Pathology Of Sickle Cell Disease Research Articles

In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation

AbstractStable, mixed-donor–recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient–red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

Exploring Mitochondrial DNA Heteroplasmy in a Mouse Model of Sickle Cell Disease

BACKGROUND: Mitochondria are important in the pathology of sickle cell disease (SCD). They are abnormally retained in sickle red blood cells (RBCs) and the likely source of cell-free mitochondrial DNA (mtDNA), an erythrocytic-DAMP that triggered formation of neutrophil extracellular traps (Tumburu et al, 2022). Acquired mutations in the mitochondrial genome, referred to mtDNA heteroplasmy, are emerging markers of aging and inflammation. Using whole genome sequence data from peripheral blood DNA, we previously established that patients with SCD not only have increased mtDNA heteroplasmy, but the burden also varied with the sickle genotype [Ahmad et. al, 2021]. The burden of mtDNA heteroplasmy is known to vary across different organs and tissues. Here, we utilized the Townes mouse model of SCD to explore differential mtDNA heteroplasmy burden across the different tissues and sickle genotypes in SCD. METHODS: Genomic DNA was extracted from 9 different organs (heart, brain, muscle, kidney, blood, bone marrow, liver, lungs, spleen) of littermate mice (3 male and 3 female) of three genotypes (HbAA, HbAS, HbSS) in three replica sets (total number of mice = 18, 9 different tissue samples per mouse, total sample number = 162). Mitochondria DNA was enriched from genomic DNA by long range PCR in 2 amplicons of 9kb and 7kb sizes (Misa Hirose et al, 2018). The enriched mtDNA PCR amplicons were mixed together and subjected to library preparation for deep sequencing using Illumina platform. Sequence reads aligned to mtDNA were mapped and called for variants using LoFreq variant calling tool. RESULTS: Of the 162 samples, 1 sample (HbAA, lung) was excluded from analysis due to low coverage. Using LoFreq variant caller tool (VAF cut-off 4% &amp; Coverage Filter 3500X), and after removing the repeating variants, 11 unique variants spanning 16kb mouse mitochondrial genome, were found in 106 of the 161 (65.8%) samples. The variants were differentially distributed across the tissues and genotypes with the variant at mito-position 9820 (MT: 9820) being most frequent, present in 19, 16 and 18 samples in AA, AS and SS mice, respectively (Fig. 1). Two variants at MT: 11866 and MT: 6881 were shared between AA and AS mice. Four variants were unique to SS mice: MT: 6753 in COX1 gene causing Phe476-Leu substitution, MT: 6794 in intergenic region upstream to COX2 gene, MT: 1220 in intergenic region upstream to ND1 gene, and MT425 in regulatory/D-Loop region (Table -1). The SS-unique variants were present in all tissues except for Cox1 Phe476-Leu (MT: 6753), a single variant that was found only in the spleen. Overall, HbSS had the highest concentration of variants distributed among 41/54 (75.9%) followed by AS 38 /54 (70.3 %) and AA 27/53 (50.9%). Female mice had higher mtDNA heteroplasmy burden compared to male mice in all genotypes. Across all genotypes and tissues, spleen had the highest number of variants (16.03%) and liver, the lowest (9.4%). CONCLUSION: In keeping with data in patients with SCD, HbSS mice had the highest mtDNA heteroplasmic burden followed by HbAS and HbAA genotypes. Four mito-variants were unique to HbSS. We also found differentially higher mtDNA heteroplasmic burden in splenic tissues and speculate that this could arise from metabolic stress conditions in the spleen.

Reticulocyte mitochondrial retention increases reactive oxygen species and oxygen consumption in mouse models of sickle cell disease and phlebotomy-induced anemia

Sickle cell disease (SCD) is caused by a mutation of the β-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets.

