Manipulation of Developmental Gamma-Globin Gene Expression: an Approach for Healing Hemoglobinopathies.
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ABSTRACT:
SUBMITTER: Venkatesan V
PROVIDER: S-EPMC7849396 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:β-hemoglobinopathies are the most common genetic disorders worldwide. In sickle cell disease (SCD) a single mutation (E6V) in the b-globin (HBB) gene results in dysfunctional hemoglobin protein, while in β-thalassemia, over 300 mutations distributed across the HBB gene are known to reduce the production of β-globin and cause severe anemia. A genetic engineering approach that replaces the whole HBB gene is an ideal strategy to rescue HBB expression for most genotypes but is technically challenging as the insert cannot be homologous to the endogenous gene and codon-optimized, intron-less sequences may not reconstitute adequate HBB levels. Here, we developed a novel approach for a “one-size-fits-all" HBB gene repair strategy using CRISPR-Cas9 which successfully addresses these problems. First, our DNA donor design avoids sequence homology through a diverged HBB coding sequence and second, incorporates heterologous introns from the fetal g-globin gene, further reducing homology to endogenous HBB while mimicking its intron composition. Screening DNA donors with various heterologous globin introns, polyadenylation signals and truncated sequences, identified a heterologous intron DNA donor that highly expresses β-globin and rescued β-globin expression in two in vitro hemoglobinopathy models in hematopoietic stem and progenitor cells (HSPCs). Furthermore, healthy donor HSPCs modified with this HBB gene replacement approach showed successful engraftment in immunodeficient mice at 16 weeks. In summary, we developed a universal HBB gene replacement strategy that results in physiological b-globin production, offering a potential differentiated approach for treating patients with β-thalassemia, SCD or compound heterozygous individuals.
2024-04-01 | GSE229212 | GEO
Project description:The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
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Project description:It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human gamma-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the gamma-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the gamma-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the gamma TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the gamma gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the gamma promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the gamma-globin gene.
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Project description:β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease.
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Project description:The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.
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Project description:Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.
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Project description:Increased fetal hemoglobin expression in adulthood is associated with acute stress erythropoiesis. However, the mechanisms underlying gamma-globin induction during the rapid expansion of adult erythroid progenitor cells have not been fully elucidated. Here, we examined COUP-TFII as a potential repressor of gamma-globin gene after stem cell factor (SCF) stimulation in cultured human adult erythroid progenitor cells. We found that COUP-TFII expression is suppressed by SCF through phosphorylation of serine/threonine phosphatase (PP2A) and correlated well with fetal hemoglobin induction. Furthermore, down-regulation of COUP-TFII expression with small interfering RNA (siRNA) significantly increases the gamma-globin expression during the erythroid maturation. Moreover, SCF-increased expression of NF-YA associated with redox regulator Ref-1 and cellular reducing condition enhances the effect of SCF on gamma-globin expression. Activation of Erk1/2 plays a critical role in SCF modulation of downstream transcriptional factor COUP-TFII, which is involved in the regulation of gamma-globin gene induction. Our data show that SCF stimulates Erk1/2 MAPK signaling pathway, which regulates the downstream repressor COUP-TFII by inhibiting serine/threonine phosphatase 2A activity, and that decreased COUP-TFII expression resulted in gamma-globin reactivation in adult erythropoiesis. These observations provide insight into the molecular pathways that regulate gamma-globin augmentation during stress erythropoiesis.
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Project description:To investigate the control of the gamma-globin gene during development, we produced transgenic mice in which sequences of the beta-gene promoter were replaced by equivalent sequences of the gamma-gene promoter in the context of a human beta-globin locus yeast artificial chromosome (betaYAC) and analyzed the effects on globin gene expression during development. Replacement of 1,077 nucleotides (nt) of the beta-gene promoter by 1,359 nt of the gamma promoter resulted in striking inhibition of the gamma-promoter/beta-gene expression in the adult stage of development, providing direct evidence that the expression of the gamma gene in the adult is mainly controlled by autonomous silencing. Measurements of the expression of the gamma promoter/beta-globin gene as well as the wild gamma genes showed that gene competition is also involved in the control of gamma-gene expression in the fetal stage of development. We conclude that autonomous silencing is the main mechanism controlling gamma-gene expression in the adult, while autonomous silencing as well as competition between gamma and beta genes contributes to the control of gamma to beta switching during fetal development.
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Project description:Induction of fetal hemoglobin (HbF) is a promising strategy in the treatment of β-thalassemia major (β-TM). The present study shows that plasma exosomal miRNAs (exo-miRs) are involved in γ-globin regulation. Exosomes shuttle miRNAs and mediate cell-cell communication. MiRNAs are regulators of biological processes through post-transcriptional targeting. Compared to HD (Healthy Donor), β-TM patients showed increased levels of plasma exosomes and the majority of exosomes had cellular origin from CD34+ cells. Further, HD and β-TM exosomes showed differential miRNA expressions. Among them, deregulated miR-223-3p and miR-138-5p in β-TM exosomes and HD had specific targets for γ-globin regulator and repressor respectively. Functional studies in K562 cells showed that HD exosomes and miR-138-5p regulated γ-globin expression by targeting BCL11A. β-TM exosomes and miR-223-3p down regulated γ-globin expression through LMO2 targeting. Importantly, miR-223-3p targeting through sponge repression resulted in γ-globin activation. Further, hnRNPA1 bound to stem-loop structure of pre-miR-223 and we found that hnRNPA1 knockdown or mutagenesis at miR-223-3p stem-loop sequence resulted in less mature exo-miR-223-3p levels. Altogether, the study shows for the first time on the important clinical evidence that differentially expressed exo-miRNAs reciprocally control γ-globin expressions. Further, the hnRNPA1-exo-miR-223-LMO2 axis may be critical to γ-globin silencing in β-TM.
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Project description:To test the role of gene order in globin gene expression, mutant human beta-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a "marked" transcript (epsilon(m), gamma(m), or beta(m)) integrated at different locations within the locus. When a beta(m)-globin gene was placed between the locus control region (LCR) and the epsilon-globin gene, beta(m)-globin expression dominated primitive and definitive erythropoiesis; only beta(m)-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an (A)gamma(m)-globin gene was placed at the same location, (A)gamma(m)-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type gamma-globin genes were not expressed. When an epsilon(m)-globin gene was placed between the delta- and beta-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type epsilon-globin gene was expressed. Placement of a beta(m)-globin gene upstream of the (G)gamma-globin gene resulted in expression of beta(m)-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type beta-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.
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