Direct correction of haemoglobin E β-thalassaemia using base editors.

Area of Science:

• Genetics and Molecular Biology
• Hematology
• Gene Therapy

Background:

• Haemoglobin E (HbE) β-thalassaemia accounts for about 50% of severe thalassaemia cases globally, impacting around 30,000 newborns annually.
• This condition arises from a specific point mutation (E26K) in the HBB gene, which, when inherited with another severe β-thalassaemia mutation, leads to a serious clinical phenotype.

Purpose of the Study:

• To develop and evaluate a base editing strategy for correcting the HbE mutation in hematopoietic stem cells.
• To investigate the potential of restoring a normal or asymptomatic trait phenotype in individuals with HbE β-thalassaemia.

Main Methods:

• A base editing strategy was employed to correct the E26K mutation to either wildtype (WT) or a normal variant (Hb Aubenas, E26G).
• Editing efficiency was assessed in primary human CD34+ cells, and editing of long-term repopulating hematopoietic stem cells (LT-HSCs) was demonstrated via serial xenotransplantation in NSG mice.
• Off-target effects were profiled using CIRCLE-seq and deep targeted capture, coupled with machine learning for predicting functional impacts of candidate off-target mutations.

Main Results:

• Editing efficiencies exceeding 90% were achieved in primary human CD34+ cells.
• Successful editing of LT-HSCs was confirmed through serial xenotransplantation studies.
• Comprehensive profiling of off-target mutations and development of predictive machine learning models were accomplished.

Conclusions:

• The described base editing strategy effectively corrects the HbE mutation in hematopoietic stem cells with high efficiency.
• This approach holds promise for developing a gene therapy to treat HbE β-thalassaemia by recreating an asymptomatic trait phenotype.
• The study also established robust methods for assessing and predicting off-target effects, crucial for therapeutic safety.