Β-globin Promoter Research Articles

Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α 2β 2). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to <20% or an α/β-globin mRNA ratio of <0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity. To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression. Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 + infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin). Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA –/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (<3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.

The Effect of Ferritin Level and Gene Expression of β-globin Promoter with β-thalassemia Patients in Al-Qadisiyah Governorate, Iraq

Abstract BACKGROUND: The genetic condition β-thalassemia causes a deficit in the β-globin chain. Goblins are produced under the supervision of at least nine different genes. Thalassemia can be distinguished from other disorders by changes in these genes, which can lead to issues with hemoglobin synthesis. A typical side effect of thalassemia syndromes is iron overload, which raises the risk of mortality and can cause organ damage. Blood ferritin levels as well as total iron of body reserves have a positive correlation when there is no inflammation. OBJECTIVE: The purpose of this study was to assess the ferritin level of an Iraqi patient and the relationship between β-thalassemia and gene expression of β-globin. MATERIALS AND METHODS: A case–control study included 60 samples with mean age (17.76 ± 0.88; 28 males and 32 females) which had been collected from patients who were diagnosed with β-thalassemia and 60 samples with mean age (22.7 ± 0.75; 29 males, 31 females) which were collected from apparently healthy individuals as a control group (CG). The procedure’s outcome is monitored using polymerase chain reaction and the Fluorecare instrument. RESULTS: Ferritin levels in thalassemia patients were higher than in CG patients. The β-globin expression in the thalassemia group was significantly lower than in the CG. The discovery of two essential sequences thymine-adenine-thymine-adenine and cytosine-adenine-thymine-adenine in the β-gene promoter that are crucial in the start of transcription can account for this downregulation. Changes made to these sequences decreased the affinity of transcription factors, which in turn restricted the transcription of the messenger ribonucleic acid. Examples of these transcription factors are erythroid Kruppel-like factor and specificity protein 1. CONCLUSION: Ferritin can be a useful indicator of severe iron overload. The results showed that the level of expression of β-globin was dramatically downregulated within the thalassemia group as compared with the CG future prospective of this study.

BACH1 Inhibitor Hpp-D Mediates γ-Globin Gene Activation in Sickle Erythroid Progenitors

Introduction: Sickle cell disease (SCD) is one of the most common β-hemoglobinopathy caused by a point mutations in the adult β-globin gene. These individuals are affected with clinical symptoms such as anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. Induction of fetal hemoglobin (HbF) using pharmacologic agents is considered as one of the best therapeutic strategies to manage clinical symptoms of SCD. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. In our previous studies we confirmed a role of NRF2 in γ-globin gene regulation and established molecular mechanisms involving competitive binding of the transcription factors NRF2 and BACH1 in the γ-globin gene promoter, antioxidant response elements (ARE) (Zhu X et al., Haematologica. 2017). Moreover, Attucks and colleagues, our collaborators at vTv Therapeutics LLC showed that HPP-D, a BACH1 inhibitor promotes nuclear export of BACH1 and translocation of NRF2 to regulate antioxidant gene expression (Attucks OC et al., PLoS One. 2014). Therefore, we tested the ability of the novel small molecule BACH1 inhibitor, HPP-D to induce HbF expression by increasing competitive binding of NRF2 in the γ-globin ARE in KU812 cells and human sickle erythroid progenitors. Methods: Human KU812 cells were cultured in Iscove's Modified Dulbecco's medium (IMDM) along with 10% fetal bovine serum, penicillin (100 unit/ml), streptomycin (0.1 mg/ml) and 4mM L-Glutamine at 37oC with 5% CO2. Human sickle cell erythroid progenitors generated from peripheral blood mononuclear cells isolated from SCD patients, were cultured using a two-phase erythroid culturing system (Li et al., Haematologica. 2018). Both cell types were treated with 0.5, 2.5, 5, 10, and 20µM of HPPD, 100µM HU or 50µM hemin for 48 hours. The HbF positive cells (F cells) were measured by flow cytometry analysis, γ-globin, β -globin gene expressions were quantified by Real Time - qPCR and BACH1, α-tubulin, and HbF protein levels were measured by Western blot analysis. ChIP assay was performed to investigate binding competition between Bach1 and NRF2 at the locus control region-hypersensitive site 2 (LCR-HS2) and γ-globin ARE in KU812 cells. The effects of HPPD on sickling of erythroid progenitors were measured by in vitro sickling assay at 1% oxygen for 6 hrs in a hypoxic chamber. Results: The KU812 cells treated with lower concentrations of HPPD (0.5, 2.5 and 5 µM) did not show toxicity to cell growth and viability. Moreover, we observed a significant increase in HbF positive cells (F-cells) by 2.5-fold (0.5μM) and 1.6-fold (5μM) when compared to the vehicle control, DMSO. In addition, Western blot analysis showed a maximal 2.7-fold induction of HbF (p<0.05) by 0.5μM HPP-D. While KU812 cells treated with 0.5µM HPP-D alone showed 20% F-cells increase, when combined with 100µM HU the F-cells increased to 46%; similar findings were observed for mean fluorescence intensity analysis by flow cytometry analysis. Simillarly, when quantifying HbF protein levels by Western blot, 0.5 μM HPP-D alone produced a 1.4-fold increase (p<0.05) of HbF and when combined with 100µM HU showed 1.7-fold HbF increase. Remarkably, enhanced binding of NRF2 were observed in the γ-globin ARE and LCR-HS2 while BACH1 binding affinity reduced in HPP-D treated cells. By contrast, no significant change was observed for NRF2 binding in the β-globin promoter by HPP-D treatment by ChIP assay. To confirm HbF induction by HPP-D, we performed similar experiments conducted at low concentrations (0.5μM and 2.5μM) in sickle erythroid progenitors without affecting cell growth and viability; HPP-D induced HbF levels by 1.6- and 1.9-fold (p<0.05) respectively, without affecting HbS protein level. Moreover, under hypoxic conditions HPP-D treatment reduced erythroid cells sickling by 50% when compared to DMSO control. Conclusions: Overall, our study findings support the ability of HPP-D to induce γ-globin protein as a single agent or in combination with HU through BACH1 silencing and enhancing NRF2 binding in the ARE region. Additionally, HPP-D induced HbF expression and mediated an anti-sickling effect in sickle erythroid progenitors. Future in vivo studies in the SCD mouse model will be conducted confirm our in vitro findings.

Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression

AbstractThe benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.

A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. DisclosuresKattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Effect of HBB: c.-121C>T Variant [–71 (C>T)] on the β-Globin Promoter: Case Series Study

One of the effective strategies in controlling thalassemia is recognition of carriers, followed by prenatal diagnosis (PND) to prevent the occurrence of new cases. There are some rare mutations and variants, for which there are not enough evidences of their effects, and can lead to misdiagnosis and even cause confusion in decision about termination of pregnancy. That is why it is very critical to know the effect of each mutation on the β chain gene. The variant of HBB: c.-121C>T [–71 (C>T)] located in the CAAT box of the promoter region, is a rare mutation. We report seven patients in Hormozagn Province, Iran, who were referred to the PND Center of Hormozgan University of Medical Science (HUMS), Bandar Abbas, Iran during 10 years (2010–2020). Briefly, this mutation causes minor changes in blood indices [mean corpuscular volume (MCV): 75.0 ± 4.0 fL; mean corpuscular hemoglobin (MCH): 25.8 ± 2.5 pg; Hb A2: 3.4 ± 0.5%] showed anemia with a trait milder than minor β-thalassemia (β-thal). Though the existence of α mutations (deletions/point mutations) along with HBB: c.-121C>T can change blood indices due to the changes in α/β ratio. The phenotype of β-thal intermedia (β-TI) was observed in one case, who was a compound heterozygosity for codon 15 (G>A)/–71(C>T) (HBB: c.48G>A/HBB: c.-121C>T. The analysis of transcription level by real-time polymerase chain reaction (real-time PCR) confirmed that this allele induces a mild β+ phenotype due to a decrease in the transcription level.

Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents &amp; Royalties; Fate Therapeutics: Patents &amp; Royalties, Research Funding; Atara: Patents &amp; Royalties, Research Funding; Takeda: Patents &amp; Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents &amp; Royalties.

