Poster Presentation Australian Society for Medical Research Annual Scientific Meeting 2016

A stem cell model for analysis and manipulation of Human globin gene switching; towards a cure for ß-Thalassemia. (#116)

Sarah T Diepstraten 1 , Chee kai Chan 2 , Jim Vadolas 3 , Adam H Hart 1
  1. La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
  2. Department of Biomedical Sciences, Nazarbayev University, Astana, Kazakhstan
  3. Murdoch Children’s Research Institute, Melbourne, Victoria, Australia

Over 7% of the global population are carriers of haemoglobinopathies. However, despite their prevalence, no effective treatments exist for β-thalassemia and sickle-cell anaemia beyond lifelong blood transfusions. The globin genes are expressed sequentially during development and nature has an inbuilt compensation mechanism when the adult β-globin gene has been deleted: the reactivation of fetal γ-globin, resulting in the benign condition hereditary persistence of fetal haemoglobin. The amelioration of the symptoms of β-globinopathies in the rare cases where these disorders are co-inherited has inspired decades of research into the molecular regulation of haemoglobin switching, with the aim of inducing therapeutic γ-globin expression. In spite of this, the mechanism remains unsolved, partly due to the lack of suitable cell and animal models available. Our lab has developed a novel system for studying and manipulating haemoglobin switching in vitro. We have generated mouse embryonic stem cell lines harbouring a 200kB bacterial artificial chromosome (BAC) containing the full human β-globin locus, including its upstream regulatory regions1. The BAC contains GFP and DsRed reporter constructs under control of the β-globin and γ-globin promoters, respectively. During haematopoietic differentiation of our embryonic stem cell lines, we have shown sequential expression of the DsRed and GFP reporter genes by fluorescence microscopy flow cytometry and transcriptional analysis, confirming that our system recapitulates haemoglobin switching during blood ontogeny. Using this system, we are unlocking the mechanisms of blood development by employing transcriptomics to identify new candidate genes for globin switch regulation and are screening chemical modifiers and CRISPR mediated gene therapies for the reactivation of y-globin expression to identify new treatment avenues for β-globinopathies.

  1. Chan KS, Xu J, Wardan H, McColl B, Orkin S, Vadolas J. (2012) Generation of a genomic reporter assay system for analysis of g- and ß-globin gene regulation FASEB J. 26(4):1736-44.