Globin Promoter Research Articles

Editing of homologous globin genes by nickase-deficient base editor mitigates large intergenic deletions in HSPCs

Recent studies have shown that base editing, even with single strand breaks, could result in large deletions of the interstitial regions while targeting homologous regions. Several therapeutically relevant genes such as HBG, HBB, CCR5, CD33 etc have homologous sites and is prone for large deletion with base editing. Although the deletion frequency and indels observed are very less than what is obtained with Cas9, it could still diminish the therapeutic efficacy. We sought to evaluate if these deletions could be overcome while maintaining the editing efficiency by the use of dCas9 fusion of ABE8e in place of nickaseCas9. Using gRNAs targeting the gamma globin promoter and the beta globin exon, we evaluated the editing outcome and frequency of large deletion using nABE8e and dABE8e in human HSPCs. We show that dABE8e can edit efficiently while abolishing the formation of large interstitial deletions. Further this approach enabled efficient multiplexed base editing on complementary strands without generating insertions and deletions. Taken together removal of nickase activity improves the precision of base editing thus making it a safer approach for therapeutic genome editing.

Screening for Multiple Organ Damage in Indian Sickle Cell Disease Cohort- a Step Towards Comprehensive Care

Introduction: Sickle cell disease (SCD) is the most common hereditary monogenic disorder and is a major health burden in India. Meagre information is available on multiple organ damage among Indian SCD population. The present study attempts to understand the spectrum of organ damage in a group of Indian SCD cohorts. Objectives: To investigate the spectrum of early organ dysfunction and chronic organ damage among SCD patients at different age groups. To study the association of haematological and biochemical parameters with organ dysfunction or chronic organ damage. To understand the implication of genetic modifies with clinical profiles of SCD patients. Methodology: Clinical investigations of SCD individuals such as MRI of brain, ultrasonography of abdomen, spirometry of lung, 2 D echo of heart and ophthalmology. Laboratory investigations of SCD individuals such as haematological parameters by cell counter (Sysmax XP100), HbF & HbS fractionization by HPLC method (Biorad Variant 2), liver function test and kidney function test by automated biochemistry analyser (Roche Cobas C111). Co-inheritance of α-thalassemia and Xmn-1 polymorphism by multiplex PCR and PCR-RFLP respectively. 100 SCD individuals were recruited with informed consent by a consecutive sampling for the study at ICMR-Centre for Research Management and Control of Haemoglobinopathies (ICMR-CRMCH), Chandrapur. SCD individuals at non-crisis, steady state; with no blood transfusion history prior 3 months or not admitted in hospital for vaso-occlusive crisis prior 1 month were enrolled in the period Jan'2021-Jan'2022. Result: One hundred SCD individuals (44 males: 56 females) were enrolled. The median age of individuals was 18 years (Interquartile range (IQR): 13-29 years). Individuals were categorized into two groups based on their ages. Children and adolescents group (6-17 years): 46 individuals, and Adults (18-52 years): 54 individuals. Out of 100, 65 were drug hydroxyurea (HU) are on HU, while remaining 35 were not taking HU. The median dosage of at 11.9 mg/kg/day (IQR: 9.17-13.5 mg/kg/day). The current study reports the spectrum of organ damage involving onset of organ dysfunction to end organ damage such as ischemia/sclerosis/atrophy 12/58 (20.7%) to stroke 2/58 (3.4%), mild restriction/obstruction 55/96 (57.3%) to mixed blockage lung disease 6/96 (6.2%), cholelithiasis 24/100 (24%) to chronic cholecystitis/cholecystectomy 6/100 (6%), shrunken spleen 16/100 (16%) to autosplenoctomy 24/100 (24%). No pulmonary hypertension determined by tricuspid valve regurgitation (TRV) >2.5 m/s was observed among 100 SCD individuals. No case of proliferative sickle retinopathy was detected in the cohort. Laboratory investigations: The mean ± standard deviation (S.D.) hemoglobin and HbF% levels among 100 SCD individuals were found to be 9.7±1.6 gm/dl and 21.2±6.6% respectively. Hematological and biochemical parameters showed association with liver and splenic dysfunction. Four out of 76 (5.3%) individuals were tested positive for microalbuminuria. No individual showed higher levels of estimated glomerular filtration rate. Molecular investigations: Ninety three HbS homozygous and 7 HbS-β thalassemia compound heterozygous were identified by ARMS-PCR method. The co-inheritance of α-gene mutations was observed in 45/94 (47.9%) SCD individuals, while the remaining 49/94 (52.3%) were normal. The Xmn1 polymorphism present in the Gγ globin promoter region -158 C→T (HBG2 c.-211 C→T) position, showed 88 as homozygous for mutant allele [+/+], while 12 as heterozygous[ +/-]. No association of α-thalassemia and Xmn1 polymorphism with organ dysfunction was observed. Conclusion: Though, vaso-occlusive crisis is the main clinical manifestation in SCD, sickle related organ damage is an important determinant for clinical severity of the disease. The progression of organ dysfunction/damage is evident as the age progresses. The comprehensive check-up at regular interval is required for the identification of early organ dysfunction and to prevent the chronic organ damage in SCD individuals.

Molecular basis of a high Hb A2/Hb Fβ-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication.

β 0-thalassemia deletion removing 5´β-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the β 0-thalassemia with 3.4 kb deletion. A total of 148 subjects, including 127 heterozygotes, 20 Hb E-β-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ-globin promoter, rs5006884 on OR51B6 gene, -158 G γ-XmnI, BCL11A binding motifs (TGGTCA) between 3´A γ-globin gene and 5´δ-globin gene. It was found that heterozygous β 0-thalassemia and Hb E-β 0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous β 0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-β 0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous β 0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain β-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´β-globin promoter may likely be responsible for this unusual phenotype. The results indicate that β 0-thalassemia with 3.4 kb deletion is a mild β-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.

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.

Structural basis for human ZBTB7A action at the fetal globin promoter.

SUMMARYElevated levels of fetal globin protect against β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at −115 and −200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of four adjacent ZFs, in complex with the −200 sequence element, which contains two copies of four consecutive C:G base pairs. ZF1 and ZF2 recognize the 5′ C:G quadruple, and ZF4 contacts the 3′ C:G quadruple. Natural non-coding DNA mutations associated with hereditary persistence of fetal hemoglobin (HPFH) impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in the base pairs recognized by ZF1 and ZF2. Our results firmly establish ZBTB7A/LRF as a key molecular regulator of fetal globin expression and inform genome-editing strategies that inhibit repressor binding and boost fetal globin expression to treat hemoglobinopathies.

