Assessing Patterns of Telehealth Use Among People with Sickle Cell Disease Enrolled in Medicaid During the Start of the COVID-19 Pandemic.

Background: Telehealth can be defined as using remote technologies to provide health care. It may increase access to care among people with sickle cell disease (SCD). This study examined (1) telehealth use, (2) characteristics of telehealth use, and (3) differences between telehealth users and nonusers among people with SCD during the COVID-19 pandemic. Methods: This was a retrospective analysis of Medicaid claims among four states [California (CA), Georgia (GA), Michigan (MI), Tennessee (TN)] participating in the Sickle Cell Data Collection program. Study participants were individuals ≥1 year old with SCD enrolled in Medicaid September 2019-December 2020. Telehealth encounters during the pandemic were characterized by provider specialty. Health care utilization was compared between those who did (users) and did not (nonusers) use telehealth, stratified by before and during the pandemic. Results: A total of 8,681 individuals with SCD (1,638 CA; 3,612 GA; 1,880 MI; and 1,551 TN) were included. The proportion of individuals with SCD that accessed telehealth during the pandemic varied across states from 29% in TN to 80% in CA. During the pandemic, there was a total of 21,632 telehealth encounters across 3,647 users. In two states (MI and GA), over a third of telehealth encounters were with behavioral health providers. Telehealth users had a higher average number of health care encounters during the pandemic: emergency department (pooled mean = 2.6 for users vs. 1.5 for nonusers), inpatient (1.2 for users vs. 0.6 for nonusers), and outpatient encounters (6.0 for users vs. 3.3 for nonusers). Conclusions: Telehealth was frequently used at the beginning of the COVID-19 pandemic by people with SCD. Future research should focus on the context, facilitators, and barriers of its implementation in this population.

Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

Characteristics of Emergency Department Visits Made by Individuals With Sickle Cell Disease in the U.S., 1999-2020.

Introduction: Individuals living with sickle cell disease experience high levels of morbidity that result in frequent utilization of the emergency department. The objective of this study was to provide updated national estimates of emergency department utilization associated with sickle cell disease in the U.S. Methods: Data from the National Hospital Ambulatory Medical Care Survey for the years 1999-2020 were analyzed. Complex survey analysis was utilized to produce national estimates overall and by patient age groups. Results: On average, approximately 222,612 emergency department visits occurred annually among individuals with sickle cell disease, a nearly 13% increase from prior estimates. The annual volume of emergency department visits steadily increased over time, and pain remains the most common patient-cited reason for visiting the emergency department. Patient-reported pain levels for individuals with sickle cell disease were high, with 64% of visits associated with severe pain and 21% associated with moderate pain. Public insurance sources continue to cover most visits, with Medicaid paying for 60% of visits and Medicare paying for 12% of visits. The average time spent in the emergency department increased from previous estimates by about an hour, rising to approximately 6 hours. The average wait time to see a provider was 53 minutes. Conclusions: Utilization of the emergency department by individuals living with sickle cell disease remains high, especially for pain. With more than half of patients with sickle cell disease reporting severe pain levels, emergency department staff should be prepared to assess and treat sickle cell disease-related pain following evidence-based guidelines and recommendations. The findings of this study can help improve care in this population.

Bach1 inhibitor HPP-D mediates γ-globin gene activation in sickle erythroid progenitors.

Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate-mediated chromatin modifications in sickle cell disease.

Sickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.

Peer mentoring to support career advancement among underrepresented minority faculty in the programs to increase diversity among individuals engaged in health-related research (PRIDE).

Although mentoring is critical for career advancement, underrepresented minority (URM) faculty often lack access to mentoring opportunities. We sought to evaluate the impact of peer mentoring on career development success of URM early career faculty in the National Heart Lung and Blood Institute-sponsored, Programs to Increase Diversity Among Individuals Engaged in Health-Related Research-Functional and Translational Genomics of Blood Disorders (PRIDE-FTG). The outcome of peer mentoring was evaluated using the Mentoring Competency Assessment (MCA), a brief open-ended qualitative survey, and a semi-structured exit interview. Surveys were completed at baseline (Time 1), 6 months, and at the end of PRIDE-FTG participation (Time 2). The following results were obtained. Between Time 1 and Time 2, mentees' self-assessment scores increased for the MCA (p < 0.01) with significant increases in effective communication (p < 0.001), aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). Mentees rated their peer mentors higher in the MCA with significant differences noted for promoting development (p < 0.027). These data suggest that PRIDE-FTG peer mentoring approaches successfully improved MCA competencies among URM junior faculty participants with faculty ranking peer mentors higher than themselves. Among URM faculty, peer mentoring initiatives should be investigated as a key strategy to support early career scholar development.

MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice.

Introduction: Therapeutic strategies aimed at reactivating HBG gene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease (SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducer via targeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF induction via targeting MYB in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction in vivo in the humanized Townes SCD mouse model. Materials and methods: Human KU812 were cultured and normal CD34 cells (n = 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) control in vitro. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR. In vivo studies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells. Results: In silico analysis predicted complementary base-pairing between MIR29B and the 3'-untranslated region (UTR) of MYB. Overexpression of MIR29B significantly reduced MYB mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length MYB 3'-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length MYB 3'-UTR as compared to cells that only expressed MYB 3'-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress MYB on HBG activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and MYB in both cell types further demonstrated the inhibitory effect of MIR29B on MYB expression, resulting in HBG reactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression of MYB and DNMT3 gene expression in the humanized Townes sickle cell mouse model. Discussion: Our findings support the ability of MIR29B to induce HbF in vivo in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate HBG transcription by MYB gene silencing in vivo. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.

Targeting Genetic Modifiers of HBG Gene Expression in Sickle Cell Disease: The miRNA Option.

Sickle cell disease (SCD) is one of the most common inherited hemoglobinopathy disorders that affects millions of people worldwide. Reactivation of HBG (HBG1, HBG2) gene expression and induction of fetal hemoglobin (HbF) is an important therapeutic strategy for ameliorating the clinical symptoms and severity of SCD. Hydroxyurea is the only US FDA-approved drug with proven efficacy to induce HbF in SCD patients, yet serious complications have been associated with its use. Over the last three decades, numerous additional pharmacological agents that reactivate HBG transcription in vitro have been investigated, but few have proceeded to FDA approval, with the exception of arginine butyrate and decitabine; however, neither drug met the requirements for routine clinical use due to difficulties with oral delivery and inability to achieve therapeutic levels. Thus, novel approaches that produce sufficient efficacy, specificity, and sustainable HbF induction with low adverse effects are desirable. More recently, microRNAs (miRNAs) have gained attention for their diagnostic and therapeutic potential to treat various diseases ranging from cancer to Alzheimer's disease via targeting oncogenes and their gene products. Thus, it is plausible that miRNAs that target HBG regulatory genes may be useful for inducing HbF as a treatment for SCD. Our laboratory and others have documented the association of miRNAs with HBG activation or suppression via silencing transcriptional repressors and activators, respectively, of HBG expression. Herein, we review progress made in understanding molecular mechanisms of miRNA-mediated HBG regulation and discuss the extent to which molecular targets of HBG might be suitable prospects for development of SCD clinical therapy. Lastly, we discuss challenges with the application of miRNA delivery in vivo and provide potential strategies for overcoming barriers in the future.

Salubrinal induces fetal hemoglobin expression via the stress-signaling pathway in human sickle erythroid progenitors and sickle cell disease mice.

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24µM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.

A randomized, placebo-controlled, double-blind trial of canakinumab in children and young adults with sickle cell anemia.

Excessive intravascular release of lysed cellular contents from damaged red blood cells (RBCs) in patients with sickle cell anemia (SCA) can activate the inflammasome, a multiprotein oligomer promoting maturation and secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). We hypothesized that IL-1ß blockade by canakinumab in patients with SCA would reduce markers of inflammation and clinical disease activity. In this randomized, double-blind, multicenter phase 2a study, patients aged 8 to 20 years with SCA (HbSS or HbSß0-thalassemia), history of acute pain episodes, and elevated high-sensitivity C-reactive protein >1.0 mg/L at screening were randomized 1:1 to received 6 monthly treatments with 300 mg subcutaneous canakinumab or placebo. Measured outcomes at baseline and weeks 4, 8, 12, 16, 20, and 24 included electronic patient-reported outcomes, hospitalization rate, and adverse events (AEs) and serious AEs (SAEs). All but 1 of the 49 enrolled patients were receiving stable background hydroxyurea therapy. Although the primary objective (prespecified reduction of pain) was not met, compared with patients in the placebo arm, patients treated with canakinumab had reductions in markers of inflammation, occurrence of SCA-related AEs and SAEs, and number and duration of hospitalizations as well as trends for improvement in pain intensity, fatigue, and absences from school or work. Post hoc analysis revealed treatment effects on weight, restricted to pediatric patients. Canakinumab was well tolerated with no treatment-related SAEs and no new safety signal. These findings demonstrate that the inflammation associated with SCA can be reduced by selective IL-1ß blockade by canakinumab with potential for therapeutic benefits. This trial was registered at www.clinicaltrials.gov as #NCT02961218.

