Globin Expression Research Articles

Induced Pluripotent Stem Cell (iPSC)-Based Mapping of Globin Expression throughout Human Erythropoietic Development

It has recently become clear that in vitro hematopoietic differentiation protocols using pluripotent stem cells mainly capture the primitive wave of hematopoietic development with limited induction of the definitive wave that gives rise to cells with adult-type characteristics such as the ability to express adult β-globin. Recent efforts from our group in optimizing our original differentiation protocol to derive more definitively patterned erythroid cells from iPSCs have resulted in a significant increase in β-globin transcripts as well as robust β-globin protein expression at the most mature stage of differentiation. To better quantify our progress in augmenting β-globin expression and to track globin ontogeny in real-time, we created a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout erythroid development.To create this tool, TALEN were used to target the β-globin locus in iPSCs where a promoterless GFP cassette was fused in frame to the first codon of the β-globin gene allowing for visualization of β-globin expression at single cell resolution via GFP expression.Interestingly, using our optimized protocol, only about 1% of cells exceeded the GFP detection threshold at the most mature stage of differentiation, suggesting that the several log-fold increase in β-globin transcripts seen at the population level as the cells progress through erythroid differentiation could be the result of high levels of β-globin transcription in just a small fraction of cells. Single cell RNA sequencing of GFP- and GFP+ sorted fractions showed significantly greater levels of β-globin transcripts in the GFP+ fraction in line with this hypothesis, thereby validating our reporter line as a tool to visualize and enrich for β-globin expressing cells.Despite lacking β-globin protein expression, the GFP- cells were not completely devoid of β-globin transcripts. As recent studies suggest that translation is dynamically controlled in maturing red blood cells, our results might indicate that posttranscriptional mechanisms could impact the translation of these globin transcripts. In fact, mining of the genes differentially expressed between both populations revealed several transcripts enriched in the GFP+ fraction that code for proteins involved in the dynamic translational control of transcripts essential for maturing red blood cells. These findings suggest advanced maturation of the GFP+ fraction, as well as a role for posttranscriptional mechanisms in the regulation of β-globin protein expression during erythroid development from iPSCs.Establishment of the developmental time frame of iPSC-derived erythroid cells is challenging as in vitro differentiation cultures lack the spatiotemporal separation of the different hematopoietic waves present in the embryo. Due to the possibility of multiple hematopoietic programs co-existing in one well, bulk expression analyses should be interpreted with caution when used to ascribe primitive or definitive characteristics to cell populations. Considering that the complexity of globin regulation in development might currently be underappreciated due to these factors, we also used single cell RNA sequencing to dissect the globin expression profiles of individual cells. Looking at the co-expression of different globins in individual cells, we found that the majority of the cells express a combination of embryonic (ε), fetal (γ) and adult (β) globins, indicative of a definitive yolk sac identity. Moreover, as β-globin transcripts increased, we observed a decrease in ε-globin transcripts, indicating that primitive/embryonic characteristics are gradually lost as cells gain more definitive/adult features.Further interrogation of the ‘β-globin expression signature’ that we distilled from the single cell RNA sequencing data will be instructive for future strategies aimed at increasing β-globin protein levels in iPSC-derived erythroid cells. Progress in this area will improve the resolution with which we can study hemoglobinopathies such as β-thalassemia and sickle cell disease and such strategies can then immediately be tested using the β-globin reporter iPSC line as a screening platform. DisclosuresNo relevant conflicts of interest to declare.

Introduction of Two Simultaneous Mutations By Genome Editing Greatly Enhances the Accumulation of the Endogenous Fetal Hemoglobin in Human Normal Erythroid Cells

Thalassemia or sickle cell patients with increased fetal hemoglobin have an ameliorated clinical picture up to transfusion independency. Genome editing with targeted nucleases of the γ-globin silencer BCL11a or of the HBG promoter has been shown to reenact an HPFH-like phenotype by significantly increasing the endogenous fetal globin (HbF) expression. In order to achieve higher efficiency in HbF reactivation we developed an innovative approach, mediated through a highly customizable helper dependent-adenoviral vector (HD-Ad5/35++) with nuclease activity. This vector bares the Cas9 gene and two sequential gRNAs, permitting simultaneous targeting of two different DNA loci, specifically, the erythroid enhancer of BCL11a and the HBG promoter to recreate the -114 to -102 HPFH deletion. This double targeting vector was compared to the respective single gRNA vectors. We observed that disruption of the BCL11a-enhancer increased Gγ globin, whereas disruption of the HBG promoter had a greater impact on the Αγ globin expression. Simultaneous disruption of both loci in human erythroid progenitors increased the overall HbF expression from less than 1% in the control to more than 20%, through a possibly synergistic effect of the two mutations (HbF:2.2% in BCL11a-enhancer only knock-out, 10.8% in HBG-only knock out) by affecting both gamma globin chains. The editing efficiency per locus was similar between the single gRNA and double gRNA vectors. Erythroid cell morphology and phenotypic profile of the double knock-out cells did not differ compared to the single knock-out cells. In addition to our in vitro experiments, we observed that mobilized peripheral blood CD34+ cells transduced and edited by the double gRNA vector, can efficiently engraft in NSG mice. Furthermore, the engrafted edited cells after erythroid differentiation expressed significantly higher levels of gamma globin compared to the control. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. DisclosuresNo relevant conflicts of interest to declare.

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.

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.

Epigenetic dysregulation of the erythropoietic transcription factor KLF1 and the β-like globin locus in juvenile myelomonocytic leukemia

ABSTRACTIncreased levels of fetal hemoglobin (HbF) are a hallmark of more than half of the children diagnosed with juvenile myelomonocytic leukemia (JMML). Elevated HbF levels in JMML are associated with DNA hypermethylation of distinct gene promoter regions in leukemic cells. Since the regulation of globin gene transcription is known to be under epigenetic control, we set out to study the relation of DNA methylation patterns at β-/γ-globin promoters, mRNA and protein expression of globins, and epigenetic modifications of genes encoding the globin-regulatory transcription factors BCL11A and KLF1 in nucleated erythropoietic precursor cells of patients with JMML. We describe several altered epigenetic components resulting in disordered globin synthesis in JMML. We identify a cis-regulatory upstream KLF1 enhancer sequence as highly sensitive to DNA methylation and frequently hypermethylated in JMML. The data indicate that the dysregulation of β-like globin genes is a genuine attribute of the leukemic cell clone in JMML and involves mechanisms not taking part in the normal fetal-to-adult hemoglobin switch.

Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells

BackgroundIn humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19–40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts.MethodsThe expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins.ResultsThe expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged.ConclusionsOverall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.

Effect of Low Temperature on Globin Expression, Respiratory Metabolic Enzyme Activities, and Gill Structure of Litopenaeus vannamei.

Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.

Long-Term Engraftment and Fetal Globin Induction upon BCL11A Gene Editing in Bone-Marrow-Derived CD34+ Hematopoietic Stem and Progenitor Cells

To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of the BCL11A gene, either within exon 2 or at the GATAA motif in the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs). Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation of BCL11A resulting from bi-allelic frameshift mutations in BCL11A exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of BCL11A did not negatively impact enucleation. Furthermore, BCL11A exon 2-edited BM-CD34+ cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed upon BCL11A erythroid-enhancer GATAA motif editing, because enhancer-edited CD34+ cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of the BCL11A erythroid-specific enhancer editing in BM-CD34+ HSPCs as an autologous stem cell therapy in SCD patients.

Development of a Predictive Pharmacophore Model and a 3D-QSAR Study for an in silico Screening of New Potent Bcr-Abl Kinase Inhibitors.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder, characterized, in most cases, by the presence of the Bcr-Abl fusion oncogene. Bcr-Abl is a constitutively active tyrosine kinase that is responsible for the malignant transformation. Targeting the Bcr-Abl kinase is an attractive treatment strategy for CML. First and second generation Bcr-Abl inhibitors have focused on targeting the ATP-binding domain of the kinase. Mutations in that region are relatively resistant to drug manipulation. Therefore, non-ATP-competitive agents have been recently developed and tested. In the present study, in an attempt to aid the design of new chemotypes with enhanced cytotoxicity against K562 cells, 3D pharmacophore models were generated and 3D-QSAR CoMFA and CoMSIA studies were carried out on the 33 novel Abl kinase inhibitors (E)-α-benzylthio chalcones synthesized by Reddy et al. A five-point pharmacophore with a hydrogen bond acceptor, two hydrophobic groups and two aromatic rings as pharmacophore features, and a statistically significant 3D-QSAR model with excellent predictive power were developed. The pharmacophore model was also used for alignment of the 33 compounds in a CoMFA/CoMSIA analysis. The contour maps of the fields of CoMFA and CoMSIA models were utilized to provide structural insight into how these molecules promote their toxicity. The possibility of using this model for the design of drugs for the treatment of β-thalassemia and sickle cell disease (SCD), since several Bcr-Abl inhibitors are able to promote erythroid differentiation and γ-globin expression in CML cell lines and primary erythroid cells is discussed.

Identifying Novel Modifiers of Embryonic Globin Expression By Combining Chipseq, Rnaseq and eQTL Mapping in the Adult Nan Mouse Model

The KLF1 transcription factor regulates a large set of genes during erythroid differentiation and promotes the developmental switch from fetal to adult globin by directly activating the adult β-globin gene as well as Bcl11a, a major repressor of fetal globin expression. Recently, LRF (leukemia/lymphoma-related factor) was also shown to be a major repressor of fetal globin. LRF participates in a NuRD complex separate from that of BCL11A. Understanding the mechanisms underlying β-like globin gene switching is essential to identify new drug targets for sickle cell disease and β-thalassemia, as increased fetal globin ameliorates disease symptoms. We previously showed that the inbred Nan (neonatal anemia) mutant mouse strain carries a missense mutation (E339D) in the second zinc finger of KLF1, which leads to aberrant binding of target genes. Nan/+ mice suffer severe, lifelong anemia accompanied by a striking failure of hemoglobin switching. Embryonic Hbb-bh1 globin expression, but not Hbb-y (εγ), is significantly upregulated in Nan E14.5 fetal liver (FL) and in adult whole spleen vs. wild-type (WT) littermates. To investigate mechanisms of Hbb-bh1 upregulation in Nan, we performed RNAseq and ChIPseq combined with genetic mapping to identify genetic modifiers of globin gene regulation, which may encode additional components of NuRD complexes or act independently. ChIPseq was performed in a Klf1 null erythroid cell line containing either a tamoxifen-inducible WT Klf1 or a Nan Klf1 transgene and revealed aberrant DNA binding, both absent and ectopic, by Nan-KLF1 vs. WT KLF1. RNAseq of WT and Nan E14.5 FLs and sorted adult spleen erythroid precursors (pro, basophilic, polychromatophilic and orthochromatic erythroblasts) confirms aberrant expression of a large set of KLF1 regulated genes. For example, only 3% of genes upregulated in Nan FL are normally activated by KLF1 (FDR 5, FC ≥ 2). Direct KLF1 targets in adult spleen have not been defined by ChIPseq; however, deregulated expression is apparent in all precursor stages and by the orthochromatic stage > 5100 genes are significanlty up- or downregulated in Nan vs. anemic (phlebotomized) WT. To identify genes potentially regulating Hbb-bh1 expression, we performed genetic mapping using two fully congenic Nan lines (Nan/+-BALB/cBy, Nan/+-129/SvImJ) intercrossed with C57BL/6J. Statistical analyses revealed two significant eQTL for Hbb-bh1 levels on Chr 7, one on Chr 11 and suggestive eQTL on Chrs 14 and 17. Combining genetic mapping data with ChIPseq and RNAseq provides a powerful tool to identify potential candidate genes regulating Hbb-bh1 expression using the criteria that (a) candidate genes must be within an eQTl 95% confidence interval (CI) and (b) show dysregulated DNA binding by Nan-KLF1 and/or abnormal expression in Nan. Using these criteria, Socs3 (Suppressor of cytokine signaling 3) emerges as one strong candidate. Socs3 is located within the CI close to the QTL peak (114.6 Mb) on Chr 11. Socs3is not bound by Nan-KLF1 but is bound by WT KLF1. Moreover, Socs3 expression is dramatically decreased in all Nan sorted spleen erythroid precursors, reaching a >100-fold decrease at the orthochromatic stage. Socs3 makes biological sense as a potential candidate. Previous studies have shown that SOCS3 negatively regulates fetal liver hematopoiesis, and conditional deletion of Socs3 in erythroid cells leads to increased cell proliferation with decreased differentiation. Socs3 is activated by cytokines and also by Sox6, a known repressor of fetal globin, but Socs3 itself has never been itself associated with the regulation of fetal globin expression. In summary, we investigated mechanisms by which Nan-KLF1 impairs β-like globin gene switching in adult mice. We found that: (1) the Nan mutation leads to aberrant binding of KLF1 and inappropriate regulation of a large set of genes, (2) eQTL mapping identified 5 eQTL containing potential modifiers of Hbb-bh1 expression, (3) strong candidate genes were identified by combining all datasets; candidates include (4) Socs3, which lies near the Chr 11 eQTL peak, is not bound by mutant Nan-KLF1 but is bound by WT KLF1, shows severely downregulated expression in erythroid precursors, and has been previously associated with erythropoiesis but not fetal globin regulation. These data suggest that Socs3 may act to suppress embryonic globin in WT adults. Definitive proof of candidate genes requires functional analysis. DisclosuresPerkins:Bristol-Myers Squibb: Honoraria; Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Therapeutic Crispr/Cas9 Genome Editing for Treating Sickle Cell Disease

IntroductionSickle cell disease (SCD) is one of the most common monogenic disorders, affecting millions worldwide. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution from A to T in the codon for the sixth amino acid in the β-globin protein converts a glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of the red blood cells (RBCs). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but it is feasible for only a small subpopulation (<15%) of patients and may be associated with a high risk. Here, we show that targeted genome editing can potentially provide a permanent cure for SCD by correcting the sickle mutation in clinically relevant hematopoietic stem and progenitor cells (HSPCs) for autologous transplantation.MethodsFor proof-of-concept, we designed CRISPR/Cas9 systems and donor templates to introduce the sickle mutation into wild-type (WT) HBB of mobilized peripheral blood CD34+ cells. To assess genome-editing outcomes mediated by CRISPR/Cas9 systems, we developed a novel digital droplet PCR (ddPCR) assay that can quantify the rates of non-homologous end joining (NHEJ) and homology directed repair (HDR) events simultaneously following the generation of DNA double strand breaks. The assay enables rapid and accurate quantification of gene modifications in HSPCs by CRISPR/Cas9 genome-editing. Specifically, Streptococcus pyogenes (Spy) Cas9 proteins, guide RNAs (gRNA), and single-stranded DNA (ssDNA) donor templates were delivered into CD34+ cells by nucleofection with optimized conditions. Different gRNAs targeting HBB near the SCD mutation site were tested, and the optimal gRNA was chosen based on high on-target activity and proximity to the mutation site. The optimal DNA donor design and concentration were determined based on the frequency of HDR events and viability/growth rate of edited cells. Treated samples and untreated controls were assayed as both single cell clones and in bulk culture. In 2-phase liquid culture, genome editing frequencies at both DNA and mRNA levels were quantified by ddPCR to confirm persistence of edited cells in the heterozygous population over time. The expression of globins and other erythroid markers were monitored using flow cytometry and real time PCR to determine if genome editing had any effect on the kinetics of erythropoiesis. Colony formation assays were used to determine the number and type of colonies following induction of differentiation. Colony ddPCR was performed to determine the genotype of edited cells. Wright/Giemsa stain was used to confirm terminal maturation of erythrocytes into enucleated RBC. Native polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) were used to confirm translation of edited β-globin protein and formation of HbS.Results and DiscussionWe found that the efficiency of site-specific gene correction could be substantially improved by optimizing the CRISPR/Cas9 systems for genome editing. For example, with optimization, we achieved ~30% HDR rates in CD34+ cells with >80% cell viability. The HDR-modified alleles persisted in the population over the course of differentiation, and the edited CD34+ cells retained differentiation potential. Genotyping of individual erythroid colonies confirmed that up to 35% of colonies are either homozygous or heterozygous for HDR alleles. Following differentiation, treated cells express modified HBB mRNA and HbS. In addition, the off-target activity of the HBB-specific gRNAs was determined using both bioinformatics tools and unbiased genome-wide mapping techniques. Ongoing work includes the validation of gene correction in SCD patient derived HSPCs, characterization of modified cells in vitro and in vivo to assess the therapeutic potential, and analysis of long-term genotoxicity.ConclusionsBased on the proof-of-concept study, we demonstrate that using the optimized CRISPR/Cas9 system and donor template, an HDR rate of ~30% can be achieved in CD34+ cells. The gene corrected cells have the potential to differentiate into erythroid cells that permanently produce WT β-globin. Our findings provide promising evidence for clinical translation of the HSPCs genome correction strategy in treating SCD patients, as well as correcting gene defects underlying other inherited single-gene disorders. DisclosuresNo relevant conflicts of interest to declare.

