900. Development of Models for RPS19 Deficient Diamond-Blackfan Anemia Using Regulatable Expression of siRNA Against RPS19
Authors: Koichi Miyake, Johan Flygare, Thomas Kiefer, Johan Richter, Isao Hamaguchi, Maciej Wiznerowicz, Didier Trono, Stefan Karlsson
Journal: Molecular Therapy (2004)

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. We have previously shown that RPS19 transgene overexpression improves erythroid development and proliferation in RPS19 deficient DBA cells (Blood 100: 2724–31, 2002; Mol Ther 7: 613–22, 2003). It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors and a model for RPS19 deficient DBA is not available for development of gene therapy. Therefore, to establish a model system for RPS19 deficient DBA, a lentiviral vector was generated where the GFP gene is controlled by an EF1a promoter and an expression cassette containing a small interfering RNA (siRNA) against RPS19 controlled by a polIII promoter and a tet operator element (siRPS19). The control vector is analogous except it expresses an siRNA against a random sequence. Suppression of expression of RPS19 was detected in siRPS19 transduced primary CD34+ cells by quantitative RT-PCR and Northern blot analysis. BFU-E and CFU-G/M colonies from siRPS19 transduced CD34+ cells were decreased compared to control vector transduced cells. Suppression of cell growth and erythroid differentiation was also observed in erythroid liquid cultures. The hematopoietic and erythropoietic phenotype of these knockdown BM cells mimics the that of BM cells from DBA patients. To establish a RPS19 deficient cell line model, inducible expression of the siRNA was achieved by co-transduction of another lentiviral vector containing the tetracycline repressor gene and red fluorescent protein (RFP) gene (J Virol 77, 8957–61, 2003). Human erythroid cell lines, TF-1 and UT-7 cells were co-transduced with siRPS19 or the control vector and the transactivator vector. To generate inducible cell lines, RFP positive and GFP negative populations were sorted. When these cells are incubated with doxycycline (Dox), more than 97% cells expressed GFP. We observed suppression of cell growth and colony formation in siRPS19 transduced cells but not in the control vector transduced cells. Significant suppression of erythroid differentiation was observed by analysis of glycohporin A and evaluation of hemoglobin content. Cell cycle analysis showed that the percentage of cells in the G0/G1 phase increased and apoptotic cells detected by Annexin-V binding analysis were also increased in siRPS19 transduced cells. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation, apoptosis and erythroid differentiation. This regulatable expression system of siRNA has therefore been successfully used to generate a cell line model for DBA. We asked whether transgene overexpression of RPS19 can rescue growth suppression in these DBA model cell lines, using engineered RPS19 cDNA which is not affected by the siRNA knockdown but produces normal RPS19 protein (eRPS19). Improvement of cell growth and colony formation was detected in eRPS19 transduced cells. These model systems can be used to identify the molecular mechanism in RPS19 deficient DBA and for development of gene therapy.