Sickle cell disease results from a homozygous missense mutation in the β-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling β-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling β-globin remained high (approximately 50% of β-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

Patients with polycythemia vera (PV) have a JAK2 (a cytosolic tyrosine kinase) mutation and an increased risk of vascular thrombosis related to red blood cell (RBC) mass and platelet activation. We investigated functional RBC abnormalities that could be involved in thrombosis. RBC adhesion to human umbilical vein endothelial cells (HUVECs) was measured by a radiometric technique and in a flow system by video microscopy, and adhesion molecule expression was determined using specific antibodies (against CD36,

Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R-RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

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Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.