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） 扩张型心肌病 (DCM) 是西方世界心脏移植最常见的适应症，RBM20（RNA 结合基序蛋白 20）是最近描述的一种心脏特异性 RNA 剪接蛋白，在 DCM20 中直接发生突变。与许多心肌病相关基因的 RNA 转录物结合，并确保心脏特异性蛋白质亚型的产生。人类研究揭示了七个紧密聚集的单氨基酸的惊人、无法解释的重复模式。 RBM20 的 RS 结构域附近的酸性 DCM 相关取代。 DCM 患者杂合突变的紧密模式和 RBM20 结合蛋白的生化研究表明，人类 DCM 突变可能对 RBM20 剪接复合物产生显性负干扰作用。 RBM20 RS 结构域突变体会引起显性负干扰，从而导致病理生理学 RNA 剪接，这与功能丧失突变不同。 我们在 iPSC 中进行了多个单碱基 RBM20 突变，无需抗生素选择（“无疤痕”），我们可以有效地生产 RBM20 RS 结构域突变体 (R636S) iPS-CM。 ，并且细胞表现出与 R636S 突变 iPS-CM 的 DCM RNA-Seq 研究一致的细胞病理学，揭示了超过 360 个选择性剪​​接事件，我们的目标是： 目标 1. 通过 RNA-Seq 分析剪接的病理变化，检验 RBM20 DCM 引起的点突变具有显性负效应的假设。 DCM 突变为同基因 iPSC，以及 RBM20 无效突变。RNA-Seq 研究将重点确定可解释 DCM 病理学的最病理性选择性剪接事件。 RBM20 RS 结构域突变 (R636S) 具有不同的 RNA 和蛋白质结合，我们将分别使用 FLAG 和 APEX 表位标记，以选择性地识别结合伙伴。我们将使用 FLAG-RBM20-RNA 结合测定 (CLIP-Seq) 来确定结合伙伴。准确的 RBM20 RNA 结合位点 FLAG- 和 APEX-APMS 将识别 RBM20 的假定结合伙伴和调节因子。 确定假定的结合伙伴是否是。我们将有条件地沉默假定的结合伴侣的表达，以确定每个蛋白质是否是潜在的治疗靶标。检验我们的假设可以解释 这些研究将提供改变 RBM20 活性的药物可以增强心脏健康和修复。为开发基于人类 iPS-CM 的平台以开发新疗法奠定了基础。