RESUMO
Fanconi anemia (FA) is a complex genetic disorder characterized by congenital abnormalities, bone marrow failure, and myeloid malignancies. Identification of 13 FA genes has been instrumental to explore gene transfer technologies aimed at correction of autologous FA-deficient stem cells. To date, 3 human FA stem cell gene therapy trials with standard 4-day transduction protocols using gammaretroviral vectors failed to provide clinical benefit. In addition, 2- to 4 day ex vivo manipulation of bone marrow from mice containing a disruption of the homologue of human FANCC (Fancc) results in a time-dependent increase in apoptosis and a risk for malignant transformation of hematopoietic cells. Here, we show that a 14-hour transduction period allows a foamyviral vector construct expressing the human FANCC cDNA to efficiently transduce murine FA stem cells with 1 to 2 proviral integrations per genome. Functionally, the repopulating activity of Fancc(-/-) stem cells from reconstituted mice expressing the recombinant FANCC transgene was comparable with wild-type controls. Collectively, these data provide evidence that short-term transduction of c-kit(+) cells with a foamyviral vector is sufficient for functional correction of a stem cell phenotype in a murine FA model. These data could have implications for future gene therapy trials for FA patients.
Assuntos
Proliferação de Células , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Células-Tronco Hematopoéticas/fisiologia , Spumavirus/genética , Transdução Genética/métodos , Animais , Células Cultivadas , Ritmo Circadiano , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Anemia de Fanconi/veterinária , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Terapia Genética , Vetores Genéticos/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-kit/genética , Fatores de TempoRESUMO
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 peripheral blood. 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.
