Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing.

Joseph Hawksworth,Ashley M Toye,Kongtana Trakarnsanga,Johannes GG Dobbe,Marjolein Meinders,David J Anstee,Jan Frayne,Deborah E Daniels,Timothy J Satchwell,Fiona Regan,Kate J Heesom,Nicole M Thornton,Geert J Streekstra,Marieangela C Wilson

doi:10.15252/emmm.201708454

Abstract

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle‐cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR‐mediated genome editing of an immortalised human erythroblast cell line (BEL‐A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K 0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof‐of‐principle demonstration of combinatorial CRISPR‐mediated blood group gene editing to generate customisable or multi‐compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

Highlights

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sicklecell disease
In order to direct the design of a bespoke or customised RBC phenotype that would be able to service existing unmet clinical transfusion needs within the blood transfusion service in England (NHSBT), a survey was conducted to collate instances where rare or problematic transfusion requirements were identified and in which the requirement for matched blood could not be fulfilled or resulted in no remaining store for subsequent patients
We decided to pursue the generation of RBCs completely deficient in surface expression of glycophorin B (GPB), Rh, Kell, Duffy and of the Bombay phenotype using CRISPR–Cas9 editing for gene knockout in a recently published immortalised erythroblast cell line: BEL-A (Trakarnsanga et al, 2017)

Summary

Introduction

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sicklecell disease. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Duffynull), GPB (SÀsÀUÀ). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements

Methods

Results

Conclusion