Dystrophin Replacement in mdx mice

描述（由申请人提供）： 杜氏肌营养不良症（DMD）是以隐性模式遗传的，这表明如果能够找到替代全身横纹肌中基因的方法，基因治疗可以提供有效的治疗。以前尝试以系统方式将基因转移到肌肉中，由于无法靶向成年哺乳动物广泛分布的肌肉而受到严重限制。例如，需要麻醉、侵入性手术和危险的辅因子来有效地维持心肌细胞群体的不同部分。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个肌肉，或者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方法，用于全身性基因转移到肌肉可能导致治疗各种各样的肌肉疾病，包括肌营养不良症。我们已经确定了一种简单而高效的方法，将基因系统地转移到成年哺乳动物的心脏和骨骼肌。该方法利用静脉内施用用6型衣壳假型化的重组腺相关病毒载体（rAAV6）。初步研究表明，rAAV6载体可以使成年小鼠全身的横纹肌中，并且这种递送可以通过共同施用血管透化剂VEGF来增强。静脉内注射表达微小肌营养不良蛋白的rAAV6载体导致成年mdx小鼠中营养不良表型的全身改善。虽然这些研究代表了一个明确的原则证据，即肌营养不良蛋白可以被递送到成年哺乳动物的所有横纹肌，但使用rAAV6将基因转移到肌肉的机制尚不清楚，并且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，然后才能在临床上进行测试。我们将研究通过循环使全身肌肉转导的机制，并将进行详细的优化方案。将对AAV载体衣壳进行修饰以改善肌肉基因转移，我们将在较大动物中进行临床前优化研究。这项工作的最终目标是为DMD和其他横纹肌疾病开发临床相关的治疗方法。

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.