Direct correction of haemoglobin E / beta-thalassaemia using base editors

Haemoglobin E (HbE)/ß-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE/ß-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamate ? GAA; lysine, E26K), and any mutation causing severe ß-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (ß-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 85% in primary human CD34+ cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of CIRCLE-seq and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations. Overall design: Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype.