HbF Induction Research Articles

Novel Conjugates of Butyrate and δ-Aminolevulinate Increase γ-Globin Gene Expression and Fetal Hemoglobin Synthesis in Erythroid Progenitors

Pharmacologic induction of fetal hemoglobin (HbF) expression reduces the clinical severity of sickle cell disease (SCD) and improves long-term survival. Research efforts to develop effective and safe therapies have focused on a wide variety of agents however, Hydroxyurea is the only FDA-approved drug proven to mediate HbF induction in about 50% of adult SCD patients. Our group previously reported potent HbF-induction by sodium butyrate through p38 MAPK activation and CREB1 binding to the Gγ-globin cyclic AMP response element (Sangerman et al., Blood 2006). However, oral administration of butyrate derivatives to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore to address this critical barrier of limited treatment options for SCD, we investigated the prodrugs 1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (AN233) and 1-(butyryloxy)butyl 5-amino-4-oxopentanoate (AN908), esters of δ-aminolevulinate (ALA) and butyric acid as HbF inducers in erythroid cells. Oral administration of AN233 and AN908 to BALB/c mice increased total hemoglobin levels without causing tissue toxicity (Rephaeli et al., Eur J Pharm Sci 2016).To test the ability of prodrugs to induce HbF, both K562 and KU812 cells and the immortalized human umbilical cord blood-derived erythroid progenitor 1 (HUDEP1) model were used. Treatment of K562 cells with 0.125-0.50 mM concentrations of AN233 produced cell toxicity at the 0.50 mM concentration, however >90% viability was observed by Trypan blue exclusion for 0.25 mM AN233 without evidence of caspace-3 cleavage. Using this concentration of AN233, a 1.5-fold and 6-fold (p<0.05) increase in γ-globin mRNA and HbF levels respectively, were observed supporting robust intracellular activation of AN233. Flow cytometry analysis confirmed a 1.5-fold increase in the percent of HbF positive cells (F-cells). Preliminary studies in HUDEP1 cells showed 0.25 mM AN233 increased γ-globin expression by 1.5 fold and HbF protein synthesis by 2.2 fold. Mechanistically, AN233 mediated a 53% decrease in phosphorylation levels of the kinase, eIF2α (eukaryotic initiation factor 2α) which favors conditions for protein synthesis. As further evidence that both prodrugs induce HbF synthesis in different cell types, KU812 cells expressing the γ-globin and β-globin genes were treated with AN908 (0.125-0.50 mM). In contrast to AN233, cell toxicity was not observed at the 0.50 mM AN908 concentration where 93% cell viability was maintained. Treating KU812 cells with 0.25 mM AN908 for 48 h produced a 1.4-fold increase in γ-globin transcription and HbF synthesis by western blot. In addition, F-cell levels increased from 45% to 70% and HbF levels (by mean fluorescence intensity) 2.2-fold (p<0.05). Interestingly, when 0.25 mM AN908 was combined with hydroxyurea (100 μM) we observed an additive increase in HbF and F-cells levels.Collectively, these findings support the oral active prodrugs AN233 and AN908 as HbF inducers in erythroid cells. Furthermore, the combination of AN908 and hydroxyurea produced an additive effect without increasing cell toxicity. Studies are underway to determine the EC50 of both prodrugs in normal and sickle primary erythroid progenitors and mechanisms of γ-globin regulation through epigenetic modifications. DisclosuresNo relevant conflicts of interest to declare.

Domain-Focused CRISPR-Cas9 Screen Identifies Hri Kinase As a Regulator of Fetal Hemoglobin in Adult Human Erythroid Cells

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit in patients with sickle cell disease (SCD) and some forms of β-thalassemia. While developing genome editing and gene replacement approaches promise long term HbF elevation in SCD patients, there remains a strong need for pharmacologic treatment of these diseases. Two transcription factors, BCL11A and LRF (ZBTB7A) and their co-regulators mediate most of fetal globin transcriptional silencing in adult erythroid cells. However, transcription factors are inherently challenging to inhibit with small molecules.To identify potentially druggable HbF regulators, we carried out a CRISPR-Cas9 based genetic screen in a human erythroid cell line targeting protein kinases as they are inherently amenable to inhibition by small molecules. Most CRISPR-screens published to date have made use of sgRNAs targeting early exons to induce frameshift mutations. It has recently been shown that targeting sgRNAs to functional protein domains significantly improves screening efficiencies, as both in-frame and frameshift mutations contribute to generating hypomorphic alleles. We thus designed a sgRNA library targeting almost all annotated kinase-domains using a newly optimized sgRNA-scaffold that improves on-target editing activity. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1) as an HbF repressor. HRI is an erythroid specific kinase known to phosphorylate the translation initiation factor eIF2α. eIF2α phosphorylation generally tempers protein synthesis, but select transcripts with upstream Open Reading Frames (uORFs) in their 5'UTR are known to escape this inhibition.HRI depletion increased the fraction of HbF+ cells and elevated γ-globin (the fetal β-type globin chain) mRNA and protein levels. These effects were surprisingly specific as evidenced by whole proteome mass spectrometry and RNA-seq. Importantly, HRI depletion did not adversely affect cell growth or maturation. Similar results were obtained in primary CD34+ derived erythroid cells that were HRI depleted. HRI deficiency in erythroid cultures from patients with SCD reduced cell sickling, suggesting that biologically relevant HbF levels might be achieved by targeting HRI.Mechanistically, HRI loss strongly reduces BCL11A expression post-transcriptionally without impacting LRF production. BCL11A appears to be a major effector of HRI function since forced expression of BCL11A in HRI-depleted cells restores HbF repression to a significant degree.Finally, we explored whether pharmacologic HbF inducers that act via distinct mechanisms might further amplify HbF production. Pomalidomide has recently been shown to lower BCL11A mRNA levels and raise HbF production. Our proof-of-concept experiments demonstrate strong cooperativity of HRI depletion and pomalidomide treatment in HbF induction. HRI emerges as an erythroid specific target for HbF induction alone or in combination with mechanistically distinct HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown, β-Globin Induction and Simultaneous Repression of β-Globin for the Potential Treatment of Sickle Cell Disease

