COMPLETE RESTORATION OF GLUCOCEREBROSIDASE DEFICIENCY IN GAUCHER FIBROBLASTS USING A BICISTRONIC MDR RETROVIRUS AND A NEW SELECTION STRATEGY

Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited an d acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a b icistronic cassette in which the selectable MDR1 cDNA encoding P-glyco protein (P-gp) a multidrug efflux pump, and the human glucocerebrosida se (GC) gene are transcriptionally fused. Transduction of human Gauche r fibroblasts with this recombinant virus allowed coordinate expressio n of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expres sion of GC, which paralleled the stringency of drug selection. Thus, s election of the genetically modified Gaucher fibroblasts in 1 mu g/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzym e deficiency present in Gaucher cells. Moreover, by simultaneously inh ibiting the P-gp pump, it was possible to use much lower concentration s of colchicine to select for high-level expression of MDR1 and GC. Th us, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency hi the CA2-transduced fibroblasts. These combination reg imens, already in clinical use for the treatment of multidrug-resistan t malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebros idase when transcriptionally fused to the MDR1 gene.