ADENO-ASSOCIATED VIRUS (AAV) VECTORS TO IMPROVE MATURE MUSCLE FUNCTION

肌营养不良是一种相对常见的遗传性疾病， 退化性肌肉疾病大多数类型是由基因突变引起的 编码肌肉中的膜相关蛋白。杜氏肌 肌营养不良症（DMD）和肢带型肌营养不良症（LGMD）通常表现为 他们在年轻的时候，导致早期发病，目前没有 有效的治疗。这些疾病是隐性的，丧失- 相应基因产物的功能，这使得它们适合 基因替代疗法重组腺相关病毒 是一种很有前途的基因置换载体， 细小病毒rAAV系统由于其非病毒性而引起关注。 致病性、基因组整合、静止细胞的转导，以及 明显缺乏细胞免疫反应与其他病毒相比， 载体，rAAV能够有效地绕过肌纤维基底膜， 板和转导成熟肌细胞。 我们已经证明，携带外源基因的rAAV载体可以 在成熟肌肉中实现高效和持续的基因表达 免疫活性动物超过1.5年，未检出 毒性最近，载体生产的显著改进 方法学使得产生高滴度和高质量成为可能 rAAV载体完全没有辅助腺病毒污染。 然而，没有使用rAAV载体来恢复功能的实验。 迄今为止已经报道了肌肉组织本身的缺陷。这里我们 建议利用rAAV载体系统，以测试两种治疗 基因（δ-肌聚糖和高度截短的肌营养不良蛋白）， 控制两种不同的启动子系统（病毒/CMV或肌肉- 特异性/MCK），在两种相关的肌营养不良动物模型中 （Bio14.6仓鼠用于LGMD，mdx小鼠用于DMD）。两个不同的向量 给药方式，局部肌肉内感染与全身给药 将被利用。我们有以下三个假设需要检验。 1）：δ-肌聚糖缺乏的肌肉可以通过以下方式在功能上拯救： 使用肌内AAV载体注射的遗传互补 LGMD仓鼠模型。2)δ-肌聚糖基因的全身递送 可以由rAAV载体通过动脉内或心室内介导 注射3)缺乏中心杆结构域的肌营养不良蛋白微型基因将 改善肌营养不良蛋白缺陷肌肉的功能， 通过AAV载体对营养不良mdx小鼠的免疫调节。

Muscular dystrophies are a relatively common group of inherited degenerative muscle disease. Most types are caused by mutations in genes coding for membrance associated proteins in muscle. Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD) often manifest themselves in young ages and lead to early morbidity with no currently available effective treatment. These diseases are recessive, loss-of- function of the corresponding gene product, which makes them suitable for gene replacement therapy. Recombinant adeno-associate virus (rAAV) is one promising gene replacement vector based on defective human parvoviruses. The rAAV system has attracted attention due to its non- pathogenicity, genomic integration, transduction of quiescent cells, and apparent lack of cellular immune reactions. In contrast to other viral vectors, rAAV is capable of efficiently bypassing the myofiber basal lamina and transducing mature muscle cells. We have demonstrated that rAAV vectors harboring a foreign gene can achieve highly efficient and sustained gene expression in mature muscle of immunocompetent animals for more than 1.5 years without detectable toxicity. Recently, significant improvement in vector production methodology has made it possible to generate high titer and high quality rAAV vectors completely free of helper adenovirus contamination. However, no experiments using rAAV vectors to restore the functional deficits in muscle tissue itself have been reported to date. Here, we propose to take advantage of rAAV vector system, to test two therapeutic genes (delta-sarcoglycan and a highly truncated dystrophin), under the control of two different promoter systems (viral/CMV or muscle- specific/MCK), in two relevant animal models of muscular dystrophies (Bio14.6 hamster for LGMD and mdx mouse for DMD). Two distinct vector delivery methods, local intramuscular infection versus systemic delivery will be utilized. We have the following three hypotheses to be tested. 1): muscle deficient in delta-sarcoglycan can be functionally rescued by genetic complementation using intramuscular AAV vector injection in the LGMD hamster model. 2) systemic delivery of the delta-sarcoglycan gene can be mediated by rAAV vectors through intra-artery or intra-ventricle injection. 3) a dystrophin mini-gene lacking the central rod domain will improve the function of dystrophin-deficient muscle when delivered into dystrophic mdx mice by AAV vectors.