CHARACTERIZATION OF A REPLICATION-COMPETENT RETROVIRUS RESULTING FROMRECOMBINATION OF PACKAGING AND VECTOR SEQUENCES

A replication-competent retrovirus (RCR) was detected by S+/L(-) assay in three lots of retroviral vector G1Na that were harvested on consec utive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RC R was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contam ination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of re troviral envelope sequences unique to pPAM3 joined to a 3' long termin al repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment o f patchy nucleotide identity (8 out of 10 bp), suggesting that these h elper and vector sequences were joined by homologous recombination. Ge neration of RCR by exchanges between helper and vector sequences under scores the necessity of testing by efficient methods all retroviral ve ctors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, inc luding 42 lots of G1Na, however, indicates that the combination of exc hanges required to generate an RCR are infrequent in this system.