In vivo Expression and functional complementation of recombinant Fancc In Fancc −/− Hematopoietic stem cells

Fanconi anemia (FA) is characterized by a progressive bone marrow (BM) failure, the acquisition of malignancies, and hypersensitivity to mitomycin C (MMC). Recently, we used a murine model containing a disruption in the Fanconi anemia group C (Fancc) gene to show that Fancc −/− hematopoietic stem cells (HSC) have a marked reduction in repopulating ability (Haneline et al, BLOOD 1999). The goal of the current studies was to determine whether expression of recombinant Fancc in vivo complements the proliferation and MMC abnormalities of Fancc −/− HSC. We utilized the murine stem cell virus (MSCV) backbone and constructed a retrovirus containing the Fancc cDNA, MSCV-Fancc. Wildtype (WT) and Fancc −/− BM cells were prestimulated for 48 hours and transduced on fibronectin. As predicted, transduction of Fancc −/− progenitors with MSCV-Fancc corrected MMC sensitivity to WT levels. To determine whether reconstituting Fancc −/− HSC expressed functional Fancc in vivo, 1×106 transduced cells were transplanted into lethally irradiated hosts and evaluated at 6 months. Fancc protein was detected in BM cells by Western analysis. Further Fancc −/− progenitors transduced with MSCV-Fancc exhibited a MMC sensitivity similar to WT cells. These data support expression of functional Fancc in vivo. Competitive repopulation studies are in progress to compare repopulating ability of cells expressing recombinant Fancc to WT cells and mock transduced Fancc −/− cells. Preliminary data 2 months after transplant suggest that short term repopulating ability was restored to WT levels with introduction of Fancc. These data have important implications regarding the application of gene transfer methodologies using Fancc −/− mice as a preclinical model.