▪Ex vivo lentiviral vector-based gene therapy has shown promise as a treatment for severe sickle cell disease (SCD) (Ribeil, NEJM 2017). This approach relies on the regulated expression of a therapeutic anti-sickling β-globin (T87Q) at sufficient levels to reduce or prevent the polymerization of mutant hemoglobin S (HbS). Reactivation of fetal hemoglobin (HbF), by reducing expression of BCL11A with a lineage-specific miRNA adapted shRNA (shRNA (miR)), is an alternative strategy to generate therapeutic hemoglobin in transduced cells (Brendel, JCI 2016). Erythroid-specific silencing of BCL11A has an added benefit of simultaneously reducing expression of adult β-globin (which is mutated to HbS in SCD). The resulting increase in HbF (which itself has anti-sickling properties) and concomitant reduction in HbS may provide an extremely powerful approach to attenuating the sickling tendency of red blood cells. Here we provide new preclinical data generated to support the initiation of a first-in-human trial studying BCL11A silencing. Specifically, we report the characterization of a lentiviral vector construct encoding an erythroid-specific BCL11A shRNA(miR), termed BCH_BBLCR-shRNA(miR), adapted from our previously reported vector, that was developed to overcome poor vector titers observed in the manufacturing scale-up of our original vector. Healthy donor human hematopoietic stem cells transduced with lentiviral vector BCH_BBLCR-shRNA(miR) produced at scale under GMP conditions achieved high vector copy numbers (VCNs; >5 copies/diploid genome) and a 5-fold higher induction of HbF than seen with mock transduced cells (65.5% vs 13.1%). SCD human HSCs transduced with high-titer BCH_BBLCR-shRNA(miR) vector achieved >80% transduction efficiency, resulting in HbF induction between 70% and 81% of all β-like globin chains (with concomitant suppression of HbS, with residual HbS ranging from 19% to 30%). In addition, we observed no impact on in vitro growth and/or differentiation of erythroid cells derived from CD34+ cells. Analysis of individual colonies showed that >96% of individual BFU-E cells have HbF >50%. Repopulation studies in NSG mice transplanted with healthy donor human HSCs transduced (>80%) with BCH_BBLCR-shRNA(miR) showed no deleterious effects in vivo of targeting BCL11A in a lineage-specific fashion even at higher VCN (>3 copies/diploid genome). Lastly, consistent with the expected behavior of current generation lentiviral vectors, in vitro immortalization assays confirmed that the BCH_BBLCR-shRNA(miR) vector did not induce immortalization in this setting. Together these data demonstrate that LVV BCH_BBLCR-shRNA(miR) is (i) safe and efficacious in preclinical studies, and (ii) can be generated at scale in a GMP setting to support a clinical study of this potentially attractive approach to the treatment of SCD. DisclosuresNegre:bluebird bio: Employment, Equity Ownership. Parsons:bluebird bio: Employment, Equity Ownership. Luo:bluebird bio Inc.: Employment. Christiansen:bluebird bio: Employment, Equity Ownership. Lewis:bluebird bio: Employment, Equity Ownership. Veres:bluebird bio: Employment, Equity Ownership. Bonner:bluebird bio: Employment, Equity Ownership. Williams:bluebird bio: Patents & Royalties: Licensed IP to bluebird, Research Funding.

Reawakening of Human Fetal Hemoglobin and an Epigenetic Path to the Clinic for Sickle Cell Disease and Beta-Thalassemia: Identification of an Orally-Available, Potent, and Selective Euchromatic Histone Lysine Methyltransferase 1 and 2 (EHMT1/2) Inhibitor

