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

复制能力强的小鼠逆转录病毒（RCRs）可以意外地自发地从用于人类基因治疗产品的基于逆转录病毒的包装细胞系统中产生。RCRs可以感染包括人类在内的多种灵长类细胞。由于与人体rcr相关的风险尚不清楚，建议对rcr进行严格检测，以排除产品可能的污染。虽然先前的研究表明，将RCR接种到正常和中度免疫抑制的猴子身上时，RCR被清除（Cornetta et al ., 1991），但随后的一项研究报告称，RCR与严重免疫抑制的恒河猴淋巴瘤的发生有关（Donahue et al ., 1992）。为了评估人类感染RCR的可能性，将4只正常的幼年恒河猴接种了含有RCR的载体病毒制剂（由马里兰州盖瑟斯堡的遗传疗法公司提供）。每种动物在感染动力学和抗体反应方面都有独特的反应。在最初建立高病毒感染的两只动物（AG6和A4W）中，在3年期间发现白细胞和淋巴细胞数量逐渐减少；初步骨髓检查未见异常。在接种后的第6年开始，A4W因腹泻和体重明显减轻而被安乐死。临床和组织学评价提示逆转录病毒介导的免疫抑制。为了研究RCR在疾病中的作用，加州灵长类动物中心的N. Lerche博士通过PCR和抗体检测证实了SRV的缺失。各种组织的原位杂交分析未检测到RCR或类人猿泡沫病毒（SFV），在接种时也发现（回顾性）存在于动物体内。其他灵敏的逆转录病毒检测方法将用于分析组织。为了研究RCR序列在宿主体内的长期持久性和潜在的种系整合，研究人员在感染后约5年半提取了AG6的精子DNA，并使用来自LTR和env区域的PCR引物分析了RCR序列。初步结果表明精子DNA中没有可检测的RCR序列。该结果有助于评估基于逆转录病毒的基因治疗产品的安全性和评估人类长期RCR感染的潜在风险。

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). 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 did not detect RCR or simian foamy virus (SFV), which was also found (retrospectively) to be present in the animal at the time of inoculation. Other sensitive methods for retrovirus detection will be used to analyze the tissues. To investigate long-term persistence of RCR sequences and potential germ-line integration in the host, sperm DNA from AG6 was obtained about 5 1/2 years post-infection and analyzed for RCR sequences using PCR primers from the LTR and env regions. Preliminary results indicate the absence of detectable RCR sequences in sperm DNA. The results contribute to safety assessment of retrovirus-based gene therapy products and evaluation of potential risk of long-term RCR infection in man.