Hemoglobin Sickle Cell Disease Research Articles

Effect of voxelotor on cardiopulmonary testing in youths with sickle cell anemia in a pilot study.

Individuals with sickle cell anemia (SCA) exhibit decreased exercise capacity. Anemia limits oxygen-carrying capacity and affects cardiopulmonary fitness. The drug voxelotor raises hemoglobin in SCA. We hypothesized that voxelotor improves exercise capacity in youths with SCA. In a single-center, open-label, single-arm, longitudinal interventional pilot study (NCT04581356), SCA patients aged 12 and older, stably maintained on hydroxyurea, were treated with 1500mg voxelotor daily, and performed cardiopulmonary exercise testing before (CPET#1) and after voxelotor (CPET#2). A modified Bruce Protocol was performed on a motorized treadmill, and breath-by-breath gas exchange data were collected. Peak oxygen consumption (peak VO2 ), anaerobic threshold, O2 pulse, VE/VCO2 slope, and time exercised were compared for each participant. The primary endpoint was change in peak VO2 . Hematologic parameters were measured before each CPET. Patient Global Impression of Change (PGIC) and Clinician Global Impression of Change (CGIC) surveys were collected. Ten hemoglobin SS patients aged 12-24 completed the study. All demonstrated expected hemoglobin rise, with average +1.6g/dL (p=.003) and P50 left shift of average -11mmHg (p<.0001) with decreased oxygen off-loading at low pO2 . The change in % predicted peak VO2 from CPET#1 to CPET#2 ranged from -12.8% to +11.3%, with significant improvement of more than 5% in one subject, more than 5% decrease in five subjects, and insignificant change of less than 5% in four subjects. All 10 CGIC and seven of 10 PGIC responses were positive. In a plot study of 10 youths with SCA, voxelotor treatment did not improve peak VO2 in 9 out of 10 patients.

The BACH1 inhibitor ASP8731 inhibits inflammation and vaso-occlusion and induces fetal hemoglobin in sickle cell disease.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

Asymmetric sickle cell retinopathy in a patient with sickle cell hemoglobin D disease: A case report

Sickle cell disease (SCD), the most commonly inherited hemoglobinopathy, can result in vision loss due to sickle cell retinopathy (SCR), vascular occlusions, and retinal atrophy. SCR is more common in heterozygous (HbSc) than homozygous (HbSS) patients. HbD (Punjab) is a less commonly reported form of hemoglobin in SCD, seen in northwestern states of India. Patients with sickle cell hemoglobin D disease (HbSD) can clinically behave like HbSS. We report a case of asymmetric SCR and multiple branch retinal artery occlusions in a patient with sickle cell hemoglobin D disease in central India.

Resveratrol-nitric oxide donor hybrid effect on priapism in sickle cell and nitric oxide-deficient mouse.

BackgroundChildren and adult with sickle cell disease (SCD) display priapism associated with low nitric oxide (NO) bioavailability and oxidative stress in penis.AimThis study aimed to evaluate the effects of hybrid compound RVT-FxMe, derived from resveratrol bearing a NO-donor subunit, on two murine model that display priapism phenotype, SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice.MethodsWild-type, SCD, and eNOS-/- mice were treated with RVT-FxMe (25 mg/kg/d, 2 weeks).OutcomesHematological parameters, concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as to electrical field stimulation (EFS), were obtained in mice corpus cavernosum strips.ResultsCorpus cavernosum relaxations to SNP and EFS were increased in eNOS-/- group, which were normalized by RVT-FxMe treatment. SCD mice exhibited an excessive CC relaxant response induced by ACh, EFS and SNP RVT-FxMe treatment did not change the increased relaxant responses to ACh, EFS and SNP in corpus cavernosum from SCD group.Clinical translationExcess of plasma hemoglobin in SCD may interfere in pharmacological activity of NO donors compounds.Strength/LimitationsWhile mechanistic data with promising potential is showed, the current study is not without limitations. RVT-FxMe effects in the mid- and long-term warrant complementary studies.ConclusionTreatment with RVT-FxMe reversed the enhanced NO-cGMP-mediated CC relaxations in eNOS-/- mice, but not in SCD mice; it is likely that excess of plasma hemoglobin in SCD mice act to inactivate NO before it reaches soluble guanylyl cyclase, avoiding restoration of NO bioavailability in penis.

