Andrews J L, Kadan M J, Gorziglia M I, Kaleko M, Connelly S
Genetic Therapy, Inc. (A Novartis Company), 9 West Watkins Mill Road, Gaithersburg, Maryland 20878, USA.
Mol Ther. 2001 Mar;3(3):329-36. doi: 10.1006/mthe.2001.0264.
The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced.
由于宿主针对载体和/或转基因的免疫反应以及载体毒性，腺病毒载体在基因治疗中的应用受到限制。为了降低腺病毒载体的免疫原性和毒性，我们通过从腺病毒骨架中去除E1、E2a、E3和E4早期基因来减弱病毒基因表达。构建了两种高度减毒的第四代（Av4）E1/E2a/E3/E4缺陷型腺病毒载体，它们在肝脏特异性白蛋白启动子的控制下编码人凝血因子VIII（FVIII）。一种Av4载体（Av4DeltaE4FVIII）在整个E4编码区存在缺陷，第二种载体除开放阅读框3（orf 3；Av4orf3FVIII）外，E4区域存在缺失。将Av4载体与一种E1/E2a/E3缺陷型第三代载体（Av3H8101）进行比较，后者含有类似的转基因表达盒，在血友病小鼠静脉注射后进行体内外研究。对Hep3B细胞的体外转导显示，所有三种载体均表达功能性FVIII。然而，Av4DeltaE4FVIII载体无法扩大规模用于体内研究。Av3H8101和Av4orf3FVIII在血友病小鼠中最初表达的FVIII水平相似。然而，在3个月时，用Av4orf3FVIII载体处理的动物不再表达FVIII，而用Av3H8101处理的小鼠则持续表达FVIII。血浆样本的肝酶分析表明，Av4orf3FVIII载体的肝毒性明显低于Av3H8101载体。这些数据表明，通过去除大部分E4编码区进一步减弱腺病毒载体骨架可显著降低载体毒性；然而，转基因表达的持续时间缩短了。
