Transduction of Hematopoietic Stem/Progenitor Cells with a Lentivirus Expressing FANCA off the Human Phosphoglycerate Kinase Promoter Corrects the Fanconi Anemia Phenotype,

Abstract 4178

Fanconi anemia (FA) is a rare recessive disease manifested by progressive bone marrow (BM) failure, congenital abnormalities and a predisposition to cancer. The only curative treatment for the pancytopenia to date is allogeneic stem cell transplantation. However, because allogeneic grafts are available to only about 30% of patients and mismatch grafts have a higher incidence of sequelae, autologous gene transfer into hematopoietic stem cells (HSCs) is a potential viable therapy. Major obstacles for gene therapy in FA include: low numbers of HSCs, an increased frequency of apoptosis and propensity for clonal selection following prolonged culture and the risks of all gene transfer technology including transactivation of proto-oncogenes from potent viral promoters such as the spleen focus-forming virus promoter (SFFV). We have recently found that a lentiviral (LV) vector containing SFFV promotor rendered a high functional and biochemical expression of the transgene in human and murine repopulating HSCs. However, given the recent concerns in the use of potent viral promoters like SFFV, in this study we developed LV vectors harboring a mammalian cell endogenous promoter human phosphoglycerate kinase (hPGK) to drive the expression of human FANCA and tested the ability of this construct to correct the phenotypes of human FANCA−/− fibroblasts and murine Fanca−/− hematopoietic stem and progenitor cells. Viral-mediated functional correction of FANCA was first evidenced by the restoration of FANCD2 mono-ubiquitination and correction of mitomycin C (MMC)-induced G2/M cell cycle arrest in human FANCA−/− fibroblast cells. In addition, the survival of genetically modified murine Fanca−/− clonogenic progenitors in the presence of 5nM MMC was improved from 30% (transduced with EGFP reporter) to 80% (transduced with FANCA cDNA), nearly equivalent to the survival of WT progenitors (90%) at that concentration. In addition, in a competitive repopulation assay, the long-term repopulating ability of Fanca−/− stem cells in the lethally-irradiated recipient mice was dramatically increased from 10% test cell chimerism (transduced with EGFP reporter) to 55% test cell chimerism (transduced with FANCA cDNA). Furthermore, the chimerism was maintained over 6 months in the primary recipients and an additional 6 months in the secondary recipients. Finally, resistance to MMC was retained in progenitors cultured from FACS sorted bone marrow test cells from the primary and secondary recipients. Collectively, these data suggest that the lentiviral construct using a mammalian cell endogenous promoter (hPGK) is sufficient to correct the phenotypes of murine FA HSC/progenitor cells. Studies are now ongoing to examine LV mediated FANCA expression in human primitive progenitor/ SCID/NOD repopulating cells.