersed short palindromic repeats (CRISPR/Cas9) have therapeutic potential for sickle cell anemia thala.
AIM: This research aimed to see if using CRISPR/Cas9 to target β-globin gene is an effective therapeutic and if it has a long-term effect on Sickle Cell Anemia.
METHODS: The method used in this study summarizes the article by looking for keywords that have been determined in the title and abstract. The authors used official guidelines from Science Direct, PubMed, Google Scholar, and Journal Molecular Biology to select full-text articles published within the last decade, prioritizing searches within the past 10 years.
RESULTS: CRISPR/Cas9-mediated genome editing in clinical trials contributes to α-globin gene deletion correcting β-thalassemia through balanced α- and β-globin ratios and inhibiting disease progression.
CONCLUSION: HBB and BCL11A targeting by CRISPR/Cas9 deletion effectively inactivate BCL11A, a repressor of fetal hemoglobin production. However, further research is needed to determine its side effects and safety.
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Nakajima I, Tsukimura T, Ono T, Shiga T, Shitara H, Togawa T, Sakuraba H, Miyaoka Y. In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells. Cell Transplant 2023; 32:9636897231173734. [PMID: 37183961 DOI: 10.1177/09636897231173734] [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: 05/16/2023] Open
Abstract
Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.
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Affiliation(s)
- Ittetsu Nakajima
- Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
- Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Takahiro Tsukimura
- Department of Functional Bioanalysis, Meiji Pharmaceutical University, Tokyo, Japan
| | - Terumi Ono
- Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
- Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Tomoko Shiga
- Department of Clinical Genetics, Meiji Pharmaceutical University, Tokyo, Japan
| | - Hiroshi Shitara
- Laboratory for Transgenic Technology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Tadayasu Togawa
- Department of Functional Bioanalysis, Meiji Pharmaceutical University, Tokyo, Japan
| | - Hitoshi Sakuraba
- Department of Clinical Genetics, Meiji Pharmaceutical University, Tokyo, Japan
| | - Yuichiro Miyaoka
- Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
- Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
- Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan
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Dashtaki ME, Ghasemi S. CRISPR/Cas9-based Gene Therapies for Fighting Drug Resistance Mediated by Cancer Stem Cells. Curr Gene Ther 2023; 23:41-50. [PMID: 36056851 DOI: 10.2174/1566523222666220831161225] [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: 03/09/2022] [Revised: 06/11/2022] [Accepted: 06/11/2022] [Indexed: 02/08/2023]
Abstract
Cancer stem cells (CSCs) are cancer-initiating cells found in most tumors and hematological cancers. CSCs are involved in cells progression, recurrence of tumors, and drug resistance. Current therapies have been focused on treating the mass of tumor cells and cannot eradicate the CSCs. CSCs drug-specific targeting is considered as an approach to precisely target these cells. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing systems are making progress and showing promise in the cancer research field. One of the attractive applications of CRISPR/Cas9 as one approach of gene therapy is targeting the critical genes involved in drug resistance and maintenance of CSCs. The synergistic effects of gene editing as a novel gene therapy approach and traditional therapeutic methods, including chemotherapy, can resolve drug resistance challenges and regression of the cancers. This review article considers different aspects of CRISPR/Cas9 ability in the study and targeting of CSCs with the intention to investigate their application in drug resistance.
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Affiliation(s)
- Masoumeh Eliyasi Dashtaki
- Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
| | - Sorayya Ghasemi
- Cancer Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
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