Human GATA1 Driven By the Human Μicro LCR/β-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced in Mice By the Gata1low Mutation

GATA1 is a member of the GATA family of transcription factors that exerts a concentration/dependent control on the differentiation of erythroid, megakaryocytic (MK), eosinophil and mast cells. Changes of GATA1 content also controls hematopoietic stem/progenitor cell proliferation and commitment. In fact, in addition to X-linked inherited disorders of the erythroid and/or MK lineage, GATA1 mutations are found in inherited and acquired MK leukaemia and myeloproliferative disorders.To study the regulation of the human β-globin locus, the Stamatoyannopoulos laboratory generated transgenic mice carrying human GATA1 driven by the human μLCR coupled with the β-globin promoter (hGATA1) (Li Q et al. PNAS 1997, 18;94:2444). These transgenic mice are born viable with normal phenotype and express hGATA1 at high levels in embryonic and fetal erythroblasts and barely detectable in the adult ones, raising the question whether in adult cells expression of the endogenous Gata1 may suppress the activity of the human μLCR- β-globin promoter.The aim of this study was to determine the effects of Gata1 on the activity of hGATA1 in mice. Since the Gata1null mutation is embryonically lethal, the hypothesis was tested by generating with standard genetic approaches double mutants carrying hGATA1 and the hypomorphic Gata1low mutation, a mutation that strongly reduces expression of Gata1 in adult hematopoietic cells (erythroblasts, MK, hematopoietic progenitors, others). If successful, these experiments would also indirectly assess the extent by which hGATA1 is capable to rescue the complex Gata1low phenotype. Gata1low mice, in fact, are born anemic and thrombocytopenic but recover from their anemia at 1 month by activating extramedullary hematopoiesis in spleen (Vannucchi et al Blood 2001;97:3040). Spleen erythropoiesis is however mostly ineffective since many of the erythroblasts present in this organ are in apoptosis (Tunel+). Gata1low mice remain thrombocytopenic all their life and with age develop myelofibrosis, a phenotype that resemble primary myelofibrosis, the most severe of the Phyladelphia-negative myeloproliferative neoplasms (Vannucchi et al Blood 2002;100:1123). In these mice, hematopoietic stem cells are detected in spleen, and not in bone marrow, and generate bipotent MK/erythroid progenitors (MEP) with a commitment program screwed toward the mast cell lineage and capable to generate in vitro at the single cell basis mast cell lines (Ghinassi et al. Blood 2007;109:1460-71).Double hGATA1Gata1low mutants were easily generated and were not anemic at birth. At 4 months, they expressed normal haematocrit but reduced blood platelet counts (Fig. 1A,B). RT-PCR analyses of prospectively isolated cell populations confermed that the transgene mRNA is barely detectable in cells from single hGATA1 mutants. By contrast, in the double mutants, hGATA1 mRNA was expressed by common myeloid progenitors, its expression increased by 10-fold in MEP, remained at the same levels expressed by MEP in erythroid cells (CD71+/Ter119+) but was barely detectable in MK (CD41+/CD61+). Expression of hGATA1 did not alter the levels of the endogenous Gata1 expressed by these populations that remained low.MEP purified from double hGATA1/Gata1low mice generated in culture more cells (fold increase=250 in Gata1low/hGATA1 cultures vs 50 in single hGATA1 or Gata1low cultures) which were mostly composed by erythroid precursors unable to generate cell lines.By contrast with single Gata1low mutants, by confocal microscopy, GATA1 was readily detectable in erythroid cells from bone marrow and spleen that these cells were not Tunel+ (Fig. 1C), indicating that hGATA1 reduced ineffective erythropoiesis.GATA1 protein remained not detectable in MK from the same tissues (Fig. 1C) and these cells displayed the abnormal morphology characteristic of the retarded maturation of Gata1low MK. Furthermore, double hGATA1/Gata1low mice, as the single Gata1low ones, developed myelofibrosis in bone marrow (by Gomori staining) at 8 months, confirming that myelofibrosis is driven by MK abnormalities.In conclusion, hGATA1 is expressed by adult CMP, MEP and erythroblasts in a genetic Gata1low background rescuing the MEP and erythroid phenotype induced by the hypomorphic mutation. hGATA1 is not expressed and does not rescue the MK phenotype of Gata1low mice, highlighting the specificity of the β-globin promoter for erythroid cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.

