Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh null syndrome. The cultured red cells generated recapitulate the major alterations of native Rh null cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh null syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh null RBCs. Together with the effects of RhAG forced expression in Rh null progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine. Am. J. Hematol. 88:343-349, 2013. V C 2013 Wiley Periodicals, Inc.
IntroductionLarge-scale approach to generate ex vivo mature erythroid cells sharing all functional characteristics with native adult RBCs [1] is well suited to investigate erythroidrestricted human disorders [2], particularly if coupled with lentiviral modification to modulate specific gene expression. The purpose of this study was to provide a proof of principle to reproduce a phenotype with functional alterations of RBC. Accordingly, we choose to mimic blood group Rhdeficiency (also called Rh null ), a rare autosomal recessive disorder affecting the RBC membrane in humans, which is associated with a hemolytic anemia of varying severity, abnormal red cell shape (stomato-spherocytosis), and osmotic fragility [3,4]. RBCs from Rh-deficient individuals basically lack Rh and LW blood group antigens, and Ss antigens expression is reduced [5]. Biochemical analysis have shown that these RBCs are severely deficient in the Rh complex, an oligomeric assembly of two erythroidspecific proteins, Rh (Rhesus), and RhAG (Rh-Associated Glycoprotein), to which accessory chains such as ICAM-4 (carrier of LW antigens), CD47 and glycophorin B (carrier of Ss antigens) are linked by noncovalent bonds. Molecular studies have revealed that Rh-deficiency is caused by different mutations that occur in either the RHAG or RH locus, but the genes encoding the accessory chains remain unaltered [3,4]. This is consistent with the inheritance of Rh null phenotypes by two distinct genetic backgrounds. The "amorph" type is caused by homozygosity for mutant alleles at the RH locus (1p34-36) and the "regulator" type by homozygosity (or composite heterozygosity) for mutants alleles at the genetically independent RHAG locus (6p12-21) encoding the RhAG protein subunit [6]. These findings suggest that when either the Rh...
