Identifying Therapeutic Targets for RNA Splicing-Related Cardiomyopathy

确定 RNA 剪接相关心肌病的治疗靶点

• 批准号: 9195146
• 负责人: Bruce R Conklin
• 金额: $ 45.65万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-14 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195146
• 关键词: Adopted; Affinity Chromatography; Alleles; Alternative Splicing; Amino Acids; Antibiotics; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiac health; Cardiomyopathies; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Dilated Cardiomyopathy; Disease; Dominant-Negative Mutation; Doxycycline; Employee Strikes; Engineering; Ensure; Epitopes; Event; Foundations; Gene Expression; Genes; Goals; Heart; Heart Transplantation; Human; Human Genetics; Letters; Life; Mass Spectrum Analysis; Modeling; Molecular; Molecular Target; Mus; Mutate; Mutation; Pathogenicity; Pathologic; Pathology; Patients; Pattern; Peroxidases; Pharmaceutical Preparations; Point Mutation; Process; Production; Protein Binding Domain; Protein Isoforms; Protein Splicing; Proteins; RNA; RNA Binding; RNA Splicing; RNA analysis; Rattus; Recurrence; Regulation; Reporting; Rodent Model; Role; RyR2; Sampling; Series; System; Techniques; Technology; Testing; Therapeutic; Tissues; Transcript; Western World; ascorbate; base; cardiac repair; cellular pathology; crosslinking and immunoprecipitation sequencing; human data; human disease; induced pluripotent stem cell; insight; loss of function mutation; mutant; novel; novel therapeutics; null mutation; protein complex; public health relevance; targeted treatment; therapeutic target; transcriptome sequencing

 DESCRIPTION (provided by applicant) Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in the western world. RBM20 (RNA binding motif protein 20) is a recently described cardiac-specific, RNA splicing protein that is mutated in 2-3% of DCM. RBM20 directly binds to the RNA transcripts of many cardiomyopathy-associated genes and ensures the production of cardiac-specific protein isoforms. Human studies revealed a striking, unexplained, recurring pattern of seven tightly clustered single amino acid DCM-associated substitutions near the RS domain of RBM20. The tight pattern of heterozygous mutations in DCM patients and biochemical studies of RBM20 binding proteins suggest that human DCM mutations could have dominant negative interfering effects on the RBM20 splicing complex. Our central hypothesis is that the RBM20 RS domain mutants cause dominant negative interference resulting in pathophysiological RNA splicing that are distinct from loss-of-function mutations. We plan to develop a series of isogenic human induced pluripotent stem cell (iPSC)-cardiomyocytes (iPS-CMs). We have made multiple single-base RBM20 mutations without antibiotic selection ("scarless") in iPSCs. We can efficiently produce RBM20 RS domain mutant (R636S) iPS-CMs, and the cells display cellular pathology consistent with DCM. RNA-Seq studies of R636S mutant iPS-CMs reveal >360 alternative splicing events, including many that have been previously reported. Our aims are: Aim 1. To test the hypothesis that RBM20 DCM-causing point mutations have a dominant negative effect, by analyzing pathological changes in splicing by RNA-Seq. We will engineer the seven recurring human DCM mutations into isogenic iPSCs, as well as RBM20 null mutations. The RNA-Seq studies will focus on identifying the most pathological alternative splicing events that could explain the DCM pathology. Aim 2. To test the hypothesis that RBM20 RS domain mutation (R636S) has different RNA- and protein- binding, we will use FLAG and APEX epitope tagging respectively, to selectively identify binding partners. We will use FLAG-RBM20-RNA binding assays (CLIP-Seq) to determine the exact RBM20 RNA binding sites. FLAG- and APEX-APMS will identify putative binding partners and regulators of RBM20. Aim 3. To determine if the putative binding partners are essential for RBM20 splice regulation. We will conditionally silence the expression of putative binding partners. We will use CRISPRi to modulate the expression of each gene, to determine if each protein is a potential therapeutic target. With the completion of these aims, we will directly test our hypothesis that would explain the pattern of human RBM20 mutations, and provide a molecular mechanism for pathological splicing. We will have defined the molecular targets and protein partners of RBM20. Drugs that alter RBM20 activity could enhance cardiac health and repair, via a novel mechanism. These studies will provide a foundation for developing a human iPS-CM-based platform to develop new therapeutics.

 描述（由申请人提供） 扩张的心肌病（DCM）是西方世界心脏移植的最常见指示。 RBM20（RNA结合基序蛋白20）是最近描述的心脏特异性RNA剪接蛋白，在DCM的2-3％中突变。 RBM20直接与许多心肌病相关基因的RNA转录本结合，并确保产生心脏特异性蛋白质同工型。人类研究表明，在RBM20的RS结构域附近，七个紧密聚集的单氨基酸DCM相关取代的罢工，无法解释的，反复出现的模式。 DCM患者中杂合突变的紧密模式以及RBM20结合蛋白的生化研究表明，人DCM突变可能对RBM20剪接复合物具有显着的负干扰作用。我们的中心假设是，RBM20 RS结构域突变体引起显性负干扰，导致病理生理RNA剪接与功能丧失突变不同。我们计划开发一系列的同源性 人类诱导的多能干细胞（IPSC） - 心肌细胞（IPS-CMS）。我们已经在IPSC中进行了多个无抗生素选择（“无疤”）的单基RBM20突变。我们可以有效地产生RBM20 RS结构域突变体（R636S）IPS-CM，并且细胞显示与DCM一致的细胞病理学。 R636S突变体IPS-CMS的RNA-seq研究显示> 360个替代剪接事件，其中包括许多先前已报道的。我们的目的是：目标1。通过分析RNA-Seq的剪接中的病理变化，测试RBM20引起的点突变具有显性的负面影响的假设。我们将把七个重复的人DCM突变设计为等生IPSC以及RBM20无效突变。 RNA-seq研究将重点介绍可以解释DCM病理的最病理替代剪接事件。目的2。为了测试RBM20 RS域突变（R636S）具有不同的RNA和蛋白质结合的假设，我们将分别使用FLAG和APEX表位标记，以选择性地识别结合伴侣。我们将使用FLAG-RBM20-RNA结合测定法（Clip-Seq）来确定精确的RBM20 RNA结合位点。 Flag-和Apex-apms将确定RBM20的假定约束伙伴和监管机构。目标3。确定推定的结合伙伴是否对于RBM20剪接调节至关重要。我们将有条件地沉默推定的结合伙伴的表达。我们将使用CRISPRI调节每个基因的表达，以确定每种蛋白是否是潜在的治疗靶标。随着这些目标的完成，我们将直接检验我们的假设，以解释 人RBM20突变的模式，并提供了病理剪接的分子机制。我们将定义RBM20的分子靶标和蛋白质伴侣。改变RBM20活性的药物可以通过一种新型机制来增强心脏健康和修复。这些研究将为开发基于人类IPS-CM的平台开发新疗法的基础。