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

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Replication-competent murine retroviruses (RCRs) can be generated in some cases from packaging cell lines used in preparation of gene therapy products intended for human use. Since the risk associated with RCR in man is unknown, rigorous testing for RCRs is recommended to exclude possible contamination of the product. Although previous studies have shown that RCRs were cleared when inoculated into normal and moderately immunosuppressed monkeys (Cornetta et al, 1991), a recent study reported RCR to be associated with development of lymphomas in severely immunosuppressed rhesus monkeys (Donahue et al, 1992). To assess the potential risk of RCRs in man, we undertook to analyze RCR infection in rhesus monkeys. Four normal juvenile animals were inoculated with a RCR preparation obtained from Genetic Therapy Inc. The monkeys were monitored for virus infection, replication, persistence and clinical changes. Although all 4 animals became infected and infectious virus was isolated the animals have remained healthy over a year. Each monkey responded differently to the inoculum with regards to kinetics of virus isolation and serological response. Generally, infectious virus was isolated early post-inoculation (PI) but only transiently. Antibodies to MuLV were detected in all 4 animals but the response varied, ranging from 2 weeks to 18 weeks PI. We have further analyzed the persistence of retroviral sequences in the infected animals. DNAs from PBMCs were prepared from different time points PI and analyzed by PCR using MuLV env primers. Viral sequences were detected reproducibly in 2 of the 4 animals at 53 weeks PI; a weak signal was detected in a third animal. The data indicates persistence of retroviral sequence in the host genome in the absence of viral replication. Further studies are directed to analyze the activation of the latent RCR sequences in the infected monkeys. The variation seen in the response to the same inoculum in different animals indicate that other host and viral factors may contribute to RCR infection in primates. During the RCR virus isolation experiments, we discovered simian foamy virus (SFV) in 3 of the 4 monkeys in our study. Since foamy virus is a retrovirus that also is found in some humans (HFV), its interactions with RCR should also be understood to evaluate the potential risk of RCR in man. Thus we have undertaken to analyze the interaction between SFV and RCR in the monkeys. The biology of SFV is poorly understood and detection assays not rigorously developed. We have initially established SFV detection assays in our laboratory. Furthermore, we have found a highly sensitive cell line for its detection. Future studies will be directed to analyze the role of the FV in RCR infection and replication in primate cells.