Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted"') adenovirus is markedly improved by trans-acting adenoviral gene products
Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted"') adenovirus is markedly improved by trans-acting adenoviral gene products
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DOI：
10.1089/104303401750476249
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发表时间：
2001-09-20
影响因子：
4.2
通讯作者：
Karpati, G
中科院分区：
文献类型：
--
作者：
Gilbert, R;Nalbantoglu, J;Karpati, G
辅助依赖性腺病毒（HDAd）是缺乏全部或大部分病毒基因的Ad载体。它们对于疾病如杜氏肌营养不良症（DMD）的基因治疗具有很大的希望，因为它们的免疫原性低于E1/E3缺失的Ad（第一代Ad或FGAd），并且可以携带全长（F1）肌营养不良蛋白（dys）cDNA（12 kb）。我们比较了HDAd（HDAdCMVDysFl）和FGAd（FGAdCMV-dys）在细胞培养物（HeLa，C2 C12肌管）和mdx小鼠（DMD的小鼠模型）的肌肉中的转基因表达。两种载体编码由相同的巨细胞病毒（CMV）启动子调控的肌营养不良蛋白。我们证明，在体外和体内，与HDAdCMVDysFl相比，用FGAdCMV-dys基因转移后表达的肌营养不良蛋白的量显著更高。然而，在FGAd存在下用HDAd-CMVDysFl的基因转移导致肌营养不良蛋白表达的显著增加，表明由FGAd合成的基因产物反式增加了所产生的肌营养不良蛋白的量。这种增强发生在细胞培养中，并在mdx小鼠和另一种DMD动物模型营养不良金毛猎犬（GRMD）的肌肉中进行基因转移后。Ad的E4区域是增强所需的，因为在体外和体内存在E1/E4缺失的Ad的情况下没有观察到来自HDAdCMVDysFl的肌营养不良蛋白表达的增加。可能需要表征这些增强基因产物，然后将其纳入HDAd中，以产生足够的肌营养不良蛋白，从而通过HDAd介导的基因转移减轻DMD的病理。
Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAd-CMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.