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.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

“…These distinct characteristics of HSCs may underlie the unexpected genotoxic outcomes of CBE and PE in HSCs 43 . In this context, the sole inhibition of p53 using p53DD represents the optimal strategy for therapeutic gene editing of HSCs in conditions such as sickle cell anemia 58 or β-thalassemia 59 , both of which are subjects of ongoing clinical trials (NCT05456880). The encouraging outcomes in gene-corrected HSCs with base editing have extended the applicability to other cell types such as hepatic progenitors 60 , keratinocytes 61 , and so on.…”

The research progress of correcting pathogenic mutations by base editing

“…or a normal variant hemoglobin (E26G). Ninety percent of primary human CD34+ cells were edited, and long-term repopulating hematopoietic stem cells (HSCs) were confirmed through xenotransplantation in NSG mice [39]. Editing the mutation in HBB represents a widespread strategy for treating SCD or β-thalassemia.…”

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

“…In Table 2 a few examples are also reported discussing the newly developed prime and base editing of β-thalassemia mutations ( Liang et al, 2017 ; Zhang et al, 2022 ; Badat et al, 2023 ; Hardouin et al, 2023 ). These novel approaches are expected to limit genotoxic of the gene editing procedures, especially those due to homology-directed repair (HDR), activated following the introduction of DNA double-strand breaks (DSB) during the conventional CRISPR approach ( Liang et al, 2017 ; Komor et al, 2018 ; Zeng et al, 2020 ; Collantes et al, 2021 ; Zhang et al, 2022 ; Badat et al, 2023 ; Carusillo et al, 2023 ; Hardouin et al, 2023 ).…”

“…In respect to the limitations of the CRISPR-Cas9 based procedures, HDR is inherently genotoxic in somatic cells; therefore, the recent development of base editing procedures that edit a target base without requiring the generation of DSB or HDR offers an alternative and very appealing approach ( Liang et al, 2017 ; Zhang et al, 2022 ; Badat et al, 2023 ; Hardouin et al, 2023 ). For instance, Hardouin et al developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor ( Hardouin et al, 2023 ).…”

“…In addition, we have to mention that novel gene-editing strategies (for instance base-priming and base-editing), characterized by lower level of genotoxicity, are available and are expected to be extensively studied and validated in the next future ( Liang et al, 2017 ; Zipkin, 2019 ; Collantes et al, 2021 ; Zhang et al, 2022 ; Badat et al, 2023 ; Hardouin et al, 2023 ). In our opinion, this last development of gene editing protocols is of great interest to propose combined treatments based on gene editing (or multiplexed gene editing) with HbF inducers.…”