Improved detection of replication-competent retrovirus

Recombinant retroviral vectors are the predominant delivery system in human gene therapy protocols. Since contaminating replication-competent retrovirus (RCR) can arise during the production of retroviral rector supernatants, sensitive assays for the screening of supernatants are necessary. In this study, we present a marker rescue assay based upon a Mns dunni cell line stably transduced with a lacZ gene. We show that detection of RCR in vector supernatants by the M. dunni lacZ marker rescue assay or PG-4 S + L - focus-forming assay is equally sensitive. By inoculating test supernatants under centrifugation (which we term spinoculation), we increased the sensitivity of detection of RCR 10 to 100-fold with the PG-4 S + L - and lacZ marker rescue assays. While the spinoculation protocol had no adverse effects on cells, spinoculation of high titer vector supernatants onto PG-4 cells resulted in some cytotoxicity, making identification of RCR positive cultures difficult. However, spinoculation of vector supernatants onto M. dunni lacZ cells resulted in no cytotoxicity, and also partially overcame inhibition of detection of low levels of RCR due to the presence of high titer replication-incompetent vector.