Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

Many mutations of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (hG6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic non-spherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the hG6PD cDNA, we have obtained stable and life-long expression of hG6PD in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and of 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. We infer that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors we were able to restore both the cloning efficiency and the ability to form embryoid bodies of embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, we have obtained expression of normal human G6PD in human G6PD deficient primary hematopoietic cells, and in human hematopoietic cells engrafted in the immunodeficient NOD-SCID mice. This approach could cure severe CNSHA caused by G6PD deficiency.