993. Activation of Gene Expression from Silenced Adenovector Genomes

Abstract

Gene therapy of beta-thalassemia requires permanent transfer of a beta-globin gene into hematopoietic stem cells (HSC), and quantitative and cell-restricted regulation of its expression in the erythroblastic cell progeny. We have developed strategies for transcriptional targeting of oncoretroviral (MLV-derived) and lentiviral (HIV-derived) vectors by replacing the viral LTR control elements with cellular enhancer/promoter sequences (HS1 and HS2 enhancers of the erythroid-specific GATA-1 gene). The modified LTRs were used to drive the expression of a c-DNA coding for an epitope-tagged beta-globin. A reporter gene (LNGFR or EGFP) was placed under the control of an internal constitutive promoter to monitor cell transduction, or isolate transduced cells, independently from the expression of the targeted promoter. Human and murine erythroblastic cell lines (HEL and MEL), expressing endogenous human alpha/gamma and murine alpha/beta globins respectively, were transduced with lentiviral or retroviral vectors. Following erythroid induction, mature human beta-globin was accumulated in both erythroblastic cell lines at level comparable to that of the endogenous globin genes. These vectors were tested on human cord blood-derived HSC. Gene expression was analyzed in the differentiated progeny of transduced stem cells in vitro, both in liquid culture and in clonogenic assay. Transcriptional targeting of the modified LTR was efficient and we obtained production of vector-derived globin in the erythroblastic progeny of transduced HSCs. For the correction of globin chains imbalance in human beta-thalassemia, a high protein output is just as important as its restricted expression. To improve the overall efficiency of beta-globin cDNA expression, different elements, which are naturally present in the beta globin gene (introns, untranslated regions), were introduced in our vector backbone. The cloning of a spliceable beta-globin gene under the control of chimeric promoter in LV/RV vectors requires to protect the introns from splicing events during vector production in packaging cells. We exploited the use of Rev/RRE system, by inserting the Packaging Signal (PSI) and the RRE into the first or the second intron of beta-globin gene. Packaging cells were co-transfected with the transfer vector and a plasmid coding for Rev protein. Following transduction of HEL cells, we observed a higher level of beta globin expression than that obtained by vectors lacking globin introns. However, the viral titer of retroviral vectors was greatly reduced, making difficult the production of optimal viral stocks. We recently moved to the development of lentiviral vectors containing the HIV-derived PSI/RRE sequences into the beta-globin introns. The activity of new vectors will be first analyzed following transduction of human and murine erythroblastic cells, and by testing the therapeutic potential of transduced cells in appropriate pre-clinical models, such as bone marrow transplantation in beta-thalassemic mice.