A randomised double-blind placebo-controlled clinical trial of oral hydroxyurea for transfusion-dependent \u03b2-thalassaemia

Hydroxyurea is an antimetabolite drug that induces fetal haemoglobin in sickle cell disease. However, its clinical usefulness in β-thalassaemia is unproven. We conducted a randomised, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of hydroxyurea in transfusion-dependent β-thalassaemia. Sixty patients were assigned 1:1 to oral hydroxyurea 10–20 mg/kg/day or placebo for 6 months by stratified block randomisation. Hydroxyurea treatment did not alter the blood transfusion volume overall. However, a significantly higher proportion of patients on hydroxyurea showed increases in fetal haemoglobin percentage (89% vs. 59%; p < 0.05) and reductions in erythropoietic stress as measured by soluble transferrin receptor concentration (79% vs. 40%; p < 0.05). Based on fetal haemoglobin induction (> 1.5%), 44% of patients were identified as hydroxyurea-responders. Hydroxyurea-responders, required significantly lower blood volume (77 ± SD27ml/kg) compared to hydroxyurea-non-responders (108 ± SD24ml/kg; p < 0.01) and placebo-receivers (102 ± 28ml/kg; p < 0.05). Response to hydroxyurea was significantly higher in patients with HbE β-thalassaemia genotype (50% vs. 0%; p < 0.01) and Xmn1 polymorphism of the γ-globin gene (67% vs. 27%; p < 0.05). We conclude that oral hydroxyurea increased fetal haemoglobin percentage and reduced erythropoietic stress of ineffective erythropoiesis in patients with transfusion-dependent β-thalassaemia. Hydroxyurea reduced the transfusion burden in approximately 40% of patients. Response to hydroxyurea was higher in patients with HbE β-thalassaemia genotype and Xmn1 polymorphism of the γ-globin gene.

Gastrointestinal Vasoocclusive Crisis in a Woman with Hemoglobin SC Disease

A 32-year-old woman with hemoglobin sickle cell (SC) disease was admitted to the hospital at 33 weeks gestation with pain in her arms and legs, consistent with vasoocclusive crisis. She was hypertensive and had proteinuria. Her platelet count decreased to 46 K/μL, and aspartate aminotransferase increased to 344 units/L, alanine aminotransferase to 92 units/L, and lactate dehydrogenase to 11,141 units/L. Peripheral blood smear showed schistocytes, in addition to the sickled and target cells seen in hemoglobin SC disease (Figure 1).

Effects of BCL11A Shmir-Induced Post-Transcriptional Silencing on Distributions of HbF in Single-RBCs and Reticulocytes

