β-haemoglobinopathies are inherited blood disorders that affect haemoglobin in erythrocytes, with hundreds of thousands of affected new-borns each year. While sickle cell disease results from a single-nucleotide substitution, β-thalassaemia is caused by hundreds of different mutations, making the direct repair of mutations challenging. As such, a universal therapeutic approach is widely sought.
Much of the effort has been put into reactivating the foetal γ-globin by various means in the adult cells. As an alternative, δ-globin is expressed in adult red blood cells and shares a much higher sequence homology to β-globin. As such, δ-globin, and the resultant haemoglobin A2 (HbA2), is biochemically more similar and fully functional as the conventional adult haemoglobin HbA. However, δ-globin is expressed at a low level and HbA2 only constitutes 2-3% of the total adult haemoglobin. The low expression is believed to be due to mutations at the promoter that disrupt transcription factor binding. Nevertheless, overexpression of δ-globin transgene is able to rescue the sickling phenotype of red cell in a mouse model. As such, we propose reactivating the δ-globin by editing the promoter as effective alternative therapeutics of SCD and β-thalassaemia.
In this project we employed CRISPR gene editing techniques to install an KLF1 motif at the δ-globin promoter in the immortalised HUDEP-2 cell line. Through RNP transfection along with an HDR repair template, we were able to drive the expression of δ-globin to ~160 fold in the HUDEP-2 cells compared to the control. The upregulated δ-globin transcript level is around 60% of that of β-globin. Upon differentiation, we observed the HbA2 level is upregulated to 20% by HPLC. Using NGS, we demonstrated the editing is highly efficient with over 50% HDR events. Lastly, we showed that this upregulation of δ-globin is highly dependent on the insertion site at the promoter.