GATA-1 Deacetylation and Interaction with HDAC1 Is Critical for GATA-1 Mediated Gene Transcription

The transcription factor GATA-1 is an essential factor for globin gene transcrption and is required for erythroid and megakaryocytic cell differentiation and maturation. GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 chromatin binding activity. However, it is not clear whether GATA-1 acetylation can be reversed by a deacetylase. It is showed that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) associated NURD corepressor complexes through binding to FOG-1. However, we found that the NURD complex does not deacetylate GATA-1. We discovered that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for the interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) blocks the interaction of HDAC1, but doesn't affect its DNA binding in vitro. Importantly, the mutation does not affect the interaction with FOG-1, indicating that GATA-1 direct interaction and indirect association via FOG-1 with HDAC1 are separate events. To further understand the role of the HDAC1-GATA-1 direct interaction in gene transcription and erythropoiesis, we introduced the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Interestingly, upon estradiol induction, GATA-1 2RA does not promote b-globin gene transcription and erythroid differentiation of G1E cells, although GATA-1 2RA is highly acetylated. Chromatin immunoprecipitation assay (ChIP) shows that GATA-1 2RA binds poorly to HS3 and β-globin promoter. Similar binding defect is also detected on GATA-1 promoter, indicating defective GATA-1 recruitment on chromatin. Interestingly, HDAC1 binding to these regions are also significantly reduced, suggesting HDAC1-GATA-1 interaction may be important for GATA-1 deacetylation, as well as stabilizing GATA-1 binding. In order to investigate the effect of GATA-1 2RA in vivo, we generated a GATA-1 2RA knock in mice. The knock in mice are viable but suffered from anemia and thrombocytopenia. β-globin expression reduced at least 50% in knock in mice compare to wild type litter mate. To further identify and compare gene expression profiles regulated by HDAC1 direct or indirect associated GATA-1 during erythroid differentiation, we performed RNA sequencing assays to study the effects of GATA-1 2RA in gene expression in comparison with wild type GATA-1 or GATA-1-V205M (a mutation abolished binding with FOG-1). Expression of GATA-1 2RA largely affects gene expression profile in both GATA-1 activated and repressed genes compared to cells expressing wild type GATA-1. The gene expression pattern in 2RA cells also is largely different from cells expressing GATA-1 V205M, indicating the direct and indirect interaction with HDAC1 may mediate differential functions. Our results indicate that HDAC1 is required for GATA-1 recruitment and GATA-1 mediated transcription regulation. Thus, this study unveils a novel regulation of GATA-1 by its direct interaction with HDAC1. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Two Novel Deletions Involving the 5′ Region of the β-Globin Gene

We report two novel β-thalassemia (β-thal) deletions involving the 5′ region of the β-globin gene (HBB). The first deletion spans 538 bp and removes the β-globin promoter, 5′ untranslated region (5′UTR) and most of exon 1. This deletion was identified in a 3-year-old Vietnamese boy with non transfusion dependent Hb E (HBB: c.79G>A)/β0-thal. The second deletion spans 1517 bp and removes the β-globin gene promoter, 5′UTR, and exons 1 and 2. This deletion was identified in two unrelated adults of European descent who had β-thal trait with unusually high Hb A2 levels. Deletions such as these are generally associated with higher levels of Hb A2 and Hb F than typical β-thal alleles, which may ameliorate the severity of the disease.

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer–promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer–promoter looping in erythroid cells independent of mediator and cohesin.

Development of a New Generation, Forward-Oriented Therapeutic Vector for Hemoglobin Disorders

