Murine retrovirus from a cell line used in gene therapy

总结： 可复制的鼠逆转录病毒（RCR）可意外地自发地从人基因治疗产品中使用的基于逆转录病毒的包装细胞系统中产生。RCR可以感染包括人类在内的多种灵长类动物细胞。由于人体中与RCR相关的风险未知，因此建议对RCR进行严格检测，以排除可能的产品污染。 尽管先前的研究表明，当接种到正常和中度免疫抑制猴中时，RCR被清除（Cornetta et al，1991），但随后的研究报告RCR与严重免疫抑制恒河猴中淋巴瘤的发生相关（Donahue et al，1992）。 为了评估RCR感染人类的可能性，用含有RCR的载体病毒制剂（由Genetic Therapy Inc.，盖瑟斯堡，马里兰州）。每只动物对感染和抗体反应动力学的反应独特。在最初建立高病毒感染的两只动物（AG 6和A4 W）中，在3年期间观察到WBC和淋巴细胞数量逐渐减少;然而，初步骨髓检查未观察到异常。在接种后第6年开始时，A4 W由于腹泻和显著体重减轻而被人道处死。临床和组织学评价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，N。Lerche（Calif.灵长类中心）。各种组织的原位杂交分析未检测到RCR或猴泡沫病毒（SFV），也发现（回顾性）在接种时存在于动物中。 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年半从AG 6获得精子DNA，并使用来自LTR和env区域的PCR引物分析RCR序列。结果表明，在精子DNA中不存在可检测的RCR序列。然而，对外周血单核细胞（PBMC）DNA的分析表明，在接种后6年，AG 6和A4 W中的LTR和env序列持续存在。这些结果表明RCR序列在灵长类动物模型中的长期持久性，并支持对基于逆转录病毒的基因治疗产品进行持续严格的监测。 正在进行体外研究以评估灵长类细胞中潜伏的逆转录病毒诱导。这些研究的结果将被应用于调查在持续感染的恒河猴RCR激活。

Summary: 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. 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. The results indicate the absence of detectable RCR sequences in sperm DNA. Analysis of DNA from the peripheral blood mononuclear cells (PBMC), however, indicated the persistence of LTR and env sequences in AG6 and A4W at 6 years post-inoculation . The results indicate long-term persistence of RCR sequences in a primate animal model and supports the continued rigorous monitoring of retrovirus-based gene therapy products. In vitro studies are underway to evaluate latent retrovirus induction in primate cells. The results of these studies will be applied to investigate RCR activation in persistently infected rhesus macaques.