O'Neal W K, Zhou H, Morral N, Aguilar-Cordova E, Pestaner J, Langston C, Mull B, Wang Y, Beaudet A L, Lee B
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Hum Gene Ther. 1998 Jul 20;9(11):1587-98. doi: 10.1089/hum.1998.9.11-1587.
Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.
与仅缺失E1的第一代载体相比，在E2a区发生突变的第二代腺病毒载体被认为可降低宿主对转导细胞的免疫反应、降低毒性并延长表达持续时间。为了进一步验证这些假设，我们构建了一种表达人α1-抗胰蛋白酶（hAAT）的缺失E2a的腺病毒载体。通过血液学指标、肝功能测试和组织学分析确定第一代和缺失E2a载体的毒性，在C3H小鼠中以1×10(12)颗粒/千克开始递增剂量给予载体后评估21天。两种载体均诱导剂量依赖性异常，包括短暂性血小板减少、血清ALT水平升高、肝细胞增殖增加，随后出现炎症，然后是肥大。载体制剂中颗粒与噬斑形成单位的比例差异导致在相似颗粒剂量下hAAT表达存在差异。当在匹配的hAAT表达水平下测量时，两种载体的毒性没有差异。然而，在中等颗粒剂量下，缺失E2a的载体显示出肝细胞毒性略有降低。这表明肝细胞毒性主要与病毒进入和表达有关，而不是与非感染性颗粒的存在有关，这意味着完全消除病毒基因表达的载体，如删除所有病毒编码序列的载体，在安全性和毒性方面可能具有实质性优势。