Ameliorating hemoglobin disorders such as sickle cell disease (SCD) using hematopoietic stem cell (HSC) gene therapy is under development. Unlike in other diseases, therapeutic globin vectors have demanding requirements including high-level β-globin expression, tissue specificity among erythroid cells, long-term persistence, and high-level modification at the HSC level. These demanding requirements necessitate the inclusion of complex genetic elements including the locus control region (LCR), β-globin promoter, β-globin gene, and the 3' untranslated region (3'UTR), all now feasible using lentiviral vectors. The additional requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent loss by RNA splicing during viral preparation. This reverse-orientation is in contrast to all other therapeutic vectors under clinical development. Current reverse-oriented globin vectors can drive phenotypic correction in mouse models for both b-thalassemia and SCD, while they are limited by lower viral titers and lower transduction efficiency in primary human HSCs, limiting their prospects, especially in SCD. We hypothesized that the reverse-orientation impedes both viral preparation and vector transduction, as despite deletion of cryptic polyadenylation (polyA) signals to optimize a conventional reverse-oriented globin vector, titers were still 10-fold lower than a standard GFP-vector.We thus designed a forward-oriented globin-expressing vector, which was further optimized by minimizing the size of the LCR, inclusion of a large segment of the β-globin promoter and an enhancer region of the 3’UTR lacking the polyA signal. Viral titers of the forward-oriented vectors (1.0±0.2x10e9 IU/mL) were 6-fold higher than the optimized vector in the reverse orientation (1.6±0.2x10e8 IU/mL, p<0.01), and comparable to a standard GFP-marking vector (1.9±0.2x10e9 IU/mL, p<0.01). The forward-oriented vector demonstrated 3-4 fold higher transduction efficiency among human erythroid cells derived from transduced CD34+ cells in in vitro culture (34±0% vs 13±1%, p<0.01) and in xenografted mice (31±9% vs 7±5%, p<0.05).To evaluate transduction efficiency for long-term HSCs, we transduced rhesus CD34+ cells with our optimized reverse-oriented vector and our forward-oriented vector including GFP or YFP genes (instead of β-globin gene) in a competitive repopulation assay following 10 Gy total body irradiation. In two animals, GFP and YFP signals from both vectors were detected exclusively in red blood cells, documenting tissue specificity. Gene marking levels were 10-fold higher out to 4 years with the forward-oriented vector, compared to the reverse-oriented vector, and were comparable to standard GFP or YFP-marking vectors in 2 other animals.We then replaced the GFP gene with the β-globin gene containing intron 2 in the forward-oriented vector construct. To positively select intron-2-containing β-globin vectors, essential viral components (packaging signal, rev response element (RRE), or central polypurine tract (cPPT)) were deleted in the backbone of the forward-oriented vector, and the deleted viral components were inserted into intron 2 of the β-globin gene. We observed that half of the forward-oriented vectors lost intron 2 during vector preparation when no elements were included into intron 2. Insertion of the RRE resulted in positive selection of intron-2-containing β-globin vectors. We confirmed β-globin expression from the forward-oriented vector by hemoglobin A production in human erythroid cells derived from transduced peripheral blood mononuclear cells from SCD patients. Finally, human β-globin expression was detected in rhesus erythroid cells following transplantation of transduced CD34+ cells in 2 animals.In summary, we have developed a clinically relevant forward-oriented globin-expressing vector, which has 6 fold higher viral titers and 4-10 fold higher transduction efficiency for hematopoietic repopulating cells, as compared to the optimized reverse-oriented vector. RRE insertion allowed positive selection of intron-2-containing β-globin vectors, and human β-globin production was observed in transplanted rhesus macaques with the forward-oriented β-globin vector transduction. These findings bring us closer to a curative gene therapy for hemoglobin disorders. DisclosuresNo relevant conflicts of interest to declare.

Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia.

β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 μg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.

230. Designing High-Titer Lentiviral Vectors for Gene Therapy of Sickle-Cell Disease

Sickle cell disease (SCD, OMIM 603903) is one the most common inherited blood disorders worldwide, caused by a single amino acid substitution (E6V) in the β-globin chain. SCD is associated with abnormal red cell morphology, hemolytic anemia, vascular occlusion, severe pain, progressive organ damage and reduced life expectancy. Gene therapy by transplantation of autologous, genetically corrected hematopoietic stem cells could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Recent attempts to treat β-thalassemia and SCD with gene therapy showed encouraging results as well as potential limits of a technology based on complex lentiviral vectors for stem cell transduction due to the vector size and complexity, and limited globin synthesis. Designing an efficient vector that combines high-level globin expression with high titer and infectivity remains a formidable challenge. The GLOBE vector carries a β-globin gene under the control of its promoter and a reduced version of the β-globin locus control region (LCR); it was designed to reduce the size and complexity of the LCR while maintaining its enhancer and chromatin opening activity (Miccio et al., PNAS 2008). Based on the GLOBE design, we developed novel LVs for SCD gene therapy, carrying a modified transgene encoding an anti-sickling β-globin gene with 3 amino acid substitutions (AS3), alternative β-globin promoters and a short sequence (FB) with insulating and enhancer-blocking activity in the LTR. The vectors are produced at high titer and reproducibly transduce 60-70% of human BM CD34+ cell-derived clonogenic progenitors at an average VCN of 3. The GLOBE vectors were compared to the Lenti/βAS3-FB vector (Romero, Urbinati et al. JCI 2013), previously developed for a gene therapy clinical trial of SCD. We will present data comparing the different vectors for transduction efficiency in BM CD34+ cells from healthy donors and SCD patients, βAS3-globin transgene expression and anti-sickling activity, in vivo repopulation activity in NSG mice and vector integration profiles in repopulating cells before and after transplantation. This study provides a comprehensive analysis of different LV vectors, useful to determine the optimal candidate for gene therapy of SCD that can provide high transduction of Hematopoietic Stem and Progenitor Cells (HSPCs) as well as maintaining an adequate expression of the anti-sickling globin gene.

Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.

In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies.

The LSD1 Inhibitor RN-1 Recapitulates the Fetal Pattern of Hemoglobin Synthesis in Anemic and Normal, Non-Anemic Baboons

Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease (SCD). Hydroxyurea (HU), the only drug approved for the treatment of sickle cell disease, increases HbF levels, reduces pain crises, and increases the lifespan of patients. Because HU is not effective in a large proportion of patients, new pharmacological agents that increase HbF levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in SCD mice. As the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon remains the best predictor of the activity of HbF-inducing agents in man. Therefore, experiments were performed to test the effect of RN-1 on HbF in anemic baboons. Anemia (Hct=20) was induced by repeated phlebotomies for 14 days prior to administration of RN-1. Five anemic baboons were treated with varying doses of RN-1(0.125-2.5 mg/kg/d; 5d; sc). Dose-dependent increases in HbF, F cells, F retics, and γ-globin chain synthesis in reticulocytes were associated with decreased neutrophils and increased monocytes. Globin chain synthesis analysis following RN-1 treatment showed that the 5' Iγ and 3' Vγ-globin genes were expressed in the ratio characteristic of fetal development (5'Iγ/3'Vγ>2) rather than that induced in adults by erythropoietic stress (5'Iγ/3'Vγ<0.5) or decitabine treatment where changes in the Iγ/Vγ ratio are observed but the fetal ratio is not attained. RT-PCR analysis of pre-and post-treatment BM showed that RN-1 increased γ-globin mRNA nearly 5 fold (p<0.05) with no effect on ε-globin mRNA. ChIP analysis of pre- and post-treatment BM erythroid cells showed increased levels of pol II, H3K9ac, H3K4me2 and H3K4me3 associated with the γ-globin but not with the ε- or β-globin promoter and IVS II regions consistent with increased γ-globin transcription. Levels of H3K9me2 were increased at the β-globin promoter and IVSII regions. Bisulfite sequence analysis showed a small decrease in level of DNA methylation of the γ-globin promoter in post-treatment (0.68+0.07% total cytosine) compared to pre-treatment (0.79+0.05%; p<0.05). To investigate whether induction of γ-globin expression was dependent on erythropoietic stress, four normal, non-anemic baboons were treated with varying doses and schedules of RN-1 (0.2-0.5mg/kg/d; 5d/wk; 1-10wks; sc). The effect of RN-1 was measured by analysis of F cells and globin chain synthesis in peripheral blood. The results (Table) show that RN-1 increases γ-globin expression and F cells in normal, non-anemic baboons in a dose-dependent manner. Analysis of globin chain synthesis showed predominant synthesis of the Iγ-globin chain with an Iγ/Vγ ratio exceeding the fetal ratio in all individuals.Effect of RN-1 in Normal, Non-Anemic Baboons TableAnimalRN-1 Dose (mg/kg/d)ScheduleGlobin chain synthesis (γ/γ+β)Iγ/Vγ chain synthesisF cells (%)ANC (/μl)Plt(X103/μl)Mono(%)PrePostPrePostNadirNadirPeak85490.55d0.030.714.63NDND191016921.685490.255d0.010.264.47NDND191021914.586980.255d/wk/2 wks0.010.2020.12.09.21210687.986960.2-0.255d/wk/10 wksND0.106.633.526.81440646.1Neutropenia following RN-1 treatment of anemic baboons (mean ANC nadir=746±156) was reduced in non-anemic animals (2 of 4 baboons >1500; mean ANC nadir=1617±350; p<0.02). Increased monocytes and decreased platelet counts were observed. Differences between anemic and non-anemic baboons may reflect perturbation of hematopoietic differentiation by phlebotomy. We conclude that RN-1 is a powerful in vivo inducer of HbF in both anemic and non-anemic baboons that preferentially increases synthesis of the 5' Iγ-globin gene and recapitulates the fetal pattern of hemoglobin synthesis. Our data predicts that RN-1 treatment will induce clinically relevant levels of HbF in SCD patients, although careful titration of