Optimising Techniques for CRISPR/Cas9 Genome Editing as Treatment for Haemoglobinopathies

The problem: Sickle cell disease and β (beta) thalassaemia are among the most common monogenic disorders worldwide, causing significant morbidity and early mortality in affected patients. Both are caused by mutations (including deletions in the case of β thalassaemia) in the β globin gene (HBB), resulting in abnormal or reduced haemoglobin, respectively. The solution: Over recent decades, genome modification strategies have been developed with the aim of providing long-term amelioration of these conditions without the need for a bone marrow (BM) transplant. One such strategy involves targeted genome editing with the aim of reinducing foetal haemoglobin, which is known to improve symptoms. Hypothesis: HbF reinduction can be maximised by application of a multiplex genome editing strategy targeting both HBG promoter and BCL11A erythroid enhancer regions. Methods: CRISPR/Cas9 editing strategies targeting the HBG-113 and BCL11A-ee regions individually, or together either simultaneously or sequentially, were compared in terms of editing efficiencies (by TIDE analysis (Tracking of Indels by Decomposition) after Sanger sequencing) and HbF reinduction (by flow cytometry and high-performance liquid chromatography). Translocation events were measured qualitatively and then quantitatively by standard polymerase chain reaction (PCR) and digital droplet PCR respectively. Experiments were conducted in vitro using human haematopoietic stem and progenitor cells (HSPCs), and in vivo using the humanised mouse model. Results and Conclusion: Maximum HbF reinduction was observed with the sequential dual editing strategy, which also demonstrated an acceptable cytotoxicity profile and engraftment in vivo. However, dual editing was associated with the development of chromosomal translocations, which persisted in vivo and preclude the further development of such a multiplex editing strategy in this context.