IGF2BP3 Moderately Increases Fetal Hemoglobin Levels in Human Adult Erythroblasts

Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; >65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders.The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis.These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Strict in vivo specificity of the Bcl11a erythroid enhancer

AbstractBCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. Bcl11a enhancer–deleted mice, Bcl11a(Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.

Evolution and Expression of Tissue Globins in Ray-Finned Fishes.

The globin gene family encodes oxygen-binding hemeproteins conserved across the major branches of multicellular life. The origins and evolutionary histories of complete globin repertoires have been established for many vertebrates, but there remain major knowledge gaps for ray-finned fish. Therefore, we used phylogenetic, comparative genomic and gene expression analyses to discover and characterize canonical “non-blood” globin family members (i.e., myoglobin, cytoglobin, neuroglobin, globin-X, and globin-Y) across multiple ray-finned fish lineages, revealing novel gene duplicates (paralogs) conserved from whole genome duplication (WGD) and small-scale duplication events. Our key findings were that: (1) globin-X paralogs in teleosts have been retained from the teleost-specific WGD, (2) functional paralogs of cytoglobin, neuroglobin, and globin-X, but not myoglobin, have been conserved from the salmonid-specific WGD, (3) triplicate lineage-specific myoglobin paralogs are conserved in arowanas (Osteoglossiformes), which arose by tandem duplication and diverged under positive selection, (4) globin-Y is retained in multiple early branching fish lineages that diverged before teleosts, and (5) marked variation in tissue-specific expression of globin gene repertoires exists across ray-finned fish evolution, including several previously uncharacterized sites of expression. In this respect, our data provide an interesting link between myoglobin expression and the evolution of air breathing in teleosts. Together, our findings demonstrate great-unrecognized diversity in the repertoire and expression of nonblood globins that has arisen during ray-finned fish evolution.

