Absence of Replication-Competent Lentivirus in the Clinic: Analysis of Infused T Cell Products

Abstract

Exposure to replication-competent lentivirus (RCL) is a theoretical safety concern for individuals treated with lentiviral gene therapy. For certain ex vivo gene therapy applications, including cancer immunotherapy trials, RCL detection assays are used to screen the vector product as well as the vector-transduced cells. In this study, we reviewed T cell products screened for RCL using methodology developed in the National Gene Vector Biorepository. All trials utilized third-generation lentiviral vectors produced by transient transfection. Samples from 26 clinical trials totaling 460 transduced cell products from 375 subjects were evaluated. All cell products were negative for RCL. A total of 296 of the clinical trial participants were screened for RCL at least 1 month after infusion of the cell product. No research subject has shown evidence of RCL infection. These findings provide further evidence attesting to the safety of third-generation lentiviral vectors and that testing T cell products for RCL does not provide added value to screening the lentiviral vector product.

Keywords: clinical gene therapy; immunotherapy; lentivirus; replication-competent virus; safety.

Figure 1

Schematic Representation of the RCL Assay T cell products are incubated with C8166-45 cells at a ratio of 1:5. Over half of test articles submitted contained approximately 1 × 10⁸ T cells and were divided into twenty-five 300-cm² flasks. Cultures were passed a minimum of 5 passages using increasingly smaller vessels. After 3 weeks, cells were placed in fresh media, and conditioned media were harvested after 24 hr. Media from all amplification cultures are pooled, and two aliquots are then incubated with naive C8166-45 cells. After 7 days, culture media were analyzed for p24 antigen by ELISA and either the psi-gag PCR assay or PERT. The figure depicts amplification of a low-titer RCL present in the test article.