Expression of genes in the β-globin locus switch via epigenetic control at key stages of early gestational development from an embryonic (HbE, α2ε2) to fetal form (HbF, α2γ2) and again around birth to the adult form (HbA, α2β2). Sickle cell disease (SCD) is a multi-organ genetic disorder caused by a point mutation in the adult β-globin gene producing sickled hemoglobin (HbS, α2βs2). It is well-established that SCD patients carrying genetic variations in HbF-regulatory loci have a concomitant increase in blood levels of HbF (ranging from 5-25% of total Hb). These individuals exhibit fewer major crises or no clinical symptoms, suggesting that re-expression of the γ-gene and increasing HbF is a potential disease modifying treatment approach for SCD patients. Hydroxyurea (HU) and L-glutamine are currently the only FDA-approved drugs for the treatment of SCD, with HU working in-part, through the re-induction of HbF. Safety and efficacy responses to HU vary widely, and there remains a strong need for more robust HbF induction strategies with improved safety profiles.Previous groups have shown that inhibitors to various epigenetic modifiers are able to overcome γ-gene silencing and induce HbF; however, associated defects in hematopoiesis have been reported. Recently, histone methytransferase enzymes EHMT1 and EHMT2, responsible for the repressive chromatin mark dimethyl-lysine 9 on histone H3 (H3K9me2), have been shown to be essential for robust repression of γ-globin expression. To-date, an in vitro tool compound UNC0638 has demonstrated activation of γ-globin expression in human adult erythroid progenitor cells but no evaluation of in vivo efficacy of EHMT1/2 inhibition on HbF induction has been possible due to a lack of drug-like properties.Here we describe the generation and characterization of a potent, selective, and orally available small molecule inhibitor of EHMT1/2, EPZ035544. EHMT2 enzymatic activity in a biochemical assay, as measured by the half-maximal inhibitory concentration (IC50) is 10nM and cellular inhibition of H3K9me2 correlated well with an IC50 of 55nM. Treatment of EPZ035544 in primary human CD34+ progenitor cells led to dose-dependent decreases in H3K9me2 with concomitant induction of γ-globin relative to β-globin expression of 10-30% as measured by mass spectrometry and qPCR leading to 100% of F-positive cells. Chromatin immunoprecipitation and sequencing studies performed on EPZ035544-treated CD34+ cells demonstrated that H3K9me2 was reduced throughout the genome with an associated increase in H3K9Ac. The γ-globin gene locus displayed a reduction of H3K9me2 and an increase of H3K9Ac and RNA Polymerase II occupancy leading to significantly increased γ-globin gene expression upon EPZ035544 treatment.The drug-like properties of EPZ035544 enabled the first prolonged in vivo investigation of an EHMT1/2 inhibitor by continuous oral dosing over 90 days in C57BL/6 mice. As mice do not have HbF, we measured the levels of embryonic hemoglobin (Hbb-εy) which is normally repressed after birth. We performed twice-daily (BID) oral dosing in two dose groups: 25 and 50 mg per kilogram of body weight (mg/kg) and once-daily (QD) oral dosing in a single dose group of 75 mg/kg. Dose-dependent increases in the absolute levels of Hbb-εy mRNA in peripheral blood mononuclear cells (PBMCs) were observed with maximal induction of ~ 100 fold achieved at the 50 mg/kg BID and 75 mg/kg QD doses. Using quantitative mass spectrometry, we also observed drug-induced increases in Hbb-εy at the protein level. A corresponding reduction in H3K9me2 levels in PBMCs and bone marrow, as measured by flow cytometry and ELISA, strongly suggests that these effects were a direct consequence of EHMT1/2 inhibition. EPZ035544 was well tolerated throughout the study with no significant changes observed in body weights, spleen weights, or complete blood cell counts. In summary, we have developed an orally bioavailable EHMT1/2 inhibitor with demonstrated on-target in vivo induction of mouse Hbb-εy expression with up to 90 days of continuous dosing. To our knowledge this is the first study to test the in vivo effects of an EHMT1/2 inhibitor on hemoglobin induction. We believe this approach holds promise as a novel treatment option for β-globinopathies, and we continue to advance our program towards the clinic. DisclosuresChan-Penebre:Epizyme Inc.: Employment, Equity Ownership. Gibaja:Epizyme Inc.: Employment, Equity Ownership. Campbell:Epizyme Inc.: Employment, Equity Ownership. Olu:Epizyme Inc.: Employment, Equity Ownership. Admirand:Epizyme Inc.: Employment, Equity Ownership. Brach:Epizyme Inc.: Employment, Equity Ownership. Tang:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Hood:Epizyme Inc.: Employment, Equity Ownership. Cocozaki:Epizyme Inc.: Employment, Equity Ownership. Karaman:Epizyme Inc.: Employment, Equity Ownership. Stickland:Epizyme Inc.: Employment, Equity Ownership. Munchhof:Michael J. Munchhof LLC: Consultancy. Armstrong:Epizyme Inc.: Employment, Equity Ownership. Riera:Epizyme Inc.: Employment, Equity Ownership. Orkin:Bioverativ: Consultancy; Epizyme Inc.: Consultancy. Boriack-Sjodin:Epizyme Inc.: Employment, Equity Ownership. Janzen:Epizyme Inc.: Employment, Equity Ownership. Blakemore:Epizyme Inc.: Employment, Equity Ownership. Ribich:Epizyme Inc.: Employment, Equity Ownership. Chesworth:Epizyme Inc.: Employment, Equity Ownership. Smith:Epizyme Inc.: Employment, Equity Ownership. Duncan:Epizyme Inc.: Employment, Equity Ownership.

Screening Fetal Hemoglobin Inducing Compounds in Human Umbilical Cord Blood-Derived Erythroid Progenitor Cells

