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

在某些情况下， 出乎意料地和自发地来自基于逆转录病毒包装细胞 用于人类基因治疗产品的系统。 RCR继续 嗜酸性鼠白血病病毒（MuLV）具有广泛的宿主 范围广泛，可感染包括人类在内的多种灵长类动物细胞。 由于与人类RCR相关的风险尚不清楚， 对于RCR，建议排除可能的产品污染。 尽管先前的研究表明，当RCR被清除时， 接种到正常和中度免疫抑制的猴（Cornetta 等，1991年），最近的一项研究报告RCR与 严重免疫抑制恒河猴淋巴瘤的发生 （Donahue等人，1992）。 评价RCR感染的可能性， 人、4只正常的幼年恒河猴用载体接种 含有RCR的病毒制剂（由GeneticTherapyInc.， 盖瑟斯堡，马里兰州）。 定期监测猴子的病毒 通过1）从PBMC中分离感染性MuLV; 2）Western 抗体应答的印迹分析;和3）DNA PCR，用于持续性的抗体应答。 宿主体内的病毒序列 通过血清学监测临床变化 化学、血液学和体格检查。 3年的结果 分析表明所有4只动物均感染了RCR。 有 早期、短暂的病毒分离; PBMC和长期抗体应答。 每只动物都有反应 在感染和抗体反应的动力学方面， （手稿正在编写中）。 目前的工作是分析 感染者中长期持续存在的RCR序列的状态 动物我们正在研究潜在序列的激活， 使用各种已知的逆转录病毒诱导剂的感染性病毒。 这些结果 在评估RCR感染的潜在风险方面将是重要的， 伙计

Replication-competent murine retroviruses (RCRs) can arise, in some case, unexpectedly and spontaneiously from retrovirus-based packaging cell systems used in human gene therapy products. RCRs continuing an amphotropic murine leukemia virus (MuLV) envelope possess a broad host range and can infect a wide variety of primate cells including human. Since the risk associated with RCRs in man is unknown, rigorous testing for RCRs is recommended to exclude possible contamination of 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 evaluate the potential of RCR infection in humans, 4 normal, juvenile rhesus monkeys were inocualted with a vector virus preparation which contained RCR (provided by Genetic Therapy Inc., Gaithersburg, MD). The monkeys were monitored regularly for virus infection by 1) isolation of infectious MuLV from the PBMCs; 2) Western blot analysis for antibody response; and 3) DNA PCR for persistence of viral sequences in the host. Clinical changes were monitored by serum chemistry, hematology and physical examination. The results of a 3-year analysis idicate that all 4 animals were infected with RCR. There was early, transient virus isolation; persistence of viral DNA sequences in the PBMCs and long-term antibody response. Each animal responded uniquely with respect to the kinetics of infection and antibody response (manuscript in prep.). Current efforts are directed towards analyzing the state of the long-term persisting RCR sequences in the infected animals. We are studying the activation of the latent sequences for infectious virus using various known retrovirus inducers. These results will be important in evaluating the potential risk of RCR infection in man.