Feasibility of Near-Infrared Spectroscopy for Monitoring Hemodynamic Changes in Patients with Sickle Cell Disease Treated with Mitapivat

Background: Continuous inflammation and vasculopathy underlies the pathology of sickle cell disease (SCD). Therapeutics intervening the different stages in the pathophysiological cascade - polymerization of hemoglobin S (HbS) and "sickling" of the red blood cells (RBCs), vaso-occlusion and inflammation- are under development. Sickled RBCs initiate vaso-occlusion and have altered biomechanical properties that impact the rheology and hemodynamics in patients with SCD. There is an unmet need for robust, compact technologies that can non-invasively characterize the microvascular hemodynamics in SCD. Such a tool would be a valuable adjunct for clinicians both for broadening understanding of as well as monitoring therapeutic changes. However current clinical approaches can be time- and resource- intensive which limit their utility. Near-infrared spectroscopy (NIRS) is a non-invasive, optical technique for quantifying microvascular hemodynamics including hemoglobin concentration, tissue oxygen saturation, and blood flow (Cerussi et al, 2005). Here, we evaluate the sensitivity of NIRS to hemodynamic changes in patients with SCD undergoing treatment with Mitapivat (AG-348), a pyruvate kinase activator under clinical development to determine how well NIRS parameters compare with standard hematology markers. Methods: We studied patients currently enrolled in a Phase I/II clinical trial (ClinicalTrials.gov, NCT04610866) that evaluates the long-term safety and tolerability of Mitapivat in the SCD population. We performed tissue hemodynamic assessments by affixing NIRS probes to the forearm and forehead of the subjects at baseline (prior to drug initiation) and over the course of three months of treatment with Mitapivat totaling five measurement visits per patient. The optical hemodynamic assessment consisted of resting baseline measurements followed by a brachial cuff occlusion and a recovery period. We acquired optical measures of hemodynamic markers, such as the concentration of oxy- and deoxy- hemoglobin and tissue oxygen saturation (StO2). Additionally, we calculated measures of vascular reactivity by analyzing the post-occlusion reactive hyperemic response. These metrics are compared for each patient across time to determine if there are any significant changes compared to baseline. Optical metrics were compared against changes in blood hemoglobin (Hb) level, an expected therapeutic effect of Mitapivat (Xu et al, 2022). Nine patients with complete NIRS data at baseline and spanning 3 months of Mitapivat were analyzed. Results: We calculated optical hemodynamic metrics for all 45 measurement visits; one visit was excluded due to poor NIRS data quality. Figure 1A shows a typical measurement setup with NIRS probes acquiring simultaneous measurements from the forehead and forearm, which reflect tissue hemodynamics in the prefrontal cortex and skeletal muscle respectively. Figure 1B compares the mean Hb level, mean forehead StO2, and mean forearm StO2 across the first three months of treatment. On average, forehead StO2 acquired by NIRS trended similarly to the Hb level acquired by blood draws over the first three months. We observed that 7/9 patients exhibited a sustained increase in blood Hb compared to baseline; the mean Hb increase for all patients compared to baseline was 1.2 g/dL at the end of three months. Similarly, forehead StO2 measured by NIRS also showed a similar, sustained increase. The mean forehead StO2 increase after three months compared to baseline was 4.0%. Discussion: We have demonstrated that NIRS could be a promising adjunct for non-invasively monitoring hemodynamics in patients with SCD undergoing treatment with Mitapivat. NIRS can provide objective measures of tissue composition and metabolism and vascular reactivity which can be performed in a simple, non-invasive measurement. Future analysis will include analyzing optical metrics of vascular reactivity and blood flow. While this initial cohort is small, the preliminary results are encouraging and warrant continued study into NIRS as a tool for monitoring hemodynamic changes promoted by other therapies for SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A PHASE 2/3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY OF MITAPIVAT IN PATIENTS WITH SICKLE CELL DISEASE