Background: Human Umbilical Cord Blood-Derived Erythroid Progenitor Cells-2 (HUDEP-2) cells are an immortalized human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroid precursor cell line that expresses BCL11A and β-globin. HUDEP-1 cells are an immortalized human CD34+ HSPC-derived erythroid precursor cell line that does not express BCL11A, predominantly expresses γ-globin, and exhibits an increase in γ-globin after differentiation. High baseline γ-globin and difficulty in distinguishing between true γ-globin induction and changes in HUDEP-1 differentiation, which increases g-globin, combined with the lack of β-globin expression, make HUDEP-1 cells a suboptimal system in which to study hemoglobin switching. HUDEP-2 cells have been used successfully to demonstrate HbF induction in several gene editing experiments involving deletion of BCL11A enhancer regions and substitution mutations in the γ-globin locus. HUDEP-2 also express very low levels of γ-globin; this has led to the hope that potential HbF inducing compounds can be screened in HUDEP-2 cells, causing them to express significant amounts of γ-globin, and potentially a reduction in β-globin expression, demonstrating hemoglobin switching.Objectives: Determine if HUDEP-2 cell lines are suitable systems in which to examine pharmacologic hemoglobin switching. We will determine if γ-globin induction can be achieved in HUDEP-2 cells from hydroxyurea, metformin, piceatannol, decitabine, or pomalidomide at doses that induce HbF in human HSPCs and raise γ-globin expression in HUDEP-1 cells.Methods: We performed drug treatment on differentiated HUDEP-1 andHUDEP-2 cells as well as hematopoietic stem and progenitor cells (HSPCs) from SCD patients with hydroxyurea, metformin, piceatannol, decitabine and pomalidomide at different doses. The effect of the drugs on HUDEP-1 differentiation was determined by measurement of Band3 by quantitative western blot. After 7 days of drug treatment, HbF was measured by western blot and HPLC in HSPCs, and by RT-qPCR (gamma globin expression) and western blot in HUDEP-1 and HUDEP-2.Results: Hydroxyurea, metformin, piceatannol, decitabine and pomalidomide all significantly increased γ-globin expression in HUDEP-1 cells, and HbF in HSPCs derived from patients with SCD. However, metformin was shown to increase HUDEP-1 differentiation, as measured by a rise in Band 3, which could explain the rise in γ-globin expression. HPLC analysis of the culture of HSPCs derived from patients with SCD showed that drug treatment with 30 µM hydroxyurea, 100 µM Metformin, 25 µM piceatannol or 200 nM decitabine produced a 2.3 fold, 2.1 fold, 3.1 fold, and 2.9 fold increase in HbF respectively. No change was observed in γ-globin expression or HbF in HUDEP-2 cells with any of the 5 drugs tested.Conclusions: γ-globin is not inducible in HUDEP-2 cells using hydroxyurea, metformin, piceatannol, decitabine and pomalidomide, known HbF inducing drugs that are believed to act through different pathways and mechanisms. It is therefore unlikely that HUDEP-2 cells will be effective in screening drug compounds for HbF induction activity. HUDEP-1 cells do show increased γ -globin levels in response to these agents, but care must be taken to show that the rise in γ-globin is not due to further differentiation of HUDEP-1 cells. DisclosuresNo relevant conflicts of interest to declare.

Metformin Induction of Fetal Hemoglobin

Background: Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. We identified FOXO3, a potentially “druggable” transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. We then treated hematopoietic stem and progenitor cells (HSPCs) derived from patients with SCD with metformin, an FDA-approved drug used for type 2 diabetes, which is known to activate FOXO3. Metformin significantly increased HbF alone, and additively with hydroxyurea.Objective: Determine the mechanism by which metformin induces HbF in SCD patient derived HSPCs, and report preliminary clinical trial results of metformin in patients with SCD.Methods: HSPCs from patients with SCD were obtained from discarded red blood cell exchange product. 100 µM Metformin was added on day 7 of two phase primary erythroid culture, and HPLC analysis of hemoglobins or RNA extraction and RT-qPCR or RNASeq was performed on day 14 of culture. BCL11A, pAKT and pAMPK were quantified by Western blot, performed in triplicate.Patients with HbSS between the ages of 16 and 40 who produced less than 20% HbF at maximum tolerated dose of hydroxyurea or were not taking hydroxyurea were recruited to participate in an IRB approved pilot clinical trial of metformin in patients with hemoglobinopathies (Fetal Hemoglobin Induction with Metformin, NCT02981329). RNA was collected from CD45+ can CD71+ cells collected from their peripheral blood at study entry and after 3 months at 1500 mg metformin, and analyzed by RNASeq.Results: Metformin significantly increased HbF production in 80% of the HSPCs derived from unique SCD patients, in contrast with hydroxyurea, which failed to increase HbF in 33% of patient derived HSPCs. When FOXO3 is knocked out in HSPCs, metformin no longer induces HbF. Expression of known gamma globin regulators BCL11A, KLF1, and MYB are not altered by metformin treatment of patient derived HSPCs, nor are they altered by metformin treatment in patient derived RNA from CD45+ or CD71+ cells. Metformin treatment of SCD patient derived HSPCs causes FOXO3 to accumulate in the nucleus, reduces the active, phosphorylated form of AKT, a FOXO3 inhibitor, and increases the active, phosphorylated form of AMPK, a FOXO3 activator. Our clinical trial is ongoing, has enrolled 10 patients to date; results will be presented at the American Society of Hematology meeting.Conclusions: Metformin induction of HbF in SCD patient derived HSPCs requires FOXO3 expression, and affects the activity of known FOXO3 activator AMPK and FOXO3 inhibitor AKT. Metformin does not alter expression levels of known regulators of HbF either in treated HSPCs from patients with SCD or in samples from clinical trial subjects treated with metformin compared to their trial entry samples. We conclude that metformin induces HbF through the FOXO3-AMPK-AKT pathway, and may be a viable pharmacologic HbF inducer alone or in combination with hydroxyurea. DisclosuresNo relevant conflicts of interest to declare.

Generation of New Cell Models to Screen Improved Vectors for Functional Hemoglobin Expression

