Retroviral Coexpression of a Multidrug Resistance Gene (MDR1) and Human α-Galactosidase A for Gene Therapy of Fabry Disease

Human α-galactosidase A (α-Gal A; EC. 3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human α-Gal A cDNA either upstream (pHa-αGal-IRES-MDR) or downstream (pHa-MDR-IRES-αGal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-αGal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-αGal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of α-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-αGal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-αGal retrovirus-transduced clones showed significantly higher activity of α-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.