The key pathology in sickle cell disease (SCD), a life-threatening, hereditary hemoglobin (Hb) disorder, is red blood cell (RBC) sickling due to polymerization of deoxygenated sickle Hb (HbS), which can be exacerbated by increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG) and decreased ATP. Sickled RBCs are rigid, non-deformable, and fragile, resulting in vaso-occlusive pain and chronic hemolysis. Current SCD treatment options are limited; therefore, there is an unmet need for safe, effective therapies that alleviate both anemia and pain. Activation of the key RBC glycolytic enzyme pyruvate kinase (PKR) by oral mitapivat decreases 2,3-DPG and increases ATP, which may reduce HbS polymerization, RBC sickling, and hemolysis in SCD. Data from the phase (ph) 1 National Institutes of Health multiple ascending dose study of up to 100 mg mitapivat twice daily (BID) in patients with SCD (NCT04000165) showed that mitapivat was safe and tolerable, increased ATP and decreased 2,3-DPG in a dose-dependent manner, and improved anemia and hemolysis. Here we report the study design of RISE UP (NCT05031780, EudraCT: 2021-001674-34), a ph 2/3, double-blind, randomized, placebo (PBO)-controlled, multicenter study evaluating the efficacy and safety of mitapivat in patients (pts) with SCD. Eligible: pts aged ≥16 yrs (≥18 yrs [France and Germany]) with documented SCD (HbSS, HbSC, HbSβ 0 /HbSβ + thalassemia, other variants), 2–10 sickle cell pain crises (SCPCs; acute pain requiring intervention, acute chest syndrome, priapism, hepatic or splenic sequestration) in the prior 12 months, and Hb 5.5–10.5 g/dL. If on hydroxyurea (HU), the dose must be stable for ≥ 90 days before starting study drug. Ineligible: pts receiving regular blood transfusions, with severe kidney or hepatobiliary disease, current SCD therapies except HU, or prior gene therapy, bone marrow or stem cell transplantation. In the double-blind ph 2 part, 69 pts will be randomized (1:1:1) to 50 mg or 100 mg mitapivat, or PBO BID for 12 weeks (wk). The primary objective is to determine the recommended ph 3 mitapivat dose by evaluating anemia and safety vs PBO via the following endpoints: Hb response (≥ 1.0 g/dL increase in average (avg) Hb concentration over Wk 10–12 vs baseline [BL]), and type, severity, and relationship of adverse events (AEs) and serious AEs. In the double-blind ph 3 part, 198 pts who did not participate in ph 2 will be randomized (2:1) to the selected ph 3 mitapivat dose or PBO BID for 52 wk. The primary objectives are to determine the effect of mitapivat vs PBO on anemia, measured by Hb response (≥ 1.0 g/dL increase in avg Hb concentration over Wk 24–52 vs BL), and the effect of mitapivat vs PBO on SCPC, measured by annualized rate of SCPCs. Key secondary endpoints: avg change from BL in Hb concentration, indirect bilirubin, percent reticulocyte, and Pt-Reported Outcomes Measurement Information System ® Fatigue 13a Short Form score over Wk 24–52; and annualized frequency of SCPC admissions. Pts who complete the double-blind period will be eligible to receive mitapivat in the open-label extension period in each ph. Pending. N/A. This ph 2/3 study will investigate the efficacy and safety of mitapivat in pts with SCD. Enrollment is currently ongoing.

THE RANDOMIZED, DOUBLE-BLIND, MULTICENTER, PLACEBO-CONTROLLED PHASE 3 RESOLVE TRIAL INVESTIGATING THE EFFICACY OF VOXELOTOR WITH STANDARD OF CARE IN THE RESOLUTION OF LEG ULCERS IN PATIENTS WITH SICKLE CELL DISEASE

