Complete Restoration of Glucocerebrosidase Deficiency in Gaucher Fibroblasts Using a BicistronicMDRRetrovirus and a New Selection Strategy

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Abstract
Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 μg/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, 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 in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene. Successful retrovirus-mediated gene transfer for correction of genetic disorders frequently depends on sustained, high-level expression of the therapeutic transgene in the target cells. To satisfy these requirements for Gaucher disease, we have designed a bicistronic vector in which a single mRNA encodes the glucocerebrosidase (GC) gene and the selectable multidrug resistance (MDR1) gene. By selecting for drug resistance, expression of GC is essentially guaranteed in Gaucher fibroblasts that have been transduced by the MDR-GC retrovirus. If the efficiency of the MDR1-encoded drug efflux P-glycoprotein (P-gp) pump is reduced by concomitant use of inhibitors of the pump, selection for higher levels of P-gp and GC can be obtained with lower amounts of cytotoxic drug. This new selection strategy combining P-gp substrates and inhibitors may prove useful in gene therapy trials using MDR1-based vectors to reduce the systemic toxicity frequently associated with cytotoxic drug treatment.