Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products
Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products
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DOI：
10.1038/sj.gt.3302984
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发表时间：
2007-09-01
期刊：
影响因子：
5.1
通讯作者：
Takeda, S.
中科院分区：
文献类型：
--
作者：
Yuasa, K.;Yoshimura, M.;Takeda, S.
利用小鼠模型，我们已经证明了重组腺相关病毒(RAAV)介导的微肌营养不良蛋白基因转移是治疗Duchenne肌营养不良症(DMD)的一种有前途的方法。为了利用更大的动物模型(如营养不良犬模型)进一步检验rAAV介导的微肌萎缩蛋白基因转移的疗效和安全性问题，我们首先研究了rAAV在野生型犬肌肉细胞中的转导效率，发现编码β-半乳糖苷酶的rAAV2在体外有效地转导了犬的初级肌管。然而，随后将rAAV2转移到正常狗的骨骼肌中，导致b-半乳糖苷酶的低表达和瞬时表达，并在体内出现强烈的细胞浸润，细胞和体液免疫反应被显著激活。相反，表达无转基因的rAAV2未引起细胞浸润。联合应用免疫抑制剂、环孢素和霉酚酸酯可部分改善rAAV2转导。综上所述，这些结果表明，对转基因产物的免疫反应导致了狗的细胞渗透并消除了转导的肌纤维。此外，体外干扰素-g释放试验表明，犬脾细胞对免疫原或有丝分裂原的反应比小鼠更敏感。我们的结果强调了在将rAAV介导的基因治疗应用于DMD患者之前，在更大的动物模型中仔细检查AAV载体的免疫反应的重要性。
Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding beta-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of b-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immuno-suppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-g release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.