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.

抽象的胚胎和新生儿心脏中的哺乳动物心肌细胞（CMS）是增生的，可以使人心脏再生发生。 CMS的这种增殖能力在出生后不久和成人心脏CMS丢失在很大程度上是有丝分裂后的，每年的更新率低于1％。成人CMS无法扩散，以及没有能力生成新CMS的常驻干细胞，限制成年哺乳动物心脏在受伤后自我修复的能力，例如心肌梗塞（MI）。初步的我在培养的新生大鼠CMS中CRISPR敲除屏幕的结果表明该基因的敲除腺苷脱氨酶（ADA-KO）导致一级啮齿动物和多能的稳健细胞周期激活干细胞衍生的人CMS体外。后续RNA测序分析表明ADA-KO改变了CM 代谢。由于已显示代谢改变是其他已知的促销行为的基础我提出的研究将确定代谢和分子信号传导机制管理ADA-KO介导的人类CMS的增殖。此外，我将测试ADA-KO的体外效应在使用多倍体人CMS制造的受伤的工程心脏组织（ECT）上，我们已证明是在体内类似于多倍体CMS的抗性。此外，在体内研究概念验证方面，我将 ADA-KO在心肌梗塞中测试治疗功效，通过AAV递送ADA-KO SGRNA 至具有CM特异性CAS9表达的转基因小鼠，然后研究心脏增殖和功能。完成后，这些在体外和体内研究中的跨物种将提供更好地了解角色心脏代谢和再生中的ADA，并将为开发新基因创造基础心脏再生的疗法。