ENDOGENOUS MURINE LEUKEMIA-VIRUS DNA-SEQUENCES IN MURINE CELL-LINES -IMPLICATIONS FOR GENE-THERAPY SAFETY TESTING BY PCR

Safety testing for replication-competent retrovirus (RCR) is an import ant requirement in gene transfer clinical trials using retroviral vect ors. A sensitive polymerase chain reaction (PCR) method is one approac h to RCR detection. Only in the presence of RCR will the pol-env encod ing sequences, necessary for viral replication and packaging, be ampli fied from proviral DNA in infected indicator cells. To avoid false-pos itive results in this assay it is crucial that indicator cell lines ar e free of endogenous retroviral sequences that could also be amplified with pol-env PCR primers. We screened candidate murine indicator cell lines and determined that while Mus dunni is free of detectable pol-e nv sequences, endogenous retroviral sequences do indeed exist in sever al cell lines and lead to false-positive results in the PCR assay for RCR. Furthermore, these endogenous retroviral sequences are expressed as RNA transcripts in NIH 3T3 and SC-1 cell lines, as determined by PC R amplification of cDNA but, nevertheless, do not give rise to replica tion-competent particles. We recognize the potential for murine cell l ines to undergo spontaneous rearrangements of endogenous viral sequenc es in culture and give rise to recombinants containing newly acquired contiguous pol-env sequences. Indicator cell lines should thus be care fully selected and monitored on an ongoing basis when used in safety t esting using PCR approaches for the detection of RCR.