Talen Mediated Therapeutic Gene Editing Strategy for ß-Hemoglobinopathies

Hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common single-gene disorders in the world and represent a major global public health concern. The unifying principle of this heterogeneous mix of gene mutations is the decreased production of wild type hemoglobin molecules either due to structural defects in the case of SCD or insufficient production of β-hemoglobin subunits. Hemoglobinopathy patients with gene variants that induce expression of fetal (γ) hemoglobin often exhibit a less severe phenotype. Recent evidence has demonstrated that the in vitro introduction of a previously described naturally occurring 13bp deletion in the promoter of the Aγ globin gene can reconstitute the induction of fetal hemoglobin seen in patients with this mutation. As temporary expression of endonuclease is a key element of a clinically viable editing technique, we have generated a set of TALENs (Transcription Activator-Like Effector Nucleases) targeting the 13bp deletion site that are transfected as mRNA. In human CD34 cells, transfection with and expression of the TALEN pair results in INDEL generation at both the Aγ and Gγ globin loci confirmed by sequencing, T7 analysis and ddPCR. INDEL generation occurs in both the Aγ and Gγ globin promoters with slightly higher rates at the Aγ globin locus (~53%) compared to the Gγ globin locus (~45%) (Figure 3). Compared to control cells, edited CD34+ cells placed in erythroid differentiation media result in an increased expression of fetal hemoglobin with a near doubling in the frequency of ‘F-Cells’ by flow cytometry and a threefold increase in γ globin protein detected by HPLC (Figure 1 & 2). These findings support a model in which TALEN induced targeted INDELs in the γ globin promoter are capable of de-repressing fetal hemoglobin in hematopoietic progenitor cells. d13 TALEN edited CD34 cells have also been transplanted into NSG-W41 mice, a strain demonstrated to be more permissive of human erythropoiesis and are capable of high rates of engraftment with minimal conditioning (66-95% human chimerism at 14 weeks). An analysis of the INDEL rates retained by the engrafted cell populations 14 weeks post-transplantation demonstrates an average gene editing retention rate of approximately 50% (individual animals ranged from 15-77%) (Figure 4). These edits are retained in multiple hematopoietic cell lineages from each mouse suggesting that editing is occurring at least in the hematopoietic progenitor population and does not rule out the possibility of edited long term repopulating stem cells. A unique feature of targeted gene editing at this locus is that therapeutic benefit can be theoretically derived both from INDEL generation as well as the introduction of synthetic sequence via the homologous repair pathway. AAV transfected homologous recombination templates have been generated with a goal of further manipulating hemoglobin expression and allowing for the selection of edited cells. A basic GFP selection template has been successfully integrated at the target site (approximately 27% efficiency) resulting in erythroid and myeloid methylcellulose progenitor colonies detectable by fluorescence microscopy. Other repair templates that either introduce the specific 13bp deletion γ-hemoglobin promoter or elements of the β-hemoglobin promoter result in higher rates of HbF expression in GFP+ cells. A T87Q anti-sickling β hemoglobin construct has also been successfully integrated and expressed in human CD34 cells resulting in both an increase in HbF expression as well as the presence of T87Q hemoglobin making up a combined 60% of the total β hemoglobin proteins expressed in edited cells. Continued efforts are underway to optimize repair template designs, editing efficiencies and selection strategies to prepare this approach for clinical applications. [Display omitted] DisclosuresScharenberg:Casebia: Employment.

In Vivo HSC Transduction and Selection Results in Long-Term, High-Level Expression of Human Gamma Globin in Peripheral Blood Erythrocytes of Mice

Current protocols for HSC gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. We have developed a new approach for in vivo gene delivery into HSCs that does not require myeloablation and HSC transplantation. It involves injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. Transgene integration is achieved (in a random pattern) using a hyperactive Sleeping Beauty transposase (SB100x). We demonstrated in adequate mouse models, using GFP as a transgene, that primitive HSCs transduced in the periphery home back to the bone marrow where they persist and stably express GFP long-term (Blood 128:2206 (2016). To achieve high level transgene marking in differentiated peripheral blood cells, we combined our in vivo HSC transduction approach with in vivo selection of transduced HSCs using the mgtmP140K mutant and low dose O6BG/BCNU treatment. Here were tested the potential of this approach for the therapy of hemoglobinopathies. Our HDAd5/35++ vector contained the human-gamma globin gene under the control of a 6kb version of the beta-globin LCR (containing HS1 to HS4 and the beta globin promoter) and the mgmtP140K gene under the control of the PGK promoter. Mobilized immunocompetent hCD46 transgenic mice were injected IV with the integrating vector system and subjected to three rounds of treatment with low-dose O6BG/BCNU followed by immunosuppression to avoid immune responses against the human transgene product. The procedure was safe and resulted in stable human gamma globin expression in >95% of erythrocytes in the peripheral blood of in vivo transduced cells mice. Human gamma globin levels were 100% of endogenous mouse beta globin levels in erythrocytes measured by HPLC. Analysis of bone marrow at week 16 after in vivo transduction showed that >60% of bone marrow LSK cells contained the integrated transgene cassette and that the beta globin LCR efficiently restricted gamma globin expression to erythroid cells. After transplantation of unsorted, lineage-depleted bone marrow cells into lethally irradiated recipients, long-term gamma-globin expression at levels seen in the primary in vivo treated mice was demonstrated. The efficacy of our approach achieved in this study indicates that it would be curative in thalassemia models or patients. Our new approach avoids the collection of HSCs, their transduction ex vivo, and subsequent transplantation into myeloablated recipients. It therefore could greatly simplify HSC gene therapy of common diseases. DisclosuresKiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Repurposing the CRISPR‐Cas9 System for Targeted Chromatin O ‐linked β‐ N ‐acetylglucosamine Editing