NH and EE equally contributed. ADW and PB co-signed.The expression of fetal hemoglobin (HbF) is one of the main targets of sickle cell disease treatment, as it inhibits the polymerization of hemoglobin S. The hypothesis of an inhibitory threshold of HbF per red blood cell (RBC) has been suggested, 1 although not well defined, as the overall percentage of HbF does not reflect the heterogeneous distribution of HbF per cell. Likewise, the qualitative analysis of RBCs containing HbF, called F cells, is neither reproducible nor clinically interpretable, due to low expression. 2 We have developed a technique for measuring the amount of HbF per cell, to determine thresholds of HbF expression per RBC correlated with clinical and biological effects. 2 Among genes controlling its expression, BCL11A has a major repressive effect on the expression of gamma globin/HbF during the fetal to adult hemoglobin switch. Post-transcriptional silencing of BCL11A, using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression, is safe and demonstrates high levels of %HbF in a pilot clinical study (NCT 03282656). 3 Here, we show the quantitative measurement of HbF per RBC and reticulocyte.Methods: During patient follow-up, HbF quantification per single cell RBC was performed using a fluorescent HbF antibody. 2 Addition of an anti-CD71 fluorescent antibody allowed selection of reticulocyte sub-populations for determining their HbF content. Fold-increase in percentage of RBC versus percentage of reticulocyte were calculated. Kinetics of HbF/RBC and HbF/Reticulocyte were modeled using mixed effects polynomial linear regression to account for the correlation between repeated data over time.Results: With a median follow-up of 15 months [12-20] after gene transfer, figure 1 shows the mathematical modeling of single-RBC HbF measurement representing RBC percentage containing at least 2, 4, 6, 8 and 10 pg of HbF. Percentage of RBC above each threshold was higher compared to 14 hydroxyurea treated patients for 6 months. Figure 2 shows fold increase between reticulocytes and RBCs with same thresholds of HbF/cell. For low thresholds, RBCs were found in same percentage as reticulocytes whereas RBCs containing increasing levels of HbF were found in higher percentage than reticulocytes, until 6pg/cell showing a clear selective advantage for red cells with a threshold ≥ 6pg/cell of HbF. Figure 3 shows different kinetics of HbF increase according to two different transduction strategies with 2 enhancers in patients 2-4 compared to one enhancer in patients 6-8.Conclusion: BCL11A down-regulation in six clinical trial subjects was associated with an in vivo selection process RBCs with ≥ 6pg HbF per cell attained with different engraftment kinetics, depending on transduction processes, and ultimately stable high level and broadly distributed HbF.1 Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014 Jan 23;123(4):481-5.2 Hebert N, Rakotoson MG, Bodivit G, et al. Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease. Am. J. Hematol.3 Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215. [Display omitted] DisclosuresEsrick: bluebird bio: Consultancy. Audureau: GBT: Honoraria. Higgins: Sebia, Inc.: Honoraria; Danaher Diagnostics: Consultancy. Williams: BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Bartolucci: AGIOS: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; Jazz Pharma: Other: Lecture fees; Emmaus: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; INNOVHEM: Other: Co-founder; Hemanext: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Fabre Foundation: Research Funding.

Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.

β-Hemoglobinopathies are the most common monogenic disorders, and a century of research has provided us with a better understanding of the attributes of these diseases. Allogenic stem cell transplantation was the only potentially curative option available for these diseases until the discovery of gene therapy. The findings on the protective nature of fetal hemoglobin in sickle cell disease (SCD) and thalassemia patients carrying hereditary persistence of fetal hemoglobin (HPFH) mutations has given us the best evidence that the cure for β-hemoglobinopathies remains hidden in the hemoglobin locus. The detailed understanding of the developmental gene regulation of gamma-globin (γ-globin) and the emergence of gene manipulation strategies offer us the opportunity for developing a γ-globin gene-modified autologous stem cell transplantation therapy. In this review, we summarize different therapeutic strategies that reactivate fetal hemoglobin for the gene therapy of β-hemoglobinopathies.

