3057 – MATURATION OF EX VIVO-CULTURED HUMAN ERYTHROCYTES AND SICKLE CELL DISEASE MODELLING USING CRISPR-CAS9

Abstract

In vitro-produced red blood cells (RBCs) could be used as cellular models for studying erythroid diseases or for providing reagent cells expressing specific blood group antigens. However, the non-fully mature state of cells achieved with current protocols is a significant barrier to their potential clinical applications. We aimed at modifying existing differentiation protocols in order to improve final erythroid maturation from human hematopoietic stem and progenitor cells (HSPCs). If successful, the method would facilitate the generation of mature erythrocytes, their preservation and allow phenotype evaluation. As a proof-of-concept using gene editing, we chose to model the "sickling" phenotype of sickle cell disease (SCD). After significant improvements of known methods, we devised an optimized culture medium for the efficient erythroid differentiation and maturation of adult HSPCs. The differentiation protocol yielded up to 95% of enucleation. More importantly, reticulocyte maturation yielded up to 80% of cells molecularly defined as mature erythrocytes devoid of any CD71 marker expression, total RNA, and mitochondria. Next, we have exploited a virus-free CRISPR-Cas9-based editing strategy to effectively introduce genetic variants in HSPCs combined with the erythroid differentiation protocol. We achieved 40% of precise gene modification within the beta-globin gene, with nearly 20% of the cells harboring the mutation on both alleles. Interestingly, when incubating cells in hypoxic conditions, we were able to visualize the "sickling" phenotype of the mature genetically-modified erythrocytes. In summary, we have successfully combined high-efficiency CRISPR-Cas9 editing of HSPCs with in vitro erythroid maturation to recapitulate SCD in vitro. This work can be applied to other genetic variants and cell types for the study of clinically relevant mutations.