Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc−/−hematopoietic stem cells and decreases the risk of clonal evolution

Abstract

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we used Fancc ؊/؊ mice to show that Fancc ؊/؊ hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinant Fancc (rFancc) would restore the repopulating ability of Fancc ؊/؊ HSC to wild-type levels. Fancc ؊/؊ HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, ϳ30% of primary recipients (7 of 22) transplanted with uncorrected Fancc ؊/؊ cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted with Fancc ؊/؊ cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-␥, TNF-␣, and MIP-1␣ compared to both Fancc ؊/؊ progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransduced Fancc ؊/؊

Oxidant Hypersensitivity of Fanconi Anemia Type C-deficient Cells Is Dependent on a Redox-regulated Apoptotic Pathway

“…Mice-Fancc ϩ/Ϫ mice in a C57Bl/6J genetic background were bred to generate Fancc Ϫ/Ϫ and wild type (WT) mice for hematopoietic progenitor assays and timed embryos for murine embryo fibroblast (MEF) cell lines as previously described (52,53). All of the studies were approved by the Indiana University Laboratory Animal Research Center.…”

“…Retroviral supernatants were harvested and utilized to transduce GPϩE86 retroviral packaging cells as previously described (52) to pseudotype viral particles with an ecotropic envelope. The MFG-FAC retrovirus encoding the FANCC cDNA was used as a control, which previously was shown to correct mitomycin C sensitivity of Fancc Ϫ/Ϫ cells to WT levels (52).…”

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice

“…Fanca −/− mice and Fancc −/− mice on a C57BL/6J background were used as detailed in previous studies [36,37]. Congenic wildtype C57BL/6J (WT) mice and wildtype B6.SJLPtrca Pep/BoyJ (BoyJ) mice were purchased from Jackson ImmunoResearch Laboratories.…”

“…Competitive repopulation experiments were conducted as previously described in primary and secondary recipients [36,38]. Briefly, LDMC from 1 ml (cohort 1) or 2 ml (cohort 2) of the PB of donor WT, Fanca −/− or Fancc −/− mice treated with G-CSF, AMD3100 or both were transplanted along with 7.5 × 10 5 BoyJ competitor BM LDMC into WT (8-12 week old) lethally irradiated recipients (1100 rads).…”

“…These two independent experiments with 3-5 recipients/genotype/treatment group were analyzed. Chimerism was measured monthly as described previously [36]. Briefly, PB cells were collected and stained with FITCconjugated anti-CD45.2 antibodies (BD Biosciences) and the percentage of positive cells was determined by flow cytometry.…”