https://orcid.org/0000-0002-7489-9894
In this issue of Blood Advances, Leonard et al1 show that red blood cell (RBC) labeling with biotin enables assessment of RBC survival in patients with sickle cell disease (SCD) in 2 novel populations, people with HbSC and HbSβ+ disease, and people with SCD before and after hematopoietic stem cell transplantation (HSCT). Methods to measure the minimum necessary therapy required for curative treatment success are critically important for several applications, such as for the use of reduced intensity bone marrow ablation for those with fully matched donors undergoing HSCT, for mismatched and haploidentical HSCT, and with the recent approvals of gene therapy. This pilot study demonstrates that biotin labeling of RBCs enables measurement of RBC survival and that RBC survival correlates multiple markers of hemolysis and rates of RBC sickling after deoxygenation, making it a technique with potentially wide applications across multiple curative therapies for SCD.
SCD is an inherited monogenic disorder that affects millions of people worldwide. It is caused by a mutation in the β-globin protein which causes hemoglobin to polymerize when deoxygenated, resulting in hemolysis and shortened RBC survival. Over the last decades, HSCT emerged as a curative therapy for individuals living with SCD. However, its use is limited by the toxicities associated with myeloablative preparation for HSCT and challenges in finding matched donors. Recent research supports that fully matched and possibly even haploidentical transplants can be done without full myeloablative conditioning and that only 20% of donor myeloid chimerism may be adequate to reverse the “sickling” phenotype in individuals who underwent transplantation.2-4 Therefore, questions remain on how to optimize the use of reduced intensity bone marrow conditioning pre-HSCT.
Now, with the recent approval of 2 gene therapies which also require myeloablation, additional research will need to determine the optimal and acceptable pretherapy conditioning regimen as well as the level of gene modification required for successful reversal of the sickling phenotype.5,6 Currently, fully myeloablative conditioning is used because a less-intensive pretherapy myeloablative conditioning regimen risks lower proportions of gene-edited RBC lineages and possibly higher rates of secondary cancers owing to DNA damage without cell death. Accurate estimation of minimum necessary therapy will be essential for the expansion of gene therapy in SCD.
Biotin is a water-soluble vitamin which was initially developed as a safer alternative to radiolabeling of RBCs and has been used for decades to tag RBCs in SCD research.7 RBCs can be extracted, washed, tagged with biotin, and reinfused; subsequent blood draws determine RBC lifespan by measuring the proportion of biotin-tagged cells. Biotin tagging in sickle-cell hemoglobin (HbSS) helped elucidate foundations of SCD pathophysiology including demonstration of decreased RBC survival and the increased survival of SCD RBCs containing fetal hemoglobin (HbF).8
This study by Leonard et al is a pilot study using biotin tagging to evaluate RBC lifespan in small but novel populations of individuals including non-HbSS genotypes of SCD (HbSβ+, HbSC) and individuals with SCD before and after fully matched and haploidentical HSCT with both full and mixed chimerism after transplant.
Previous modeling of acceptable levels of chimerism did not look directly at RBC survival.2,9 Studies such as this one allow direct measurement of this parameter of interest. The authors confirmed differences in RBC lifespan between a group comprising patients who underwent transplant (including 1 patient with donor myeloid chimerism of only 28%), individuals with HbAS, and healthy controls by comparing them to a group with multiple genotypes of SCD who had not undergone HSCT. The differences between these groups were also apparent in several laboratory markers, including hemoglobin and markers of hemolysis such as total bilirubin and lactate dehydrogenase, and these markers correlated with RBC survival. The authors showed that over time, biotin-tagged cells were more likely to contain non-HbSS in both patients with pretransplant and posttransplant mixed chimerism, indicating a shorter lifespan for RBCs with HbS. In patients who underwent transplantation with full myeloid chimerism, proportions of HbS and HbA remained relatively stable over time. Finally, the authors showed during deoxygenation that pretransplant HbSS RBCs sickled rapidly, whereas full-chimerism posttransplant RBCs demonstrated minimal sickling, similar to HbAS cells. Interestingly, mixed (28%)-chimerism RBCs showed that sickling in response to hypoxia was reduced but present but at a reduced level similar to those of 8 patients whose results were previously published who received gene therapy addition of HbAT87Q.5 Rate of sickling in response to deoxygenation also correlated with RBC survival.
One advantage of using biotin labeling to measure RBC survival over other methods of determining minimum effective protocols for gene therapy is a lack of bias related to heterocellular expression of the target gene. As Leonard et al noted, many gene therapies target the upregulation of γ-globin (ie, HbF) but measuring concentrations of HbF or the number of cells containing HbF may not reliably predict clinical outcomes because expression may not be pancellular, particularly when editing is <100%. Heterocellular expression of the target gene may reduce clinical efficacy; therefore, measuring RBC survival could aid in guiding adjunctive therapies to increase HbF in cases in which expression may be heterocellular.
Direct measurement of RBC lifespan using biotin tagging is a clinically meaningful technique that was safe in this pilot study. The authors convincingly demonstrate that RBC tagging with biotin can be used to measure RBC survival in non-HbSS genotypes and after both fully matched and haploidentical HSCT. Future work should examine this method in people with SCD who undergo posttreatment with gene therapy. The method could help to assess the success of HSCT or gene therapy in SCD and to refine protocols to find a threshold level of therapy required to prevent development of the sickling phenotype while minimizing toxicity.
Conflict-of-interest disclosure: S.C. served as a consultant to Pfizer. S.M. declares no competing financial interests.
