331. Human T Cells Efficiently Abort RCR-Infection through a CEM15/APOBEC3G-Mediated Mechanism

Abstract

Top of pageAbstract Replication competent retrovirus (RCR) may arise through homologous recombination in virus producer cells. A seminal study by Donahue et al. showed that infection with RCR may lead to development of cancer in primates. Therefore, rigorous testing of clinical grade preparations of retroviral vectors is required to exclude the presence of RCR. Many regulatory agencies require RCR-testing of ex vivo transduced cells as well. The latter requirement originates from the presumed amplification of RCR during culture, when cells are infected by very low levels of RCR. In a previous study we challenged the requirement of RCR-testing of ex vivo transduced T cells (Ebeling et al., Gene Ther. 10: 1800, 2003). We found that T cells have a very low capacity to produce RCR. In this study we present the mechanism, which allow T cells to efficiently abort RCR-infection. Individual clones were established from the bulk cultures of RCR-infected T cells. None of the 19 clones tested produced RCR, but all clones had been infected initially, as evidenced by an LTR-specific PCR. Intriguingly, only two clones contained the env gene, suggesting that part of the proviral genome had been deleted. To establish the extent of the deletions, four PCR assays were designed, which together cover the entire proviral genome. Twelve clones were negative in all four PCR assays, suggesting that a very large part of the proviral genome had been deleted. One clone, #27, was positive in the PCR assay, which covers almost the entire env gene. A second clone, #36, was positive in two PCR assays, which together cover half of the pol and the entire env gene. These deletions were consistent with the activity of the recently discovered CEM15/APOBEC3G gene. The CEM15 gene product has been shown to be co-packaged in retro- and lentiviral particles produced by T cells. After infection of target cells with such CEM15+ virions, CEM15 converts C into U residues in the negative cDNA strand during viral replication. As a consequence, this negative strand is degraded. Alternatively, the mutated negative strand may remain (partly) intact and serve as a template for synthesis of the positive strand, resulting in G to A mutations in this strand (e.g. Harris et al., Cell 2003, and Mangeat et al., Nature 2003). Therefore, the provirus in clones #27 and #36 and the LTR of four other clones were sequenced. We found that the 3' end of the PCR product from clone #27 contained numerous G to A substitutions. We therefore conclude that RCR-infection in human T cells is aborted by the CEM15 protein. Additional analysis of the proviral integration sites through molecular cloning only revealed the 3' LTR. The 5' LTR was never found. These data add to our conclusion that the major part of the proviral genome has been deleted from these clones.Together, the results broaden our understanding of the precise mechanism of action of CEM15. In conclusion, we believe that these results will be valuable in evaluation of the safety profile of retro- and lentiviral vectors. In addition, these experiments provide sufficient reason to reconsider the necessity of RCR-testing of ex vivo transduced T cells.