不同血清型的重组腺相关病毒载体对成熟肌纤维转导的研究

目的探讨不同血清型对成熟肌纤维的转导效率.方法我们用不同的血清型AAV（rAAV-1,rAAV-2,rAAV-3和rAAV-5）表达LacZ和GDNF基因研究在小鼠骨骼肌转导效率,转基因表达用组化方法或ELISA来评定. 结果在3周龄的小鼠中,LacZ组化染色显示rAAV5在慢和快的肌纤维中都有效的转导,而rAAV2和rAAV3优先在慢纤维中转导.在8周龄的小鼠（成年鼠）中,rAAV3和rAAV5在慢纤维和快纤维中都有效的转导.用rAAV3-LacZ或rAAV5-LacZ优先转导的慢纤维与rAAV2相比没有显著性差异.用8周龄的小鼠肌肉注射不同血清型的rAAV-GDNF后,结果显示rAAVl-GDNF表达最高;rAAV2、rAAV3和rAAV5在骨骼肌的表达也有效.结论在肌肉基因转导中,rAAV3和rAAV5介导的载体都是有效的,能克服rAAV2所受到的限制.

Efficacy of recombinant adeno-associated virus vectors with different serotypes in the transduction of mature muscle fibers

Objective Recombinant vectors based on adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV-2) are the most thoroughly characterized and frequently employed. Here, we investigate the role of rAAV vectors with other different serotypes in the transduction of mature skeletal muscle fibers. Methods rAAV vectors of different serotypes (rAAV-1, rAAV-2, rAAV-3 and rAAV-5) that express the report gene LacZ or GDNF were adopted to investigate the transduction efficacy of slow or fast skeletal muscle fibers. The transgene expression was evaluated by histochemical method or ELISA. Results In 3-week old mice, histochemical staining for LacZ showed that rAAV5 efficiently transduced both slow and fast myofibers while slow fibers were preferentially transduced by rAAV2 and rAAV3. In 8-week old (adult) mice, rAAV3 and rAAV5 efficiently transduced both slow and fast myofibers. Preferential transduction of slow myofibers with rAAV3-lacZ or with rAAV5-lacZ was not remarkable in contrast to transduction with rAAV2-lacZ. We also explored the gene expression of intramuscular delivery of the GDNF gene mediated by rAAVs in 8-week old mice. rAAV1-GDNF is the most efficient serotype for the expression. rAAV-2, rAAV-3 and rAAV5 were demonstrated also efficient for transgene expression in skeletal muscle. Conclusion These results demonstrate that rAAV-3 and rAAV-5 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.