FAT EMBOLISM SYNDROME AS THE PRESENTING SYNDROME OF UNDIAGNOSED SICKLE CELL DISEASE IN A MIDDLE-AGED ADULT

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Fat embolism syndrome (FES) represents a myriad of clinical signs and symptoms that occur following the release of fat emboli into systemic circulation including hypoxemia, neurologic changes, petechial rash, and multi-organ dysfunction. FES often occurs post-orthopedic trauma but can be a complication of sickle cell disease (SS) [1]. CASE PRESENTATION: An otherwise healthy 44-year-old female presented to our emergency department (ED) with worsening, atraumatic back and knee pain after an ED visit three days prior where x-rays and blood-work were unremarkable. She was short of breath, hypoxic, tachycardic and hypotensive. Her mental and respiratory status deteriorated requiring intubation. Laboratory work was significant for hemoglobin 8.5 g/dL, hematocrit 24.0%, WBC 14.8x10⁹/L, and platelets 59x10⁹/L including 11% bands and 15% nRBCs (baseline CBC had no abnormalities). A chest x-ray revealed bilateral, patchy infiltrates consistent with pulmonary edema. Further blood-work revealed acute renal and liver failure, lactic acidosis, and troponinemia. CT imaging revealed patchy, widespread pulmonary disease/edema and splenomegaly. She underwent resuscitation with fluid, blood products, vasopressor support, and broad spectrum antibiotics. All infectious disease workups were negative. The patient was ultimately transferred elsewhere for whole blood exchange but expired. Autopsy revealed vertebral and long-bone marrow infarction and evidence of FES. She was discovered to be hemoglobin SS post-mortem, and no trigger for this episode, including infectious disease, was identified. DISCUSSION: Sickle cell disease screening is routine in the United States but less standardized in other nations [2]. Most sickle cell disease patients exhibit disease signs by age 4-5 months making her middle-aged presentation uncommon. Our patient, a Haitian immigrant, as well as her husband, adamantly denied prior SS symptoms or crisis. Moreover, according to a recent systematic review, FES is a rare complication of sickle cell disease with a predilection for heterozygous patients [2]. Of all FES cases, only 15% of patients were homozygous SS (n = 13) [3]. It is rare for FES to be the presenting syndrome of a previously undiagnosed, middle-aged, homozygous SS disease patient. CONCLUSIONS: We present a rare case of FES as the presenting syndrome of a previously undiagnosed, middle-aged, SS disease patient. Prompt consideration and treatment of FES should be initiated to avoid complications. Reference #1: Akhtar S. Fat embolism. Anesthesiol. Clin. 2009; 27 (3):533–550. Reference #2: Center for Disease Control Sickle Cell Disease. Oct 2019. Retrieved from https://www.cdc.gov/ncbddd/sicklecell Reference #3: Tsitsikas DA, May JE, Gangaraju R, Abukar J, Amos RJ, Marques MB. Revisiting Fat Embolism in Sickle Syndrome: Diagnostic and Emergency Therapeutic Measures. British Journal of Haematology. 2019 Aug; 186(4), 112-115. DISCLOSURES: No relevant relationships by Osman Abbasi, source=Web Response No relevant relationships by Jamie Allen, source=Web Response No relevant relationships by Jeffrey Gesell, source=Web Response No relevant relationships by Benjamin Lin, source=Web Response No relevant relationships by Louis Morolla, source=Web Response No relevant relationships by Andres Zirlinger, source=Web Response

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania

BackgroundSickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation.MethodsWe investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1.ResultsResults revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis.ConclusionsThis study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.

Pathophysiology and recent therapeutic insights of sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive blood disorder which occurs due to point mutation in the β-globin chain of hemoglobin. Since the past decades, various therapies have been put forth, which are based on obstructing pathophysiological mechanisms of SCD including inhibition of Gardos channel and cation fluxes which in turn prevents sickle erythrocyte destruction and dehydration. The pharmacological approaches are based on the mechanism of reactivating γ-globin expression by utilizing fetal hemoglobin (HbF)-inducing drugs such as hydroxyurea. In SCD, gene therapy could be considered as a promising tool which involves modifying mutation at the gene-specific target by either promoting insertion or deletion of globins. Although there are various therapies emerged so far in the treatment of SCD, many of them have faced a major setback in most of developing countries in terms of cost, unavailability of expertise, and suitable donor. Therefore, in addition to pathophysiological aspects, this review will discuss new advancements and approaches made in the therapeutic domain of SCD including a viewpoint of modulating hemoglobin in SCD by the intervention of probiotics.

A Rare Case of Rapidly Enlarging Myelolipoma in Sickle Cell Disease

Adrenal myelolipoma (AM) is a benign tumor composed of mature fat cells and hemopoietic elements. Most AMs are incidental findings on imaging and clinically asymptomatic. The purpose of this case report is to describe a rare case of AM and explore its clinical manifestations, imaging features, and treatment. In this study, we report a case of a rapidly growing right AM in a patient with uncontrolled hemoglobin sickle cell disease. A 38-year-old male presented to our institution's endocrine surgery clinic for evaluation of an enlarging right adrenal mass. This mass was incidentally found during an abdominal ultrasound performed for transaminitis and thrombocytopenia. Patient was asymptomatic without any abdominal discomfort, back pain, nausea, or vomiting. Patient was lost to follow up until 2018. Follow-up computed tomography scan in 2018 showed the right adrenal mass measuring 12.3 cm in greatest dimension with significant macroscopic fat. Given the imaging features, AM was the presumed diagnosis. However, with a medical history of uncontrolled sickle cell disease, extra-medullary hematopoiesis and rapidly growing liposarcoma could not be ruled out. Surgical excision was performed due to size and significant tumor growth. Diagnosis was confirmed with histopathology and revealed myelolipoma. Image characteristics can be helpful in diagnosis of AM; however, the appearance of this lesion on computed tomography can be similar to other adrenal gland pathologies such as liposarcoma and mass-forming extramedullary hematopoiesis. Percutaneous needle biopsy may be indicated if the diagnosis remains unclear.

