EFFECT OF CELLULAR TYPE ON EXPRESSION OF ACID BETA-GLUCOSIDASE - IMPLICATIONS FOR GENE-THERAPY IN GAUCHER DISEASE

The effects of cellular type on the expressed activity of acid beta-gl ucosidase were evaluated using retroviral constructs containing the hu man cDNA. MFG retroviral ecotropic and amphotropic vectors containing the human acid beta-glucosidase cDNA were produced and used to infect different murine cell lines (fibroblast, neuronal and monocytic) and h uman cells (HL60 and cord blood CD34(+)), respectively. The expression of human acid beta-glucosidase was evaluated by enzyme activity assay s, quantitative Western blots and immunofluorescence. All cells perman ently integrated viruses and expression of enzyme protein was achieved in all cell lines, but cellular transduction efficiency differed even between different neuronal cell lines (eg N18S>PC12). In most cell li nes acid beta-glucosidase activity was increased between two- and thre e-fold with concomitant signal increases by Western blot and immunoflu orescence. N18S cells had poor transduction efficiency, but high cellu lar expression in transduced cells. In NIH3T3 and MC3T3-E1 cells acid beta-glucosidase protein was expressed in 2-, 7- and 14-day cultures a fter infection and at least to passage four. The expressed acid beta-g lucosidase in NIH3T3 cells was at two to three times normal activity l evels, and was processed similarly to the human fibroblast enzyme. Ina ctive human acid beta-glucosidase was expressed in MC3T3-E1 preosteobl astic cells and this was maintained during differentiation to osteobla sts. These results indicate that gene transfer results in cell lines m ay not be generally extrapolated to all cells in tissues or to differe ntiated progeny.