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

人类基因治疗产品中使用的基于逆转录病毒的包装细胞系统可能会意外和自发地产生具有复制能力的小鼠逆转录病毒(RCR)。RCRs可以感染包括人类在内的各种灵长类细胞。由于人体内与RCRs相关的风险尚不清楚，建议对RCRs进行严格的检测，以排除可能的产品污染。尽管以前的研究表明，当RCR接种到正常和中度免疫抑制的猴子中时，RCR被清除(Cornetta等人，1991)，但随后的研究报告RCR与严重免疫抑制的猕猴的淋巴瘤的发展有关(Donahue等人，1992)。为了评估RCR在人类中感染的可能性，将含有RCR的载体病毒制剂(由马里兰州盖瑟斯堡的遗传治疗公司提供)接种给4只正常的幼年恒河猴。每种动物对感染的动力学和抗体反应都有独特的反应。在最初建立高病毒感染的两只动物(AG6和A4W)中，在3年期间，白细胞和淋巴细胞数量逐渐减少；但初步骨髓检查未发现异常。在接种后第6年初，A4W因腹泻和体重显著下降而被安乐死。临床和组织学评估表明，逆转录病毒介导的免疫抑制。为了研究RCR在疾病中的作用，通过聚合酶链式反应和抗体测试证实了SRV的缺失。灵长类中心)。各种组织的原位杂交分析没有检测到RCR或猴泡沫病毒(SFV)，这也被发现(回溯)在接种时存在于动物体内。其他敏感的逆转录病毒检测方法将被用于分析组织。为了研究RCR序列在宿主中的长期持久性和潜在的生殖系整合，在感染后约5年半的时间里，从AG6中提取精子DNA，并使用来自LTR和env区的聚合酶链式反应(PCR)引物对RCR序列进行分析。初步结果表明，精子DNA中没有可检测到的RCR序列。这些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，并评估人类长期感染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). 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.