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

可复制的小鼠逆转录病毒(RCR)可能会意外出现 并自发地从基于逆转录病毒的包装细胞系统中 人类基因治疗产品。RCR可以感染多种灵长类动物 包括人类在内的细胞。因为与男性RCR相关的风险是 建议对RCR进行未知的严格测试，以排除可能的 产品的污染。尽管之前的研究表明 RCR在正常和中度接种时被清除 免疫抑制猴子(Cornetta等人，1991)，随后的一项研究 据报道，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RCR在人类中感染的可能性，4名正常的青少年 给恒河猴接种一种载体病毒制剂，该病毒制剂 包含RCR(由马里兰州盖瑟斯堡的基因治疗公司提供)。这个 定期对猴子进行病毒感染监测，方法是：1)分离 外周血单核细胞来源的传染性小鼠白血病病毒；2)抗体的免疫印迹分析 响应；以及3)DNA聚合酶链式反应，用于病毒序列在宿主中的持久性。 临床变化通过血清化学、血液学和 体检。一项为期3年的分析结果表明， 4只动物感染RCR。有一种早期的、短暂的病毒 分离；病毒DNA序列在PBMC中的持久性和长期 抗体反应。每一种动物都对 感染和抗体反应的动力学。在其中两种动物中(AG6 和A4W)，在那里最初建立了高病毒感染，逐渐 白细胞和淋巴细胞的数量在三年中都有所下降。 期间；然而，初步骨髓没有发现异常 考试。 在接种后的第6年开始，A4W被安乐死 腹泻和显著的体重减轻。临床和组织学 评估表明，逆转录病毒介导的免疫抑制。至 研究RCR在疾病中的作用，SRV的缺失 由N.Lerche博士(加利福尼亚州)通过聚合酶链式反应和抗体检测确认灵长类 居中)。各种组织的原位杂交分析目前正在进行 正在进行RCR和猿猴泡沫病毒(SFV)的检测 它也被发现(回溯)存在于动物体内 接种时间。这些结果将对评估 男性长期感染RCR的潜在风险。

Replication-competent murine retroviruses (RCRs) can arise unexpectedly and spontaneously from retrovirus-based packaging cell systems used in human gene therapy products. RCRs 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 subsequent 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 inoculated 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 indicated 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. In two of the animals (AG6 and A4W), where high virus infection was initially established, a gradual decrease in the number of WBC and lymphocytes was seen over the 3 year period; however, no abnormalities were noted on preliminary bone marrow examination. In the beginning of the 6th year post-inoculation A4W was euthanized due to diarrhea and significant weight loss. Clinical and histological evaluation indicated retroviral-mediated immune suppression. To investigate the role of RCR in the disease, the absence of SRV was confirmed by PCR and antibody testing by Dr. N. Lerche (Calif. Primate Center). In situ hybridization analysis of various tissues are currently underway for the detection of RCR as well as for simian foamy virus (SFV) which was also found (retrospectively) to be present in the animal at the time of inoculation. The results will be important in evaluating the potential risk of long-term RCR infection in man.