Allele Specific Knockdown for LGMD-D1

项目摘要 基于基因的治疗正在成为一个现实致残神经肌肉疾病。然而，肢带 肌营养不良症（LGMD）基因治疗领域几乎完全集中在复发性遗传性疾病上， 将显性遗传的LGMD远远抛在了治疗的后面。最近，DNAJB 6突变， HSP-40共分子伴侣被发现引起LGMD-D1，一种成人发病的显性遗传性肌病。 LGMD-D1背后的疾病机制尚未完全了解，阻碍了治疗策略 发展解决这一未满足的医疗需求将有助于我们更好地了解不仅如何治疗LGMD- D1，以及如何有效地接近治疗策略的发展，为其他显性遗传 肌病已知显性肌病具有复杂的异质性疾病机制，包括 毒性功能获得、显性阴性和单倍性不足。每一种都需要独特的治疗方法， 例如整体敲低、等位基因特异性敲低（ASKD）、敲低和置换或简单基因 在单倍不足的情况下进行替换。几条证据表明DNAJB 6的突变发挥了 通过功能的毒性获得的显性效应。此外，DNAJB 6敲除（KO）小鼠是胚胎性的， 由于客户蛋白质的聚集而致死，表明整体敲除处理可能是有害的。 然而，单倍不足似乎是耐受的，因为杂合KO小鼠是可行的并且能够繁殖。 因此，突变等位基因的靶向敲低是解决LGMD-D1治疗的理想策略。 功能机制的毒性获得，同时避免完全DNAJB 6敲除的破坏作用。这 该提案旨在验证ASKD对LGMD-D1具有治疗潜力。由于几种siRNA目前已被FDA 该项目将优化siRNA以实现突变DNAJB 6的ASKD（目的1），并验证是否 ASKD改善小鼠和人LGMD-D1模型中的疾病表型（目的2）。这些基础 研究，是一种在野生型等位基因上含有flag标签的敲入LGMD-D1小鼠模型，允许大小- 基于等位基因特异性变化的检测。成功完成这些研究将确定一种治疗方法， 靶向LGMD-D1，这反过来也将为疾病机制提供额外见解。LGMD-D1是 这只是几种可能适用于本提案所述策略的疾病之一。

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.