Characterization of a Replication-Competent Retrovirus Resulting from Recombination of Packaging and Vector Sequences

Abstract

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

Feasibility of multidrug resistance (MDR-1) gene transfer in patients undergoing high-dose therapy and peripheral blood stem cell transplantation for lymphoma

“…29 The master cell bank and retroviral supernatants were shown to be free of contaminating replication competent retrovirus by Mus dunni and S+L− assay. 30,31 Following CD34 selection, the enriched product was added to a 2 l intravenous feeding bag containing 1 l of retroviral supernatant and protamine sulphate 4 g/ml. The cells and virus were incubated for 6 h at 37°C then transferred to a COBE 2991 cell washer (COBE BCT, Lakewood, CO, USA) and washed with 3 l of tissue culture grade phosphate-buffered saline containing 1% human serum albumin.…”

Enhancer-deficient amphotropic murine leukemia virus and recombinants with heterologous transcription elements can be efficiently amplified and detected in Mus dunni fibroblasts

“…6 Recombinant, replication-competent amphotropic murine leukemia virus is a potential contaminant and safety risk in these vector preparations. 7,8 Replication-competent virus can potentially arise at all stages of vector production by recombination between vector and packaging constructs or host DNA, such as endogenous retroviruses. The probability of such a recombination and the exact genotype of the recombinant virus depends on the design of the production process and the nature of packaging-and vector constructs.…”

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells

“…Human gene transfer studies frequently use MLV-based vectors with amphotropic host range to introduce and express marker or therapeutic genes (5). Recombinant, replication-competent amphotropic MLV is a potential contaminant and safety risk in these vector preparations (6,7). However, only very limited information is available about the replication and gene expression of amphotropic MLV in human cells (8).…”