Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

To understand the role of sickle cell adherence to the vascular endothelium in the pathophysiology of sickle cell anemia (SS) vasoocclusion, we have carried out a microcirculatory study utilizing the ex vivo mesocecum vasculature of the rat. A single bolus of washed oxy-normal (AA) erythrocytes or oxy-SS cells (unseparated or density-defined SS cell classes) was infused. Hemodynamic monitoring and intravital microscopic observations of the microvascular flow revealed higher peripheral resistance for SS erythrocytes and adherence of these cells exclusively to the venular endothelium but rare or no adherence of AA cells. The extent of adhesion was inversely correlated with venular diameters (r = -0.812; P < 0.00001). The adhesion of SS erythrocytes is density-class dependent: reticulocytes and young discocytes (SS1) > discocytes (SS2) > irreversible sickle cells and unsicklable dense discocytes (SS4). Selective secondary trapping of SS4 (dense cells) is found in postcapillary venules where deformable SS cells are preferentially adhered. We conclude that in the oxygenated condition, vasoocclusion can be induced by two events: (i) random precapillary obstruction by a small number of SS4 cells; (ii) increased adhesion of SS1 and SS2 cells in the immediate postcapillary venules. A combination of precapillary obstruction, adhesion in postcapillary venules, and secondary trapping of dense cells may induce local hypoxia, increased polymerization of hemoglobin S, and rigidity of SS erythrocytes, thereby extending obstruction to nearby vessels.The polymerization of hemoglobin S, which results in increased rigidity of erythrocytes, is central but may not be the exclusive causal factor responsible for the onset of a sickle cell (SS) induced vasoocclusive episode. The lack of correlation between the percent dense cells and incidence of painful crisis (1) suggests that factors other than intracellular polymerization might be involved (2). Increased adhesion of sickle cells could be an additional factor and was first demonstrated by Hebbel et al. (3,4) and later confirmed in both static and flowing systems with cultured endothelial cells obtained from human and other mammalian sources by several investigators (5-10). Nevertheless, whether the increased adherence of sickle cells to the endothelium may potentially contribute to vasoocclusion has not yet been evaluated in a living microvascular network.The erythrocytes in sickle cell anemia patients are heterogeneous in density, morphological characteristics, and function (11-15), but the relative contribution of individual sickle cell classes to adhesion and obstructive events has not been demonstrated in a perfused microvasculature.Since sickle cell vasoocclusion in a microcirculatory event, contributions from microvascular factors such as the vessel wall features, topography, and prevailing wall shear rates will be critical to the microvascular obstruction (16) and to secondary trapping or adhesion of individual sickle cell classes. No direct microcirculatory study has ...

No abstract