Novel histone deacetylase inhibitor CT-101 induces γ-globin gene expression in sickle erythroid progenitors with targeted epigenetic effects.

Induction of fetal hemoglobin (HbF) expression ameliorates the clinical severity and prolong survival in persons with sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved HbF inducer however, additional therapeutics that produce an additive effect in SCD are needed. To this end, development of potent Class I histone deacetylase inhibitors (HDACi) for HbF induction represents a rational molecularly targeted approach. In studies here, we evaluated CT-101, a novel Class I-restricted HDACi, a Largazole derivative, for pharmacodynamics, cytotoxicity, and targeted epigenetic effects. In SCD-derived erythroid progenitors, CT-101 induced HbF expression with additive activity in combination with HU. CT-101 preferentially activated γ-globin gene transcription, increased acetylated histone H3 levels, and conferred an open chromatin conformation in the γ-globin promoter. These data indicate CT-101 represents a strong potential candidate as a molecularly targeted inducer of HbF.

Exploring epigenetic and microRNA approaches for γ-globin gene regulation.

Therapeutic interventions aimed at inducing fetal hemoglobin and reducing the concentration of sickle hemoglobin is an effective approach to ameliorating acute and chronic complications of sickle cell disease, exemplified by the long-term use of hydroxyurea. However, there remains an unmet need for the development of additional safe and effective drugs for single agent or combination therapy for individuals with ß-hemoglobinopathies. Regulation of the γ-globin to ß-globin switch is achieved by chromatin remodeling at the HBB locus on chromosome 11 and interactions of major DNA binding proteins, such as KLF1 and BCL11A in the proximal promoters of the globin genes. Experimental evidence also supports a role of epigenetic modifications including DNA methylation, histone acetylation/methylation, and microRNA expression in γ-globin gene silencing during development. In this review, we will critically evaluate the role of epigenetic mechanisms in γ-globin gene regulation and discuss data generated in tissue culture, pre-clinical animal models, and clinical trials to support drug development to date. The question remains whether modulation of epigenetic pathways will produce sufficient efficacy and specificity for fetal hemoglobin induction and to what extent targeting these pathways form the basis of prospects for clinical therapy.

Effect of Poloxamer 188 vs Placebo on Painful Vaso-Occlusive Episodes in Children and Adults With Sickle Cell Disease: A Randomized Clinical Trial.

Importance: Although effective agents are available to prevent painful vaso-occlusive episodes of sickle cell disease (SCD), there are no disease-modifying therapies for ongoing painful vaso-occlusive episodes; treatment remains supportive. A previous phase 3 trial of poloxamer 188 reported shortened duration of painful vaso-occlusive episodes in SCD, particularly in children and participants treated with hydroxyurea. Objective: To reassess the efficacy of poloxamer 188 for vaso-occlusive episodes. Design, Setting, and Participants: Phase 3, randomized, double-blind, placebo-controlled, multicenter, international trial conducted from May 2013 to February 2016 that included 66 hospitals in 12 countries and 60 cities; 388 individuals with SCD (hemoglobin SS, SC, S-ß0 thalassemia, or S-ß+ thalassemia disease) aged 4 to 65 years with acute moderate to severe pain typical of painful vaso-occlusive episodes requiring hospitalization were included. Interventions: A 1-hour 100-mg/kg loading dose of poloxamer 188 intravenously followed by a 12-hour to 48-hour 30-mg/kg/h continuous infusion (n = 194) or placebo (n = 194). Main Outcomes and Measures: Time in hours from randomization to the last dose of parenteral opioids among all participants and among those younger than 16 years as a separate subgroup. Results: Of 437 participants assessed for eligibility, 388 were randomized (mean age, 15.2 years; 176 [45.4%] female), the primary outcome was available for 384 (99.0%), 15-day follow-up contacts were available for 357 (92.0%), and 30-day follow-up contacts were available for 368 (94.8%). There was no significant difference between the groups for the mean time to last dose of parenteral opioids (81.8 h for the poloxamer 188 group vs 77.8 h for the placebo group; difference, 4.0 h [95% CI, -7.8 to 15.7]; geometric mean ratio, 1.2 [95% CI, 1.0-1.5]; P = .09). Based on a significant interaction of age and treatment (P = .01), there was a treatment difference in time from randomization to last administration of parenteral opioids for participants younger than 16 years (88.7 h in the poloxamer 188 group vs 71.9 h in the placebo group; difference, 16.8 h [95% CI, 1.7-32.0]; geometric mean ratio, 1.4 [95% CI, 1.1-1.8]; P = .008). Adverse events that were more common in the poloxamer 188 group than the placebo group included hyperbilirubinemia (12.7% vs 5.2%); those more common in the placebo group included hypoxia (12.0% vs 5.3%). Conclusions and Relevance: Among children and adults with SCD, poloxamer 188 did not significantly shorten time to last dose of parenteral opioids during vaso-occlusive episodes. These findings do not support the use of poloxamer 188 for vaso-occlusive episodes. Trial Registration: ClinicalTrials.gov Identifier: NCT01737814.

Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of ß-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human ß-globin gene (ß-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in ß-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of ß-hemoglobinopathies.

Sickle cell disease: progress towards combination drug therapy.

Dr. John Herrick described the first clinical case of sickle cell anaemia (SCA) in the United States in 1910. Subsequently, four decades later, Ingram and colleagues characterized the A to T substitution in DNA producing the GAG to GTG codon and replacement of glutamic acid with valine in the sixth position of the ßS -globin chain. The establishment of Comprehensive Sickle Cell Centers in the United States in the 1970s was an important milestone in the development of treatment strategies and describing the natural history of sickle cell disease (SCD) comprised of genotypes including homozygous haemoglobin SS (HbSS), HbSß0 thalassaemia, HbSC and HbSß+ thalassaemia, among others. Early drug studies demonstrating effective treatments of HbSS and HbSß0 thalassaemia, stimulated clinical trials to develop disease-specific therapies to induce fetal haemoglobin due to its ability to block HbS polymerization. Subsequently, hydroxycarbamide proved efficacious in adults with SCA and was Food and Drug Administration (FDA)-approved in 1998. After two decades of hydroxycarbamide use for SCD, there continues to be limited clinical acceptance of this chemotherapy drug, providing the impetus for investigators and pharmaceutical companies to develop non-chemotherapy agents. Investigative efforts to determine the role of events downstream of deoxy-HbS polymerization, such as endothelial cell activation, cellular adhesion, chronic inflammation, intravascular haemolysis and nitric oxide scavenging, have expanded drug targets which reverse the pathophysiology of SCD. After two decades of slow progress in the field, since 2018 three new drugs were FDA-approved for SCA, but research efforts to develop treatments continue. Currently over 30 treatment intervention trials are in progress to investigate a wide range of agents acting by complementary mechanisms, providing the rationale for ushering in the age of effective and safe combination drug therapy for SCD. Parallel efforts to develop curative therapies using haematopoietic stem cell transplant and gene therapy provide individuals with SCD multiple treatment options. We will discuss progress made towards drug development and potential combination drug therapy for SCD with the standard of care hydroxycarbamide.

NRF2 mediates γ-globin gene regulation through epigenetic modifications in a ß-YAC transgenic mouse model.

IMPACT STATEMENT: Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human ß-like globin gene locus of ß-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Conjugate prodrug AN-233 induces fetal hemoglobin expression in sickle erythroid progenitors and ß-YAC transgenic mice.

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical ß-YAC mouse model. We observed significantly increased γ-globin gene transcription and HbF expression mediated by AN-233 in K562 cells. Moreover, AN-233 stimulated mild heme biosynthesis and inhibited expression of heme-regulated eIF2α kinase involved in silencing γ-globin expression. Studies using primary erythroid precursors generated from sickle peripheral blood mononuclear cells verified the ability of AN-233 to induce HbF, increase histone H3 and H4 acetylation levels at the γ-globin promoter and reduce erythroid precursor sickling by 50%. Subsequent drug treatment of ß-YAC transgenic mice confirmed HbF induction in vivo by AN-233 through an increase in the percentage of HbF positive red blood cells and HbF levels measured by flow cytometry. These data support the potential development of AN-233 for the treatment of SCD.

MIR29B mediates epigenetic mechanisms of HBG gene activation.

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.