Inflammation Response Cytokines IFN-γ and IL-10 Regulate Monocyte Subset Differentiation

Circulating monocytes comprise of a heterogeneous and functionally-diverse cell population which based on surface markers can be divided into three subsets: classical (CMo), intermediate (IMo), and non-classical monocytes/patrolling monocytes (PMo). The frequency/number, gene expression profile and activity of IMo and PMo significantly change in a variety of inflammatory diseases with the changes associated with disease risk and severity as well as response to treatment. While it is believed that CMo differentiate into IMo and that IMo further differentiate into PMo, there is paucity of data on the mechanisms that alter CMo to IMo/PMo differentiation profiles in these conditions. In addition, factors which induce human and mouse IMo and PMo differentiation have yet to be identified. To screen cytokine/chemokine candidates affecting IMo/PMo differentiation, human monocytes were isolated from healthy donors (HD) and cultured with candidates (22 cytokines/chemokines) for 3 days. On day 3, IMo/PMo marker expression was examined by flow cytometry focusing on candidate molecules that led to increased expression of markers (CD16, CX3CR1, CD11c, HO-1, HLA-DR) whose levels are normally found to be higher in IMo/PMo and to decrease in expression of markers (CD14, CD36, CCR2) which are expressed at higher level in CMo as measured by mean fluorescence intension (MFI). We found that of all molecules tested, only two , IFN-γ and IL-10, had significant effects: IFN-γ (10ng/ml) increased expression of CX3CR1 (20 fold), CD16 (60%), HLADR (15%) and inhibited CD36 (42% inhibition) and CD14 expression (45% inhibition) while IL-10 (10ng/ml) increased CD16 (3.3 fold), CD11c (45%), HO-1 (59%) and CX3CR1 (6 fold) expression and inhibited CCR2 (60% inhibition) expression. These data suggest that IFN-γ and IL-10, two key cytokines involved in sterile and infectious inflammation, induce IMo and PMo differentiation. To test whether the effect of IFN-γ and IL-10 in human in vitro cultures can be replicated in vivo, wildtype B6 mice were I.V. injected with IFN-γ or IL-10 for 3 days: IFN-γ, IL-10, or the same volume of PBS. The frequencies of monocyte subsets in blood (gated on CD45+Ly6G-CD11bhighCD115+ for total monocyte population and CMo/IMo/PMo based on Ly6C expression level) on day 4 were analyzed by flow cytometry. We found that IFN-γ (2.5μg/injection/mice twice/day) significantly increased IMo frequencies (from 12% to 35%) but decreased PMo frequencies (from 38% to 26%) while IL-10 (0.25μg/injection/mice twice/day) significantly induced PMo differentiation (from 38% to 63%) without effect on IMo frequencies. The data suggest that IFN-γ increases IMo frequency by simultaneously inducing CMo differentiation into IMo and inhibiting IMo differentiation into PMo. We have previously reported lower PMo frequency in patients with sickle cell disease (SCD), considered an inflammatory disease with altered immune profiles. To test whether altered differentiation programming of IMo/PMo may contribute to reduced PMo frequency in SCD, we analyzed the frequency of CMo/IMo/PMo at baseline and after IFN-γ or IL-10 injection to mimic an inflammatory response in AA mice (expressing normal human hemoglobin) and SS mice (expressing human SCD hemoglobin). We found significantly lower IMo frequency before treatment (AA vs SS:15.0% vs 9.2%) but also lower induction of IMo following IFN-γ treatment in SS mice (18%) relative to AA mice (35%), suggesting that IFN-γ inhibition of IMo differentiation into PMo in SCD is impaired. Furthermore, IL-10 was less effective in inducing PMo in SS as compared to AA mice (SS vs AA: 40% vs 60%). These data suggest that IFN-γ or IL-10-mediated monocyte differentiation in SCD is altered. Altogether, these data have unraveled a novel role for IFN-γ or IL-10, two key cytokines known to be induced during an inflammatory response, in monocyte differentiation, and suggest that IMo/PMo differentiation in a chronic inflammatory disease such as SCD may be defective due to altered response to IFN-γ and IL-10, opening up the potential for identification of novel therapeutic targets for IMo/PMo associated diseases including SCD. Disclosures No relevant conflicts of interest to declare.

