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

可复制的鼠逆转录病毒（RCR）可以在一些细胞中产生。 用于制备基因治疗的包装细胞系的病例 供人类使用的产品。 由于RCR相关风险 男性未知，建议对RCR进行严格检测，以排除 产品的可能污染。 以前的研究中， 中度免疫抑制猴接种RCR 表明病毒快速清除且无病理学（Cornetta et al，1991）; 然而，最近的一项研究表明，RCR与发育有关， 在严重免疫抑制的恒河猴中的淋巴瘤（Donahue等， 1992年）。为了评估RCRs在人体中的潜在风险，我们承诺 用猴子作为灵长类动物分析RCR感染和淋巴瘤发生 模型系统4只正常幼年恒河猴接种 从Genetic Therapy Inc. 血样 接种后每两周获得一次：检测PBL的病毒 分离;血浆用于通过蛋白质印迹分析进行抗体检测;和 PBL DNA用于PCR病毒序列。此外，临床血液学和 进行血清化学。 结果表明，所有四只猴子 血清转化早期从PBLs中回收感染性RCR PI点（PI 4 - 6周）;然而， 感染（PI 53周）。 PBL DNA的PCR分析表明存在 PI第69周时的RCR序列。 这些结果表明，RCR可以 在正常恒河猴中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也会持续存在于宿主DNA中。 电流 研究的目的是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.