Abstract:
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Almost one fifth of clinical trials of human gene therapy is based on the use of retroviral vectors for gene transfer. Their integration ensures stable transmission of the transgene to progeny cells. However, the provirus may influence host gene expression potentially leading to insertional oncogenesis. Moreover, transgene need to be expressed at proper level in order to avoid a potential toxicity. The understanding of determinants of exogenous DNA epigenetic regulation and the elaboration of tools to control transgene expression will prove important in various investigative aims concerning gene technology. We characterized a collection of vectors with various designs [lentiviral or gammaretroviral nature, native LTR or self-inactivating i.e. SIN-LTR (self inactivating long terminal repeat), an internal cellular or viral promoter, with or without modulating sequences within SIN-LTR] whose impact on transgene expression was assessed over the time. We have highlighted a phenomenon of reduction of transgene expression, depending on the sequence inserted into SIN-LTR and its orientation. A cost-benefice balance associated to the introduction of sequences at vector extremities could then explain that cHS4 avian insulator has a positive effect on transgene expression only in one given orientation. The cellular status also needs to be considered for gene transfer analysis: several DNA repair proteins seem to be implicated at different steps of retroviral cycle (genome circularization, integration, expression). Our vector collection was used to conducted a systematic and comparative analysis of retroviral transduction within cellular models whose status in DNA repair (Ku80, BRCA1) or epigenetic related factors (H2AX, PHF1) has been modified. Our experimental design could serve as a model for gene therapeutic trials that target cells with DNA repair defaults. Ku80 status had an important impact on expression from HIV-LTR, but not on expression from others lentiviral or gammaretroviral vectors, nor endogenous retroviruses. Moreover, preliminary results have revealed a positive role of BRCA1 and H2AX in lentiviral transduction efficiency. The impact of sequences cloned into SIN-LTR on these phenomena is under analysis. This study will provide further insights on the impact of retroviral vector design and retroviral transduction in cells with DNA repair defaults.
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