Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene. (Blood. 2011;118(23): 6087-6096) IntroductionDiamond-Blackfan anemia (DBA; Online Mendelian Inheritance in Man [OMIM] no. 105650) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. 1 During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. 2 However, DBA is a developmental disease because ϳ 30%-47% of patients show a broad spectrum of physical abnormalities including craniofacial, heart, and upper limb malformations, and short stature. 1,[3][4][5] All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for ϳ 55% of DBA cases. [6][7][8][9][10][11] Twenty-five percent of the patients have mutations in a gene coding for ribosomal protein S19 (RPS19) making it the most common DBA gene. 6 The majority of the mutations completely disrupt the expression of the RPS19, whereas the rest are missense mutations interfering with the assembly of RPS19 into 40S ribosomal subunits. [12][13][14] All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as the basis for disease pathology.Despite of the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation 15 have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors with substantially reduced clonogenic output that correlates with the age of the patient. 2,16-19 A similar age-dependent decrease in granulocyte-macrophage progenitor (GMP) numbers has been reported. 20 Although present at normal freq...

Gene therapy cures the anemia and lethal bone marrow failure in a mouse model of RPS19-deficient Diamond-Blackfan anemia

“…Administration of doxycycline to the recipients with D/D BM results in acute and lethal BM failure, while recipients with D/+ BM develop a mild, chronic phenotype (Online Supplementary Figure S1). 21 Since the D/D mice develop lethal BM failure shortly after doxycycline administration, we chose this model to rigorously test whether gene correction can rescue the lethal phenotype and cure the disease. The D/+ model was used in parallel to test the vectors upon Rps19 haploinsufficiency, 21 a condition that is not lethal and allows long-term monitoring of experimental animals.…”

“…21 Briefly, this model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of the Collagen A1 gene ( Figure 1A). Experimental animals were bred to be either heterozygous (D/+) or homozygous (D/D) for the shRNA in order to generate two models with intermediate or severe Rps19 deficiency, respectively ( Figure 1B).…”

“…21 Briefly, this model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of the Collagen A1 gene. Rps19 deficiency was induced by feeding the mice with doxycycline-containing food pellets (200 mg/kg doxycycline; Bio-Serv), with the exception of the recipients transplanted with D/D BM which were given doxycycline in the drinking water (2 mg/mL doxycycline; SigmaAldrich) supplied with 10 mg/mL sucrose (Sigma-Aldrich) for the first 2 weeks of induction.…”

“…To genetically correct the Rps19 deficiency, we developed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the internal SFFV promoter, followed by IRES and GFP (SFFV-RPS19) ( Figure 1C). 21 The codon-optimized RPS19 cDNA was further modified to prevent its recognition and downregulation by the Rps19-targeting shRNA used. A similar vector without the RPS19 cDNA was used as a control vector (SFFV-GFP).…”

Ribosome biogenesis dysfunction leads to p53-mediated apoptosis and goblet cell differentiation of mouse intestinal stem/progenitor cells

“…9,10 Studies on cellular and animal models suggest that unscheduled upregulation of p53 may account for many clinical symptoms associated with ribosomopathies. [11][12][13][14][15][16] There are now evidences that ribosome biogenesis dysfunction also triggers p53-independent mechanisms. [17][18][19] Because bone marrow defects is a frequent clinical manifestation of ribosomopathies, most studies focused on the hematopoietic tissue and less is known about the impact of ribosome biogenesis dysfunction in other organs.…”