An Investigation on the Association Between HIV-1 Integrase and Gene Knock-out

Lentivirus-mediated gene therapy has shown promise to provide therapeutic genes for permanent treatment of several diseases. This process relies on integration of the virus genome into that of the host using the virus integrase, which cuts open host DNA for insertion before repair by the cell’s non-homologous end joining (NHEJ) pathway. NHEJ can result in insertions or deletions (indel mutations), which may result in genotoxicity. Vector integration is known to activate oncogenes, primarily due to the interaction of the lentivirus vector (LV) genome with genes found near to the site of vector integration. Inactivation of tumor suppressor genes, or gene knockout is also known to occur. In the production of virus particles, genome empty particles are also generated alongside full vector genome containing LV, each still carry the integrase enzyme. Hence, the frequency of genotoxicity associated with NHEJ activity following vector integration or integrase activity alone is difficult to quantify, particularly for gene knock-out. In this study, we used the HPRT locus as a genetic target to investigate gene disruption by genome-containing and empty LV particles to identify the integrase contribution to HPRT mutagenesis. The HPRT gene is large at 35 kb in size and has been used for several mutagenesis studies by carcinogens. It offers forward and reverse selection, where cells with functional HPRT selected for and then cells with inactivated HPRT caused by mutagenesis can be selected to quantify the mutagenic effects of the carcinogen. We firstly used V79 and MRC5 cell lines and primary human T cells derived from healthy donors (male and female) with functional NHEJ DNA repair pathways and AT5BIVA cells with impaired DNA damage repair to follow the cleavage and repair process associated with integrase activity using a fluorescent marker against γH2AX that binds to broken DNA, which is then released after repair. By this analysis, the activity of the virus integrase was also compared for LV with and without virus genome and cells not exposed to virus infection. We next used forward and reverse selection to measure the presence and loss of HPRT activity in infected cells that was observed for genome full and empty LV particles. To further support our findings that integrase activity can cause gene knockout, we expressed HIV-1 integrase in yeast to measure knockout of the CAN-1 gene and found this also occurs. These data show genotoxicity by LV particles is possible even for particles not carrying a therapeutic vector genome and suggest to reduce this potential, empty particles should be removed during LV during manufacture.