Gene addition of a functional copy of β-globin and reactivation of fetal hemoglobin (HbF) are promising therapeutical approaches for β-globinopathies such as Sickle Cell Disease (SCD) and β-thalassemia (β-thal).Results from the ongoing clinical trials for β-globinopathies, based on lentiviral delivery of the β-globin gene, indicate that a successful outcome is genotype-dependent. So far, the vectors used in the trials failed to produce curative Hb levels in most of the patients whose genotype leads to null adult hemoglobin (HbA) synthesis, like the β0/β0. Therefore, to achieve HbA synthesis at therapeutic levels in the most severe of genotypes, and with minimal vector copy number (VCN) per cell, more powerful and versatile vectors are required.Our lab has engineered a new lentiviral vector, ALS10, that carries the β-globin gene, including the non-coding regions. In erythroblasts from patients with SCD and β-thal ALS10 induces HbA synthesis of 26.8% and 68.6%, respectively, with an average VCN=1.In the present study we aim to streghten the efficacy of our gene addition approach. To do so, a shRNAmiR targeting the transcription factor BCL11A, a known repressor of γ□-globin, was incorporated into ALS10. The shRNAmiR sequences targeting BCL11A (Guda et. al. Molecular Therapy 2015) were flanked by an optimized backbone termed “miR-E” (Fellmann et. al. Cell Reports 2013) for the purpose of knocking down BCL11A and increasing HbF levels.With this approach we expect to overcome some of the limitations of the vectors presently on clinical trials by simultaneous 1) production of transgenic HbA, 2) reactivation of endogenous HbF and 3) decreased production of endogenous mutant protein.The development of gene addition and RNAi based therapies mainly relies on the isolation of cells from volunteer donors. Which is dependent on the establishment of ethical protocols and on the availability of donors. Moreover, the cells isolated by this procedure have a limited number of cycles before undergoing full differentiation into red blood cells and consequent death. The HUDEP-2 cell line (Kurita et. al. Plos One 2013) under the appropriate conditions, can be propagated indefinitely and, upon induction, differentiated into enucleated RBCs. Although it offers an efficient alternative system to overcome some of the limitations of using donor samples, HUDEP-2 are derived from healthy cells and produce almost exclusively HbA, which is a limitation in approaches based on the addition of a wt β-globin gene.To overcome this limitation we used a CRISPR/Cas9 system to mutagenize the β-globin gene of the HUDEP-2 cell line. We have established 2 clones targeted in both alleles named M#9 and M#13. Clone M#9 is homozygous for the deletion of the triplet that encodes Glu6 and was predicted to translate into a shorter variant of the β-globin protein, made of 146 instead of 147aa. Clone M#13 presents the same deletion on 1 allele and a single nucleotide deletion on the second allele, which, by frameshift, causes a downstream premature stop codon that was predicted to encode a 19aa long protein. Chromatographic separation of the hemolysates of clones M#9 and M#13 confirmed the presence of a new hemoglobin variant that is eluted at a different time, distinguishable from both HbA and sickle hemoglobin (HbS). As expected, neither M#9 nor M#13 produce any HbA. From these properties, we predict that these 2 mutant HUDEP clones can be used as a a new cell model for the β-globinopathies, by providing a tool to measure the levels of HbA or F generated after lentiviral treatment.We transduced M#9 and M#13 with a combined lentiviral vector with the miR-E-BCL11A sequence cloned in the 5' region of β-globin intron 2. Our preliminary results show that, upon integration of the dual lentiviral vector, both clones M#9 and M#13 reach levels of 41% HbF and 36% HbA with a simultaneous decrease in the endogenous mutant form to 17% of the total Hb produced. Western blot analyses confirmed a reduction of BCL11A protein levels and concurrent increase of γ-globin levels.Our results show that M#9 and M#13 are a suitable platform to test both HbF and HbA induction therapies and confirm their versatility as a screening system for the various emerging therapeutic approaches for β-globinophaties. In addition, we show for the first time, that both HbA and F can be elevated using a single lentiviral construct, which could maximize the rate of Hb correction in patients with β-globinopathies. DisclosuresNo relevant conflicts of interest to declare.

Piceatannol Induces Fetal Hemoglobin in Erythroid Progenitor Cells from Patients with Sickle Cell Disease

Background: We have identified the FOXO3-AMPK-AKT pathway as important to HbF regulation through unbiased analysis of whole exome sequencing from patients with sickle cell disease (SCD). Both AMPK and AKT are activated by phosphorylation; AMPK is a FOXO3 activator; AKT inhibits AMPK and FOXO3. We have also treated hematopoietic stem and progenitor cells (HSPCs) from patients with SCD with metformin, a known AMPK/FOXO3 activator, and produced a significant rise in HbF from 17.6% at baseline, to 44.9%. This data is the foundation for an IRB approved clinical trial of metformin in patients with hemoglobinopathies, enrolling patients now.Objectives: We seek to investigate other drugs which act on the FOXO3-AMPK-AKT pathway as possible HbF inducing agents. Resveratrol and piceatannol are both known activators of the pathway; however, resveratrol is not bioavailable. We will investigate piceatannol as a HbF inducing agent, assess toxicity, and verify its action is via the FOXO3-AMPK-AKT pathway.Methods: HSPCs from 5 patients with SCD were treated with serial doses of piceatannol at 5, 12.5, 25, 50 and 100 µM on day 7 of two phase erythroid culture. Cell lysate was collected on day 14, and hemoglobin profile analyzed by HPLC. 2 µM Compound C was added with piceatannol to block AMPK activity, and the effect on HbF measured by HPLC. The effect of piceatannol on the amount of phosphorylated AMPK and AKT in HSPCs was measured by quantitative western blot in triplicate, and the effect of piceatannol on FOXO3 cellular localization was determined by fluorescent microscopy and fractionation of the cells into nuclear and cytoplasmic compartments, followed by quantitative western blot analysis.Results: Erythroid progenitor cells from 5 unique patients with SCD treated with 25 µM piceatannol all exhibited a 3-4 fold rise in HbF as measured by HPLC. Toxicity studies showed that 12.5-50 µM of piceatannol induced HbF without significantly altering CFU generation of human CD34+ HSPCs; treatment with 100 µM piceatannol showed a reduction in CFU. In the presence of Compound C, piceatannol induction of HbF was attenuated. 5-25 µM piceatannol markedly increased the amount of phosphorylated AMPK present in the cell, caused an increase in FOXO3 localization in the nucleus, and effectively inhibited AKT phosphorylation.Conclusions: Piceatannol induces HbF in SCD patient derived HSPCs. While piceatannol has been reported to have HDAC inhibitor activity, the fact that pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction by piceatannol supports the hypothesis that piceatannol acts through the FOXO3-AMPK-AKT pathway. Additionally, piceatannol reduced phosphorylation of a known FOXO3 inhibitor, AKT, and increased phosphorylation of AMPK, a FOXO3 activator, thereby increasing FOXO3 activity and localization to the nucleus. With further safety studies, piceatannol could be a novel HbF inducing agent for patients with hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Characterization of a HBB -Locus Fetal Chromatin Domain Region during Adult Erythropoiesis

The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching.Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core.To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested.Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. DisclosuresNo relevant conflicts of interest to declare.

