RECOMBINANT RETROVIRAL VECTOR INTERFERES WITH THE DETECTION OF AMPHOTROPIC REPLICATION COMPETENT RETROVIRUS IN STANDARD CULTURE ASSAYS

Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as g ene delivery vehicles. The use of retroviral vectors for human gene th erapy requires careful screening of vector-producing cell lines and cu lture supernatants to ensure the absence of replication competent retr ovirus (RCR) in clinical products. In this study we have examined seve ral different culture assays routinely used to test for the presence o f RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level o f contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retrovirual vector ca n interfere, to varying degrees, with the detection of low-level RCR i n culture supernatants when a marker rescue assay, an extended mink S( +)L(-) assay or a PG-4 S(+)L(-) assay is used. Interference can be par tially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other c ulture assays routinely used for the detection of RCR.