Mean Corpuscular Hemoglobin Is an Important Modulator of HbF/F-Cell and Response to Gene Therapy and Hydroxyurea in Sickle Cell Disease

Gene therapy for sickle cell anemia induced high levels of fetal hemoglobin (HbF) or a fetal-like adult hemoglobin (HbA^T87Q) accounting for the outstanding clinical results. This is because HbF and its hybrid tetramer (α2γβ^S) are excluded from the sickle hemoglobin (HbS) polymer phase preventing erythrocyte injury that drives disease pathophysiology. F-cells are erythrocytes with HbF detectable by florescence activated cell sorting. Four-6 pg. of HbF/cell is needed to detect F-cells; 9-10 pg. of HbF appear to protect the cell almost fully from polymer induced injury. The distribution of HbF concentrations among F-cells is the critical determinant of the protection HbF provides against vasoocclusive and hemolysis-associated disease complications. Different HbF distributions may explain why the clinical effects of approved gene therapies are far superior to those of hydroxyurea (HU) even when HU-treated patients achieve similar levels of HbF. We propose that this distribution is regulated by mean corpuscular hemoglobin (MCH).

The first gene therapy trials reported HbF or HbA^T87Q levels of about 40% with near normal hemoglobin concentration. MCH or MCV were not increased from baseline. Patient's clinical “cure” suggested that nearly all sickle erythrocytes had sufficient HbF to reverse the disease phenotype. To model the underlying physiology, we used the hematologic parameters reported in 2020 after CRISPR/Cas9 disruption of the BCL11A enhancer and estimated the distribution of HbF concentrations among F-cells. We imposed constraints allowing no more than 10% of cells with complete inactivation of BCL11A expression that might be accompanied by as much as 20 pg. of HbF/F-cell and 2% non-F-cells with less than 5 pg. of HbF. We showed that by achieving 40% HbF or HbA^T87Q without an increase in MCH or MCV, and with >95% F-cells, most F-cells should contain at least 10 pg. of HbF. We now contrast this outcome with HU treatment of adults who had 36.9±3.4% HbF and MCH of 41.7±4.8 pg., the latter representing a 25-30% increase from baseline. Clinically, hemolytic anemia and acute vasoocclusive episodes continued, albeit at a reduced rate. Even with HbF levels >40% and 98% F-cells some children continued having hospitalizations and ED visits. Based on this analysis we investigated gene therapy with 10% to 20% increases in MCH from normal baseline where there were 95% F-cells and >40% HbF. With a 10% increase in MCH from the baseline and the induction of HbF >40%, most gene-edited erythrocytes would have to contain 9-10 pg. of HbF/F-cell, sufficient to prevent nearly all erythrocyte damage and reverse the disease phenotype. In HU treated patients, maximum number of F-cells was set at 80% with >20<30% HbF and 85% in patients with >30% HbF. MCH increases up to 30% in HU treated patients resulted in some cells with very high HbF levels, but simultaneously a population of erythrocytes with less than 9-10 pgs. of HbF levels. Our analysis suggests that up to 50% of erythrocytes could have <5 pgs. of HbF. Such cells could be short-lived and initiate acute vasoocclusive episodes. We hypothesize that sickle cell disease therapeutics that do not increase stress erythropoiesis and maintain MCH/MCV should result in more homogeneous (pancellular) HbF/F-cell distributions with fewer cells prone to hemolysis or provoking acute vasoocclusion. Increased MCH may be detrimental and blunt the therapeutic response because of a population of cells with a low content of HbF. Normal or low MCH is also likely to explain the benign phenotype of HbS-HPFH where HbF averages 30%. MCH and HbF might provide a biomarker for HbF-induction therapeutics and be prognostically useful.

Steinberg:Vertex Pharmaceuticals: Consultancy, Honoraria; Fulcrum Therapeuticc: Honoraria; Edital Medicine: Honoraria. Brugnara:Pfizer: Consultancy, Honoraria. Kutlar:NIH/NHLBI: Other: Sickle cell disease Implementation Consortium; REACH and PUSH-UP Studies: Other: DSMB MEMBER (NHLBI); Akira Bio: Consultancy, Research Funding; Global Blood Therapeutics/ Pfizer: Other: EAC Member; blue bird bio: Consultancy, Other: DMC; Novo-Nordisk: Consultancy, Research Funding; Vertex: Consultancy, Other: Evebt adjudication committee; Novartis: Consultancy, Research Funding.