STUDY OF A MURINE RETROVIRUS FROM A PACKAGING CELL LINE USED IN GENE THERAPY

可复制的小鼠反转录病毒(RCR)可在某些 用于基因治疗准备的细胞系包装案例 生产供人类使用的产品。由于与RCR相关的风险 MAN未知，建议对RCR进行严格测试以排除 产品可能受到污染。以前的研究中，正常的 中度免疫抑制的猴子接种RCR 表明病毒迅速清除且无病理改变(Cornetta等人，1991年)； 然而，最近的一项研究表明，RCR与发育有关 严重免疫抑制的恒河猴中的淋巴瘤(Donahue等人， (1992年)。为了评估人类再生障碍性贫血的潜在风险，我们承诺 以猴为灵长类动物分析RCR感染和淋巴肿大 模型系统。将4只正常幼年恒河猴接种于 从基因治疗公司获得的RCR制剂的血液样本 接种后每两周获取一次：对PBL进行病毒检测 分离；血浆进行免疫印迹分析以检测抗体；以及 用聚合酶链式反应检测病毒序列的PBL DNA。此外，临床血液学和 进行血清化学检测。结果表明，这四只猴子 血清转换。感染性RCR早期从PBL中恢复 PI点(4-6周PI)；然而，在晚期没有恢复病毒 感染(53周PI)。PBL DNA的聚合酶链式反应分析表明存在 在69周PI时的RCR序列。这些结果表明，RCR可以 在正常恒河猴体内感染和复制以及病毒序列 即使在没有活动性感染的情况下，宿主DNA也会持续存在。当前 研究旨在了解宿主和病毒因素 可能激活潜伏的RCR序列及其对宿主的影响 猴子。

Replication-competent murine retroviruses (RCRs) can be generated in some cases from packaging cell lines used in preparation of gene therapy products generated for human use. Since the risk associated with RCR in man is unknown, rigorous testing for RCRs is recommended to exclude possible contamination of the product. Previous studies in which normal and moderately immunosuppressed monkeys were inoculated with RCR indicated rapid virus clearance and no pathology (Cornetta et al, 1991); however, a recent study has shown RCR to be associated with development of lymphomas in severely immunosuppressed rhesus monkeys (Donahue et al, 1992). To assess the potential risk of RCRs in man, we undertook to analyze RCR infection and lymphomagenesis using monkeys as a primate model system. Four normal juvenile rhesus monkeys were inoculated with a RCR preparation obtained from Genetic Therapy Inc. Blood samples were obtained every two weeks post-inoculation: PBLs were tested for virus isolation; plasma for antibody detection by Western blot analysis; and PBL DNA for viral sequences by PCR. In addition clinical hematology and serum chemistry was carried out. Results indicated that all four monkeys seroconverted. Infectious RCR was recovered from PBLs at early time points PI (4 - 6 weeks PI); however, no virus was recovered late in the infection ( 53 weeks PI). PCR analysis of PBL DNAs indicated presence of RCR sequences at 69 weeks PI. These results indicated that RCR can infect and replicate in normal rhesus monkeys and that viral sequences persist in the host DNA even in absence of active infection. Current studies are directed towards understanding host and viral factors which may activate the latent RCR sequences and its consequences on the host monkey.