701. A SIN Lentiviral Vector That Allows In Vivo Drug-Selection of Hematopoietic Stem Cells yet Retains Erythroid-Specific Expression of the Ferrochelatase Gene and Results in Cure of Murine Protoporphyria

Abstract

Erythropoietic Protoporphyria (EPP) is an inherited defect of the ferrochelatase (FC) gene characterized by accumulation of toxic protoporphyrin mainly in the bone marrow (BM) erythroid lineage. We previously described successful gene therapy of EPP mice with erythroid-specific (ES) SIN lentiviral vectors without pre-selection of corrected cells (Richard E et al Mol Ther 2001). However, high-level gene transfer in mice is not translatable to large animal models and humans if there is no selective advantage to genetically modified hematopoietic stem cells (HCSs) in vivo. We first developed a bipromoter vector, with erythroid specific human ankyrin promoter/HS-40 enhancer driving EGFP expression and the MND promoter driving the mutant methylguanine-methyltransferase (MGMT) expression (HAGMM vector). Although we achieved in vivo selection of GFP+ RBC in mice transplanted with HAGMM transduced cells, there was loss of ES. We next developed bipromoter-SIN-lentiviral vectors encoding EGFP/FC based on human PGK promoter driving MGMT expression (HAGPM/HAFPM). We hypothesized that these vectors would allow ubiquitous expression of MGMT and yet retain ES expression of EGFP/FC genes. We also developed bicistronic vectors with the MND promoter driving expression of both genes (EGFP/FC and MGMT linked with an IRES) as control. Normal or EPP HSCs were transduced with EGFP or FC-expressing bicistronic vectors, respectively, and transplanted into non-myeloablated (4 Gy irradiation) EPP mice. Following a low-level of engraftment into EPP mice, we were able to expand the transduced cell population in vivo by 3 cycles O6-benzylguanine (BG)/ BCNU administration, resulting in a 10-fold increase of normal or corrected EPP cells in the bone marrow (BM) and peripheral blood (PB) (8 ± 5 vs 87 ± 21% EGFP+ normal RBCs without or with BG/BCNU, respectively). Selection of EPP HSC transduced with the FC expressing vector led to 4 fold increase of BM FC activity as compared with normal mice [22 ± 14 vs. 5 ± 2 FC specific activity (SA)] and complete correction of the disease phenotype. Southern blot analysis of secondary transplant and tertiary CFU-S confirmed that HSCs were selectively expanded. However, expression was not lineage-specific. EPP HSCs were then transduced with the HAFPM vector and BM transplants were performed. After BG/BCNU injection, we observed a 25-fold increase of PB corrected RBCs associated with phenotypic correction of the disease. A 4-fold increase of FC activity was seen but limited to BM Ter119+ lineage (2.65 ± 0.5 vs. 0.7 ± 0.3 SA in Ter119+ cells for transduced EPP/BG/BCNU vs. untransduced EPP respectively). Experiments with HAGPM vector demonstrated also ES expression. In conclusion, although MGMT-based in vivo selection can efficiently expand lentiviral vector transduced-HSCs transplanted without myeloablation in a murine model of EPP, we can demonstrate MGMT-based HSC selection associated with ES expression of a therapeutic gene. These results can form the basis of new gene therapy strategies for other RBC disorders such as hemoglobinopathies.