dose may be required to minimize effects on hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Changes Accompany Disordered Hemoglobin Regulation in Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by massive proliferation of not only the monocytic and granulocytic lineage but also of erythropoietic precursors. Elevated levels of fetal hemoglobin (HbF) are found at time of diagnosis in more than half of JMML cases and are associated with poor outcome. We and others previously found that a key molecular feature of many JMML cases is DNA hypermethylation of distinct target genes, which also has adverse prognostic relevance. It is long known that epigenetic processes are involved in hemoglobin regulation. We therefore hypothesized that epigenetic dysregulation would also be involved in aberrant hemoglobin expression in JMML erythroblasts.ɣ-globin silencing, which is completed one year after birth under physiological conditions, is associated with dense CpG methylation of the ɣ-globin promoter. Consistent with re-expression of ɣ-globin in JMML cases with elevated HbF, CpG methylation at the ɣ-globin promoter was decreased in purified erythroblasts from JMML patients with elevated HbF compared to JMML with normal HbF (average methylation 40.0% ±4.2%, N=6 vs. 72.0% ±3.5%, N=4) (P<0.001). CpG methylation at the ɣ-globin promoter inversely correlated with the relative amount of ɣ-globin expression (ɣ/(β+ɣ)) as determined by RT-qPCR in JMML erythroblasts. Correspondingly, the β-globin promoter is normally unmethylated in erythroblasts from healthy adults, and was also found to be unmethylated in erythroblasts from a JMML patient with normal HbF, whereas it was hypermethylated in JMML with elevated HbF (average methylation 67.0% ±26.0%, N=2). We also assessed CpG methylation at the KLF1 gene which encodes an erythroid-specific transcription factor and strong β-globin activator. We found that the KLF1 enhancer (-593 to -312 relative to the transcription start site of KLF1) and promoter (-182 to +203) regions were unmethylated in healthy erythroblasts irrespective of neonatal or adult origin (N=11; enhancer, 4.5% ±0.9%; promoter, 2.2% ±0.6%), whereas both regions were densely methylated (>80%) in healthy non-erythroid cell populations. By contrast, the KLF1 enhancer and promoter were aberrantly methylated in JMML erythroblasts (N=11; enhancer, 21.2% ±4.7%, P=0.002; promoter, 11.5% ±2.9%, P=0.004). This suggested a regulatory role of KLF1 methylation in aberrant globin expression in JMML. To explore this functionally, we used a dual luciferase reporter assay, ligating the KLF1 enhancer into a CpG-free reporter vector and comparing the reporter activity of unmethylated vector with that of in vitro methylated vector after transfection into K562 cells. CpG methylation at the KLF1 enhancer abolished its activity, supporting methylation-associated silencing of KLF1 in JMML erythroblasts. Compatible with this, KLF1 transcript levels were reduced 2.1-fold in JMML erythroblasts (N=12) compared to healthy erythroblasts (N=9) as assessed by RT-qPCR (P=0.018). We additionally assessed the protein expression of the direct KLF1 target CD44 on the surface of JMML erythroblasts by FACS analysis. CD44 expression was 2.4-fold lower in JMML erythroblasts with high KLF1 enhancer methylation compared to JMML erythroblasts with normal KLF1 enhancer methylation.In summary, we show that multiple layers of hemoglobin regulation are affected by epigenetic changes in JMML erythroblasts. The globin genes themselves are targets of aberrant DNA methylation in JMML erythroblasts with elevated HbF. Moreover, the β-globin activator KLF1 is aberrantly methylated and repressed in JMML erythroblasts. These findings provide for the first time a mechanistic explanation for the strong correlation and uniform prognostic relevance of elevated HbF and gene hypermethylation in JMML. DisclosuresNo relevant conflicts of interest to declare.

