Dystrophin Replacement in mdx mice

描述(由申请人提供)： Duchenne肌营养不良症(DMD)是以隐性模式遗传的，这表明，如果能找到方法取代全身横纹肌中的基因，基因疗法可能会提供一种有效的治疗方法。以前系统地将基因转移到肌肉的尝试由于无法针对广泛分布的成年哺乳动物肌肉而受到严重限制。例如，麻醉、侵入性手术和危险的辅助因子已经被要求有效地转导不同部分的心肌细胞。同样，使用质粒或病毒载体将基因转移到四肢肌肉需要直接注射单独的肌肉，或者需要在麻醉下使用危险的辅助因子进行复杂的外科手术。开发一种有效和安全的系统基因转移到肌肉的方法可能导致治疗包括肌肉营养不良在内的各种肌肉疾病。我们已经确定了一种简单而高效的方法来系统地将基因转移到成年哺乳动物的心肌和骨骼肌中。这种方法利用静脉注射假型为6型衣壳的重组腺相关病毒载体(RAAV6)。初步研究表明，rAAV6载体可以转导成年小鼠全身的横纹肌，联合应用血管通透剂VEGF可以增强这种转导作用。静脉注射表达微肌营养不良蛋白的rAAV6载体导致成年mdx小鼠营养不良表型的全身改善。虽然这些研究清楚地证明了营养不良蛋白可以传递到成年哺乳动物的所有横纹肌，但使用rAAV6将基因转移到肌肉中的机制尚不清楚，在可以在临床上进行测试之前，有必要提高该方法的效率，解决安全问题，并将该方法应用于更大的动物模型。我们将研究通过循环实现全身肌肉转导的机制，并将对方案进行详细的优化。AAV载体衣壳将被修改以改进肌肉基因转移，我们将在更大的动物身上进行临床前优化研究。这项工作的最终目标是开发一种临床相关的治疗DMD和其他横纹肌疾病的方法。

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is inherited in a recessive pattern, suggesting that gene therapy could offer an effective treatment if methods can be found to replace the gene in striated muscles throughout the body. Previous attempts to transfer genes to muscles in a systemic manner have been severely limited by an inability to target widely dispersed muscles of adult mammals. For example, anesthesia, invasive surgery, and hazardous co-factors have been required to transduce varying fractions of the cardiomyocyte population efficiently. Similarly, the transfer of genes to the muscles of limbs using plasmids or viral vectors has required either direct injection of individual muscles, or complex surgical procedures performed under anesthesia using hazardous co-factors. Development of an efficient and safe method for systemic gene transfer to muscle could lead to a treatment for a wide variety of muscle diseases including the muscular dystrophies. We have identified a simple and highly efficient method to transfer genes systemically to cardiac and skeletal muscles of adult mammals. This approach utilizes intravenous administration of recombinant adeno-associated viral vectors pseudotyped with the type 6 capsid (rAAV6). Preliminary studies show that rAAV6 vectors can transduce striated muscles throughout the body of adult mice, and that this delivery can be enhanced by co-administration of the vascular permeabilizing agent VEGF. Intravenous injection of rAAV6 vectors expressing micro-dystrophin results in a whole body amelioration of the dystrophic phenotype in adult mdx mice. While these studies represent a clear proof of principle that dystrophin can be delivered to all the striated muscles of an adult mammal, the mechanisms that enable gene transfer to muscle using rAAV6 are unclear, and it will be necessary to increase the efficiency of the approach, address safety issues and apply the methods to larger animal models before they could be tested in the clinic. We will study the mechanisms that enable systemic muscle transduction via the circulation and will conduct a detailed optimization of the protocols. The AAV vector capsids will be modified for improved muscle gene transfer, and we will perform preclinical optimization studies in larger animals. The ultimate goal of this work is to develop a clinically relevant treatment for DMD and other diseases of striated muscle.