Direct correction of haemoglobin E β-thalassaemia using base editors

Abstract

Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-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 (β-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 90% 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 circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.

Fig. 1. Base editing of the Haemoglobin E.

a ABE editing strategy to repair the HbE mutation. The target adenine in HbE (A₅) lies at position 5 of the protospacer, with a bystander adenine (A₆) at position 6. The HbE codon (AAG) can be edited with one of three outcomes depending on which adenine has been deaminated: editing A₅ alone reverts the codon to WT (GAG), both A₅ and A₆ converts it to a normal variant that codes for β^Aubenas (GGG) and A₆ alone converts it to a previously undescribed codon (AGG). b Adenine base editing using ABE8e V106W highly efficiently converts the HbE codon to normal or a normal variant (n = 6 biologically independent samples). c Codon editing outcomes on the HbE allele and the non-target thalassaemic allele (where the thalassaemic allele was not sequenced editing at HBD was used as a surrogate) (n = 6 biologically independent samples). d Increase in β-globin expression as assessed by the β/α ratio in edited erythroid cells from patients with HbE β-thalassaemia compared to unedited controls (n = 4 biologically independent samples). e, f Haemoglobin variants in control and edited erythroid cells measured by CE-HPLC. HbE and HbA₂, and HbA and Hb Aubenas are given together as they run in the same window and cannot be resolved using CE-HPLC (n = 3 biologically independent samples; n = 2 for unedited controls). g IEF showing haemoglobin variants in control and edited cells. Hb Aubenas is clearly detected but no other novel haemoglobins are observed. All error bars represent the standard error of the mean. Source data are provided as a Source Data file.

Fig. 2. Editing of long term haemopoietic stem cells and off target effects.

a Engraftment of edited human cord blood HSCs in NSG mice in primary and secondary transplants (n = 3 biologically independent samples). b Editing efficiencies for the cells prior to transplantation and following the primary and secondary transplants (n = 3 biologically independent samples). These cells were edited with ABEmax, due to the lag time on these experiments and this has lower editing efficiencies than the ABE8 editors, which are also reflected in the in vitro (Supplementary Fig. 2d). c Location of all potential off-target effects identified by CIRCLE-seq combined with two in silico approaches (Cas-OFFinder and CRISPOR). d Targeted sequencing through oligonucleotide capture at the top 250 sites identified, which confirmed likely low level off target editing at 70 sites. e Predictions from DeepHaem of the effects of all possible off-target edits in the non-coding genome on chromatin accessibility. f MA plots based on ATAC-seq data comparing WT vs base edited cells, showing that there were no significant differentially accessible peaks detected (DESeq2, alpha 0.05). Source data are provided as a Source Data file.