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

肌营养不良症是一组相对常见的遗传性 退行性肌肉疾病。大多数类型是由基因突变引起的 肌肉中膜相关蛋白的编码。杜兴肌肉 营养不良症(DMD)和四肢带状肌营养不良症(LGMD)常表现为 并导致早期发病，目前没有 可获得有效的治疗。这些疾病是隐性的，丢失的 相应基因产物的功能，这使它们适合 用于基因替代疗法。重组腺相关病毒 是一种很有前途的基于缺陷人类的基因替换载体 细小病毒。RAAV系统由于其非对称性引起了人们的关注 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。与其他病毒不同 载体，rAAV能够有效地绕过肌纤维基础 板层和转导成熟肌肉细胞。 我们已经证明了携带外源基因的rAAV载体可以 在成熟肌肉中实现高效和持续的基因表达 免疫活性动物已超过1.5年而未被检测到 毒性。最近，媒介生物产量的显著改善 方法学使产生高滴度和高质量成为可能 重组腺病毒载体完全没有辅助腺病毒的污染。 然而，没有实验使用rAAV载体来恢复功能性 到目前为止，有报道称肌肉组织本身存在缺陷。在这里，我们 建议利用rAAV载体系统，检测两种治疗方法 基因(德尔塔-肌聚糖和高度截断的肌营养不良蛋白)，在 控制两个不同的启动子系统(病毒/CMV或肌肉- 在两种相关的肌营养不良动物模型中 (LGMD为Bio14.6仓鼠，DMD为MDX小鼠)。两个不同的向量 分娩方式、局部肌肉内感染与全身分娩 将会被利用。我们有以下三个假设需要检验。 1)：通过以下方法可以从功能上挽救缺乏三角洲-肌聚糖的肌肉 肌肉注射AAV载体用于遗传互补的研究 LGMD仓鼠模型。2)系统递送德尔塔-肌聚糖基因 可通过动脉内或脑室内由rAAV载体介导 注射。3)缺乏中央杆状结构域的抗肌营养不良蛋白微基因将 改善肌营养不良蛋白缺陷肌的功能 携带AAV载体的营养不良mdx小鼠。

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.