Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses
Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses
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DOI：
10.1128/jvi.71.6.4626-4637.1997
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发表时间：
1997-06-01
影响因子：
5.4
通讯作者：
Yeh, P
中科院分区：
文献类型：
--
作者：
Dedieu, JF;Vigne, E;Yeh, P
我们比较了一组表达大肠杆菌劳斯肉瘤病毒长末端重复序列lacZ基因的同基因E1和E1/E4缺陷型腺病毒的体外和体内行为。用双重缺陷腺病毒感染人源的肿瘤来源的已建立细胞系导致（i）与E2 A特异性RNA和蛋白质水平降低相关的病毒骨架的较低复制，（ii）晚期基因和蛋白质表达的显著关闭，和（iii）没有明显的病毒诱导的细胞毒性。因此，与缺失程度无关，病毒骨架中E4的额外失活在体外显著地使病毒失活，对转基因表达没有明显影响。使用lacZ转基因模型来比较C57 BL/6小鼠肝脏中的不同重组腺病毒。在该模型中，对病毒编码的β-半乳糖苷酶的免疫应答最小，因为输注E1缺陷型腺病毒导致转基因表达的时间过程与免疫缺陷（nu/nu）小鼠相似，肝脏中几乎没有炎症和坏死。给转基因动物施用双重缺陷的腺病毒导致病毒DNA在肝脏中的长期染色体外持续存在，没有检测到CPG二核苷酸的甲基化。然而，观察到瞬时转基因表达与E4缺失的程度无关，表明启动子的选择对于维持来自这些减毒腺病毒载体的转基因表达至关重要。
We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CPG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter mag be critical to maintain transgene expression from these attenuated adenovirus vectors.