In sickle cell disease (SCD), polymerization of deoxygenated sickle hemoglobin (HbS) is the major pathological process that leads to red blood cell sickling and hemolysis. In addition to the major complications of SCD, including hemolytic anemia, inflammation, and vascular occlusion, patients often experience secondary complications such as stroke, organ damage, shortened lifespan, and painful and debilitating skin ulcers. Given the lack of registries and large prospective studies examining leg ulcers, their prevalence among individuals with SCD is likely underestimated but varies widely by geographic region: 43% in Brazil, 30% in Jamaica, 27% in Nigeria, 19% in Ghana, 13% in Sierra Leone, 8% in Saudi Arabia, and 1% to 5% in the US. Voxelotor, a HbS polymerization inhibitor that targets the underlying SCD pathology, is approved in the US for treatment of SCD in patients aged ≥4 years and in the UK, EU, and UAE for patients aged ≥12 years. In a post hoc analysis of voxelotor-treated patients from the pivotal HOPE trial (NCT03036813), leg ulcers resolved within 24 weeks in 10 out of 14 patients and improved or resolved by week 72 in 13 out of 14 patients. RESOLVE is an ongoing phase 3, randomized, double-blind, placebo-controlled, multicenter trial investigating the efficacy of voxelotor with standard of care (SOC) in the resolution of leg ulcers in patients with SCD. The study will be conducted at sites in Brazil (9), Nigeria (7), and Kenya (5). Target study enrollment is 80 patients aged ≥ 12 years with a confirmed diagnosis of SCD (HbSS or HbSβ 0 genotype) and ≥1 cutaneous ulcer on the lower extremity (leg, ankle, or dorsum of foot) that meets the following criteria: ≥ 2 weeks’ and < 6 months’ duration at screening and >2 cm 2 in area before randomization. After a 2-week run-in period, participants are randomized 1:1 to receive once-daily oral voxelotor 1500 mg or placebo in addition to SOC for 12 weeks. After the randomized treatment period, all participants enter an open-label treatment period and receive voxelotor 1500 mg plus SOC for an additional 12 weeks. Participants will have the option to enroll in a separate open-label extension study after completing the 24-week treatment period. All study sites in Brazil will be activated by the end of 2022, with the first patient evaluation in Brazil anticipated to occur in the first week of December 2022. The primary objective of this study—to assess the efficacy of voxelotor plus SOC compared with placebo plus SOC in healing leg ulcers—is measured by the proportion of patients with resolution of target ulcer(s) in each treatment group by week 12. Key secondary endpoints include days to resolution of target ulcer(s) up to week 12, change from baseline in total surface area of target ulcer(s) at week 12, and incidence of new ulcers by week 12. Exploratory analyses include correlation of target ulcer(s) healing with change in hemoglobin and hemolytic parameters. The study will also assess patient-reported outcome measures, including health-related quality of life and pain. Findings from a post hoc analysis of participants with leg ulcers in the HOPE trial suggest that voxelotor may be an effective treatment for leg ulcers in patients with SCD. Voxelotor directly targets the underlying mechanism of disease (namely HbS polymerization) and may have a therapeutic effect on the clinical complications of SCD associated with anemia and hemolysis, including leg ulcers. Results from this study will advance our understanding of the potential effects of voxelotor on leg ulcers in patients with SCD in global populations.

Precision Medicine and Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by variable clinical outcomes, with some patients suffering life-threatening complications during childhood, and others living relatively symptom-free into old age. Because of this variability, there is an important potential role for precision medicine, in which particular different treatments are selected for different groups of patients. However, the application of precision medicine in SCD is limited by difficulties in identifying different prognostic groups and the small number of available treatments. The main genetic determinant of outcomes in SCD is the underlying β-globin genotype, with sickle cell anemia (HbSS) and hemoglobin SC disease (HbSC) forming the 2 major forms of the disease in most populations of African origin. Although there are clear differences in clinical outcomes between these conditions, treatments approaches are very similar, with little evidence on how to treat HbSC in particular. Other genomic information, such as the co-inheritance of α-thalassemia, or high fetal hemoglobin (HbF) levels, is of some prognostic value but insufficient to determine treatments. Precision medicine is further limited by the fact that the 2 main drugs used in SCD, penicillin and hydroxyurea, are currently recommended for all patients. Newer treatments, such as crizanlizumab and voxelotor, raise the possibility that groups will emerge who respond best to particular drugs or combinations. Perhaps the best current example of precision medicine in SCD is the selective use of blood transfusions as primary stroke prevention in children with evidence of cerebral vasculopathy. More precise treatments may emerge as we understand more about the pathology of SCD, including problems with erythropoiesis.

Under the hood: The molecular biology driving gene therapy for the treatment of sickle cell disease

Gene therapy will soon become the dominant modality for treating of sickle cell disease (SCD). Currently, three technologies are the most promising: expression of transgenic globin genes via a lentiviral vector, controlled mutation of the β-globin control cluster by transgenic CRISPR-based ribonucleoprotein, and suppression of BCL11a mRNA by shRNA. In this review, we discuss the mechanism of each technology and how they correct the SCD pathology at the molecular level. We conclude by discussing potential directions future therapy may take.

