Reduced-intensity conditioning (RIC) regimens have the potential to decrease toxicities related to hematopoietic stem cell transplantation (HCT) in patients with sickle cell disease (SCD) and thus make HCT a more acceptable therapeutic option for this group of patients. We report the results of 7 patients enrolled on a study to evaluate safety and efficacy of HCT using bone marrow from an HLA matched sibling donor following an RIC regimen for patients with high-risk SCD. The conditioning regimen consisted of busulfan, fludarabine, equine antithymocyte globulin, and total lymphoid irradiation with shielding of the liver, lungs, heart, and gonads on day 1. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mycophenolate mofetil. The regimen was well tolerated, and all patients had hematopoietic recovery. Six of 7 patients are stably engrafted off immunosuppression and without sickle cell-related symptoms at 2 to 8.5 years after HCT. Consistent with the complete resolution of SCD related symptoms observed in the 6 engrafted patients, erythropoiesis of complete or predominantly donor origin was detected by red blood cell-specific chimerism assays, despite their having persistent mixed chimerism in the mononuclear and lymphoid compartments. These findings demonstrate the curative potential of allogeneic HCT after an RIC regimen in patients with SCD.

IntroductionSickle cell disease (SCD) is a common and severe disorder in which a missense mutation in the ␤-globin gene results in polymerization of hemoglobin S under deoxygenating conditions, leading to chronic peripheral hemolysis and intravascular vaso-occlusion. Less fully appreciated is the extent to which sickle hemoglobin in committed erythroid precursor cells might also affect earlier events in erythropoiesis. Improved understanding of the impact of SCD on intramedullary erythropoiesis is relevant to the development of new therapeutic strategies for this chronic debilitating disease.One approach to directly assess the contribution of ineffective erythropoiesis to the pathophysiology of sickle cell disease is to analyze erythropoiesis in sickle cell disease patients who have undergone matched related donor allogeneic stem cell transplantation (SCT) following a nonmyeloablative (NMA) conditioning regimen. A variety of nonmyeloablative conditioning regimens have been developed to facilitate engraftment of donor stem cells and reduce the treatment-related toxicity of hematopoietic SCT. [1][2][3][4] In this nonmyeloablative setting, host hematopoiesis frequently persists following transplantation and coexists with engrafted donor cells within the marrow. Depending on the underlying disease and the intensity of the conditioning regimen, stable mixed hematopoietic chimerism occurs frequently and can persist for long intervals. Mixed hematopoietic chimerism has been well documented after allogeneic hematopoietic SCT for SCD, and this provides a unique opportunity to perform a direct side-by-side comparison of normal and sickle erythropoiesis in vivo. 5,6 To distinguish between donor and host erythropoiesis, we previously developed methods to specifically measure erythroid lineage chimerism in both nucleated and non-nucleated erythroid elements. Although molecular methods for measurement of donor chimerism are well established, they are based on analysis of genomic DNA, and thus measure nucleated-cell chimerism, which is predominantly leukocyte derived in peripheral blood. 7-9 Therefore, in addition to measuring overall engraftment by analysis of genomic DNA, the present studies also use two alternate methods to distinguish between donor and host-derived erythropoiesis. One method relies on direct enumeration of donor-derived erythroid precursors on paraffin-imbedded marrow sections following staining with a donor-specific marker, such as red blood cell (RBC) isohemagglutinin antigens in an ABO disparate setting. A second method uses a novel molecular assay, RNA ␤-globin pyrosequencing, which directly measures erythroid lineage-specific chimerism from total RNA by quantifying the amount of RBC-specific RNA transcripts that bear a donor or host-specific single nucleotide polymorphism (SNP) or mutation. 6 In addition to using the sickle mutation as an informative ␤-globin SNP, we also previously identified an alternate ␤-globin SNP, 3H3, that occurs at high minor allele frequency. 6 Since ␤-globin RNA is expressed...