Group members: Pekka Jaako, Karin Olsson and Stefan Karlsson
Diamond-Blackfan anemia (DBA) is a congenital anemia characterized by reduced frequency of erythroid progenitor cells in the bone marrow. Children are usually diagnosed within the first year after birth. Some patients respond to corticosteroid treatment, while twenty percent recover spontaneously. A second alternative treatment is bone marrow transplantation (BMT). While BMT is curative when successful, most patients do not have access to a suitable BMT donor within their family and the procedure frequently causes severe morbidity and lethal complications may occur. Forty percent of DBA patients are dependent on frequent blood transfusions, which eventually cause iron overload and premature death, resulting in a median survival of 31 years.
In 1999, our collaborator in Uppsala, Niklas Dahl, identified RPS19 as the first DBA disease gene, making DBA the first human disease linked to a mutation in a ribosomal gene. Currently, at least 10 additional DBA genes have been identified that all encode for ribosomal proteins and together these genes cover approximately 50 % of DBA cases. The fact that all DBA genes encode for ribosomal proteins strongly suggests that defective ribosomal synthesis or function is responsible for the DBA pathogenesis. Moreover, other congenital syndromes, such as Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage hair hypoplasia and Treacher-Collins syndrome, have been linked to defective ribosome biogenesis. In addition, in the 5q-syndrome, acquired haploinsufficiency for RPS14 results in a highly similar erythroid phenotype to DBA.
In order to understand DBA pathogenesis it is important to locate the exact stage of erythroid defect. Early in vitro studies show that DBA patients have normal or
decreased number of BFU-E and CFU-E erythroid progenitors, although both colony types are smaller in size compared to controls. In addition, some studies have also found a decrease in the number of megakaryocytic and myeloid colony-forming progenitors. One convincing study demonstrated that the expansion of DBA erythroid progenitors was comparable to controls, while their terminal EPO-dependent differentiation was compromised. However, in order to confirm these findings, animal models recapitulating the erythroid defect of DBA must be generated.
The generation of models for RPS19-deficient DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. A mouse model for RPS19 deficiency has been made which exhibits no hematologic phenotype in the heterozygous state. However, the RPS19 null embryos die before implantation (Mattsson et al, 2004).
RNA interference (RNAi) provides an alternative means to downregulate gene
expression. We have established human erythroid leukemia cell lines that
express RPS19-targeting short interfering RNAs (siRNA) upon doxycycline
induction (Miyake et al, 2005). Moreover, we have demonstrated further that RNAi could be used to induce DBA?like phenotype in human CD34+ hematopoietic cells (Flygare et al, 2005).
Since RNA interference -mediated RPS19 downregulation results in a DBA phenotype in human cells in vitro, we have used the short hairpin RNA (shRNA) technology to create an RPS19-deficient mouse models for DBA. In these models, the expression of RPS19-targeting shRNAs is induced by doxycycline administration allowing a reversible and dose-dependent RPS19 downregulation.
The purpose of this research program is to ask what molecular mechanism is responsible for the hematopoietic and erythroid deficiencies, i.e. how the RPS19 deficiency causes the hematopoietic phenotype. Secondly, we are developing a gene therapy protocol for RPS19-deficient DBA.
Our unpublished studies demonstrate that the location of the main erythroid defect is at the preCFU-E/CFU-E stage or the transition from CFU-E to Pro-Erythroblasts as shown in the figure (Jaako et al, unpublished)
By characterizing the mouse models we want to pinpoint the location of the erythroid defect within the hematopoietic hierarchy, and study the underlying molecular mechanisms leading to the disease at this particular stage. We hope these studies will lead to the identification of genes and pathways that play an important role in DBA pathogenesis, and could provide a basis for designing novel therapies.
Our laboratory has shown that it is possible to partially correct the defect in RPS19 deficient DBA patient erythroid progenitor cells by using lentiviral and retroviral vectors to transfer the RPS19 gene into bone marrow progenitors from DBA patients in culture (Hamaguchi et al, 2002; Hamaguchi et al, 2003). Furthermore, we have shown that gene corrected CD34+ bone marrow cells from DBA patients have improved engraftment and erythroid development following transplantation (Flygare et al. 2008). Currently we are generating new RPS19 expressing vectors in collaboration with Christopher Baum, and testing these vectors in the mouse models.
• David Bryder, Lund University
• Steven Ellis, University of Louisville, USA
• Johan Flygare, Whitehead Institute for Biomedical Research, Cambridge, USA.
• Marieke von Lindern, Erasmus Medical Centre, Rotterdam, The Netherlands.
• Christopher Baum, Hannover Medical School, Germany
Hamaguchi, I, Ooka, A, Richter, J, Dahl, N, Karlsson, S. Gene transfer improves erythroid developent in ribosomal protein S19-deficient Diamond-Blackfan anemia. Blood. (2002) vol. 100(8) pp.2724-31.
Hamaguchi I, Flygare J, Nishiura H, Brun AC, Ooka A, Kiefer T, Ma Z, Dahl N, Richter J, Karlsson S. Proliferation deficiency of multipotent hematopoietic progenitors in ribosomal protein S19 (RPS19)-deficient diamond-Blackfan anemia improves following RPS19 gene transfer. Mol Ther. (2003) May; 7(5 Pt 1) pp.613-22.
Matsson H, Davey EJ, Draptchinskaia N, Hamaguchi I, Ooka, A, Levéen P, Forsberg E, Karlsson S, Dahl N (2004). Targeted disruption of the ribosomal protein S19 is lethal prior to implantation. Mol Cell Biol. (2004) vol 24(9) pp. 4032-7.
Miyake et al. Development of cellular models for ribosomal protein S19 (RPS19)?
deficient diamond?blackfan anemia using inducible expression of siRNA against
RPS19. Mol Ther (2005) vol. 11(4) pp. 627?37
Flygare et al. Deficiency of ribosomal protein S19 in CD34+ cells generated by
siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan
anemia. Blood (2005) vol. 105(12) pp. 4627-34
Flygare J, Karlsson S (2007). Diamond Blackfan anemia: Erythropoiesis lost in translation. Blood 109: 3152-3160.
Flygare J, Olsson K, Richter J, Karlsson S. Gene therapy of Diamond-Blackfan anemia 34(+) cells leads to improvederythroid development and engraftment following transplantation. Exp Hematol (2008) vol 36(11) pp. 1428-35.
Miyake K, Utsugisawa T, Flygare J, Kiefer T, Hamaguchi I, Richter J, Karlsson S (2008). RPS19 deficiency leads to reduced proliferation and increased apoptosis but does not affect terminal erythroid differentiation in a cell line model of Diamond Blackfan anemia. Stem Cells 26:323-329.
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Last modified: 2010-07-30