G(HbF): a genetic model of fetal hemoglobin in sickle cell disease

AbstractFetal hemoglobin (HbF) is a strong modifier of sickle cell disease (SCD) severity and is associated with 3 common genetic loci. Quantifying the genetic effects of the 3 loci would specifically address the benefits of HbF increases in patients. Here, we have applied statistical methods using the most representative variants: rs1427407 and rs6545816 in BCL11A, rs66650371 (3-bp deletion) and rs9376090 in HMIP-2A, rs9494142 and rs9494145 in HMIP-2B, and rs7482144 (Xmn1-HBG2 in the β-globin locus) to create g(HbF), a genetic quantitative variable for HbF in SCD. Only patients aged ≥5 years with complete genotype and HbF data were studied. Five hundred eighty-one patients with hemoglobin SS (HbSS) or HbSβ0 thalassemia formed the “discovery” cohort. Multiple linear regression modeling rationalized the 7 variants down to 4 markers (rs6545816, rs1427407, rs66650371, and rs7482144) each independently contributing HbF-boosting alleles, together accounting for 21.8% of HbF variability (r2) in the HbSS or HbSβ0 patients. The model was replicated with consistent r2 in 2 different cohorts: 27.5% in HbSC patients (N = 186) and 23% in 994 Tanzanian HbSS patients. g(HbF), our 4-variant model, provides a robust approach to account for the genetic component of HbF in SCD and is of potential utility in sickle genetic and clinical studies.

ExVivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide.

Improved solubility of deoxygenated hemoglobin in sickle cell disease: Correlations between erythrocytic Ca2+-ion concentration and hemoglobin solubility

Improved solubility of deoxygenated hemoglobin in sickle cell disease: Correlations between erythrocytic Ca2+-ion concentration and hemoglobin solubility

Original Research: A case-control genome-wide association study identifies genetic modifiers of fetal hemoglobin in sickle cell disease

Sickle cell disease (SCD) is a group of inherited blood disorders that have in common a mutation in the sixth codon of the β-globin (HBB) gene on chromosome 11. However, people with the same genetic mutation display a wide range of clinical phenotypes. Fetal hemoglobin (HbF) expression is an important genetic modifier of SCD complications leading to milder symptoms and improved long-term survival. Therefore, we performed a genome-wide association study (GWAS) using a case-control experimental design in 244 African Americans with SCD to discover genetic factors associated with HbF expression. The case group consisted of subjects with HbF≥8.6% (133 samples) and control group subjects with HbF≤£3.1% (111 samples). Our GWAS results replicated SNPs previously identified in an erythroid-specific enhancer region located in the second intron of the BCL11A gene associated with HbF expression. In addition, we identified SNPs in the SPARC, GJC1, EFTUD2 and JAZF1 genes as novel candidates associated with HbF levels. To gain insights into mechanisms of globin gene regulation in the HBB locus, linkage disequilibrium (LD) and haplotype analyses were conducted. We observed strong LD in the low HbF group in contrast to a loss of LD and greater number of haplotypes in the high HbF group. A search of known HBB locus regulatory elements identified SNPs 5' of δ-globin located in an HbF silencing region. In particular, SNP rs4910736 created a binding site for a known transcription repressor GFi1 which is a candidate protein for further investigation. Another HbF-associated SNP, rs2855122 in the cAMP response element upstream of Gγ-globin, was analyzed for functional relevance. Studies performed with siRNA-mediated CREB binding protein (CBP) knockdown in primary erythroid cells demonstrated γ-globin activation and HbF induction, supporting a repressor role for CBP. This study identifies possible molecular determinants of HbF production.

