Genome editing of haemopoietic stem cells for treatment of thalassaemia

Abstract

AIM: Thalassaemia is commonly due to mutations at the beta globin (HBB) locus, and this causes transfusion dependent anaemia in severe cases. A key pathophysiological factor is the imbalance of alpha and beta globin production. This results in accumulation of excess alpha globin chains, which are toxic and cause cell death. Patients who co-inherit partial deletions of the alpha globin genes with beta thalassaemia usually have a mild phenotype and are transfusion independent. We aim to develop genome editing strategies of haemopoietic stem cells to exploit this for use as part of an autologous transplant to treat thalassaemia. METHODS: CRISPR-Cas9 was used to edit the most important enhancer of the alpha globin gene to elicit a controlled reduction in alpha globin expression. In silico methods were used to define the key sequences to delete to abrogate transcription factor binding. This allowed us to develop a strategy to disrupt single transcription factor binding sites using Cas9 ribonucleoprotein. RESULTS: Our in silico approaches allowed us to define three key transcription factor binding sites within the enhancer. We were able to achieve indel efficiencies in excess of 75% as measured by next generation sequencing. This resulted in a much more controlled reduction in alpha globin expression than was achieved by deletion of the whole enhancer. DISCUSSION: In silico prediction allows the identification of the sites within enhancers that allow genome editing to be used to reduce gene expression in a highly controlled manner.