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...

Biologic and clinical observations suggest that combining imatinib with IFN-␣ may improve treatment outcome in chronic myeloid leukemia (CML). We randomized newly diagnosed chronic-phase CML patients with a low or intermediate Sokal risk score and in imatinib-induced complete hematologic remission either to receive a combination of pegylated IFN␣2b (Peg-IFN-␣2b) 50 g weekly and imatinib 400 mg daily (n ‫؍‬ 56) or to receive imatinib 400 mg daily monotherapy (n ‫؍‬ 56). The primary endpoint was the major molecular response (MMR) rate at 12 months after randomization. In both arms, 4 patients (7%) discontinued imatinib treatment (1 because of blastic transformation in imatinib arm). In addition, in the combination arm, 34 patients (61%) discontinued Peg-IFN-␣2b, most because of toxicity. The MMR rate at 12 months was significantly higher in the imatinib plus Peg-IFN-␣2b arm (82%) compared with the imatinib monotherapy arm (54%; intention-to-treat, P ‫؍‬ .002). The MMR rate increased with the duration of Peg-IFN␣2b treatment (< 12-week MMR rate 67%, > 12-week MMR rate 91%). Thus, the addition of even relatively short periods of Peg-IFN-␣2b to imatinib markedly increased the MMR rate at 12 months of therapy. Lower doses of Peg-IFN-␣2b may enhance tolerability while retaining efficacy and could be considered in future protocols with curative intent. (Blood. 2011;118(12):3228-3235)

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34 ؉ CD38 lo nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells.

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9 -11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5-6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD in vivo. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA. (Blood. 2012;120(11):2225-2228)