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia have identified O-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, chromatin dynamics, and cofactor activity. Decoding the precise function of all the intracellular O-GlcNAc-regulated elements identified in these studies remains challenging. Technologies to manipulate O-GlcNAcylation at specific DNA loci for functional analysis without pleiotropic consequences were lacking, but the advent of CRISPR-Cas9-based technologies has enabled the precise perturbation of DNA sequences and delivery of proteins to specific cis-acting DNA regulatory elements to elucidate their role in transcription. We reasoned that a CRISPR-Cas9 system could be developed to study the function of O-GlcNAcylation in transcription, which would circumvent the problems inherent in conventional gene manipulation and pharmacological approaches. We developed a programmable CRISPR-Cas9-based system that allows for O-GlcNAc manipulation of transcriptional complex function at specific cis-regulatory elements. The tools consist of nuclease-null Cas9 (dCas9) fused to O-GlcNAc transferase (OGT), which adds the O-GlcNAc modification, or O-GlcNAcase (OGA), which removes the modification. Previously, we demonstrated that O-GlcNAc plays a role in regulating human □-globin gene transcription. Increasing fetal hemoglobin (HbF) via activation of □-globin chain synthesis is widely accepted as the most effective treatment for SCD and certain types of β-thalassemias. O-GlcNAcylation modulates the formation of a GATA-1-FOG-1-NuRD repressor complex that binds the -566 GATA site of the A□-globin promoter when □-globin gene expression is silent. OGT and OGA interact with GATA-1 and CHD4, a component of the NuRD complex. O-GlcNAcylation of CHD4 stimulates the formation of this repressor complex, whereas removing this PTM results in activation of A□-globin gene expression. As proof of principle to demonstrate the utility of our system, we targeted dCas9-OGT or dCas9-OGA fusion proteins to DNA sequences flanking the -566 GATA site of the A□-globin gene promoter. Increased □-globin gene transcription was measured by RT-qPCR when dCas9-OGA was targeted to this area, whereas □-globin gene expression decreased when dCas9-OGT was targeted to the identical sequences. These data support our published work, confirming that O-GlcNAcylation regulates A□-globin gene transcription. RT-qPCR data coupled with Cut and Run assays demonstrate the robust and highly specific nature of our targeting system, which can be employed as a generally applicable tool to study the role of O-GlcNAcylation in transcriptional regulation.

A human H1-HBB11-GFP reporter embryonic stem cell line (WAe001-A-2) generated using TALEN-based genome editing.

Hemoglobin production during mammalian development is characterized by temporal switches of the genes coding for the α- and β-globin chains. Defects in this controlled process can lead to hemoglobinapathies such as sickle cell disease and β-thalassemia. The ability of human embryonic stem cells (hESC) to proceed through hematopoiesis could provide a clinically useful source of red blood cells. However, hESC-derived red cells exhibit an embryonic/fetal, but not adult, mode of hemoglobin expression. The resource described here is a hESC line engineered to express a reporter from its adult globin promoter, providing a screening platform for small molecules that lead to efficient induction of adult globin.

Combining HPFH Mutations in Human Adult HSCs to Enhance Reactivation of Fetal Hemoglobin

Despite the significant advances in developing novel therapies for hemoglobinopathies, developing a universal and robust therapeutic approach, able to achieve an event-free result in all β0/β0 thalassemic and sickle cell patients is a work in progress. It is known that increase of Fetal hemoglobin (HbF) can lead to an ameliorated clinical picture up to transfusion independency. Inactivation via genome editing of γ-globin suppressors or introduction of Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the promoter of hemoglobin gamma (HBG) have been shown to significantly increase the endogenous HbF expression. Here we studied the effect in HbF reactivation by targeting the binding site of the two main HbF silencers, BCL11a and LRF and how it compares to the well-established editing of the BCL11a erythroid enhancer. Furthermore, in order to achieve higher levels of fetal hemoglobin, we assayed the effect of all combinations of these cis and trans acting mutations. We first observed that all single knock outs had similar effects in HbF reactivation. Interestingly, by targeting both sites in the gamma globin promoter we observed a decrease in HbF induction compared to each single edit, suggesting a possible binding of a gamma globin activator in the in-between region. However, editing of the BCL11a erythroid enhancer and either of the two silencer binding sites yielded maximum levels of fetal hemoglobin, with pancellular HbF expression, both in vitro and in vivo following xeno-transplantation in NSGW mice. Collectively, our data suggest that the combination of two fetal globin reactivation mutations, one cis and one trans, have the potential to significantly increase HbF both totally and on per cell basis. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site