Serum Copper Level Significantly Influences Platelet Count, Lymphocyte Count and Mean Cell Hemoglobin in Sickle Cell Anemia

Background: Changes in serum micro nutrients levels affect a number of critically important metabolic processes; these could potentially influence blood counts and ultimately disease presentation in patients with sickle cell anemia (SCA). Objectives: To evaluate the influence of serum micro-nutrients levels; zinc, copper, selenium and magnesium on blood counts in steady state SCA patients. Methods: A cross sectional study that involved 28 steady state adult SCA subjects. Seven milliliters (mls) of blood was collected; 3 mls was for hemoglobin electrophoresis and full blood count determination while 4 mls was for measurement of serum micro nutrients levels, by the atomic absorption spectrophotometry. Correlation between serum micro-nutrient levels and blood counts was done by the Pearson’s linear regression. Ethical approval was obtained from the institutional review board and each participant gave informed consent. All data was analyzed by SPSS software version 20. Results: There was a significant correlation between serum copper levels and mean cell hemoglobin (MCH), platelet and lymphocyte counts (r = 0.418; P = 0.02, r = -0.376; P = 0.04 and r = -0.383; P = 0.04, respectively). There were no significant correlations between serum levels of other micro nutrients (selenium, zinc and magnesium) and blood counts. Conclusions: Copper influences blood count in SCA patients probably by inducing red cell haemolysis, oxidant tissue damage and stimulating the immune system.

Dimethyl Fumarate Induces Fetal Hemoglobin in Sickle Cell Disease

Sickle cell disease (SCD) is caused by a point mutation in the β-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive crises (VOC) and chronic hemolytic anemia. Fetal hemoglobin (HbF) is the major species of hemoglobin during fetal and neonatal development, the expression of which is replaced by the adult beta globin perinatally. Reactivation of HbF in adult SCD patients is considered beneficial in ameliorating the symptoms of the disease. This is exemplified by a condition known as hereditary persistence of fetal hemoglobin (HPFH) in heterozygous sickle disease patients where symptoms of SCD are absent and the typical HbF level is over 30% (Steinberg, M.H. et al, 2014). Strategies to reactivate HbF have been used successfully in mouse models of SCD and led to amelioration of the disease phenotype, which is also partly the mechanism of action of hydroxyurea, the only FDA approved drug for SCD. Up-regulation of HbF can be achieved by several different approaches including pharmacologic or genetic manipulation of transcription activators or repressors of HbF. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a basic leucine zipper transcription factor that has been shown to activate γ-globin transcription and increase HbF levels in cultured CD34+ erythroid cells. Nrf2 is well established for its role in cytoprotective and anti-oxidant actions by transcriptionally activating target genes that confer protection from oxidative damage triggered from injury and inflammation. Nrf2 is normally sequestered in the cytoplasm by Keap1, a Kelch-domain protein. Release of Nrf2 from Keap1 allows nuclear translocation of Nrf2 and activation of target genes via its binding to an anti-oxidant response element (ARE) in the promoter region of Nrf2 target genes. Previous studies (Macari and Lowrey, 2011) have demonstrated that the γ-globin promoter contains an ARE sequence supporting Nrf2 as an inducer of HbF. We investigated the role of dimethyl fumarate (DMF), a small molecule Nrf2 agonist, in activating γ-globin transcription and enhancing levels of HbF in tissue culture and murine SCD models. Delayed-release DMF, approved by the FDA as Tecfidera, is an oral therapeutic for the treatment of relapsing multiple sclerosis (MS). After oral administration of DMF, human exposure occurs to both DMF and the bioactive primary metabolite, monomethyl fumarate (MMF). We assessed the ability of increasing concentrations of DMF to induce γ-globin mRNA in human erythroleukemia cells, CD34+ cells isolated from bone marrow of non-SCD volunteer donors, and peripheral blood mononuclear cells from SCD volunteer donors. γ-globin mRNA increased by 3-4 fold compared to control as quantitated using real-time PCR. DMF incubation also resulted in 2-fold upregulation in HbF protein levels as demonstrated by UPLC and western blotting analysis and also a 2-fold induction in percentage of RBC containing HbF (F-cells) by flow cytometry. In addition, co-incubation of DMF with hydroxyurea produced an additive effect on γ-globin mRNA (8 - 10 fold in non-SCD; 2-4 fold in SCD) and F-cell induction in CD34+ stem cell derived erythroid progenitors (3-4 fold in both SCD and non-SCD). DMF treatment of KU812 erythroleukemia cells induced Nrf2 activation resulting in Nrf2 nuclear translocation and occupancy of the ARE in the γ-globin promoter by ChIP assay. Moreover, siRNA based knockdown of Keap1 resulted in up-regulation of HbF by flow cytometry, demonstrating that activation of the Nrf2 pathway in erythroid cells can result in the up-regulation of HbF. The effect of DMF on HbF induction was assessed in the Townes SCD mouse model. Townes mice were administered DMF at 100 mg/kg IP for 5 day per week for one month duration and compared to treatment with vehicle control and to treatment with hydroxyurea. This dose and route of administration of DMF resulted in clinically relevant plasma levels of MMF in Townes mice. After a month of dosing, there was a 3- to 4-fold increase in circulating F-cells in the DMF treated mice which was comparable to the hydroxyurea treated mice. Based on these findings, DMF may have potential as an HbF inducer in the therapy of SCD, in addition to its vascular protective and heme detoxifying properties (Belcher et al, ASH 2014). Further clinical studies are needed to evaluate the safety and efficacy of DMF in SCD. DisclosuresKrishnamoorthy:Biogen: Employment, Equity Ownership. Gupta:Biogen: Employment, Equity Ownership. Sturtevant:Biogen: Employment. Li:Georgia Regents University: Employment. Makala:Georgia Regents University: Employment. Hobbs:Biogen: Employment, Equity Ownership. Light:Biogen: Employment, Equity Ownership.

