Ex Vivo CRISPR-Based Sickle Cell Modeling

Abstract

Autologous hematopoietic stem cell transplantation combined with genome editing holds promise for curing patients with hemoglobinopathies such as sickle cell anemia (SCA). Hence, experimental models to assess the impact of genome editing modalities (i.e. nucleases, base editors, and prime editors) are important tools that could complement the development of safe curative approaches for hemoglobinopathies. The culture and ex vivo engineering of red blood cells (RBC) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements in the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis - i.e., the terminal maturation of reticulocytes into erythrocytes - so that the RBCs produced ex vivo resemble mature circulating blood cells. Completing their maturation will also be required in some instances to recapitulate specific RBC phenotypes. Here we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in feeder-cell-free and animal-protein-free conditions. The approach improves post-enucleation cell integrity and survival, and enables subsequent storage of cRBCs for up to 42 days in classical nutritive solution conditions, without any specialized equipment. We next used a non-viral, selection-free CRISPR-Cas9-based genome editing to efficiently insert the HBB-βs mutation at the endogenous locus in HSPCs. From this process, mature RBCs with crescent shape, a hallmark of SCA, where readily obtained. The sickle phenotype was recapitulated at high frequencies - comparable to the levels of biallelic HBB-βs modification obtained. Importantly, the sickle shape was observed exclusively in RBCs that underwent the complete cRBC culture protocol - consistent with the deformability properties acquired during the late-stages of RBC maturation. Moreover, a β-globin variant forming an in-frame, 9bp deletion (HBBΔ9), was frequently observed following the targeting of HBB. Contrary to usual editing by-products creating β-globin knockout alleles, this variant was expressed in erythroid cells and formed a hemoglobin tetramer, suggesting that its potential impact should be further studied in the context of clinical trials relying on this editing strategy. We foresee that this system, combining efficient genome editing of HSPCs and production of mature cRBCs, will support the functional study of various erythroid traits and could be exploited to chaperone the development of gene-modified HSPC transplants for hemoglobinopathies.