2003; baxter - Supplements - Haematologica

118T. VandenDriessche et al.Figure 1. Genetic heterogeneity model. Hemophilia A mice are not genetically identical. As a consequence, some of the micemay be prone to develop inhibitory antibodies to FVIII and/or CTLs to the FVIII-transduced cells following in vivo gene therapywith FVIII-vectors. This would result in the neutralization of circulating FVIII proteins and/or the immune rejection of the FVIIIexpressingcells in an MHC class I-restricted fashion. Conversely, some of the mice may have a distinct genotype allowingimmune tolerance to be established to FVIII proteins and FVIII-transduced cells. This genetic heterogeneity hypothesis mayexplain the heterogeneous immune response in the neonatal hemophilic mice that received FVIII-retroviral vectors 21 but mayapply to other vector systems as well in adult recipient mice.essary for integration, reverse transcription andexpression are incorporated into the vector backbone,along with the gene of interest, in casu, theFVIII gene.Although many different types of non-dividingcells in different tissues can be transduced withlentiviral vectors, not all cells are permissive andin particular, there may be unknown limitingfactors that preclude efficient gene transfer intonon-dividing hepatocytes. 28,31,32 In anticipationof using lentiviral vectors for FVIII gene deliveryinto the liver, it was therefore important to firstdemonstrate that lentiviral vectors can transducenon-dividing hepatocytes. An improved lentiviralvector expressing the green fluorescent reporterprotein (GFP) was constructed. The vector containedthe central DNA flap which facilitatesintra-nuclear transport of the lentiviral pre-integrationcomplex and enhances transduction efficiency.33Lentiviral transduction was analysed afterintravenous injection of 109 transducing units oflentiviral-GFP vectors into adult mice (Vanden-Driessche et al., in press). Confocal microscopyrevealed intense GFP fluorescence in hepatocytes(5-10% GFP + ) and non-parenchymal cells in theliver of immuno-deficient Scid recipient mice,mostly around the vasculature. In addition,splenocytes were efficiently transduced (20-30%GFP + ) whereas no transduced GFP + cells couldbe detected in other organs. Transduction resultedin stable genomic integration of the lentiviralvector leading to long-term transgene expressionin liver and spleen (> 3 months). To assess thedependence of transduction on cell division,BrdU was continuously administered. Analysis ofGFP and BrdU colocalisation showed that >95%of transduced hepatocytes and splenocytes werenon-dividing. Liver toxicity was assessed by measuringserum-transaminases which were moderatelyelevated 24 hr post-injection but returnedto normal levels the next day. This confirms thatlentiviral vectors can be used to transduce nondividinghepatocytes in adult mice, obviating theneed to artificially induce hepatocyte proliferation.haematologica vol. 88(supplement n. 12):september 2003