Umbilical Cord Blood-derived Erythroid Progenitor Research Articles

Long Non-Coding RNA H19 Leads to Upregulation of γ-Globin Gene Expression during Erythroid Differentiation

Long noncoding RNAs (lncRNAs) are important because they are involved in a variety of life activities and have many downstream targets. Moreover, there is also increasing evidence that some lncRNAs play important roles in the expression and regulation of γ-globin genes. In our previous study, we analyzed genetic material from nucleated red blood cells (NRBCs) extracted from premature and full-term umbilical cord blood samples. Through RNA sequencing (RNA-Seq) analysis, lncRNA H19 emerged as a differentially expressed transcript between the two blood types. While this discovery provided insight into H19, previous studies had not investigated its effect on the γ-globin gene. Therefore, the focus of our study was to explore the impact of H19 on the γ-globin gene. In this study, we discovered that overexpressing H19 led to a decrease in HBG mRNA levels during erythroid differentiation in K562 cells. Conversely, in CD34+ hematopoietic stem cells and human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, HBG expression increased. Additionally, we observed that H19 was primarily located in the nucleus of K562 cells, while in HUDEP-2 cells, H19 was present predominantly in the cytoplasm. These findings suggest a significant upregulation of HBG due to H19 overexpression. Notably, cytoplasmic localization in HUDEP-2 cells hints at its potential role as a competing endogenous RNA (ceRNA), regulating γ-globin expression by targeting microRNA/mRNA interactions.

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

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

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

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

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

Induction of therapeutic levels of HbF in genome-edited primary β0 39-thalassaemia haematopoietic stem and progenitor cells.

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of β-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δβ0 -thalassaemia, is able to completely rescue the β-major thalassaemia phenotype caused by the β0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from β0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous β0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of β-thalassaemia irrespective of the β-globin gene (HBB) mutations.

Control of Fetal Hemoglobin Levels By NFI Transcription Factors

Sickle cell disease and some forms of b-thalassemia are disorders, which can be improved by therapies that elevate HbF, the fetal form of hemoglobin. It is currently thought that in adult type erythroid cells, silencing of the fetal type b-globin-like genes HBG1 and HBG2 is accomplished predominantly by two major transcription factors: BCL11A and ZBTB7A. However, it is unknown whether there are additional transcription factors that contribute to the repression of the HBG1/2 genes. Here, using a DNA-binding domain focused CRISPR-Cas9 screening approach, we identified NFIA as a novel regulator of HbF expression. NFIA belongs to the NFI transcription factor family, which is composed of NFIA, NFIB, NFIC and NFIX. NFIA and NFIX are the predominantly expressed forms in human erythroid cells, and their expression is higher in adult erythroblast cells when compared to fetal erythroid cells. CRISPR-Cas9 mediated disruption of NFIA in an immortalized human umbilical cord blood-derived erythroid progenitor cell line 2 (HUDEP2) or CD34+ hematopoietic stem and progenitor cell-derived primary erythroblast cells led to a modest reactivation of HBG1/2 mRNA expression, whereas disruption of NFIX had little effect. However, combined NFIA and NFIX disruption produced a substantial increase in HBG1/2 expression, suggesting that these factors function in a partially redundant manner. ChIP-seq and RNA-seq studies showed that NFIA and NFIX have comparable chromatin binding and activity profiles in human erythroid cells. ChIP-seq failed to detect NFI protein occupancy at or near the HBG1/2 genes. However, given the known difficulty in detecting repressor molecules by ChIP at the silent HBG1/2 genes [Martyn et al., Nature Genetics 2018; Liu et al., Cell 2018], we tested a direct involvement of NFI proteins by disrupting putative NFI binding sites near the HBG1/2 genes. Perturbation of one such NFI motif residing upstream of the transcription start site in the HBG1/2 promoters both in HUDEP2 and primary human erythroid cells markedly increased HBG1/2 mRNA levels, comparable to those achieved by combined disruption of NFIA and NFIX. Mutation of another putative NFI motif within intron1 in the HBG1/2 gene also significantly raised HbF levels. While these results implicate NFI proteins in the direct silencing of the HBG1/2 genes, the identity of the bound factors at the NFI motifs remains to be established. Studies are currently ongoing that use alternative approaches such as Cleavage Under Target &amp; Release Using Nuclease (CUT &amp; RUN) to map the chromatin occupancy of NFI factors at the HBG1/2 genomic region. We will also discuss results from ongoing studies of NFI factors in NBSGW mice models. In sum, we uncovered NFI transcription factors as a novel HbF regulator suggesting that the silencing of HbF involves a transcription factor network that is more complex than previously appreciated. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Pfizer: Research Funding; Fulcrum Therapeutics: Consultancy.

High level of fetal-globin reactivation by designed transcriptional activator-like effector

AbstractThe fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and β thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the Gγ and Aγ-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetal-globin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the γ-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of β-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the β-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetal-globin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.

Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed &gt;1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p&lt; 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Molecular Characterization and Novel Therapeutic Strategy for X-Linked Sideroblastic Anemia Associated with ALAS2 Missense Variants

Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated &amp;gt;2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p &amp;lt; 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Regulating Transcriptional Co-Activator PGC-1α for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is the FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU).We recently identified PPARγ co-activator (PGC-1α) as a new protein involved in the regulation of the fetal globin genes. Forced overexpression of PGC-1α in vitro by adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. Furthermore, up-regulation of PGC-1α by a small molecular agonist (Compound Z) in human umbilical cord blood-derived erythroid progenitor (HUDEP-1) cells markedly increases γ-globin gene expression and HbF synthesis. The highest response was achieved when HUDEP-1 cells were treated with 5µM Compound Z for 2 days, which results in 66.6% HbF+ cells compared to vehicle control (29.5% HbF+ cells). The effect of Compound Z in inducing HbF was further validated in erythroblasts derived from cultures of normal adults' CD34+ cells as well as in iPSC-derived sickle erythroblasts (SS24 cells). These data suggest that modulating PGC-1α activity may be effectively applied to the treatment of SCD since enhanced HbF synthesis would alleviate pathophysiological effects of SCD. DisclosuresNo relevant conflicts of interest to declare.

