Modeling Diamond-Blackfan Anemia in the Mouse: Disease Pathogenesis and Evaluation of Novel Therapies

Abstract: Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia that presents early in infancy. The main hematopoietic symptoms include macrocytic anemia with reticulocytopenia and selective absence of erythroid precursors in a normocellular bone marrow. In addition to the hematopoietic symptoms, DBA is characterized by the presence of physical abnormalities and cancer predisposition. Mutations in genes encoding ribosomal proteins have been identified in approximately 60-70% of DBA patients. Among these genes, ribosomal protein S19 (RPS19) is the most common disease gene (25% of the cases). All reported patients are heterozygous for the mutations and in most cases the mutations are predicted to result in haploinsufficiency of the respective ribosomal protein. Knowledge about DBA pathophysiology has been limited due to lack of appropriate animal models. The aim of this thesis was to generate mouse models for RPS19-deficient DBA and to use these models to study DBA pathophysiology and to evaluate novel therapies. In article I we generated and characterized novel mouse models for RPS19-deficient DBA. These models contain a doxycycline-regulatable Rps19-targeting shRNA that allows an inducible and graded downregulation of Rps19. We demonstrate that Rps19-deficient mice recapitulate many of the phenotypic and molecular features seen in patients, and are therefore well suited for the evaluation of novel therapies. In article II we used these mouse models to assess the therapeutic potential of the amino acid L-leucine in the treatment of DBA. We show that L-leucine treatment improves the anemia and alleviates the stress hematopoiesis in Rps19-deficient mice. In article III we evaluated the therapeutic efficacy of gene therapy using our mouse models for RPS19-deficient DBA. Using lentiviral vectors we demonstrate that enforced expression of RPS19 cures the lethal bone marrow failure in recipient mice transplanted with Rps19-deficient bone marrow cells. Taking together the generated mouse models provide novel tools to study DBA pathophysiology and to evaluate novel therapies. Our studies strengthen the rationale for clinical trials of L-leucine and provide a proof of principle for the development of clinical gene therapy in the treatment of RPS19-deficient DBA.