Lentiviral-Mediated Gene Correction of Mobilized Peripheral Blood Progenitors and Repopulating Cells from FA-A Patients

Fanconi anemia (FA) is an inherited disease mainly characterized by congenital abnormalities, progressive bone marrow failure and cancer predisposition. Based on previous FA gene therapy studies we aimed at improving the therapeutic efficacy of gene therapy in FA-A patients using optimized protocols of hematopoietic stem cell (HSC) harvesting and transduction. The mobilization regimen consisted on the administration of G-CSF (neupogen; 12 µg/Kg/12 hours) and plerixafor (mozobil; 240 µg/kg body weight/day). Two out of six patients with 15 and 16 years old did not reach the threshold level of CD34+ cells in PB, and therefore, apheresis was not conducted. In the four youngest patients, the total number of collected CD34+ cells/kg ranged between 1.6×106 to 7.6×106 (8.6×105 to 5.1×106 CD34+ cells/Kg after purification), and were cryopreserved for clinical use. The short transduction of small aliquots of mPB CD34+ samples from these patients with a GMP-produced lentiviral vector that harbors the FANCA therapeutic gene showed transduction efficacies between 20-40%, measured by the survival of transduced colony forming cells (CFCs) to mitomycin C (MMC). To assess the repopulating ability of transduced FA-A CD34+ cells, aliquot samples were transplanted into NSG mice conditioned with 1.5 Gy. Remarkably, most of the transplanted samples engrafted into the NSG mice (1-10% of the BM cells were hCD45+/mCD45−). Moreover, a selection advantage of corrected CD34+ FA-A cells was observed in engrafted mice, as deduced from the high MMC-resistance of hCFCs obtained from recipients’ BM. Our results show for the first time that clinically applicable transduction protocols can correct the phenotype of human FA-A hematopoietic repopulating cells. Currently a gene therapy trial of FA-A patients based on the ex vivo transduction of mPB CD34+ cells with lentiviral vectors is open in our Institutions.