Comparative Studies Reveal Robust HbF Induction By Editing of HBG1/2 Promoters or BCL11A Erythroid-Enhancer in Human CD34+ Cells but That BCL11A Erythroid-Enhancer Editing Is Associated with Selective Reduction in Erythroid Lineage Reconstitution in a Xenotransplantation Model

▪Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells.Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential.All groups achieved high levels of human chimerism (>70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model.Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. DisclosuresChang:Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

AMPK Regulates the Expression of the Fe-S Cluster Assembly Enzyme (ISCU) and ALAS2, Modulating Cellular Iron Metabolism and Increasing Hemoglobin Synthesis

Iron-sulfur (Fe-S) clusters are iron-containing prosthetic groups and enzymatic cofactors. They are strong oxidants when unbound yet essential in many processes like facilitating ATP production in mitochondria, promoting DNA, RNA and protein syntheses during cell proliferation and enhancing DNA repair in antioxidant defense. In particular, Fe-S clusters are indispensable in erythropoiesis, where the majority of physiological iron is utilized and where Fe-S clusters are required for the heme synthesis. Deficient Fe-S cluster synthesis predisposes individual to various diseases, such as cancer, metabolic and neurodegeneration diseases and blood disorders. However, it is unclear how Fe-S cluster synthesis is regulated and coordinates with environmental and developmental needs to prevent oxidative damage.The 5' AMP-activated protein kinase (AMPK) is a kinase activated by oxidative stress and energy starvation and critical for maintaining redox and energy homeostasis. In this study, we investigated the role of AMPK on Fe-S clusters synthesis and function and extended our findings in normal and thalassemic erythroid cells.Through bioinformatic analysis, we found that the Fe-S cluster assembly enzyme (ISCU), a scaffold protein indispensable for Fe-S cluster biogenesis, contains putative AMPK phosphorylation motifs at serine (S) residues 14 and 29 (human numbering). Using the human cell line 293T, we confirmed that AMPK phosphorylates ISCU, while point mutations in these residues prevented this activity. Moreover, AMPK-mediated phosphorylation promoted ISCU binding to 14-3-3s, a family of proteins that, once associate with phosphorylated residues, modulates the stability and function of targeted proteins. Indeed, increased association with 14-3-3s stabilized ISCU proteins, corroborating the observation that AMPK promotes the activity of ISCU proteins.We extended our studies using A549 cells that do not have AMPK activity since they harbor a mutant LKB1 kinase, which is responsible for activating AMPK. By overexpression of wild type (WT)-LKB1 and LKB1 kinase-dead mutant (KDM), we found that only WT-LKB1 restored AMPK activity, binding of ISCU to 14-3-3s and stability of ISCU. Moreover, under hydrogen peroxide incubation and glucose starvation, ISCU protein levels and Fe-S cluster synthesis were both increased only in the presence of LKB1-WT, but not in cells harboring KMD. LKB1-WT overexpressed cells also survived hydrogen peroxide incubation and glucose starvation better than those with KMD. Together, these data suggest that AMPK activation stabilizes ISCU protein and preserves Fe-S cluster synthesis to maintain a healthy redox and energy homeostasis.We then explored the effect of AMPK on Fe-S cluster synthesis in erythropoiesis by using the drug AICAR, an AMPK activator, in murine erythroleukemia (MEL) cells. We found that in MEL cells, AICAR treatment stabilized ISCU, increased Fe-S cluster levels and promoted the synthesis of the aminolevulinic acid synthase 2 (ALAS2) protein, which represents the rate-limiting enzyme in erythroid heme synthesis. Furthermore, this was associated with increased heme and globin chain synthesis, with a trend in increasing β-globin mRNA and proteins more than α-globin. We further confirmed these observations in Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP-2) and CD34+ cells derived from peripheral blood isolated from both healthy individuals and ß-thalassemic patients. In these cells, we found that AMPK upregulation by AICAR administration not only increased ALAS2 expression and erythroid heme levels, but also enhanced the synthesis of both a- and ß-globin chains, though with a preference for increasing β-globin levels. Analysis using specimens from thalassemic mice is in progress.In conclusion, our work demonstrates that under redox and energetic stress, activated AMPK phosphorylates and stabilizes ISCU protein, thereby enhancing Fe-S cluster synthesis and maintaining their function. Moreover, AMPK activation with AICAR treatment increases erythroid heme synthesis and hemoglobin expression. Given that AMPK is the major kinase that responds to oxidative and energetic cues, our work provides a mechanistic explanation for how erythropoiesis responds to energy starvation and redox stress as well as a potential novel therapeutic target to treat blood and metabolic disorders. DisclosuresRivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Protagonist: Consultancy; Disc Medicine: Consultancy.

Growing and Genetically Manipulating Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP) Cell Lines.

The recently established human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines have equipped red blood cell researchers with valuable in vitro models of erythroid development. Of the three established HUDEP cell lines, HUDEP-2 cells express predominantly adult β-globin and most closely resemble adult erythroid cells. This chapter describes culture protocols for the maintenance and erythroid differentiation of HUDEP-2 cells. Methods to genetically manipulate HUDEP-2 cells using a CRISPR/Cas9 nuclease-based approach are also discussed.