Novel Epigenetic Modulators That Promote Fetal Hemoglobin Production for the Prevention of Sickle Cell Disease Related Complications

Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Affected patients are at risk of multi-organ complications, significant morbidity and early mortality with an average age at death of 42 and 48 years for men and women, respectively. Complications include acute and chronic pain due to vascular occlusion, end organ damage, acute chest syndrome and increased risk of sepsis. SCD is caused by a point mutation on chromosome 11, which codes for the beta chain of adult hemoglobin. Fetal hemoglobin is not affected by the mutation. Persons with SCD who maintain persistent fetal hemoglobin demonstrate a decreased number of pain crises and acute chest episodes along with a decreased risk of mortality compared to other patients with SCD. The only FDA approved fetal hemoglobin inducer, Hydroxyurea, remains highly underutilized due to the risk of complications and unwanted side effects. The discovery of a new agent that promotes re-expression of fetal hemoglobin could revolutionize the care of affected patients by reducing morbidity and mortality. Epigenetic modulation is one possible mechanistic approach to accomplish this goal. Recent studies with lysine specific demethylase-1 (LSD1) inhibitors, including tranylcypromine (TCP) have shown promising results. Unfortunately, TCP also affects monoamine oxidase and thus has significant side effects. Developing a molecule with high specificity, low cross reactivity and negligible toxicity would increase desired effects while reducing unwanted side effects.Objectives: To discover small-molecule inhibitors of LSD1 that exhibit high selectivity for LSD1 and low in-vivo toxicity that can be used to promote re-expression of fetal hemoglobin in SCD patients.Design/Methods: Our laboratory at the Medical University of South Carolina has recently described a library of potent, non-toxic LSD1 inhibitors. Our preliminary results suggest that these inhibitors show promise as fetal hemoglobin inducers. Using K562 cells, an erythroleukemic cell line known to model in-vivo hemoglobin production, we are screening this library to identify the most effective compounds using Western blotting for the gamma globin chain that is unique to fetal hemoglobin. Following identification of the most effective compounds in initial screens, we will confirm results with RT-qPCR. Subsequent studies will involve culturing and treating erythroid progenitor cells from healthy subject samples and from patients with SCD obtained through bone marrow sampling. HPLC will be used for identification and quantification of hemoglobin chains, including fetal hemoglobin, after ex-vivo treatment with selected compounds.Results: Initial results from Western blots for fetal hemoglobin (gamma globin protein) demonstrate that TCP, as well as the experimental compounds C1, 107-3 and 107-15, promote the re-expression of fetal hemoglobin in K562 cells. Experimental compounds were compared to DMSO as negative control, and hemin (10 mM) and hydroxyurea (200 mM) as positive controls. All experimental compounds evaluated are significantly less toxic than hydroxyurea, and do not impact cell viability in trypan blue exclusion studies.Conclusion: Initial studies show promising results for fetal hemoglobin production using these novel LSD-1 inhibitors. Viability data is also encouraging. Continued investigation is warranted to further investigate the efficacy of these compounds. DisclosuresNo relevant conflicts of interest to declare.