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism.

Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia, and progressive organ failure. Chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism can also occur with the loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The association of laboratory parameters with the spleen status including hyposplenism, asplenia, and splenomegaly/hypersplenism, and their implication in vaso-occlusive crisis and long-term complications in SCD remain to be determined. We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients: Group a) Patients with asplenia/hyposplenism (N = 22) (including auto-splenectomy and splenectomized patients) vs. Group b) patients with splenomegaly and or hypersplenism (N = 9). Laboratory studies included: Complete Blood Count, reticulocyte count, iron metabolism parameters, C Reactive Protein (CRP), Hb variant distribution, and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release, and K+ leakage, fetal RBC (F-Cells) and F-Reticulocytes, annexinV+, CD71+, oxidative stress measured by GSH presence in RBC and finally Howell Jolly Bodies count were all analyzed by Flow Cytometry. Scanning electron microscopy analysis of RBC was also performed. Patients with asplenia/hyposplenism showed significantly higher WBC, platelet, Hematocrit, hemoglobin S, CRP, D-dimer, Gamma Glutamyl Transferase (GGT), cholesterol, transferrin, annexin V+ RBCs, CD71+ RBCs, together with a markedly lower F Reticulocyte levels in comparison with splenomegaly/hypersplenism patients. In summary, important differences were also found between the groups in the studied RBCs parameters. Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia on laboratory parameters and in clinical manifestations, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach managing hypersplenism in SCD patients should be re-evaluated.

PB2222: THE PROTECTIVE EFFECT OF THE SPLEEN IN SICKLE CELL PATIENTS. A COMPARATIVE STUDY BETWEEN PATIENTS WITH ASPLENIA AND HYPERSPLENISM.

Background: Sickle cell disease (SCD) is caused by a point mutation in the beta globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia and progressive organ failure. A chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from a mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism, can also occur with loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The implications of the spleen status including asplenia and splenomegaly/ hypersplenism in vaso- occlusive crisis and long-term complications in SCD remain to be determined. Aims: In this study, we compared blood parameters and RBC properties of SCD patients and compared the results between patients with asplenia, to patients with splenomegaly/hypersplenism. In addition, we implemented novel, non-routinely used methods for the evaluation of the functional status of the spleen in those patients. Methods: We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients divided into two groups: Group a) Patients with asplenia (N=22) (including auto-splenectomy and patients that were splenectomized) vs. Group b) patients with Splenomegaly and or Hypersplenism (N=9). Laboratory studies included: CBC, reticulocyte count, iron metabolism parameters, CRP, Hb variant distribution and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release and K+ leakage; fetal RBC (F-Cells) and F Reticulocytes, annexin V+, CD71+, oxidative stress measured by GSH presence in RBC and finally HJB count were all analyzed by FC. SEM analysis of RBC was also performed. Results: Patients in group (a) showed significantly higher WBC, platelet, HCT, hemoglobin S, CRP, D-dimer, GGT, cholesterol and transferrin levels than patients in group (b). The percent of hyperchromic RBCs, F-Reticulocytes were lower in patients with asplenia. Important differences were also found between the groups in the studied RBCs metabolic parameters. Summary/Conclusion: Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia in vaso-occlusive crisis, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach to manage hypersplenism in SCD patients should be re-evaluated.

Considerations for Selecting Cognitive Endpoints and Psychological Patient-Reported Outcomes for Clinical Trials in Pediatric Patients With Sickle Cell Disease.