Abstractβ-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations in the adult β-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The −113A>G HPFH mutation falls within the −115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the −113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the −113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the −113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Ourfindings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

KLF1 drives the expression of fetal hemoglobin in British HPFH

Abstractβ-Hemoglobinopathies are among the most common single-locus inherited diseases. In this condition, high fetal hemoglobin (HbF) levels have been found to be beneficial, and boosting HbF expression is seen as an attractive therapy. Naturally occurring mutations in the fetal globin promoter can result in high HbF persisting into adulthood in a benign condition known as hereditary persistence of fetal hemoglobin (HPFH). Individuals with one form of HPFH, British HPFH, carry a T to C substitution at position −198 of the fetal globin gene promoter. These individuals exhibit HbF levels of up to 20%, enough to ameliorate the symptoms of β-hemoglobinopathies. Here, we use clustered regularly interspaced short palindromic repeat–mediated genome editing to introduce the −198 substitution into human erythroid HUDEP-2 cells and show that this mutation is sufficient to substantially elevate expression of HbF. We also examined the molecular mechanism underlying the increase in fetal globin expression. Through a combination of in vitro and in vivo studies, we demonstrate that the mutation creates a de novo binding site for the important erythroid gene activator Krüppel-like factor 1 (KLF1/erythroid KLF). Our results indicate that introducing this single naturally occurring mutation leads to significantly boosted HbF levels.

Characterisation of Novel Hypomorphic and Null Mutations in Klf1 Derived from a Genetic Screen for Modifiers of a-Globin Transgene Variegation

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers. DisclosuresPerkins:Novartis Oncology: Honoraria.

DOCK8-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. The relative role of dendritic cells, B cells and macrophages in the induction of RBC alloimmunization remain unclear. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused RBCs. A particularly useful transgenic "HOD mouse" has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffyb blood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune cells or receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs.Using these models, we recently discovered that mice lacking the GEF (guanine nucleotide exchange factor) DOCK8 fail to develop alloimmunity to transfused RBCs. Dendritic cells in these knockout mice fail to migrate to T cells due to lack of coordinated actin rearrangement governed by this GEF. Both B cell and T cell activation in the spleen to the transgenic transfused RBCs is abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in DOCK8-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. Our preliminary data now suggest that DOCK8-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The need for dendritic cell migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed; these mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion and the specific role of splenic dendritic cell subsets in CD4+ T cell priming to allogeneic RBCs. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias.

NLRP10-Deficient Mice Reveal a Crucial Role for Dendritic Cell Activity in the Initiation of the Allogeneic Response to Transfused Red Blood Cells