Animal models of &amp;beta;-hemoglobinopathies: utility and limitations

The structural and functional conservation of hemoglobin throughout mammals has made the laboratory mouse an exceptionally useful organism in which to study both the protein and the individual globin genes. Early researchers looked to the globin genes as an excellent model in which to examine gene regulation – bountifully expressed and displaying a remarkably consistent pattern of developmental activation and silencing. In parallel with the growth of research into expression of the globin genes, mutations within the β-globin gene were identified as the cause of the β-hemoglobinopathies such as sickle cell disease and β-thalassemia. These lines of enquiry stimulated the development of transgenic mouse models, first carrying individual human globin genes and then substantial human genomic fragments incorporating the multigenic human β-globin locus and regulatory elements. Finally, mice were devised carrying mutant human β-globin loci on genetic backgrounds deficient in the native mouse globins, resulting in phenotypes of sickle cell disease or β-thalassemia. These years of work have generated a group of model animals that display many features of the β-hemoglobinopathies and provided enormous insight into the mechanisms of gene regulation. Substantive differences in the expression of human and mouse globins during development have also come to light, revealing the limitations of the mouse model, but also providing opportunities to further explore the mechanisms of globin gene regulation. In addition, animal models of β-hemoglobinopathies have demonstrated the feasibility of gene therapy for these conditions, now showing success in human clinical trials. Such models remain in use to dissect the molecular events of globin gene regulation and to identify novel treatments based upon the reactivation of developmentally silenced γ-globin. Here, we describe the development of animal models to investigate globin switching and the β-hemoglobinopathies, a field that has paralleled the emergence of modern molecular biology and clinical genetics.

Defining the minimal factors required for erythropoiesis through direct lineage conversion

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Bi-functional prodrugs of 5-aminolevulinic acid and butyric acid increase erythropoiesis in anemic mice in an erythropoietin-independent manner

Anemia is a major cause of morbidity and mortality worldwide resulting from a wide variety of pathological conditions. In severe cases it is treated by blood transfusions or injection of erythroid stimulating agents, e.g., erythropoietin (Epo), which can be associated with serious adverse effects. Therefore, there is a need to develop new treatment modalities. We recently reported that treatment of erythroleukemic cells with the novel the bi-functional prodrugs of 5-aminolevulinic acid (ALA) and butyric acid (BA), AN233 and AN908, enhanced hemoglobin (Hb) synthesis to a substantially higher level than did ALA and BA individually or their mixture. Herein, we describe that these prodrugs when given orally to mice induced histone deacetylase inhibition in the kidneys, bone marrow and spleen, thus, indicating good penetrability to the tissues. In mice where anemia was chemically induced, treatment with the prodrugs increased the Hb, the number of red blood cells (RBCs) and the percentage of reticulocytes to normal levels. The prodrugs had no adverse effects even after repeated treatment at 100–200mg/kg for 50days. The lack of increased levels of Epo in the blood of mice that were treated with the prodrugs suggests that AN233 and AN908 affected the Hb and RBC levels in an Epo-independent manner. Taken together with our previous studies, we propose that the prodrugs increase globin expression by BA inhibition of histone deacetylase and elevation heme synthesis by ALA. These results support an Epo-independent approach for treating anemia with these prodrugs.

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.

SummaryErythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.

Use of genome-editing tools to treat sickle cell disease.

Recent advances in genome-editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next-generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). SCD is an inheritable monogenic disorder which is caused by a point mutation in the β-globin gene. Substantial success has been achieved in the development of supportive therapeutic strategies for SCD, but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Hence, a universally applicable therapy is highly desirable. In this review, we will discuss the three programmable nucleases that are commonly used for genome-editing purposes: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). We will continue by exemplifying uses of these methods to correct the sickle cell mutation. Additionally, we will present induction of fetal globin expression as an alternative approach to cure sickle cell disease. We will conclude by comparing the three methods and explaining the concerns about their use in therapy.