An investigation on the relationship between DNA double strand breaks in the mammalian genome and gene therapy retrovirus vector genotoxicity

Abstract

Gene therapy uses viral vectors to transfer therapeutic genes to host cells for permanent expression. Retroviral vector (RV) integrase causes double-strand-breaks (DSBs) in the host genome for viral cDNA integration and utilises host repair of these breaks via nonhomologous- end-joining (NHEJ). This thesis hypothesises that cells lacking NHEJ are susceptible to retrovirus-mediated genotoxicity. To determine if presence of the retrovirus genome is required for DSBs, cells were infected with replication defective RV either containing vector genomes or without genomes (“empty”). DSB frequency was measured using immunocytochemistry of phosphorylated histone H2AX, formed when DSBs occur and eliminated post-NHEJ repair. DSBs were measured in infected cell-lines with NHEJ function (MRC5-SV1) and without (AT5-BIVA and XP14BRneo17) to determine DSB and repair profiles during infection. This showed cells competent for NHEJ have rapid DSB repair whilst cells lacking NHEJ pathways have DSBs remaining high. Insertional mutagenesis was measured using the hypoxanthine-phosphoribosyltransferase (HPRT) mutagenesis assay for RV carrying genomes versus empty RV. This analysis relies on HPRT knock-out using male V79 and MRC5-SV1 cell lines, carrying one X chromosome, as the HPRT gene resides on this chromosome. In each cell-line, empty vectors showed little genotoxic readout compared to the genome carrying particles. However, expression of the viral integrase alone in these cells exhibited the highest mutagenesis. The potential for genotoxicity by RV due to packaging human-endogenous-retroviruses (HERVs) was evaluated in empty vectors by PCR for HERV-K113 (known to be full length and packaged by retroviruses). Primers designed to amplify a 7.5 kb portion of HERVK113 gave positive bands, corroborated by restriction digests, and presence in 293T human cells, but not in hamster V79 or mouse 3T3 cells. Further work to determine whether HERV sequence are transferred to infected cells sensitively was not completed in this thesis but discussed as future investigation.