Personalized Hydroxyurea Dosing to Reduce Time to MTD and Optimize the HbF Response: Results from the TREAT Study

▪Background: Hydroxyurea is well established as the standard of care for children with sickle cell anemia (SCA) in the US, but current dosing schemes and dose responses for individual patients are highly variable. Traditionally, hydroxyurea is started at a low dose of 15-20 mg/kg/day, then slowly escalated to maximum tolerated dose (MTD) based upon hematological parameters to avoid toxicities. Using this approach, the average hydroxyurea MTD is about 25 mg/kg/day and yields an average HbF induction of 20-25%, but the time to achieve MTD is more than 6 months. We developed and prospectively evaluated a population pharmacokinetics (PK)-based dosing model of hydroxyurea for children with SCA, aiming to initiate hydroxyurea at a personalized dose that achieves MTD quickly and achieves a more robust HbF response.Methods: The Therapeutic Response Evaluation and Adherence Trial (TREAT, ClinicalTrials.gov NCT02286154) is a prospective sudy with a primary objective to develop and evaluate a population PK-based model to predict the hydroxyurea MTD through an individualized dosing strategy. The study recruited patients (age 6 months to 18 years) who were initiating hydroxyurea therapy. Prior to starting hydroxyurea, each participant took a single oral dose of 20 mg/kg with subsequent post-dose sampling at 15, 60, and 180 minutes. This sparse sampling strategy was based upon our previously described population PK model that allows accurate estimation of hydroxyurea exposure using only three post-dose blood samples. Plasma hydroxyurea concentrations were measured by HPLC. Published PK data from children who had reached MTD demonstrated that the mean hydroxyurea exposure (AUC) at MTD was 115 mg×h/L. TREAT incorporated individual PK data for each participant using population PK model based Bayesian estimation, to generate a starting hydroxyurea dose that would target this AUC goal in an attempt to start at a dose near or at MTD. Clinical follow-up and dose adjustments were subsequently provider-dependent and based upon institutional clinical guidelines recommending dose escalation to MTD, based on a target ANC of less than 3.0 x10^6/L. The official determination of MTD was declared by two independent hematologists blinded to patient identity; MTD was defined as a stable daily dose without dose escalation, holds, or decreases for at least 8 weeks, along with laboratory values within the target range. The primary outcome of TREAT was to reduce the time to MTD by at least two months from an average of published studies demonstrating a mean (±SD) of 9±4 months.Results:A total of 38 children enrolled in TREAT had PK studies performed to generate an individualized starting dose. Only three participants (7.9%) had incomplete PK studies that did not allow for an individualized starting dose and were started at 20 mg/kg. The enrolled children were young with an average (mean±SD) age of 3.1±4.3 years. Most participants (26/38=68%) were less than two years of age at enrollment, while half (19/38=50%) were less than one year of age. For participants starting with PK-based dosing (n=35), the average starting hydroxyurea dose was 27.5±4.5 mg/kg/day, with 12/35 children (34%) starting hydroxyurea at doses ≥30 mg/kg/day. Despite the high starting doses, there were very few hematological toxicities, with only 2/35 participants requiring a dose decrease from the starting dose due to moderate neutropenia. The individualized starting dose was the actual MTD for 73% of participants without the need for a single dose escalation. The mean time to MTD was 6.2 months (median 4 months) compared to a historical baseline of 9 months. Even with high starting HbF (24.9±11.6%), response was excellent with all participants who have completed at least 6 months of hydroxyurea demonstrating an increase in HbF from baseline. The mean maximum achieved HbF is 35.5±9.5% with 74% achieving HbF>30% and 35% achieving HbF>40%.Conclusions: TREAT has demonstrated the feasibility of individualized, PK-guided hydroxyurea therapy and suggests that early initiation of hydroxyurea for young children with SCA is both safe and effective, achieving HbF responses (>30-40%) beyond which has been seen with standard dosing. This precision medicine approach to hydroxyurea therapy suggests that it is possible to exceed the traditional therapeutic goals with individualized dosing and could be transformational for the treatment of young children with SCA. DisclosuresQuinn:Global Blood Therapeutics: Research Funding. Ware:Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Improved Clinical Outcomes in Adults with Sickle Cell Disease and Diabetes Treated with Metformin

