307. A Mechanism for AZT Mediated Enhancement of Transgene Expression

Abstract

Recombinant adeno-associated viral (rAAV) vectors have been used to treat hemophilia B by efficient transfer of F.IX into liver and muscle in animals; human trials are currently underway. In a recent dose-escalation study investigating muscle directed F.IX gene transfer using rAAV vectors, an HIV-positive subject who received the lowest vector dose showed highest F.IX expression levels. Coadministration of Zidovudine (3'-Azido-3'-deoxythymidine or AZT) during the trial was suggested as an explanation for this observation (Manno et al, Blood 101:8, 2003). AZT is a nucleoside analog that inhibits HIV reverse transcriptase activity. It has also been implicated in inhibiting DNA replication, in generation of oxygen radicals and in induction of transcription factors. We hypothesized a role for the AZT induced nuclear transcription factor, NF-kappaB, in enhancing gene expression by binding to the four consensus NF-kappaB binding sites in the CMV promoter of the vector. Previously, we have shown an AZT dependent increase (30-fold) in expression of a CMV promoter-driven transgene following transduction of HepG2 cells with an AAV vector. Similar experiments using a vector with an EF1alpha promoter showed no change in levels of transgene expression in response to AZT, indicating a CMV promoter-specific response. Western analysis of total cellular extracts from transduced cells cultured for up to 72h in the presence of different levels of AZT showed an increase in NF-kappaB (p65) levels with increasing AZT. NF-kappaB consensus sequence binding assays using nuclear extracts from AZT treated (0.4, 4 and 40mM) cells showed 1.5, 4.1 and 4.3-fold increase, respectively, in NF-kappaB levels over untreated samples. Reporter assays were performed using plasmids with a luciferase gene driven by either a TK (thymidine kinase) minimal promoter or an NF-kB4-TK promoter. Our results show increasing reporter expression (8-fold in 40mM AZT treatment) with increasing concentrations of AZT with NF-kB4-TK promoter, but not with the TK-minimal promoter. We further confirmed our findings by silencing the ATM gene, an activator of NF-kappaB, using siRNA. ATM silenced cells produced significantly lower levels of active NF-kappaB in nuclear extracts of cells treated with AZT. Moreover, the activity of the NF-kB4-TK luciferase promoter decreased 7-fold, indicating a specific role for ATM in CMV driven gene expression via NF-kappaB activation pathway. Our findings thus provide a molecular explanation for enhanced CMV-promoter driven gene expression in a clinical trial subject treated with Zidovudine and provide a novel avenue towards exploring the use of host transcription factors to significantly enhance gene transfer treatment strategies.