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.