ASH1L: A Novel Beta-Globin Gene Regulator in Humans?

BACKGROUNDWe have previously described a unique English family with beta-thalassemia trait which was not linked to the β-globin gene locus (Thein, Wood, Wickramasinghe, & Galvin, 1993). This suggested involvement of a trans-acting factor required for full activation of the β-globin gene locus. Such a factor is likely to be a modulator of disease severity in sickle cell disease and beta-thalassemia which could provide insights for novel therapeutic targets in the beta-globinopathies. RESULTSWe applied whole genome scan (WGS) to 2 affected and 2 unaffected subjects of the English family. The familial segregation suggested a dominant transmission mode; WGS identified 15 genes as potentially causative to the phenotype, with four genes located on chromosome 1, four on chromosome 3, three on chromosome 20, and one on chromosome 6, chromosome 8, chromosome 10 and chromosome 19.Sanger sequence analysis on 23 family members spanning three generations, including the 4 individuals that were subjected to WGS, revealed that the 15 variants were not artefacts of the WGS and that all variants were present in the 2 affected but not in the 2 unaffected individuals. Furthermore we found that 4 of the 15 variants were consistently and uniquely present in all 9 affected but absent in the unaffected family members. We performed association linkage analysis using the 15 markers in the whole family, and confirmed that the phenotype was closely linked to the 4 genes that were inherited as a block spanning the centromere on chromosome 1. We concluded that the region containing these 4 genes most likely harbours the mutation causing the phenotype. Among the 4 candidate genes, 2 were not expressed in erythroid cells, but the other 2 - one encoding an integral membrane protein (LRIG2) and the other one encoding a methyl transferase (ASH1L)- were expressed in erythroid cells.Functional studies for these two genes were performed on primary human erythroid progenitor cells (hEPCs) in culture. In following the kinetics of the 2 candidates during differentiation of hEPCs, we observed that the expression of ASH1L increased at later stages of differentiation, where LRIG2 displayed a less dramatic change of expression. Moreover, ASH1L has previously been found to occupy transcribed chromatin domains and methylate histone tails in vitro (Gregory et al., 2007; Miyazaki et al., 2013; Tanaka et al., 2011). In undifferentiated mouse embryonic stem cells there is no ASH1L recruitment to the β-globin gene locus but upon erythroid differentiation the protein is recruited to the transcribed portion of the gene (Gregory et al., 2007). This suggests an involvement of ASH1L in beta-globin activation in erythroid lineages. We used shRNA lentiviruses to generate knock-down (KD) of ASH1L and obtained over 65-75% KD of the gene. In hEPCs treated with the shRNA lentivirus, we observed a slight decrease in beta-globin expression compared to the control hEPCs. The α/β-globin and α/(β+γ) globin ratios were also affected by the gene knock-down. ChIP-qPCR was performed to assess the enrichment of the ASH1L protein at β-globin promoter region. The results show that enrichment of ASH1L at the β-globin promoter correlates with the β-globin expression in cells. CONCLUSIONSThese results suggest that ASH1L is responsible for the phenotype observed in the English family and act in differentiating hEPCs as a trans-acting factor for full beta-globin gene activation. Further ChIP analysis to assess the binding of the protein to the beta-globin locus during hEPCs differentiation and under KD condition will provide us with a better understanding of the influence of the methyl transferase on β-globin activation. The replication of the patient mutation in vitro using CRISPR technology will provide the model to study fully the impact of the mutation on the phenotype described in the original paper. These findings could provide new insights for therapeutic targets for beta-globinopathies. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Activity of Erythroid Kruppel-like Factor (EKLF/KLF1) Modulated by PIAS3 (Protein Inhibitor of Activated STAT3)

Erythroid Kruppel-like factor (EKLF or KLF1) is a transcription factor crucial for red cell development that is directly involved in regulation of a large number of erythroid genes. EKLF serves mostly as an activator of expression of these genes; however, it can act also as a repressor. Here, we present evidence that EKLF interacts with proteins from the PIAS (protein inhibitor of activated STAT) family that convey repressive activity to EKLF in the absence of sumoylation. Our studies identify PIAS3 as a transcriptional corepressor of EKLF for at least a subset of its target genes during erythropoiesis (e.g. β-globin, α-hemoglobin stabilizing protein). We demonstrate an interaction between EKLF and PIAS proteins confirmed by in vivo coimmunoprecipitation assays with both exogenous and endogenous proteins. We identified an LXXLL signature motif located near the N terminus of PIAS proteins that, although not involved in the EKLF-PIAS3 interaction, is required for the transrepression activity. Knockdown of endogenous PIAS3 accelerates differentiation of both murine erythroleukemia cells, as well as fetal liver cells, whereas an increase in PIAS3 levels inhibits this increase. Using chromatin immunoprecipitation assays, we show that PIAS3 preferentially occupies the β-globin promoter in undifferentiated murine erythroleukemia cells. Together these results demonstrate that an interaction between EKLF and PIAS3 provides a novel mode of regulation of EKLF activity in the absence of sumolylation and furthermore shows an important involvement of PIAS proteins in erythropoiesis.