IntroductionFanconi anemia (FA) is a complex recessive inherited disorder that is clinically characterized by variable congenital abnormalities, progressive bone marrow (BM) failure, and a high propensity to develop myeloid and epithelial malignancies. 1-5 On a cellular level, FA is characterized by a profound hypersensitivity upon exposure to DNA cross-linking agents such as mitomycin-C (MMC) or diepoxybutane (DEB). [6][7][8][9] Genetically, germ-line mutations in 13 genes (FANCA/B/C/D1/ D2/E/F/G/I/J/L/M/N) result in the clinical phenotype of FA. 2,8,[10][11][12][13][14] Spontaneous genetic correction of a germ-line mutation leading to repopulation of the entire hematopoietic system with normal progeny has been identified in a few FA patients. [15][16][17][18][19] These observations, in combination with the fact that the hematopoietic system can be functionally corrected in mice with targeted disruptions of FA genes by retroviral vectors expressing human analogues of the targeted mouse genes in stem cells, [20][21][22] have led to 3 clinical stem cell gene therapy phase 1 studies in FA-A and FA-C patients. So far, neither long-term marking/correction of cells nor clinical benefits for the patients were observed. 23,24 Due to the biologic characteristics of the gammaretroviral vectors used for transduction of stem cells, 25,26 optimal gene transfer protocols for delivery of genes to mammalian stem cells require a prestimulation period of 1 to 2 days with cytokines that promote the proliferation and survival of stem/progenitor cells. This is followed by a 2-to 3-day exposure of the target cells to vector containing supernatant on the recombinant fibronectin fragment 28 This gene transfer protocol was successful in transducing hematopoietic stem cells in humans, primates/monkeys, and mice. [29][30][31] However, in murine FA models, prolonged in vitro culture of Fancc Ϫ/Ϫ BM results in a length-of-culture-dependent reduction in myeloid progenitors and repopulating ability, 32,33 and the surviving untransduced Fancc Ϫ/Ϫ repopulating cells have an increased risk of developing cytogenetic abnormalities and myeloid malignancies. 22 Therefore, limiting the in vitro culture would be predicted to enhance both the efficacy and safety for genetic therapies of FA stem cells.Wild-type foamy viruses are the only retroviruses that are not associated with any disease in their natural hosts or in accidentally infected human beings. [34][35][36] It has been shown that vectors based on the prototype (formerly human) foamy virus (FV) can efficiently transduce hematopoietic stem cells from mice, 37 dogs, 38 and nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) repopulating human cells. [39][40][41] Further, FV vectors are at least equally efficient at transduction of CD34 ϩ umbilical cord blood cells engrafting in NOD/SCID mice as lentiviral vectors based on HIV-1. 40 In the present study, we demonstrated for the first time the ability of FV vectors encoding the human FANCC transgene to completely correct Fancc Ϫ/Ϫ myelo...

Fanconi anemia (FA) is a heterogeneous inherited disorder characterized by a progressive bone marrow (BM) failure and susceptibility to myeloid leukemia. Genetic correction using gene transfer technology is one potential therapy. A major hurdle in applying this technology in FA patients is the inability of granulocyte colony-stimulating factor (G-CSF) to mobilize sufficient numbers of hematopoietic stem (HSC)/progenitor cells (HPC) from the BM to the PB. Whether the low number of CD34 + cells is a result of BM hypoplasia or an inability of G-CSF to adequately mobilize FA HSC/HPC remains incompletely understood. Here we use competitive repopulation of lethally irradiated primary and secondary recipients to show that in two murine models of FA, AMD3100 synergizes with G-CSF resulting in a mobilization of HSC, whereas G-CSF alone fails to mobilize stem cells even in the absence of hypoplasia.

