Allele Specific Knockdown for LGMD-D1

LGMD-D1 等位基因特异性敲低

• 批准号: 10666694
• 负责人: Andrew Findlay
• 金额: $ 7.87万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10666694
• 关键词: Address; Adult; Alleles; Breeding; Cell Line; Cells; Client; Clinical; Coculture Techniques; Complex; Data; Detection; Development; Disease; Dominant-Negative Mutation; Embryo; FDA approved; Future; Goals; Haploidy; Hereditary Disease; Heterozygote; Human; In Vitro; Infrastructure; Inherited; Intramuscular Injections; Knock-in; Knock-out; Knockout Mice; Length; Limb-Girdle Muscular Dystrophies; Location; Medical; Modeling; Molecular Chaperones; Mus; Muscle Fibers; Mutation; Myoblasts; Myopathy; Neuromuscular Diseases; Patient Care; Patients; Phenotype; Pilot Projects; Point Mutation; Proteins; Qualifying; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Small Interfering RNA; Specificity; Testing; Therapeutic; Translations; United States National Institutes of Health; Western Blotting; disease phenotype; experimental study; gain of function; gene correction; gene replacement; gene therapy; improved; in vivo; induced pluripotent stem cell; insight; knock-down; lead candidate; lead optimization; mouse model; mutant; neuromuscular; prevent; therapeutic target; tool; treatment strategy

PROJECT SUMMARY Gene-based therapies are becoming a reality for disabling neuromuscular disorders. However, the limb girdle muscular dystrophy (LGMD) gene therapy field is focused almost entirely on recessively inherited disorders, leaving dominantly inherited LGMDs far behind in the path towards a cure. Recently mutations in DNAJB6, an HSP-40 co-chaperone, were discovered to cause LGMD-D1, an adult onset, dominantly inherited myopathy. The disease mechanism behind LGMD-D1 is incompletely understood, preventing therapeutic strategy development. Addressing this unmet medical need will help us better understand not only how to treat LGMD- D1, but also how to efficiently approach therapeutic strategy development for other dominantly inherited myopathies. Dominant myopathies are known to have complex, heterogenous disease mechanisms, including toxic gain of function, dominant negative, and haploinsufficiency. Each requires a unique therapeutic approach, such as global knockdown, allele specific knockdown (ASKD), knockdown and replace, or simply gene replacement in the case of haploinsufficiency. Several lines of evidence indicate mutations in DNAJB6 exert a dominant effect through a toxic gain of function. Additionally, DNAJB6 knockout (KO) mice are embryonic lethal due to aggregation of client proteins, indicating a global knockdown treatment may be deleterious. However, haploinsufficiency appears to be tolerated as heterozygous KO mice are viable and able to breed. Targeted knockdown of the mutant allele is therefore an ideal LGMD-D1 treatment strategy to address the toxic gain of function mechanism, while avoiding the damaging effects of complete DNAJB6 knockout. This proposal aims to validate ASKD has therapeutic potential for LGMD-D1. As several siRNAs are now FDA approved, this project will optimize siRNAs to achieve ASKD of mutant DNAJB6 (aim 1), and validate whether ASKD improves disease phenotypes in mouse and human LGMD-D1 models (aim 2). Foundational to these studies, is a knock-in LGMD-D1 mouse model containing a flag tag on the wild type allele, allowing for size- based detection of allele specific changes. Successful completion of these studies will identify a therapeutic target for LGMD-D1, which will in turn, also provide additional insights into disease mechanism. LGMD-D1 is only one of several disorders that may be amenable to the strategies described in this proposal.

项目摘要 基于基因的疗法已成为致残神经肌肉疾病的现实。但是，肢体腰带 肌肉营养不良（LGMD）基因治疗领域几乎完全集中在隐性遗传疾病上， 在通往治愈的道路上，遗传了远处的LGMD。最近在DNAJB6中突变 发现HSP-40的伴侣，发现引起LGMD-D1（成人发作，主要遗传肌病）。 LGMD-D1背后的疾病机制尚未完全理解，以防止治疗策略 发展。解决这种未满足的医疗需求将有助于我们更好地了解如何治疗LGMD- D1，以及如何为其他主要继承的其他主要继承的治疗策略开发 肌病。众所周知，主要的肌病具有复杂的异质疾病机制，包括 功能的有毒毒性增益，主导性负和单倍弥补。每个都需要一种独特的治疗方法， 例如全球敲低，等位基因特定的敲低（ASKD），敲低和替换，或者简单的基因 在单倍不足的情况下取代。几条证据表明DNAJB6突变施加A 通过功能的有毒物质获得的主要作用。此外，DNAJB6敲除（KO）小鼠是胚胎 由于客户蛋白的聚集而导致的致命，表明全球敲低处理可能是有害的。 然而，由于杂合的KO小鼠可行并且能够繁殖，因此单倍性似乎被耐受性。 因此 功能机制的有毒增益，同时避免了完全DNAJB6敲除的破坏作用。这 提案旨在验证ASKD具有LGMD-D1的治疗潜力。由于几个sirnas现在是fda 已批准，该项目将优化SIRNA，以实现突变DNAJB6的AskD（AIM 1），并验证是否是否验证 ASKD改善了小鼠和人LGMD-D1模型中的疾病表型（AIM 2）。这些基础 研究是一种敲入LGMD-D1小鼠模型，该模型在野生型等位基因上包含标志标签，从而使大小 - 基于等位基因特定变化的检测。这些研究的成功完成将确定治疗性 LGMD-D1的靶标也将依次提供对疾病机制的更多见解。 LGMD-D1是 可能适合本提案中描述的策略的几种疾病中只有一种。