Retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with viral particles and are devoid of viral vector RNA

Obtaining high expression levels of a therapeutic gene in target cells coul d be achieved by integrating multiple copies of a recombinant retrovirus, H owever, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copi es, suggesting that maximum retroviral transduction was achieved at relativ ely low MOIs, The same results were obtained when purified virus, free of m ost medium components, was used, Retroviral infection was shown to be inhib ited by supernatants of other viral producer cell lines, and this inhibitio n could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparatio ns showed that the amount of retroviral vector RNA present was similar to t he amount expected on the basis of virus titers. Our data suggest that retr oviral preparations derived from PA317 packaging cells contain inhibitors t hat copurify with retroviruses and do not contain viral vector RNA. We post ulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more ef fective packaging cell lines.