Lochmüller H, Petrof B J, Pari G, Larochelle N, Dodelet V, Wang Q, Allen C, Prescott S, Massie B, Nalbantoglu J, Karpati G
Neuromuscular Research Group, Montreal Neurological Institute, Quebec, Canada.
Gene Ther. 1996 Aug;3(8):706-16.
Adenovirus (AV)-mediated gene transfer into skeletal muscles of adult immune-competent animals has been limited by the fact that a cell-mediated immune attack of the host against transduced muscle fibers prevented efficient long-term transgene expression. More recently, various immunomodulating strategies have been shown to improve the longevity of transgene expression after AV-mediated gene transfer. In this study we treated adult dystrophic (mdx) mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 5, 10, 30 and 60 days after AV-mediated dystrophin gene transfer and compared the transduction level with saline-injected mdx controls. We show that daily FK506 treatment after AV-mediated dystrophin gene transfer into adult mdx muscle results in the maintenance of the initial transgene expression for at least 2 months, even when FK506 treatment was discontinued after 1 month. This is in keeping with the marked reduction of inflammatory infiltrates and the reduced activation level (inducible nitric oxide synthase) of macrophages in adenoviral recombinant (AVR)-injected muscles of FK506-treated animals. Moreover, we find that FK506 efficiently suppresses the humoral immune response against both the vector proteins and the transgene protein product (dystrophin). Furthermore, we demonstrate that continuous FK506 treatment over 30 days significantly improves the efficiency of gene transfer when the same vector is readministered to an animal which had been transduced 20 days earlier. In conclusion, the data suggest that sensitization by the initial antigenic load of the AVR application plays a pivotal role in triggering the humoral and cellular immune response of the host, which can be significantly counteracted by relatively short-term immunosuppressive treatment. These findings have important implications for the design of future human trials for gene replacement therapy in Duchenne muscular dystrophy.
腺病毒（AV）介导的基因转移到成年免疫健全动物的骨骼肌中受到限制，原因是宿主对转导的肌纤维进行细胞介导的免疫攻击会阻碍有效的长期转基因表达。最近，各种免疫调节策略已被证明可改善AV介导的基因转移后转基因表达的持久性。在本研究中，我们在AV介导的肌营养不良蛋白基因转移后的5、10、30和60天，每天皮下注射免疫抑制药物FK506（他克莫司）治疗成年营养不良（mdx）小鼠，并将转导水平与注射生理盐水的mdx对照进行比较。我们发现，在AV介导的肌营养不良蛋白基因转移到成年mdx肌肉后，每天用FK506治疗可使初始转基因表达维持至少2个月，即使在1个月后停止FK506治疗也是如此。这与FK506治疗动物的腺病毒重组体（AVR）注射肌肉中炎性浸润的显著减少以及巨噬细胞激活水平（诱导型一氧化氮合酶）的降低相一致。此外，我们发现FK能有效抑制针对载体蛋白和转基因蛋白产物（肌营养不良蛋白）的体液免疫反应。此外，我们证明，当将相同载体重新给予20天前已转导的动物时，连续30天的FK506治疗可显著提高基因转移效率。总之，数据表明，AVR应用的初始抗原负荷致敏在触发宿主的体液和细胞免疫反应中起关键作用，相对短期的免疫抑制治疗可显著抵消这种反应。这些发现对未来杜兴氏肌营养不良症基因替代疗法的人体试验设计具有重要意义。
