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 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。尽管之前的研究已经 结果表明，在正常和中度接种RCR时，RCRs被清除 免疫抑制的猴子(Cornetta等人，1991)，最近的一项研究报告 RCR与重症淋巴瘤的发生有关 免疫抑制的恒河猴(Donahue等人，1992)。 为了评估人类RCR的潜在风险，我们承诺分析RCR 恒河猴的感染。给四只正常的幼年动物接种 使用来自Genetic Treatment Inc.的RCR制剂 监测病毒感染、复制、持久性和临床 改变。尽管4只动物都被感染了，传染性病毒也是 与世隔绝，这些动物一年多来一直保持健康。每只猴子 在病毒动力学方面，对接种剂的反应不同 分离和血清学反应。一般来说，传染性病毒是 接种后早期分离(PI)，但只是短暂的。抗体 在所有4种动物中都检测到对MuLV的反应，但反应各不相同，范围 术后2周至18周。我们进一步分析了这种持续性 受感染动物体内的逆转录病毒序列。从PBMC中提取的DNA是 不同时间点PI的制备及多病毒环境下的聚合酶链式反应分析 底漆。在4例中有2例可重复检测到病毒序列 53周龄的动物；在第三只动物身上检测到微弱的信号。 这些数据表明逆转录病毒序列在宿主基因组中持续存在 在没有病毒复制的情况下。进一步研究的方向是 分析感染者体内潜伏的RCR序列的激活 猴子。 不同品种对相同接种量反应的差异 动物表明，其他宿主和病毒因素可能与RCR有关 灵长类动物的感染。在RCR病毒分离实验中，我们 在我们研究的4只猴子中有3只发现了猴泡沫病毒(SFV)。 由于泡沫病毒是一种逆转录病毒，也在一些人类中发现 (HFV)，它与RCR的相互作用也应该被理解为评估 人类RCR的潜在风险。因此，我们已承诺分析 猴的SFV和RCR之间的相互作用。SFV的生物学特性是 人们对此知之甚少，检测分析也没有得到严格的发展。我们有 本实验室初步建立了SFV检测方法。 此外，我们还发现了一种对其高度敏感的细胞系 侦测。未来的研究将致力于分析FV的作用 在灵长类细胞中的RCR感染和复制。

Replication-competent murine retroviruses (RCRs) can be generated in some cases from packaging cell lines used in preparation of gene therapy products intended 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. Although previous studies have shown that RCRs were cleared when inoculated into normal and moderately immunosuppressed monkeys (Cornetta et al, 1991), a recent study reported 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 in rhesus monkeys. Four normal juvenile animals were inoculated with a RCR preparation obtained from Genetic Therapy Inc. The monkeys were monitored for virus infection, replication, persistence and clinical changes. Although all 4 animals became infected and infectious virus was isolated the animals have remained healthy over a year. Each monkey responded differently to the inoculum with regards to kinetics of virus isolation and serological response. Generally, infectious virus was isolated early post-inoculation (PI) but only transiently. Antibodies to MuLV were detected in all 4 animals but the response varied, ranging from 2 weeks to 18 weeks PI. We have further analyzed the persistence of retroviral sequences in the infected animals. DNAs from PBMCs were prepared from different time points PI and analyzed by PCR using MuLV env primers. Viral sequences were detected reproducibly in 2 of the 4 animals at 53 weeks PI; a weak signal was detected in a third animal. The data indicates persistence of retroviral sequence in the host genome in the absence of viral replication. Further studies are directed to analyze the activation of the latent RCR sequences in the infected monkeys. The variation seen in the response to the same inoculum in different animals indicate that other host and viral factors may contribute to RCR infection in primates. During the RCR virus isolation experiments, we discovered simian foamy virus (SFV) in 3 of the 4 monkeys in our study. Since foamy virus is a retrovirus that also is found in some humans (HFV), its interactions with RCR should also be understood to evaluate the potential risk of RCR in man. Thus we have undertaken to analyze the interaction between SFV and RCR in the monkeys. The biology of SFV is poorly understood and detection assays not rigorously developed. We have initially established SFV detection assays in our laboratory. Furthermore, we have found a highly sensitive cell line for its detection. Future studies will be directed to analyze the role of the FV in RCR infection and replication in primate cells.