Petrof B J, Acsadi G, Jani A, Massie B, Bourdon J, Matusiewicz N, Yang L, Lochmüller H, Karpati G
Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada.
Am J Respir Cell Mol Biol. 1995 Nov;13(5):508-17. doi: 10.1165/ajrcmb.13.5.7576685.
The protein dystrophin is absent in muscles of patients with Duchenne muscular dystrophy (DMD) as well as in mdx mice. The mdx mouse diaphragm closely resembles the human DMD phenotype and should serve as an appropriate model for future studies of dystrophin gene replacement. In this regard, recombinant adenovirus (AV) holds great promise as a vector for delivering a functional dystrophin gene to muscle. However, the use of AV is hampered by the development of an immune response against transduced cells, resulting in short-lived transgene expression as well as possible adverse effects on organ function. In the present study, sensitive reporter genes were employed to determine the efficiency and functional consequences of AV-mediated gene transfer to the diaphragm in both normal and mdx adult mice. One week after direct intramuscular injection of AV into the diaphragm, the level of transgene expression was significantly increased in mdx compared with normal diaphragms. In addition, small-caliber fibers (< 500 microns2) demonstrated preferential transduction in both groups of mice. Normal diaphragms receiving AV exhibited a substantial reduction in maximal twitch and tetanic force generation, whereas no significant effect on diaphragm contractility was found in the mdx group at 1 wk after injection. At 1 mo after AV administration, however, there was a significant decrease in force production by both normal and mdx diaphragms. Immunosuppression with cyclosporine A over 1 mo did not augment the level of transgene expression, but a beneficial effect on diaphragm force-generating capacity was observed in both groups of animals. We conclude the following: (1) short-term transduction of the diaphragm is more efficient in mdx than in normal mice; (2) AV leads to reduced force production by the diaphragm, with this effect being more pronounced in normal than in mdx in the early (but not the late) postinjection period; and (3) immunosuppressive therapy with cyclosporine has a partially protective effect on muscle function after AV administration, which is apparently unrelated to sparing of transduced fibers from elimination by the host immune system. These findings have important implications for the application of AV-mediated dystrophin gene transfer to the treatment of DMD.
杜兴氏肌营养不良症（DMD）患者以及mdx小鼠的肌肉中缺乏抗肌萎缩蛋白。mdx小鼠的膈肌与人类DMD表型极为相似，应作为未来抗肌萎缩蛋白基因替代研究的合适模型。在这方面，重组腺病毒（AV）作为将功能性抗肌萎缩蛋白基因传递至肌肉的载体具有巨大潜力。然而，对转导细胞产生免疫反应阻碍了AV的使用，导致转基因表达短暂以及可能对器官功能产生不良影响。在本研究中，使用敏感的报告基因来确定AV介导的基因转移至正常和mdx成年小鼠膈肌的效率及功能后果。将AV直接肌内注射至膈肌一周后，与正常膈肌相比，mdx小鼠中转基因表达水平显著增加。此外，两组小鼠中直径小的纤维（<500平方微米）均表现出优先转导。接受AV的正常膈肌在最大抽搐和强直力产生方面大幅降低，而注射后1周时mdx组中未发现对膈肌收缩力有显著影响。然而，在给予AV 1个月后，正常和mdx膈肌的力产生均显著下降。用环孢素A进行1个月以上的免疫抑制并未提高转基因表达水平，但在两组动物中均观察到对膈肌力产生能力有有益影响。我们得出以下结论：（1）短期转导膈肌时，mdx小鼠比正常小鼠更有效；（2）AV导致膈肌力产生减少，在注射后早期（而非晚期），这种影响在正常小鼠中比在mdx小鼠中更明显；（3）用环孢素进行免疫抑制治疗在给予AV后对肌肉功能有部分保护作用，但这显然与使转导纤维免受宿主免疫系统清除无关。这些发现对应用AV介导的抗肌萎缩蛋白基因转移治疗DMD具有重要意义。