▪BackgroundComplications of sickle cell disease (SCD) include pain episodes, chronic anemia, cardiopulmonary disease, and long-term end-organ damage. Hydroxyurea is efficacious in preventing these complications by increasing fetal hemoglobin levels (HbF); however, adherence is limited. Metformin, a Food and Drug Administration-approved drug used for type-2 diabetes mellitus (DM), was recently found to induce HbF through upregulation of FOXO3 and gamma-globin expression. However, the relation of metformin utilization to clinical outcomes and healthcare utilization in adults with SCD and DM has not been studied.ObjectivesOur specific aims were to 1) estimate the prevalence of DM in a cohort of adult patients with SCD and 2) assess the association of metformin with clinical outcomes and healthcare utilization in this population. We hypothesized that adults with SCD and DM on metformin have less frequent SCD-related complication and lower utilization of hospital care for those complications.MethodsWe analyzed subjects with SCD and DM ≥18 years of age from the Truven Health MarketScan® Multi-State Medicaid Database from 2009-2015 with continuous enrollment of ≥365 days. Subjects with SCD and DM were identified through the presence of ≥1 inpatient or ≥2 outpatient disease-specific billing codes or ≥1 outpatient DM code and ≥1 DM-related drug claim. Subjects with drug claims for insulin, hydroxyurea, or iron chelation were excluded. Metformin use was defined as having ≥1 metformin-related drug claim in the study period. Outcomes included annual rates of all-cause inpatient hospitalizations, SCD-related hospitalizations, all-cause emergency department (ED) visits, SCD-related ED visits, vaso-occlusive episodes (VOE), strokes, acute chest syndrome (ACS) episodes, avascular necrosis (AVN) events, and gallstone events. Outcomes were identified based on claims records after the subject's first DM diagnosis record in the dataset. Clinical events among metformin users and non-users were compared using chi square tests. Rate ratios (RR) comparing annual rates of clinical events among metformin users to non-users were estimated using negative binomial regression, controlling for age, sex, and claims-data-defined Charlson Comorbidity Index (CCI) score.ResultsWe identified 7,617 subjects with SCD and 1,296 (17%) of them also had a diagnosis of DM. Our final cohort included a total of 375 subjects who met all our pre-defined criteria; of them, 126 (34%) were treated with metformin. Subjects were 72% female and 82% Black, and had a median age of 44 years (interquartile range (IQR) 33-52) and CCI score of 2 (IQR 0-4). All clinical events were less frequent in metformin users when compared to non-users, summarized in Table 1a, and subjects in both groups were similar in age, sex, race and CCI score. Using negative binomial regression, adjusted for age, sex and CCI score, metformin users had significantly lower all-cause inpatient stays (rate ratio (RR) 0.55; 95% confidence interval (CI) 0.42-0.72), SCD-related inpatient stays (RR 0.36; 95% CI 0.24-0.54), all-cause ED visits (RR 0.65; 95% CI 0.50-0.86), SCD-related ED visits (RR 0.43; 95% CI 0.27-0.68), VOE (RR 0.15; 95% CI 0.08-0.27), ACS (RR 0.14; 95% CI 0.03-0.59) and AVN (RR 0.29; 95% CI 0.09-0.91), compared to metformin non-users (Table 1b). Similar trends were also seen when patients were stratified by age as young adults (<40 years) vs. adults (≥40 years), as shown in Table 1b.ConclusionsThis study is the first to examine clinical associations of metformin use with SDC-related complications in adults with SCD and DM. These findings strongly suggest that metformin has important protective effects on all-cause and SCD-related hospital-treated clinical events. Among adults with SCD and DM, metformin users had 45% fewer SCD-related inpatient stays and roughly 85% fewer VOE or ACS episodes after controlling for confounding variables. These benefits are consistent with the effects of metformin on HbF induction observed in earlier laboratory data. An analysis of the effects of adherence to metformin on clinical outcomes and healthcare utilization in our cohort is ongoing. Given our findings and the known safety profile of metformin, future prospective studies are warranted to evaluate its efficacy and cost-effectiveness in relation to clinical outcomes, quality of life, mortality and healthcare utilization in adults with SCD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

Fetal hemoglobin (HbF) modifies the severity of sickle cell disease (SCD) and β-thalassemia. One of the major quantitative trait loci (QTL) associated with HbF level is the HBS1L-MYB intergenic polymorphisms (HMIP) on chr6q23. MYB regulates proliferation and maturation of erythroid cells, and modulates gene expression within the HBB gene cluster. A distal enhancer located at ~84 kb upstream of MYB has been shown by GWAS (Farrell et al , Blood 2011), insertional mutation (Suzuki et al, Mol Cell Biol 2013), long-range interaction by 3C analysis (Stadhouders et al, J Clin Invest 2014), and gene editing with Cas9 nucleases (Canver et al, Nat Genet 2017). This enhancer encompasses a 3-bp deletion polymorphism (rs66650371), which is surrounded by binding sites for erythroid-specific transcription factors, and is likely the functional motif to account for most of the effect upon HbF level by this HbF QTL. Furthermore, ENCODE datasets annotated RNA polymerase 2 occupancy and a 50-bp RNA transcript adjacent to rs66650371, and led us to hypothesize that this transcript is part of a long noncoding RNA (lncRNA). LncRNAs are greater than 200 nucleotides long, are transcribed throughout the genome, and have broad functionality.We characterized a novel 1283 bp lncRNA (HMI-LNCRNA) which was transcribed from this MYB enhancer and contained binding sites for TAL1/E47, GATA, and RUNX1. Its expression in 25 human tissues and cells was examined by qPCR. The thymus and testis had by far the highest expression. Increased expression was seen in erythroblasts from cultured cord blood CD34+ cells, followed by fetal liver, bone marrow, spleen and K562 cells. Other non-hematopoietic tissues had mostly minimal expression. The expression pattern of HMI-LNCRNA displayed similar features of most lncRNAs, including being located almost entirely within nuclei in erythroid cells, suggesting that it might regulate gene expression; and being less abundant than the protein-coding gene, MYB . HMI-LNCRNA expression was significantly higher in erythroblasts from cultured adult peripheral blood CD34+ cells, which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells, which expressed much more HBG . Similarly, HMI-LNCRNA expression was higher in HUDEP-2 cells, an immortalized human adult-like erythroid cells that expressed only HBB, compared to HUDEP-1 cells that expressed predominantly HBG .Transduction of HUDEP-2 cells with HMI-lncRNA shRNA led to downregulation of HMI-LNCRNA expression to 50% of normal level, and resulted in 200-fold increase in HBG mRNA but no change in HBB or HBS1L mRNA. HBG mRNA expression in these cells amounted to 20% of the total HBB and HBG mRNA levels. The changes in HBG mRNA levels were associated with corresponding changes in HBG protein levels documented by Western blot analysis and immunofluorescence staining using anti-HBG antibody. Similar changes in mRNA and protein levels of HBG were seen when HUDEP-2 cells transduced with HMI-lncRNA shRNA were allowed to mature in culture for up to 7 days. There was no change in the expression of BCL11A and other transcription factors known to modulate HBG expression in HUDEP-2 cells transduced with HMI-lncRNA shRNA. FACS analysis of CD71 and CD235 expression showed that 55-65% of HUDEP-2 “naïve” cells or cells transduced with scrambled shRNA were double positive for both markers, while 85% of HUDEP-2 cells transduced with HMI-lncRNA shRNA were double positive, suggesting that downregulation of HMI-LNCRNA promoted maturation of HUDEP-2 cells.HMI-LNCRNA is likely an enhancer RNA (eRNA), and facilitates the interaction between the distal enhancer with the MYB promoter. Our experimental results indicate that it has an important role in regulating HBG expression. Its downregulation can result in markedly increased HBG expression. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in SCD and β-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