Pediatric patients with sickle cell disease (SCD) experience a range of medical complications that result in significant morbidity and mortality. Recent advances in prophylactic and curative treatment approaches have highlighted the need for sensitive and clinically-meaningful trial endpoints. The detrimental effects of cognitive and psychological difficulties on social and economic mobility are well described. Although numerous reviews have assessed cognitive outcomes in other rare genetic disorders, SCD has not received the same focus. This review describes the cognitive (i.e., executive function and processing speed) and psychological domains (i.e., depression and anxiety) that are consistently associated with SCD pathology and, therefore, may be of particular interest as clinical trial endpoints. We then discuss corresponding well-validated and reliable cognitive tests and patient-reported outcomes (PROs) that may be appropriate for clinical trials given their robust psychometric properties, ease of administration, and previous use in the SCD population. Further, we provide a discussion of potential pitfalls and considerations to guide endpoint selection. In line with the move toward patient-centered medicine, we identify specific tests (e.g., NIH Toolbox Cognition Module, Wechsler Cancellation Test) and psychological PROs (e.g., PROMIS depression and anxiety scales) that are sensitive to SCD morbidity and have the potential to capture changes that are clinically meaningful in the context of patients' day to day lives. In particularly vulnerable cognitive domains, such as executive function, we highlight the advantages of composite over single-test scores within the context of trials. We also identify general (i.e., practice effects, disease heterogeneity) and SCD-specific considerations (i.e., genotype, treatment course, and disease course, including degree of neurologic, pain, and sleep morbidity) for trial measures. Executive function composites hold particular promise as trial endpoints that are clinically meaningful, amenable to change, relatively easy to collect, and can be incorporated into the routine care of patients with SCD in various settings and countries.

Dynamics of Individual Red Blood Cells Under Shear Flow: A Way to Discriminate Deformability Alterations.

In this work, we compared the dynamics of motion in a linear shear flow of individual red blood cells (RBCs) from healthy and pathological donors (Sickle Cell Disease (SCD) or Sickle Cell-β-thalassemia) and of low and high densities, in a suspending medium of higher viscosity. In these conditions, at lower shear rates, biconcave discocyte-shaped RBCs present an unsteady flip-flopping motion, where the cell axis of symmetry rotates in the shear plane, rocking to and fro between an orbital angle ±ϕ observed when the cell is on its edge. We show that the evolution of ϕ depends solely on RBC density for healthy RBCs, with denser RBCs displaying lower ϕ values than the lighter ones. Typically, at a shear stress of 0.08 Pa, ϕ has values of 82 and 72° for RBCs with average densities of 1.097 and 1.115, respectively. Surprisingly, we show that SCD RBCs display the same ϕ-evolution as healthy RBCs of same density, showing that the flip-flopping behavior is unaffected by the SCD pathology. When the shear stress is increased further (above 0.1 Pa), healthy RBCs start going through a transition to a fluid-like motion, called tank-treading, where the RBC has a quasi-constant orientation relatively to the flow and the membrane rotates around the center of mass of the cell. This transition occurs at higher shear stresses (above 0.2 Pa) for denser cells. This shift toward higher stresses is even more remarkable in the case of SCD RBCs, showing that the transition to the tank-treading regime is highly dependent on the SCD pathology. Indeed, at a shear stress of 0.2 Pa, for RBCs with a density of 1.097, 100% of healthy RBCs have transited to the tank-treading regime vs. less than 50% SCD RBCs. We correlate the observed differences in dynamics to the alterations of RBC mechanical properties with regard to density and SCD pathology reported in the literature. Our results suggest that it might be possible to develop simple non-invasive assays for diagnosis purpose based on the RBC motion in shear flow and relying on this millifluidic approach.

P107: A PHASE 2/3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY OF MITAPIVAT IN PATIENTS WITH SICKLE CELL DISEASE

