470. Foamy Viral Mediated Transduction of Recombinant FANCC in the Absence of Prestimulation Is Sufficient To Restore the Long Term Repopulating Activity of Fancc -/- Stem Cells and Prevent the Acquisition of Myeloid Malignancies
Authors: Yue Si, Cordula Leurs, Edward F. Srour, Jin Yuan, Helmut Hanenberg, D. Wade Clapp
Journal: Molecular Therapy (2005)

Fanconi anemia (FA) is a complex genetic disorder characterized by bone marrow aplasia, a high propensity to develop acute myeloid leukemia, and hypersensitivity to mitomycin c (MMC). The identification of individual FA genes raises the potential of using gene transfer technology to express the functional cDNAs into FA deficient autologous stem cells. Current strategies for genetic therapy using Moloney retroviruses require a 2-4 day ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. However, culture of Fancc -/- bone marrow (BM) cells for 2-4 days results in a time-dependent increase in apoptosis of primitive stem/ progenitor cells. Further, prolonged ex vivo culture provides a selective pressure for Fancc -/- BM cells that are resistant to apoptosis and mice reconstituted with the surviving uncorrected cells have a propensity to acquire myeloid malignancies (Li, Blood 2005). Therefore we examined the potential of a helper-free human foamy virus derived construct (MD9-FANCC-EGFP) that encodes the rFANCC and the EGFP transgenes to transduce murine Fancc -/- stem and progenitor cells (HSPC) in the absence of prestimulation. In initial experiments, we determined that 40-80% of progenitors were transduced following a single 12-14 hr transduction. Subsequent studies demonstrated that MD9-FANCC-EGFP efficiently transduced primitive and mature myeloid hematopoietic progenitors that were in G0 and G1 at the time of transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice. Four months following transplantation, BM cells were isolated from the reconstituted recipients and clonogenic assays were established in a range of MMC concentrations. In these experiments, Fancc-/- progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wild-type controls while Fancc -/- progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess quantitatively the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. In two replicate experiments, we determined that the repopulating activity of MD9-FANCC-EGFP-transduced Fancc-/- stem cells was comparable to wildtype controls 18-24 months following transplantation in primary and secondary recipients. Importantly, while mice reconstituted with Fancc -/- cells transduced with the reporter construct only had reduced repopulating ability as compared to the other groups as expected, none of these animals acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral vector allows efficient transduction of Fancc -/- HSPC and diminishes the selection pressure that occurs during ex vivo culture.