Center for Gene Therapy, Nationwide Children's Hospital Columbus, Ohio.
Center for Gene Therapy, Nationwide Children's Hospital Columbus, Ohio ; Biomedical Sciences Graduate Program, The Ohio State University Columbus, Ohio.
Ann Clin Transl Neurol. 2015 Mar;2(3):256-70. doi: 10.1002/acn3.172. Epub 2015 Jan 20.
Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.
Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.
Treated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.
Treated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.
肌营养不良蛋白病是一组由肌营养不良蛋白基因突变引起的不可治愈的肌肉疾病。肌营养不良蛋白的缺乏导致进行性肌营养不良，伴有慢性肌纤维丧失、炎症、脂肪替代和纤维化，导致肌肉无力恶化。本工作的目的是证明基因治疗后肌营养不良蛋白表达的有效和安全性恢复。
传统的基因治疗受到腺相关病毒（AAV）包装容量限制，然而，使用双载体策略可以递送超大基因，包括肌营养不良蛋白。该双载体系统（AAV.DYSF.DV）通过使用两个离散载体来包装肌营养不良蛋白 cDNA，这两个载体通过 1kb 的同源区域定义。通过肌肉内和血管递送途径在肌营养不良蛋白缺陷小鼠和非人灵长类动物中比较了 AAV.DYSF.DV 的递送效率和安全性。
对接受治疗的肌肉进行了肌营养不良蛋白表达、整体肌肉组织学以及损伤后修复能力的检测。所有接受治疗的肌肉群均显示出高水平的肌营养不良蛋白过表达，并且功能恢复测量（膜修复能力和膈神经特异性力量）恢复到野生型水平。在灵长类动物中，也证明了强肌营养不良蛋白的表达，且无安全性问题。
治疗后的肌肉显示出高水平的肌营养不良蛋白表达，并具有功能恢复，没有毒性或免疫反应的证据，为向肌营养不良蛋白病患者转化提供了原理证明。
