Christ M, Louis B, Stoeckel F, Dieterle A, Grave L, Dreyer D, Kintz J, Ali Hadji D, Lusky M, Mehtali M
Transgène SA, Strasbourg, France.
Hum Gene Ther. 2000 Feb 10;11(3):415-27. doi: 10.1089/10430340050015888.
Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.
在C57BL/6、CBA和BALB/c小鼠中评估肝脏毒性和炎症，这些小鼠通过静脉注射一系列在E1/E3、E1/E3/E2A或E1/E3/E4中同时缺失的重组腺病毒。所有载体要么不含转基因，要么在E1中携带在巨细胞病毒（CMV）启动子控制下的人囊性纤维化跨膜传导调节因子（CFTR）cDNA。注射缺失E1/E3的载体诱导了显著的肝营养不良和炎症反应，同时伴有血清转氨酶浓度升高。在进一步缺失E2A基因时，载体毒性仍然升高，而当表达hCFTR时毒性进一步增强。相比之下，E4的额外缺失导致肝毒性降低，这表明E4基因产物在肝损伤中起积极作用。然而，E4的缺失也导致转基因表达丧失。为了确定参与肝毒性和转基因表达调控的单个E4产物，在体外和体内评估了一系列等基因的缺失E1/E3的载体，这些载体有或没有hCFTR转基因，并包含功能性E4开放阅读框（ORF）的各种组合。我们证明，单独含有ORF3或含有ORF3、4的载体可使肝损伤显著减轻，而含有ORF4、ORF6、7或ORF3、6、7的载体仍表现出升高的肝毒性和炎症反应。此外，当载体中保留ORF3、4或ORF3、6、7时，转基因表达得以恢复。这些结果突出了E4基因产物在设计改进的体内基因转移载体中的重要性。
