The Study Of The Specificity And The Stability Of The Developed HDAd Gene-Transfer Vector

Background Gutless adenovirus (HDAd) and lentiviral vectors (LV) are attractive vectors for gene therapy of muscle diseases. Because the organization of their DNA after transduction (episomal vs. integrated) differs, we have investigated if the strength, specificity and the stability of△USEx3, a novel muscle-specific promoter previously tested with plasmid, were maintained in the context of these vectors. Scaffold/matrix-attached regions (S/MAR) implicated in a variety of important functions related to gene expression Furthermore, the presence of this S/MAR in the plasmid backbone has been shown to increase transgene expression level and to prevent silencing. We will test the effects of incorporating into the HDAd a S/MAR, to test if it still prevent transgene silencing.Methods Two HDAds expressing β-galactosidase regulated either by△USEx3or the CMV enhancer/β-actin promoter (CAG), and two LVs expressing the green fluorescent protein regulated by△USEx3or CMV were constructed. Gene expression was compared in non-muscle cells (HeLa, Jurkat,293A, A549), C2C12myoblasts and myotubes. HDAds were also tested in mice after injection in the tibialis anterior muscle and intravenous injection. The Lvs was tested in different time point. Two HDAds,expressing GFP, with or without S/MAR, were transfected in HeLa cells and compared the GFP expression.Results In non-muscle cells, gene expression from△USEx3was1%or less the value of CAG and CMV, except in A549where it ranged between5-12%. In myoblasts, it was0.5%and29%the value of CAG and CMV respectively. In myotubes, AUSEx3was as strong as CMV, but10times weaker than CAG, a powerful promoter in muscle. In vivo, the activity of AUSEx3in the lung, liver and spleen was2%or less the CAG value, whereas in skeletal muscle it was10%. Despite the lower activity of AUSEx3,28%of muscle fibres of injected tibialis anterior were positive for β-galactosidase. The curve of the△USEx3is more stable in the context of LVs. With the time passed the HeLa cells with S/MAR decreased expression of the GFP protein was Slower and less pronounced.Conclusions△USEx3is a robust muscle-specific and stable promoter in the context of LV and HDAd.This, in conjunction with its small size (504bp), make it very attractive for gene therapy of muscle diseases. S/MAR in the HDAd still could prevent transgene silencing...