The generation of antibodies against transfused red blood cells (RBCs) can pose a serious health risk, especially in chronically transfused patients requiring life-long transfusion support; yet our understanding of what immune signals or cells dictate when someone will become alloimmunized is lacking. Every non-autologous red cell unit has multiple antigens foreign to the transfused recipient; some people respond to these foreign antigens with an adaptive immune response and some do not. Given the now well established role of innate immune signals in regulating adaptive immunity, understanding if and how innate immunity is triggered during transfusion may allow development of therapies to prevent alloimmunization in chronically transfused subjects such as those with myelodysplasia or hemoglobinopathies. We have established a murine model system in which we can evaluate both the role of particular innate immune stimuli as well as particular cells of the immune system in regulating the allogeneic response to transfused red blood cells. A particularly useful transgenic “HOD mouse” has been engineered, which encodes a triple fusion protein and provides a unique tool to directly assess both RBC-specific T and B cell responses. This RBC-specific antigen contains the model protein antigen hen egg lysozyme (HEL) fused to chicken ovalbumin (OVA) fused to the human Duffybblood group antigen (HEL-OVA-Duffy) as an integral membrane protein under control of the beta globin promoter. Transfusion of genetically targeted mice lacking various innate immune receptors allows us to screen for important immune pathways regulating the response to allogeneic RBCs.Using these models, we recently discovered that mice lacking the NOD-like receptor NLRP10 fail to develop alloimmunity to transfused red blood cells. Surprisingly, the early innate immune cytokine response, including IL-6, IL-1beta and TNF-alpha, was unaffected in mice lacking NLRP10. Yet both B cell and T cell activation in the spleen to the transgenic transfused RBCs was abrogated. Inclusion of OVA in the alloantigen of the HOD mice allows us to readily study naïve CD4+ T cell activation following transfusion by using the OTII T cell receptor (TCR) transgenic mice in which essentially all T cells express one antigen receptor specific for a peptide of OVA. By tracking rounds of cell division we found that adoptively transferred OTII undergo more than 5-8 rounds of division in the spleen three days following transfusion of HOD RBCs in WT recipients. In contrast, no OTII proliferation was observed in NLRP10-deficient mice following OTII adoptive transfer and HOD RBC transfusion, suggesting that T cells are failing to receive activation signals by splenic antigen presenting cells. We have previously demonstrated that NLRP10-deficient dendritic cells fail to migrate from peripheral tissues such as the skin to draining lymph nodes. Our preliminary data now suggest that NLRP10-deficient dendritic cells are able to process and present RBC-derived antigens, but do not migrate to T cell zones in the spleen to prime naïve RBC-specific T cells. The relative role of dendritic cells, B cells and macrophages in the induction of erythrocyte alloimmunization remain unclear. Further, the need for DC migration within the spleen in the induction of alloimmunity to transfused RBCs has not been addressed. These mice allow us for the first time to answer these fundamental immunologic questions during transfusion. Future work will aim to determine how dendritic cell movement within the spleen is regulated during transfusion in NLRP10-deficient mice and the specific role of splenic dendritic cells in CD4+ T cell priming to allogeneic RBCs. DisclosuresNo relevant conflicts of interest to declare.

Role of c-Jun in Tumor Necrosis Factor-alpha Inhibition of Activin A-mediated Erythroid Gene Expression in Erythroleukemia K562 Cells

Background The activation of the tumor necrosis factor-alpha (TNF-α)/nuclear factor kappa B (NF-κB) pathway inhibits the expression of erythroid genes, whereas activin A, a member of the transforming growth factor-β superfamily, induces erythroid differentiation. The effect of TNF-α on activin A-induced erythroid gene expression has not been elucidated. Methods Luciferase reporter assay and reverse transcription-polymerase chain reaction (PCR) or quantitative PCR were used to investigate globin promoter activity and globin gene expression in the hematopoietic progenitor cell line K562, respectively. Results TNF-α inhibited the activin A-induced promoter activity of α-globin and ζ-globin in a concentration-dependent manner in K562 cells. Activin A could reverse the TNF-α-inhibited promoter activity of α-globin and ζ-globin in a concentration-dependent manner. TNF-α decreased the mRNA levels of α-globin, ζ-globin, GATA-1, and NF-E2 p45 induced by activin A. The NF-κB inhibitor, Bay117082, inhibited the TNF-α-increased c-Jun level. NF-κB p65 overexpression increased c-Jun protein and enhanced the TNF-α-increased c-Jun level. Furthermore, TNF-α inhibition of activin A-induced promoter activity and mRNA expression of α-globin and ζ-globin were abolished in cells expressing dominant-negative c-Jun. TNF-α inhibition of activin A-induced mRNA expression of GATA-1 and NF-E2 p45 was also abolished in cells expressing dominant-negative c-Jun. Conclusion TNF-α may inhibit activin A-induced erythroid gene expression via increases of c-Jun in K562 cells.

