Optimizing and validation of gene therapy vectors to treat limb girdle muscular dystophy

项目总结 我们的目标是开发一种治疗常染色体隐性遗传性肢带型2A型肌营养不良症(LGMD2A)的基因疗法 (AR)CAPN3基因突变导致的肌肉萎缩障碍。LGMD2A被认为是最普遍的 AR LGMD，但目前还没有针对患者的治疗，这些患者通常依赖轮椅 确诊十年后。我们和其他人已经证明，CAPN3的过度表达可以在 骨骼肌无毒性；这一发现使LGMD2A基因治疗的可行性成为现实 目的：然而，LGMD2A与大多数其他LGMDs是独一无二的，这一事实需要仔细开发基因 治疗载体。一种考虑是，由于心脏毒性，CAPN3不能在心脏表达， Duchenne肌营养不良症和其他LGMD并非如此。尽管有一些临床前的证据， 概念研究已经成功地在小鼠中实现了AAV-Capn3的过表达，目前还没有 对任何使用LGMD2A的人类基因治疗结构进行系统优化，特别是考虑到 相对骨骼肌和心脏的基因表达问题。此外，由于LGMD2A优先考虑 影响慢纤维，关键是最终进入临床试验的治疗结构是 针对慢速纤维表达进行了优化。因为每个病人只能服用一次，所以当务之急是 用于基因传递的结构是理想的。在本应用程序中，我们将使用迭代和系统方法来 优化这些向量。我们组建了一支专家协作团队，在以下领域拥有丰富的经验 调节盒，AAV载体的发展和LGMD2A的发病机制和钙化病动物模型。 该团队将共同努力，生成并测试一系列AAV载体的安全性和有效性，以开发 LGMD2A的治疗。这些载体将针对慢纤维表达进行优化，同时避免心脏毒性。 团队成员豪施卡博士在很大程度上被认为是创造了绝大多数监管盒的功臣 目前用于杜氏肌营养不良症的AAV基因治疗试验。张伯伦博士设计并制作了 优化了第一个微型和微型营养不良蛋白，这构成了目前基因治疗中所有结构的基础 DMD的临床试验。Spencer博士和Kramerova博士创造了大量的小鼠模型，这些模型导致了 确定对测试LGMD2A的治疗干预有用的结果衡量标准。坎农博士，是一位 国际知名的肌肉生理学家，将对收缩功能进行生理学评估。 该团队将应用他们对LGMD2A、AAV载体、调节盒的广泛和积累的知识 和小鼠肌肉测试，以创造这种针对LGMD2A的基因疗法。

PROJECT SUMMARY We aim to develop a gene therapy for limb girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive (AR) muscle wasting disorder due to mutations in CAPN3. LGMD2A is considered to be the most prevalent of the AR LGMDs and yet there is currently no treatment for patients, who are usually wheelchair dependent a decade after diagnosis. We and others have shown that overexpression of CAPN3 can be accomplished in skeletal muscle without toxicity; a finding which makes the feasibility of gene therapy for LGMD2A a realistic goal; however, LGMD2A is unique from most other LGMDs and this fact warrants careful development of gene therapy vectors. One consideration is that CAPN3 cannot be expressed in the heart, due to cardiac toxicity, which is not the case with Duchenne muscular dystrophy and other LGMDs. Although a few pre-clinical, proof of concept studies have successfully accomplished AAV-Capn3 overexpression in mice, there has not been a systematic optimization of any gene therapy construct for humans with LGMD2A, especially one that considers the relative skeletal muscle vs cardiac gene expression issues. Furthermore, because LGMD2A preferentially impacts slow fibers, it is critical that the therapeutic construct that is ultimately carried into clinical trials is optimized for slow fiber expression. Because each patient can only be dosed one time, it is imperative that the construct used for gene delivery is ideal. In this application, we will use an iterative and systematic approach to optimize these vectors. We have assembled an expert, collaborative team with deep experience in the area of regulatory cassettes, AAV vector development and LGMD2A pathogenesis and calpainopathy animal models. The team will work together to generate and test the safety and efficacy of a series of AAV vectors to develop a treatment for LGMD2A. These vectors will be optimized for slow fiber expression while avoiding cardiac toxicity. One team member, Dr. Hauschka, is largely credited with creating the vast majority of regulatory cassettes being used in the current AAV-gene therapy trials for Duchenne muscular dystrophy. Dr. Chamberlain designed and optimized the first micro and mini dystrophins, which formed the basis for all constructs currently in gene therapy clinical trials for DMD. Drs. Spencer and Kramerova generated numerous mouse models that have led to the identification of outcome measures useful for testing therapeutic interventions for LGMD2A. Dr. Cannon, is an internationally known muscle physiologist who will carry out physiological assessments of contractile function. The team will apply their extensive and cumulative knowledge of LGMD2A, AAV vectors, regulatory cassettes and mouse muscle testing to create this gene therapy for LGMD2A.