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Investigating Mitogenic Mechanism and Therapeutic Efficacy of Adenosine Deaminase Knockout in Human and Murine Cardiomyocytes

研究人和小鼠心肌细胞中腺苷脱氨酶敲除的有丝分裂机制和治疗效果

基本信息

批准号：
10815529
负责人：
Sophia Bunnell DeLuca
金额：
$ 4.25万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2024-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10815529
关键词：
3-Dimensional Adenosine Adult Affect Biological Assay Birth Breeding Calcium Cardiac Cardiac Myocytes Cardiovascular Physiology Cell Cycle Chemicals Cicatrix Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Consumption Coronary artery Culture Media Custom Cyclic AMP Development Echocardiography Embryo Ethanol Metabolism Event Fibroblasts Flow Cytometry Foundations Future Gap Junctions Generations Genetic Genetic Screening Genus Hippocampus Glucose Glucosephosphate Dehydrogenase Heart Heart Injuries Human Hypertrophy Image Immunity In Vitro Injections Injury Inosine Ischemia Knock-out Letters Ligation MAP Kinase Gene MAPK3 gene Mediating Metabolic Metabolism Mitogens Modeling Molecular Mus Muscle function Myocardial Infarction Myocardium Natural regeneration Neonatal Nucleotides Pathway interactions Pentosephosphate Pathway Ploidies Pluripotent Stem Cells Polyploidy Production Proliferating Purine-Nucleoside Phosphorylase Purinergic P1 Receptors Rattus Receptor Signaling Recovery of Function Reperfusion Injury Reperfusion Therapy Reporting Research Resistance Respiration Ribose Rodent Role Sarcomeres Signal Transduction Stains Study models Tail Testing Therapeutic Therapeutic Effect Time Tissue Engineering Transgenic Mice Treatment Efficacy Veins Western Blotting Work Zebrafish adeno-associated viral vector adenosine deaminase aurora B kinase cardiac regeneration cardiac tissue engineering cardioprotection follow-up gene therapy genetic manipulation heart damage heart function heart metabolism improved in vitro Model in vitro testing in vivo induced pluripotent stem cell induced pluripotent stem cell derived cardiomyocytes inducible Cre injured insight knockout gene lipid metabolism mouse model muscle form neovascularization novel nucleotide metabolism p38 Mitogen Activated Protein Kinase pharmacologic postmitotic purine metabolism ratiometric regeneration potential repaired stem cells sugar nucleotide therapeutic evaluation transcriptome sequencing

项目摘要

Abstract Mammalian cardiomyocytes (CMs) in embryonic and neonatal hearts are proliferative, which allows for heart regeneration to occur. This proliferative capacity of CMs is lost shortly after birth and in the adult hearts CMs are largely post-mitotic, renewing at very low rates of less than 1% per year. The inability of adult CMs to proliferate, along with the absence of resident stem cells capable of robust generation of new CMs, limit the ability of adult mammalian hearts to self-repair following injury such as myocardial infarction (MI). Preliminary results from my CRISPR knockout screen in cultured neonatal rat CMs show that knockout of the gene adenosine deaminase (ADA-KO) results in robust cell cycle activation in both primary rodent and pluripotent stem cell-derived human CMs in vitro. Follow-up RNA sequencing analysis suggests that ADA-KO alters CM metabolism. As metabolic alterations have been shown to underlie the pro-proliferative actions of other known cardiac mitogens, my proposed studies will determine the metabolic and molecular signaling mechanisms governing ADA-KO mediated proliferation in human CMs. Additionally, I will test the in vitro effects of ADA-KO on injured engineered cardiac tissues (ECTs) made using polyploid human CMs, which we have shown to be resistant to division similarly to polyploid CMs in vivo. Furthermore, in proof-of-concept in vivo studies, I will test therapeutic efficacy of ADA-KO in the setting of myocardial infarction by AAV delivery of ADA-KO sgRNAs to transgenic mice with CM-specific Cas9 expression, followed by studies of cardiac proliferation and function. When completed, these cross-species in vitro and in vivo studies will provide better understanding of the roles of ADA in cardiac metabolism and regeneration and will create a basis for the development of novel gene therapies for cardiac regeneration.

摘要胚胎和新生儿心脏中的哺乳动物心肌细胞(CMS)是增殖的，这允许心脏再生才会发生。CMS的这种增殖能力在出生后不久就会消失，在成人心脏中也是如此大部分是有丝分裂后，以每年不到1%的非常低的速度更新。成年CMS不能增殖，加上缺乏能够健壮生成新的CMS的常驻干细胞，限制了成年哺乳动物心脏在心肌梗死(MI)等损伤后自我修复的能力。初步我的CRISPR基因敲除筛查结果显示，在培养的新生大鼠CMS中，该基因被敲除腺苷脱氨酶(ADA-KO)在原代啮齿动物和多能性动物中都能导致强大的细胞周期激活干细胞来源的人类CMS在体外。后续的RNA测序分析表明ADA-KO改变了CM 新陈代谢。由于新陈代谢的改变已被证明是其他已知的促增殖作用的基础心脏有丝分裂原，我提议的研究将确定代谢和分子信号机制控制ADA-KO介导的人CMS增殖。另外，我将测试ADA-KO的体外作用用多倍体人类CMS制备的损伤工程心脏组织(ECTs)，我们已经证明这是体内对分裂的抗性类似于多倍体不育系。此外，在活体概念验证研究中，我将 AAV法检测ADA-KO sgRNAs治疗心肌梗死的疗效转至具有CM特异性Cas9表达的转基因小鼠，随后进行心脏增殖和功能研究。完成后，这些跨物种的体外和体内研究将提供更好的理解 ADA在心脏代谢和再生中的作用，为新基因的开发奠定了基础心脏再生的疗法。