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

治疗肢带型肌营养不良症的基因治疗载体的优化和验证

• 批准号: 10400144
• 负责人: JEFFREY S CHAMBERLAIN
• 金额: $ 49.6万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10400144
• 关键词: Affect; Animal Model; Antibodies; Area; Binding Sites; Calpain; Cardiac; Cardiotoxicity; Clinical Trials; Code; Codon Nucleotides; Collaborations; Complementary DNA; Development; Diagnosis; Disease; Dose; Duchenne muscular dystrophy; Dystrophin; Exercise Tolerance; Fiber; Gene Delivery; Gene Expression; Gene Transduction Agent; Gene therapy trial; Genes; Goals; Heart; Heart Block; Human; International; Knockout Mice; Knowledge; Limb structure; Limb-Girdle Muscular Dystrophies; Longitudinal Studies; MicroRNAs; Modification; Mus; Muscle; Muscle Weakness; Muscular Atrophy; Muscular Dystrophies; Mutation; Myocardium; Neuromuscular Diseases; Outcome; Outcome Measure; Pathogenesis; Patients; Peptide Hydrolases; Phenotype; Physiological; Property; Proteins; Series; Skeletal Muscle; Testing; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Time; Toxic effect; Transgenes; Treatment Efficacy; Universities; Untranslated Regions; Validation; Variant; Viral Vector; Washington; Wasting Syndrome; Western Blotting; Wheelchairs; Work; adeno-associated viral vector; design; differential expression; experience; gene therapy; gene therapy clinical trial; in vitro testing; in vivo; laser capture microdissection; member; micro-dystrophin; mini-dystrophin; mitochondrial dysfunction; mouse model; novel; overexpression; pre-clinical; safety testing; therapeutic evaluation; vector

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.

项目摘要 我们的目标是开发一种基因治疗肢带型肌营养不良症2A型（LGMD 2A），一种常染色体隐性遗传病， (AR)由于CAPN 3突变引起的肌肉萎缩症。LGMD 2A被认为是最普遍的 AR LGMD，但目前还没有治疗的病人，谁通常是轮椅依赖， 诊断后十年。我们和其他人已经表明，CAPN 3的过表达可以在细胞内完成。 骨骼肌无毒性;这一发现使LGMD 2A基因治疗的可行性成为现实 然而，LGMD 2A与大多数其他LGMD不同，这一事实保证了基因的谨慎开发。 治疗载体一个考虑是由于心脏毒性，CAPN 3不能在心脏中表达， 这与杜氏肌营养不良症和其他LGMD的情况不同。虽然一些临床前， 概念研究已经成功地在小鼠中实现了AAV-Capn 3过表达，但还没有发现AAV-Capn 3过表达。 系统优化用于患有LGMD 2A的人类的任何基因治疗构建体，特别是考虑到 相对的骨骼肌与心脏基因表达问题。此外，由于LGMD 2A优先 影响慢纤维，至关重要的是，最终进行临床试验的治疗结构， 针对慢纤维表达进行优化。因为每个病人只能给药一次，所以必须 用于基因递送的构建体是理想的。在本申请中，我们将使用迭代和系统的方法， 优化这些载体。我们组建了一支在以下领域拥有丰富经验的专家协作团队： 调节盒、AAV载体开发和LGMD 2A发病机制和钙蛋白病动物模型。 该团队将共同努力，产生和测试一系列AAV载体的安全性和有效性，以开发一种新的病毒载体。 治疗LGMD 2A。这些载体将被优化用于缓慢的纤维表达，同时避免心脏毒性。 一个团队成员，Hauschka博士，在很大程度上归功于创造了绝大多数的监管卡匣， 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 优化了第一个微型和迷你抗肌萎缩蛋白，这构成了目前基因治疗中所有构建体的基础 DMD的临床试验Spencer和Kramerova博士建立了许多小鼠模型，这些模型导致了 确定对检测LGMD 2A治疗干预有用的结果指标。坎农医生是 国际知名肌肉生理学家，他们将对收缩功能进行生理评估。 该团队将运用他们在LGMD 2A、AAV载体、调控盒和基因表达方面的广泛和积累的知识， 和小鼠肌肉测试来创造这种LGMD 2A的基因疗法。