Browsing by Subject "Genome editing"

Now showing 1 - 7 of 7
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    Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing
    (bioRxiv, 2024-12-04) Zheng, Yanjiang; Mayourian, Joshua; King, Justin S.; Li, Yifei; Bezzerides, Vassilios J.; Pu, William T.; VanDusen, Nathan J.; Pediatrics, School of Medicine
    The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.
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    Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes
    (American Heart Association, 2022) Zheng, Yanjiang; VanDusen, Nathan J.; Butler, Catalina E.; Ma, Qing; King, Justin S.; Pu, William T.; Pediatrics, School of Medicine
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    Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy
    (Cell Press, 2022) Chang, Yun; Syahirah, Ramizah; Wang, Xuepeng; Jin, Gyuhyung; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Hummel, Sydney N.; Wang, Tianqi; Li, Can; Lian, Xiaojun; Deng, Qing; Broxmeyer, Hal E.; Bao, Xiaoping; Microbiology and Immunology, School of Medicine
    Neutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.
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    Genetics of paediatric cardiomyopathies
    (Wolters Kluwer, 2017-10) Ware, Stephanie M.; Pediatrics, School of Medicine
    PURPOSE OF REVIEW: Paediatric cardiomyopathy is a rare disease with a genetic basis. The purpose of this review is to discuss the current status of genetic findings in the paediatric cardiomyopathy population and present recent progress in utilizing this information for management and therapy. RECENT FINDINGS: With increased clinical genetic testing, an understanding of the genetic causes of cardiomyopathy is improving and novel causes are identified at a rapid rate. Recent progress in identifying the scope of genetic variation in large population datasets has led to reassessment and refinement of our understanding of the significance of rare genetic variation. As a result, the stringency of variant interpretation has increased, at times leading to revision of previous mutation results. Transcriptome and epigenome studies are elucidating important pathways for disease progression and highlight similarities and differences in pathogenesis from adult cardiomyopathy. Therapy targeted towards the underlying cause of cardiomyopathy is emerging for a number of rare syndromes such as Pompe and Noonan syndromes, and genome editing and induced pluripotent stem cells provide promise for additional precision medicine approaches. SUMMARY: Genetics is moving at a rapid pace in paediatric cardiomyopathy. Genetic testing is increasingly being incorporated into clinical care. Although interpretation of rare genetic variation remains challenging, the opportunity to provide management and therapy targeted towards the underlying genetic cause is beginning to be realized.
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    Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases
    (Springer Nature, 2018-04-17) Chun, Young Wook; Durbin, Matthew D.; Hong, Charles C.; Pediatrics, School of Medicine
    PURPOSE OF REVIEW: The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs. RECENT FINDINGS: The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies. The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.
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    Investigating pediatric disorders with induced pluripotent stem cells
    (Springer Nature, 2018-10) Durbin, Matthew D.; Cadar, Adrian G.; Chun, Young W.; Hong, Charles C.; Pediatrics, School of Medicine
    The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients.
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    Novel methods for the generation of genetically engineered animal models
    (Elsevier, 2023) Cassidy, Annelise; Onal, Melda; Pelletier, Stephane; Medical and Molecular Genetics, School of Medicine
    Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.