Allele Specific Knockdown for LGMD-D1

项目总结 基于基因的疗法正在成为致残神经肌肉疾病的现实。然而，四肢腰带 肌营养不良症(LGMD)基因治疗领域几乎完全集中在隐性遗传性疾病上， 将以遗传为主的LGMD远远抛在治愈的道路上。最近DNAJB6的突变，一个 HSP-40辅助伴侣被发现导致LGMD-D1，一种成人起病，主要是遗传性肌病。 LGMD-D1背后的发病机制尚不完全清楚，预防治疗策略 发展。解决这一未得到满足的医疗需求将有助于我们不仅更好地了解如何治疗LGMD- 也是如何有效地为其他以遗传为主的患者制定治疗策略 肌病。众所周知，显性肌病具有复杂的、异质性的疾病机制，包括 功能毒性增强、显性负性和单倍体功能不全。每一种都需要一种独特的治疗方法， 例如全局敲除、等位基因特异性敲除(ASKD)、敲除和替换或简单基因 在单倍体功能不全的情况下进行替换。几条证据表明，DNAJB6的突变对 通过有毒的功能获得的显性效果。此外，DNAJB6基因敲除(KO)小鼠是胚胎 由于客户蛋白的聚集而致死，表明全球击倒治疗可能是有害的。 然而，单倍体不足似乎是可以容忍的，因为杂合子KO小鼠是活的和能够繁殖的。 因此，靶向敲除突变等位基因是一种理想的LGMD-D1治疗策略，以解决 功能机制的毒性获得，同时避免完全DNAJB6基因敲除的破坏性影响。这 该提案旨在验证ASKD对LGMD-D1的治疗潜力。因为现在有几个siRNAs是FDA 批准，该项目将优化siRNA以实现突变DNAJB6(目标1)的ASHD，并验证是否 ASKD改善小鼠和人类LGMD-D1模型的疾病表型(目标2)。以此为基础 是一种在野生型等位基因上含有标志标签的敲入LGMD-D1小鼠模型，考虑到大小- 基于对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 LGMD-D1的靶点，反过来也将为疾病机制提供更多的见解。LGMD-D1IS 可能符合本提案中所述策略的几种障碍中的唯一一种。

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.