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

用于基因治疗的来自包装细胞系的鼠逆转录病毒的研究

• 批准号: 6101181
• 负责人: Arifa S Khan
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6101181
• 关键词: Macaca mulatta; Retroviridae; Retroviridae disease; antibody titering; antiviral antibody; communicable disease transmission; disease /disorder proneness /risk; host organism interaction; juvenile animal; latent virus infection; longitudinal animal study; murine leukemia virus; polymerase chain reaction; simian virus; virus genetics; virus replication; western blottings

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.

可复制的小鼠逆转录病毒（RCR）可能会意外出现 并自发地从逆转录病毒为基础的包装细胞系统， 人类基因治疗产品。RCR可以感染多种灵长类动物 细胞，包括人类。由于与男性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（Cornetta et al，1991），随后的研究 据报道，RCR与严重患者淋巴瘤的发生相关。 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， 用载体病毒制剂接种恒河猴， 包含RCR（由Genetic Therapy Inc.，盖瑟斯堡，马里兰州）。的 通过以下方式定期监测猴子的病毒感染：1）隔离 来自PBMC的感染性MuLV; 反应;和3）DNA PCR用于病毒序列在宿主中的持久性。 通过血清化学、血液学和 体检3年的分析结果表明，所有 4只动物感染RCR。有早期的，短暂的病毒 分离; PBMC中病毒DNA序列的持久性和长期 抗体反应。每只动物对 感染和抗体应答的动力学。在两只动物（AG 6）中， 和A4 W），在最初建立高病毒感染的地方， 3年内观察到WBC和淋巴细胞数量减少 阶段;然而，在初步骨髓中未观察到异常 考试 在接种后第6年开始时，A4 W因 腹泻和体重显著下降。临床和组织学 评价表明逆转录病毒介导的免疫抑制。到 为了研究RCR在疾病中的作用， 经N.博士的PCR和抗体检测确认。Lerche（Calif.灵长 中）。各种组织的原位杂交分析目前是 正在进行的RCR和猴泡沫病毒（SFV）检测 也被发现（回顾性）存在于动物中， 接种时间。结果将是重要的评估 人类长期RCR感染的潜在风险。