A Novel Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia Locates the Erythroid Defect at CFU-E / Proerythroblast Transition.

Abstract 178

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Presently, many different DBA disease genes are known that all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). In order to study pathophysiology and to evaluate novel therapies, DBA animal models are needed. Since RNA interference -mediated RPS19 down regulation has been shown to result in a DBA phenotype in human cells in vitro, we decided to use the short hairpin RNA (shRNA) technology to create an RPS19-deficient mouse model for DBA. We designed miR30 -styled shRNAs against RPS19 and introduced them into mouse embryonic stem (ES) cells downstream of the collagen A1 locus using site-specific recombination. The resulting ES cell clones contain a single RPS19-targeting shRNA under the control of a doxycycline-responsive promoter. We have generated and characterized two mouse models expressing different RPS19-targeting shRNAs (shRNA-B and shRNA-D). In general, this system allows an inducible and dose-dependent regulation of shRNA expression providing an ideal tool to study conditions like DBA that are caused by haploinsufficient expression of a protein. To induce the expression of RPS19-targeting shRNAs mice were fed with doxycycline administered in drinking water. Induction of the shRNA-B construct had no effect on the erythrocyte level, hemoglobin concentration or hematocrit, although we saw a gradual elevation in mean corpuscular volume (MCV) and a decrease in platelet number. However, after 25–35 days of doxycycline treatment the mice homozygous for the shRNA-B underwent severe weight loss accompanied with a reduction in erythrocyte number and ultimately died. In contrast, shRNA-D mice exhibited decreased erythrocyte number, hemoglobin and hematocrit already after a 10 day doxycycline treatment. When the induction was kept on longer, the homozygous mice developed a more severe anemia and died around day 20, while the heterozygous mice were able to compensate the blood indices back to normal level. In spite of the differences in blood phenotypes, both models had a similar FACS phenotype revealing a profound decrease in the number of proerythroblasts, while the levels of erythroid and bipotential megakaryocytic-erythroid (MegE) progenitors were normal or increased. We also saw an accumulation of the late erythroid precursors. Proliferative potential of erythroid progenitors was evaluated at a clonal level in vitro. When MegE or CFU-E progenitors from induced mice were cultured in presence of doxycycline, the number and size of the erythroid clones were decreased compared to controls. However, when RPS19 expression was restored by culturing the cells without doxycycline, the observed proliferation defect of MegE clones was completely restored while the rescue of CFU-E clones was only partial. We also noticed that RPS19-deficient megakaryocytes appeared smaller in size compared to controls. Importantly, transduction of RPS19-deficient cells with a lentiviral vector overexpressing sequence-modified RPS19 cDNA rescued the proliferation and colony-forming defects in vitro demonstrating that the erythroid phenotype is specifically due to down regulation of RPS19. In summary, we have generated two novel mouse models for RPS19-deficient DBA that recapitulate the key erythroid phenotype seen in patients based on both FACS analysis and single-cell proliferation assays. These models will serve as a good tool to determine the molecular mechanisms responsible for DBA and also to test gene replacement therapies.