Sastry, LakshmiXu, YiJohnson, TerryDesai, KunalRissing, DavidMarsh, JonathanCornetta, Kenneth2022-12-082022-12-082003-11-01Sastry, L., Xu, Y., Johnson, T., Desai, K., Rissing, D., Marsh, J., & Cornetta, K. (2003). Certification assays for HIV-1-based vectors: Frequent passage of gag sequences without evidence of replication-competent viruses. Molecular Therapy, 8(5), 830–839. https://doi.org/10.1016/j.ymthe.2003.08.0031525-0016https://hdl.handle.net/1805/30689A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection ∼10–100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity ∼5–50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi–gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi–gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.enAttribution-NonCommercial-NoDerivatives 4.0 Internationallentivirusp24 ELISAPCRArticle