Transfer and expression of hG6PD in human hematopoietic cells.

(A) Expression in short-term and long-term cultures in vitro. Human BM cells were transduced with retrovirus harboring the cDNA of hG6PD A, and then processed for methylcellulose colony assays and long-term cultures. 1, Untransduced human BM cells. Transduced human BM cells: 2, cells after 7 days in liquid culture; 3, pooled white and erythroid colonies; 4, adherent cells obtained at week 6 of a long-term BM culture (the m band derives from mouse MS-5 stromal cells). Controls: 5, mixture of human RBC G6PD B and of murine MS-5 cells; 6, RBCs from a woman heterozygous for G6PD B and G6PD A. hB, human G6PD B. hA, human G6PD A; hA-B, heterodimer of human G6PD A and B; m, murine G6PD. (B) Transfer and expression of hG6PD in human HSCs in vivo. RT-PCR analysis for the presence of hG6PD mRNA transcribed from integrated provirus in BM and human BM colonies (CFC) from 3 NOD-SCID mice transplanted with human HSCs.1,2 3 Positive control: transduced (T) human K562 cells. Negative controls: untransduced (U) K562 cells; untreated NOD/SCID mouse BM cells (N/S); R: reagent control; −RT: omission of reverse transcriptase. M, molecular weight marker. (C) PCR analysis of individual hematopoietic colonies from mouse 1 (Figure 4B): integrated provirus was present in 5 of 25 colonies. MC indicates methylcellulose control. (D) Expression of functional G6PD in vivo and correction of human G6PD deficiency. (Left) Electrophoretic analysis of human erythroid colonies (BFU-E) obtained from NOD/SCID mice 8 weeks after engraftment of G6PD B human cord blood transduced with an hG6PD A vector. (Right) Electrophoretic analysis of human HSCs from G6PD-deficient cord blood samples (G6PD A⁻and G6PD Mediterranean, respectively) after transduction with an hG6PD vector (G6PD B and G6PD A, respectively). In the case of G6PD Mediterranean, which entails a more severe quantitative defect than G6PD A⁻, the activity from the transferred gene is clearly higher than from the endogenous gene.