Background: The key pathology in sickle cell disease (SCD), a life-threatening, hereditary hemoglobin (Hb) disorder, is red blood cell (RBC) sickling due to polymerization of deoxygenated sickle Hb (HbS), which can be exacerbated by increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG), and decreased ATP.1–3 Sickled RBCs are rigid, not deformable, and fragile, resulting in vaso-occlusion triggering pain and chronic hemolysis.4–6 SCD treatment options are limited, with an unmet need for safe and effective therapies to improve anemia and reduce pain. Mitapivat is an oral, activator of RBC pyruvate kinase (PKR), a key glycolytic enzyme. PKR activation decreases 2,3-DPG and increases ATP, which may reduce HbS polymerization, RBC sickling, and hemolysis in SCD.3,7–9 Data from the phase (ph) 1 National Institutes of Health multiple ascending dose study of up to 100 mg mitapivat twice daily (BID) in SCD (NCT04000165) showed that mitapivat was safe and tolerable, increased ATP and decreased 2,3-DPG in a dose-dependent manner, and improved anemia and hemolysis.8,10 Aims: To report the study design of RISE UP (NCT05031780, EudraCT: 2021-001674-34), a ph 2/3, double-blind, randomized, placebo-controlled, multicenter study evaluating the efficacy and safety of mitapivat in patients (pts) with SCD. Methods: Eligible: pts aged ≥16 yrs with documented SCD (HbSS, HbSC, HbSβ0/HbSβ+ thalassemia, other SCD variants), 2–10 sickle cell pain crises (SCPCs; acute pain needing medical contact, acute chest syndrome, priapism, hepatic or splenic sequestration) in the prior 12 months, and Hb 5.5–10.5 g/dL. If taking hydroxyurea (HU), the dose must be stable for ≥90 days before starting study drug. Ineligible: pts receiving regularly scheduled blood transfusions, with severe kidney disease or hepatobiliary disorders, currently receiving SCD therapies (excluding HU) or who have received gene therapy, bone marrow or stem cell transplantation. In the double-blind ph 2 part, 69 pts will be randomized (1:1:1) to 50 mg or 100 mg mitapivat, or placebo BID for 12 weeks (wks). The primary objective of ph 2 is to determine the recommended ph 3 mitapivat dose by evaluating anemia and safety vs placebo via the following endpoints: Hb response (≥1.0 g/dL increase in average Hb concentration over Wks 10–12 vs baseline [BL]), and type, severity, and relationship of adverse events and serious AEs. In the double-blind ph 3 part, 198 pts who did not participate in ph 2 will be randomized (2:1) to the selected ph 3 mitapivat dose or placebo BID for 52 wks, stratified by number of SCPCs in the prior yr (<5, ≥5) and HU use. The primary objectives of ph 3 are to determine the effect of mitapivat vs placebo on anemia, measured by Hb response (≥1.0 g/dL increase in average Hb concentration over Wks 24–52 vs BL), and the effect of mitapivat vs placebo on SCPC, measured by annualized rate of SCPCs. Key secondary endpoints include average change from BL in Hb concentration, indirect bilirubin, percent reticulocyte, and Pt-Reported Outcomes Measurement Information System® Fatigue 13a Short Form score over Wks 24–52; and annualized frequency of hospitalizations for SCPC. Pts who complete the double-blind period will be eligible to receive mitapivat in the open-label extension period in each ph. Results: Not yet available. Conclusion: This ph 2/3 study will investigate the efficacy and safety of mitapivat in pts aged ≥16 yrs with SCD. Enrollment is currently ongoing.

CRISPR/Cas9 Genome Editing to Treat Sickle Cell Disease and B-Thalassemia: Re-Creating Genetic Variants to Upregulate Fetal Hemoglobin Appear Well-Tolerated, Effective and Durable.

