RNAi Therapy for Dominant Limb Girdle Muscular Dystrophy Type 1A

描述（由申请人提供）：肢带肌营养不良症（LGMD）是指以骨盆和肩带肌肉进行性萎缩和无力为特征的一组19种疾病。通常，患者需要轮椅辅助，并且患有某些形式的LGMD的个体也可能涉及心脏和呼吸肌肉。对相对有限的一组肌肉的适度改善可能会显著改善患者的生活质量，但目前尚无有效的治疗LGMD的方法。基因治疗方法有望成为未来的LGMD治疗方法。在过去的二十年里，由于在载体设计、生产和递送方面取得了重要的技术进步，使用基因疗法治疗肌肉萎缩症的可行性得到了改善，特别是LGMD。这些进步几乎完全用于开发旨在替换隐性疾病中有缺陷或缺失基因的策略。然而，基因替代策略并不适用于治疗显性肌营养不良症，包括7种显性形式的LGMD（称为LGMD1）。相反，显性LGMD1患者可能会受益于致病等位基因的减少，这在历史上是不可能的。近年来，RNA干扰（RNAi）已成为抑制显性疾病基因的有力工具。这一发现开创了基因治疗的一个新领域，在该领域中，治疗性抑制microrna被设计成在递送到受影响组织后减少毒性显性疾病基因的表达。本提案的具体目的旨在测试使用RNAi治疗LGMD1A的可行性，LGMD1A是由肌动素（MYOT）基因的一个等位基因突变引起的。MYOT是rnai介导的显性LGMD1A基因治疗原理验证的理想靶点，因为正常肌肉不受其缺失的影响，而且我们的研究有一个优秀的LGMD1A小鼠模型。前者的特点消除了区分突变和正常等位基因的需要（这是可行的，但比非等位基因特异性沉默方法更具挑战性），因此简化了策略。myotilin导向的microrna将使用腺相关病毒载体（AAV）系统和已经优化的肌肉基因转移方法（包括在临床试验中）递送到LGMD1A小鼠的肌肉中。我们将评估microrna介导的MYOT敲低对lgmd1a相关的组织病理学和功能异常的影响。这些研究代表了LGMD1A靶向RNAi基因治疗方法的重要的第一步，并且可能具有超出此建议的意义。重要的是，由于显性肌病是由至少37种不同基因的突变引起的，这些基因总共影响2400到3200个个体中的1个，因此我们的RNAi策略可以广泛地影响一大类显性肌肉疾病。）

DESCRIPTION (provided by applicant): Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 19 disorders characterized by progressive wasting and weakness of pelvic and shoulder girdle muscles. Commonly, patients require wheelchair assistance, and individuals with some forms of LGMD may also have cardiac and respiratory muscle involvement. Modest improvements in a relatively limited set of muscles may dramatically improve patients' quality of life, but there is currently no effective treatment for LGMD. Gene therapy approaches may hold promise as future LGMD treatments. Over the last two decades, the feasibility of using gene therapy to treat muscular dystrophy in general, and LGMD in particular, has improved, due to important technical advancements made in vector design, production, and delivery. These advancements have almost exclusively been used to develop strategies aimed at replacing defective or missing genes underlying recessive disorders. However, gene replacement strategies are not feasible for treating dominant muscular dystrophies, including the 7 dominant forms of LGMD (called LGMD1). Instead, patients with dominant LGMD1 would likely benefit from reduction of their pathogenic alleles, which was historically not possible. Recently, RNA interference (RNAi) has emerged as a powerful tool to suppress dominant disease genes. This discovery has ushered in a new arm of gene therapy, in which therapeutic inhibitory microRNAs are engineered to reduce expression of toxic dominant disease genes, following delivery to affected tissues. The Specific Aims of this proposal were designed to test the feasibility of using RNAi to treat LGMD1A, which is caused by mutations in one allele of the myotilin (MYOT) gene. MYOT is an ideal target to demonstrate proof-of-principle for RNAi-mediated gene therapy of dominant LGMD1A because normal muscles are unaffected by its absence, and because an excellent LGMD1A mouse model is available for our studies. The former feature obviates the need to discriminate between mutant and normal alleles (which is feasible but more challenging than a non-allele- specific silencing approach) and therefore simplifies the strategy. Myotilin-directed microRNAs will be delivered to muscles of LGMD1A mice using adeno-associated viral vector (AAV) systems and delivery methods that have already been optimized for muscle gene transfer, including in clinical trials. The effects of microRNA-mediated MYOT knockdown on LGMD1A-associated histopathological and functional abnormalities will be assessed. These studies represent important first steps toward targeted RNAi gene therapy approaches for LGMD1A and may have implications beyond this proposal. Importantly, since dominant myopathies arise from mutations in at least 37 different genes that collectively affect ~1 in 2,400 to 3,200 individuals, our RNAi strategies could be adapted to broadly impact a large class of dominant muscle disorders. )) PUBLIC HEALTH RELEVANCE: Patients with dominant Limb Girdle Muscular Dystrophy Type 1A (LGMD1A) have one normal, and one mutated, copy of the myotilin gene. We propose that reducing mutant myotilin in muscles will improve muscular dystrophy in LGMD1A patients. In this proposal, we will investigate a strategy to reduce mutant myotilin using gene therapy.