Keeping fetal hemoglobin in the loop

Keeping fetal hemoglobin in the loop

Fetal Hemoglobin Expression Is Differentially Affected by Inhibition of the Proposed Dred Complex Constituents, LSD1 and DNMT1

AbstractAbstract 3263 IntroductionSickle cell disease and beta thalassemia are disorders caused by mutations in adult hemoglobin (HbA) or defects in HbA expression. A potential therapeutic solution is reactivation of fetal hemoglobin (HbF) expression. Although HbF, comprising two alpha and two gamma globin chains, is the primary form of hemoglobin expressed in utero, gamma globin expression is silenced in adults. One proposed mechanism of gamma globin silencing involves binding of the direct repeat erythroid definitive (DRED) repressor complex to sequences in the gamma globin promoter. The DRED complex is reported to include the orphan nuclear hormone receptors TR2 and TR4, lysine specific demethylase (LSD1) and DNA methyltransferase (DNMT1). As both LSD1 and DNMT1 are epigenetic modifiers, gamma globin repression is proposed to be mediated by LSD1- and DNMT1-induced epigenetic changes. To investigate the role of DNMT1 and LSD1 in HbF silencing, HbF expression was evaluated in an erythroid differentiation model where hematopoietic progenitor cells were treated with either DNMT1 or LSD1 small molecule inhibitors or siRNA. MethodsHuman hematopoietic progenitor cells from healthy donors were induced to become erythroid using a two step protocol including erythropoietin, SCF, IL-3 and hydrocortisone for days 1–7 and erythropoietin and SCF for days 8–14. Cultures were treated with a range of concentrations of either tranylcypromine or S2101 (LSD1 inhibitors) or 5-azacytidine (DNMT1 inhibitor) and compared to HbF-inducing, positive control small molecules pomalidomide and lenalidomide. Cultures were also treated with LSD1 siRNAs and compared to controls. The effect of treatment on gamma, beta and alpha globin transcription was determined by qRT-PCR. The effect of treatment on HbA and HbF levels was determined by ELISA, HPLC, flow cytometry and imaging. Differentiation was characterized by morphology and flow-based detection of CD34 and glycophorin. Effects on viability were characterized by ViCell and flow cytometry. ResultsTreatment with a concentration range of 5-azacytidine increased the rate of red blood cell differentiation as measured by daily changes in CD34 and glycophorin and hemoglobinization. Quantitative ELISA demonstrated that HbF expression increased two-fold. In contrast, LSD1 inhibition reduced both the rate of proliferation and differentiation of erythroid progenitors. Consistent with impaired differentiation, both beta globin transcription and HbA expression were reduced by up to 84% (qRT-PCR) and 65% (quantitative ELISA), respectively. No increase in gamma globin transcription or HbF expression was observed in response to LSD1 inhibition. Control cultures differentiated as expected: after 14 days of treatment the majority of vehicle-, lenalidomide- or pomalidomide-treated cells were glycophorin-positive and enucleation was readily apparent. Both lenalidomide and pomalidomide treatment induced a two-fold increase in HbF expression, as previously reported. ConclusionsAlthough both LSD1 and DNMT1 are reported to be components of the DRED complex and are proposed to be jointly responsible for epigenetically modifying the gamma globin promoter to silence HbF expression, inhibition of the two proteins had different outcomes on HbF expression. DNMT1 inhibition upregulated HbF expression to a similar extent as pomalidomide (currently in Phase 1 clinical trials for HbF induction), whereas LSD1 inhibition impaired erythroid differentiation and hemoglobinization. These results suggest that the mechanism of gamma globin silencing and the proposed role of the DRED complex require further evaluation. Furthermore, this work also suggests that LSD1 inhibition is not a therapeutic strategy for HbF induction in patients with sickle cell disease or beta thalassemia. Disclosures:Arvedson:Amgen: Employment. Tran:Amgen: Employment. Ross:Amgen: Employment. Yoder:Amgen: Employment. Hertz:Amgen: Employment. Hale:Amgen: Employment. Eschelbach:Amgen: Employment. Dineen:Amgen: Employment. Matyas:Amgen: Employment. Hartley:Amgen: Employment. Morgenstern:Amgen: Employment. Winters:Amgen: Employment. Cindy:Amgen: Employment. Molineux:Amgen: Employment. Coxon:Amgen: Employment.