Fanconi anemia (FA) is a rare inherited chromosomal instability syndrome characterized by bone marrow failure and a high relative risk of MDS. Eight FA proteins associate in a core nuclear complex and function at least in part to catalyze the monoubiquitination of the downstream target protein, FANCD2 in response to DNA damage. In this nuclear pathway the FA proteins are epistatic in the activation of FANCD2 since inactivation of any one of the eight FA proteins results in failure of FANCD2 monoubiquitination and hypersensitivity to cross-linking agents. Although biochemical studies have attributed additional survival signaling functions to the FA proteins, these functions have not been evaluated using a genetic model. Murine models of FA have been established using homologous recombination for gene disruption. Although all strains of knockout mice are hypersensitive to mitomycin c, none of the single gene knockout mice display bone marrow failure, MDS, or myeloid leukemia. Seeking to develop such a model, we utilized a genetic intercross to generate mice that harbor disruptions in both Fancc and Fancg. Genetic disruption of both Fancc and Fancg predispose Fancc−/−;Fancg −/− mice or recipients adoptively transferred with Fancc −/−; Fancg −/− hematopoietic stem cells to MDS analogous to the disease phenotype in FA patients as defined histologically and by cytogenetic analysis. Genome wide transcriptomal analysis and hierarchical clustering by genotypic group of bone marrow cells from wild type, single knockout, and double knockout mice (n=3 each) confirmed substantial differences between hematopoietic cells of Fancc, Fancg and double knockout (DKO) mice. Serial pairwise analysis and gene pattern analyses (GeneSifter) showed that of the 1190 genes expressed differentially (by a factor of >1.5, FDR adjusted p<0.05) in Fancc−/− marrow cells only 134 were differentially expressed in Fancg−/− cells. Of the 524 genes expressed differentially in Fancg−/− marrow compared to WT, 277 were not expressed differentially in Fancc−/− marrow compared to WT. In pairwise analysis of Fancc−/− vs. Fancg−/− gene expression, ontologies of those genes more highly expressed in Fancc −/− cells included responses to biotic stress, defense and immune response. The most over-represented ontological category of those genes more highly expressed in Fancg−/− cells was response to oxidative stress. Since these genes are not epistatic in regards to the hematopoietic phenotype, and the transcriptomal consequences of their loss-of-function in marrow cells are significantly different, this genetic model confirms that the Fancc and Fancg proteins are multi-functional. Transcriptosomal analyses were conducted on DKO mice that contained MDS and DKO mice with no overt disease. The transcriptome of DKO marrow cells was unique in that 152 suppressed and 687 activated gene products relative to WT samples were not found in either Fancc−/− or Fancg−/− samples. Furthermore, there are distinct transcriptomal differences between the DKO mice with MDS and those that do not have MDS. These data suggest that some of these changes may be adaptive and involved in the molecular pathogenesis of MDS. The DKO model provides the first preclinical platform to systematically evaluate the molecular pathogenesis of bone marrow failure and myelodysplasia in the setting of Fanconi anemia.

Fanconi Anemia (FA) is an autosomal recessive DNA repair disorder clinically characterized by congenital abnormalities, progressive bone marrow (BM) failure and a propensity for malignancies. It is advantageous to bank FA patients’ cells for autologous treatment of pancytopenia, transplantation following chemotherapy or future gene therapy trials. Because the amount of cells able to be collected by BM harvesting is limited and the process is often problematic in children, mobilization of stem cells into the circulation would be an ideal means of collecting repopulating hematopoietic stem and progenitor cells (HSPC). Traditional mobilization protocols utilizing granulocyte colony-stimulating factor (G-CSF) as a mobilizing agent result in poor yields of phenotypic HSPC in FA patients. We have shown that analogous to the human patients with FA, administration of recombinant G-CSF is not sufficient to effectively mobilize myeloid progenitors from the BM of Fancc −/− mice (Broxmeyer et al, JEM, 2005). Antagonists to chemokine receptors, together with G-CSF, can enhance mobilization of murine wild type (WT) HSPC. AMD3100 is a selective antagonist of CXCR4, which is thought to function in the retention of HSPC in the BM. We hypothesized that the combination of AMD3100 and G-CSF would enhance mobilization of HSPC in Fanca −/− and Fancc −/− mice. Peripheral blood was collected to assess the number of myeloid progenitors and HSPC repopulating ability. Myeloid progenitor numbers were assessed using progenitor assays. Either G-CSF or AMD3100 alone resulted in poor mobilization of hematopoietic progenitors. In contrast, administration of AMD3100 and G-CSF together increased the number of colonies of myeloid progenitors up to 18 fold above that of either G-CSF or AMD3100 alone in both Fanca −/− and Fancc −/− mice. The stem cell activity of mobilized peripheral blood cells was evaluated using competitive repopulation assays, in which a common pool of isogenic ‘competitor cells’ and ‘test cells’ from mice of different genotypes or treatment conditions are co-transplanted. Fanca −/− and Fancc −/− cells mobilized with both G-CSF and AMD3100 yielded a 3–4 fold increase in peripheral blood chimerism as compared to the cells mobilized with G-CSF or AMD3100 alone. The latter mice also had a decreased survival. Phenotypic analysis of the peripheral blood of primary recipients confirmed the engraftment of multi-lineage test cells. Transplantation of chimeric bone marrow into secondary recipients maintained the test cell chimerism, confirming that long-term repopulating cells were mobilized. Collectively, these data from two FA murine models demonstrate that the AMD3100 and G-CSF protocol is an effective strategy to mobilize FA deficient repopulating HSPC. This has potential implications for more efficient banking of FA patients’ HSPC for future use or for use in gene transfer protocols.