Novel Therapeutic Advances in β-Thalassemia.

The main characteristic of the pathophysiology of β-thalassemia is reduced β-globin chain production. The inevitable imbalance in the α/β-globin ratio and α-globin accumulation lead to oxidative stress in the erythroid lineage, apoptosis, and ineffective erythropoiesis. The result is compensatory hematopoietic expansion and impaired hepcidin production that causes increased intestinal iron absorption and progressive iron overload. Chronic hemolysis and red blood cell transfusions also contribute to iron tissue deposition. A better understanding of the underlying mechanisms led to the detection of new curative or "disease-modifying" therapeutic options. Substantial evolvement has been made in allogeneic hematopoietic stem cell transplantation with current clinical trials investigating new condition regimens as well as different donors and stem cell source options. Gene therapy has also moved forward, and phase 2 clinical trials with the use of β-globin insertion techniques have recently been successfully completed leading to approval for use in transfusion-dependent patients. Genetic and epigenetic manipulation of the γ- or β-globin gene have entered the clinical trial setting. Agents such as TGF-β ligand traps and pyruvate kinase activators, which reduce the ineffective erythropoiesis, have been tested in clinical trials with favorable results. One TGF-β ligand trap, luspatercept, has been approved for use in adults with transfusion-dependent β-thalassemia. The induction of HbF with the phosphodiesterase 9 inhibitor IMR-687, which increase cyclic guanosine monophosphate, is currently being tested. Another therapeutic approach is to target the dysregulation of iron homeostasis, using, for example, hepcidin agonists (inhibitors of TMPRSS6 and minihepcidins) or ferroportin inhibitors (VIT-2763). This review provides an update on the novel therapeutic options that are presently in development at the clinical level in β-thalassemia.

Synthesis and pharmacological evaluation of pomalidomide derivatives useful for sickle cell disease treatment

Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide–nitric oxide (NO) donor derivatives (3a–f) and evaluated their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging 0.3–30.3%. Compound 3d was the most effective HbF inducer for CD34+ cells, exhibiting an effect similar to that of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-α levels in human mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer prototype with the potential to treat SCD symptoms.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with β-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat β-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with β-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34+ cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with β-thalassaemia.

Acyclovir induces fetal hemoglobin via downregulation of γ-globin repressors, BCL11A and SOX6 trans-acting factors

Pharmacological reactivation of developmentally silenced fetal hemoglobin (HbF) is an attractive approach to ameliorate the clinical manifestations of β-thalassemia and sickle cell anemia. Hydroxyurea, the only HbF inducer, has obtained regulatory approval. However, hydroxyurea non-responders and associated myelosuppression making its widespread use undesirable. A high level of HbF with safe and effective agents remains an elusive therapeutic goal for this global health burden. This study demonstrated the effect of acyclovir on γ-globin expression and erythropoiesis, associated with increased HbF production. In vitro, human erythroleukemia cells and human CD34+ erythroid progenitors, and in vivo β-YAC transgenic mice were used as experimental models. We found that acyclovir significantly induces expression of the γ-globin gene and HbF synthesis in CD34+ erythroid progenitors, without affecting terminal erythroid differentiation and erythroid cell proliferation. In contrast to other HbF inducers, no associated cytotoxicity with acyclovir was observed. Further, we reported the effect of acyclovir on γ-globin gene transcriptional regulators including BCL11A, FOP1, KLF1 SOX6, and GATA-1. Significant downregulation of the γ-globin repressors BCL11A and SOX6 was observed at both mRNA and protein levels. Whereas, GATA-1, a master erythroid transcription factor, was upregulated in acyclovir treated human CD34+ erythroid culture. Similarly, the HbF inducing effect of acyclovir in β-YAC transgenic mice revealed a good in vitro correlation, with a substantial increase in fetal globin mRNA, and F cells population. These findings collectively suggest acyclovir as an effective HbF inducer and pave the way to evaluate its clinical efficacy in treating β-globin disorders.

Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at G\u03b3-globin gene promoter

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

ZNF410 represses fetal globin by singular control of CHD4.

Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.