Persistent expression of fetal hemoglobin (HbF) beyond the neonatal period is a rare, naturally-occurring condition, referred to as the Hereditary Persistence of Fetal Hemoglobin, which substantially ameliorates the pathology of Sickle Cell Disease (SCD) and β-thalassemia (β-thal). Extensive human genetic and epidemiologic studies have demonstrated that this condition is associated with one of several specific point mutations or deletions that lead to expression of γ-globin, resulting in upregulation of HbF. Our strategy is to use the CRISPR/Cas9 genome editing technology in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) to efficiently re-create specific genetic variants associated with elevated HbF and to evaluate their safety, effectiveness and durability as a potential therapeutic strategy to treat SCD and β-thal.We have optimized CRISPR/Cas9 editing of human primary HSPCs mobilized from healthy donors to routinely achieve editing efficiencies of greater than 80%. Improved viability with no reduction in editing efficiency was achieved with Cas9 delivered as a recombinant protein as compared to Cas9 delivered as mRNA. We have further optimized the process at clinical scale in our manufacturing facility to achieve similar viability and editing efficiency with a GMP-compatible process. The high rate of editing in human primary HSPCs mobilized from healthy donors was associated with robust HbF expression following erythroid differentiation. In patient samples, clinically relevant increases in γ-globin mRNA to 39% (as a ratio of γ/α) in one β-thal patient sample and to 38±5% (Mean±SD, as a ratio of γ/(γ+β)) in six SCD patient samples were observed. Similarly high rate of editing was observed in the CD34+CD38-CD90+CD45RA- long-term repopulating subset of human primary HSPCs, as in the overall HSPC population (88±6% vs 90±4% Mean±SD, n=4). We have demonstrated the long-term persistence of transplanted cells in a NOD-SCID-Gamma (NSG) xeno-transplant mouse model at 16 weeks (44±12% of cells in controls versus 44±14% and 41±13% for two target modifications, Mean±SD, n=44-47 for each), and that the proportion of the various hematopoietic lineages derived from the transplanted edited human primary HSPCs was not affected by the editing process. Finally, the high editing rate of 85-90% persisted in human bone marrow cells at 16 weeks, demonstrating durability of the edited cells. Because the NSG model does not appropriately replicate erythroid development, we have also demonstrated normal erythroid potential of the engrafted human CD34+ cells isolated from mouse bone marrow in ex vivo differentiation assays, and the editing level is maintained in the erythroid lineage. In-depth analysis confirmed no detectable off-target sequence disruption at over 5,000 candidate off-target sites that were identified bioinformatically through homology to the on-target site or experimentally through a double strand break identification method. In vivo safety and toxicology studies are ongoing.Taken together, these findings show that a CRISPR/Cas9-based therapy has the potential to upregulate HbF and treat diseases of b-globin. We have optimized editing conditions to achieve high efficiency and viability of editing in human primary HSPCs without detectable off-target sequence disruption, and in turn, demonstrate the durability of this approach in vivo. We believe that the results to date support the initiation of clinical studies for a CRISPR/Cas9 treatment for patients with SCD and β-thal. DisclosuresLin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Burkhardt:CRISPR Therapeutics: Employment. Kernytsky:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Chen:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Cortes:CRISPR Therapeutics: Employment. Tan:CRISPR Therapeutics: Employment. Borland:CRISPR Therapeutics: Employment. Klein:CRISPR Therapeutics: Employment. Yen:Vertex: Employment. Mahajan:Vertex: Employment. Chan:Vertex: Employment. Eustace:Vertex: Employment. Porteus:CRISPRTx: Equity Ownership. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment. Novak:CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment.

A red-letter day for RBC research

Hematology The study of primary human red blood cell (huRBC) disorders such as sickle cell disease (SCD) and infectious diseases such as malaria has been hampered by a lack of in vivo models of human erythropoiesis. Song et al. transferred human fetal liver cells into MISTRG mice, which are immunodeficient and are genetically engineered with several human genes involved in hematopoiesis. This approach was unsuccessful because mature huRBCs are rapidly destroyed in the mouse liver. They then used CRISPR-Cas9 to mutate these mice into a fumarylacetoacetate hydrolase–deficient strain, allowing them to replace the mouse liver with engrafted human hepatocytes. These mice exhibited enhanced human erythropoiesis and circulating huRBC survival and could recapitulate SCD pathology when reconstituted with SCD-derived HSCs. Science , this issue p. [1019][1] [1]: /lookup/doi/10.1126/science.abe2485

Combined liver-cytokine humanization comes to the rescue of circulating human red blood cells.

In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (Fah) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)-derived hematopoietic stem cells in huHepMISTRGFah -/- mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver-cytokine-humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.

Hydroxyurea Adherence Strategies for Persons with Sickle Cell Disease: A Systematic Review.

Adherence to hydroxyurea is essential to modify the pathology of sickle cell disease. To identify best strategies to support adherence to hydroxyurea in persons with sickle cell disease. A systematic review was conducted. PubMed, Cumulative Index of Nursing and Allied Health Literature, PsycArticles, PsycInfo, Embase databases and Cochrane Library were searched for studies between 1998 to 2018. Six studies met inclusion criteria: two randomized controlled trials, three prospective studies, and one retrospective chart review. Few studies addressed strategies that tested adherence interventions primarily composed of behavioral change resulting in medication adherence. More studies on hydroxyurea adherence are needed.