Gene transfer to ankyrin-deficient bone marrow corrects spherocytosis in vitro

Objective. The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achi eve erythroid expression of functional ankyrin protein, Materials and Methods. A minigene composed of the human ankyrin promoter, m urine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2. 2 isoform was assembled in the retroviral vector, pG1, Murine erythroleukem ia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral su pernatant, Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed bu colony polymerase chain reaction (PC R) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, No rthern, and Western blot analysis. Results. MEL cells, normal bone marrow progenitors, and nb/nb cells were al l successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane st aining by immunofluorescence. Colony RT-PCR demonstrated dependence of expr ession on erythropoietin. In vitro, the transduced nb/nb cells matured to p olychromatophils, whereas nontransduced nb/nb cells matured to microspheroc ytes. Conclusion. Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from th e nb/nb mutant. The human ankyrin promoter conferred erythropoietin-depende nt expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias. (C) 2000 Inte rnational Society for Experimental Hematology. Published by Elsevier Scienc e Inc.