Heart Failure Therapy by CRISPR-Mediated Upregulation of Mitochondrial Biogenesis

通过 CRISPR 介导的线粒体生物发生上调治疗心力衰竭

基本信息

批准号：
9189490
负责人：
Deepthi Ashok
金额：
$ 4.36万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2019-08-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9189490
关键词：
Acute Adenovirus Vector Adrenergic Agents Affect Angiotensin II Receptor Angiotensin-Converting Enzyme Inhibitors Behavioral Mechanisms Binding Proteins Biogenesis Biological Assay Biological Models Bypass CRISPR/Cas technology Cardiac Cardiac Myocytes Cavia Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Cyclic AMP Cyclic AMP Response Element Cyclic AMP-Dependent Protein Kinases Cyclic AMP-Responsive DNA-Binding Protein DNA Data Deterioration Dimerization Disease Diuretics Down-Regulation EP300 gene Embryo Future Gene Activation Gene Expression Gene Proteins Gene Targeting Gene Transduction Agent Gene Transfer Genes Genetic Transcription Genome Guide RNA Heart Heart Ventricle Heart failure Image Immunoblotting Injection of therapeutic agent JUN gene Knowledge Luc Gene Luciferases Mediating Membrane Potentials Metabolic Methods Mitochondria Mitochondrial DNA Modeling Molecular Profiling Muscle Cells Myocardial Neonatal Oxidative Phosphorylation PPAR gamma Pathway interactions Pharmacotherapy Phosphoenolpyruvate Carboxylase Phosphorylation Precipitating Factors Promoter Regions Proteins Pyruvate Kinase Quality Control Rattus Reporter Reporter Genes Rodent Model Role Signal Transduction Site System Technology Testing Therapeutic Transcription Coactivator Transcription Repressor/Corepressor Transcriptional Activation Transcriptional Activation Domain United States Up-Regulation Validation Ventricular beta-adrenergic receptor cofactor effective therapy improved in vivo meetings mitochondrial dysfunction novel novel strategies nuclease overexpression prevent protein activation protein metabolite receptor research study success tool transcription factor vector

项目摘要

Mitochondrial dysfunction has been implicated as a contributing factor for heart failure. Genes involved in mitochondrial biogenesis and quality control such as the peroxisome proliferator- activated receptor gamma coactivator 1α (PGC-1α) have been severely downregulated in rodent models of heart failure. Transcription of PGC-1α can be promoted when cAMP Response Element Binding Protein (CREB) is phosphorylated at Ser-133 by PKA upon beta-adrenergic signaling. However, recent evidence shows that CREB itself has numerous factors modulating it, including phosphorylation on other sites (e.g Ser-142), availability of dimerization partners (c- Jun, CREB subtypes) and other co-factors (e.g. TORCs) that may be altered in heart failure. Here we will test whether bypassing the complex regulatory steps involved in PGC-1α activation and directly targeting cAMP Response Elements (CRE) known to regulate PGC-1α gene expression can have beneficial effects on expression of PGC-1α. Importantly, we also propose to test whether mitochondrial biogenesis can be increased via CRE-mediated PGC-1α upregulation. I intend to utilize the newly developed CRISPR/Cas9 technology as a transcriptional activator (instead of editing genes, with inactivated Cas9 fused to transcriptional activation domains) to target CRE sequences to promote transcription of PGC-1α. Use and efficacy of these gene modulating tools has not been evaluated in cardiomyocytes. With this project, I will establish if metabolic reprogramming and enhanced mitochondrial biogenesis can be effected with CRISPR-mediated activation of PGC-1α while providing fundamental knowledge of the role of CRE in PGC-1α activation and mitochondrial function. I would have also developed a streamlined and generalizable method for using gene modulating tools for expression and begun to test its efficacy in preventing or reversing heart failure.

线粒体被认为是心力衰竭的一个促成因素，参与线粒体生物发生功能障碍和质量控制的基因，例如过氧化物酶体增殖物激活受体γ共激活剂1α（PGC-1α），在心力衰竭的啮齿动物模型中被严重下调。当 cAMP 响应元件结合蛋白 (CREB) 在 β-肾上腺素能信号传导下被 PKA 在 Ser-133 位点磷酸化时，PGC-1α 可以得到促进。然而，最近的证据表明，CREB 本身有许多调节因素，包括其他位点的磷酸化（例如 Ser-142）、二聚化伙伴的可用性（c-Jun、CREB 亚型）和其他可能与其他辅助因子（例如 TORC）相关的因素。在这里，我们将测试绕过 PGC-1α 激活中涉及的复杂调节步骤并直接靶向已知调节 PGC-1α 基因表达的 cAMP 反应元件 (CRE) 是否可以改变。重要的是，我们还建议测试是否可以通过 CRE 介导的 PGC-1α 上调来增加线粒体生物合成，我打算利用新开发的 CRISPR/Cas9 技术作为转录激活剂（而不是编辑）。尚未在心肌细胞中评估这些基因调节工具的使用和功效。在该项目中，我将确定是否可以通过 CRISPR 介导的 PGC-1α 激活来实现代谢重编程和增强线粒体生物合成，同时提供 CRE 在 PGC-1α 激活和线粒体功能中的作用的基础知识。使用基因调节工具进行表达的通用方法，并开始测试其预防或逆转心力衰竭的功效。