Evaluation of the role of RNA toxicity in SCA2 pathogenesis using genome editing in patient iPSCs

使用患者 iPSC 基因组编辑评估 RNA 毒性在 SCA2 发病机制中的作用

基本信息

批准号：
9803833
负责人：
Lisa M Ellerby
金额：
$ 50.58万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9803833
关键词：
Alleles Antibodies CAG repeat Cell model Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Disease Epitopes Evaluation Exposure to Fibroblasts Fractionation Future Generations Genes Goals Human Herpesvirus 4 Huntington Disease In Situ Lead Length MJD1 protein Machado-Joseph Disease Metabolism Microsatellite Repeats Modeling Mutation Neurodegenerative Disorders Neurons Neurotoxins Nuclear RNA Pathogenesis Pathology Pathway interactions Patients Phenotype Play Property Proteins Protocols documentation RNA RNA Processing RNA Splicing RNA-Binding Proteins Ribosomal RNA Role SCA2 protein Shapes Structure Techniques Testing Therapeutic Toxic effect Transcript Translating Type 2 Spinocerebellar Ataxia base experimental study genome editing homologous recombination induced pluripotent stem cell mutant neurotoxic neurotoxicity novel polyglutamine protein expression therapy development tool vector

项目摘要

Spinocerebellar ataxia type 2 (SCA2) is a devastating neurodegenerative disease caused by a CAG repeat expansion in the gene ataxin-2 (ATXN2). The CAG repeat is translated into a polyglutamine (polyQ) tract in the mutant ATXN2 protein that has neurotoxic properties. Current therapeutic efforts are focused at suppressing the expression of the mutant ATXN2 protein, or targeting downstream pathways of neurotoxicity. We and others have shown that mutant CAG/CTG repeat-containing transcripts also contribute to pathogeneses of several repeat diseases, including Huntington's disease (HD), Huntington's disease-like 2 (HDL2) and spinocerebellar ataxia type 3 and 8 (SCA3 and SCA8). Therefore, in addition to targeting the neurotoxicity of mutant proteins, a successful therapy may require targeting the mutant transcripts and/or pathways downstream of the mutant transcripts. Our preliminary evidence supports the idea that in SCA2, in addition to toxic ATXN2 protein, both sense ATXN2 RNA and a transcript antisense to ATXN2 (ATXN2-AS) containing an expanded CUG repeat, contribute to SCA2 pathogenesis. We therefore propose to use genome editing approach to modify normal and SCA2 iPSC line into novel, isogenic iPSC lines that model either protein or RNA-induced mechanisms of SCA2 pathogenesis. These lines will be used to (1) further test the novel hypothesis of SCA2 by which mutant ATXN2/ATXN2-AS transcripts disrupt RNA processing in SCA2, including RNA export, rRNA metabolism and splicing, and (2) determine the relative contribution of RNA neurotoxicity to SCA2 pathogenesis. The results obtained from this study will not only facilitate our understanding of the increasingly complex pathogenesis of SCA2, but also that of other CAG/CTG repeat diseases in which both bi-directionally encoded transcripts and protein contribute to pathology. The results will help guide future development of SCA2 therapy, as well as therapies of other microsatellite repeat expansion diseases.

脊髓小脑共济失调 2 型 (SCA2) 是一种由 CAG 重复引起的破坏性神经退行性疾病基因 ataxin-2 (ATXN2) 的扩增。 CAG 重复序列被翻译成多聚谷氨酰胺 (polyQ) 区域具有神经毒性特性的突变 ATXN2 蛋白。目前的治疗努力集中于抑制突变 ATXN2 蛋白的表达，或针对神经毒性的下游途径。我们和其他人已经表明，含有突变的 CAG/CTG 重复转录物也有助于多种疾病的发病机制重复疾病，包括亨廷顿病 (HD)、亨廷顿病样 2 (HDL2) 和脊髓小脑病共济失调 3 型和 8 型（SCA3 和 SCA8）。因此，除了针对突变蛋白的神经毒性之外，成功的治疗可能需要针对突变转录本和/或突变下游途径成绩单。我们的初步证据支持这样的观点：在 SCA2 中，除了有毒的 ATXN2 蛋白之外，有义 ATXN2 RNA 和含有扩展 CUG 重复序列的 ATXN2 反义转录本 (ATXN2-AS)，有助于 SCA2 发病机制。因此，我们建议使用基因组编辑方法来修改正常和 SCA2 iPSC 系转化为新型同基因 iPSC 系，模拟 SCA2 的蛋白质或 RNA 诱导机制发病。这些品系将用于 (1) 进一步测试 SCA2 突变体的新假设 ATXN2/ATXN2-AS 转录物破坏 SCA2 中的 RNA 加工，包括 RNA 输出、rRNA 代谢和 (2) 确定 RNA 神经毒性对 SCA2 发病机制的相对贡献。结果从这项研究中获得的结果不仅有助于我们了解日益复杂的发病机制 SCA2，以及其他 CAG/CTG 重复疾病，其中双向编码转录本和蛋白质有助于病理学。结果将有助于指导 SCA2 疗法的未来发展，以及其他微卫星重复扩增疾病的治疗。