Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy Restore α-Globin Expression in α-Thalassemia Red Blood Cells
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PROVIDER: PRJNA1240240 | ENA |
REPOSITORIES: ENA
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Project description:Alpha thalassemia major (ATM) is an inherited blood disorder caused by the absence of all four α-globin genes (HBA2/1), resulting in severe anemia and lifelong transfusion dependence. While allogeneic hematopoietic stem cell transplant (HSCT) offers a potential cure, donor availability remains limited. We present a gene therapy approach for autologous HSCT, using lentiviral vectors (LVs) to deliver HBA2 under the regulation of optimized β-globin locus control region (LCR) enhancers, restoring α-globin expression. The best-performing LVs, EV-α and EV-α-UV, achieved 90-100% transduction efficiency in human hematopoietic stem and progenitor cells (HSPCs), optimal vector copy numbers, and a safe integration profile. ATM-derived HSPCs from three donors treated with these LVs yielded α/β-globin mRNA and chain ratios within the therapeutic range (~0.5+), and restored hemoglobin levels by 50 to 100%. These findings establish the safety and clinical potential of EV-α and EV-α-UV as a promising autologous stem cell gene therapy for ATM.
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Project description:The histone variant macroH2A generally associates with transcriptionally inert chromatin, however the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase, ATRX, as a novel macroH2A interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2A’s chromatin association. In human erythroleukemic cells deficient for ATRX, ChIP-sequencing studies reveal that macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of α globin expression. Collectively, our results implicate deregulation of macroH2A’s distribution as a contributing factor to the α thalassemia phenotype of ATRX syndrome. Mononucleosomes from K562 cells bearing integrated lentiviral shRNA constructs targeting either luciferase (shluc) or ATRX (sh92) were isolated and ChIP'd with mH2A1 antibody. DNA from shluc Input and the two mH2A1 ChIPs were isolated and sequenced on Illumina's Hiseq.
2012-03-08 | E-GEOD-35339 | biostudies-arrayexpress
Project description:beta-Thalassemia is a prevalent anemia caused by mutations in the HBB (beta-globin) gene. We show that the severity of beta-thalassemia in the frequently studied Hbb(th3/+) mouse model is influenced by ancestral beta-globin gene (Hbb) haplotypes that differ in common strains.
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Project description:β-Thalassemia is a prevalent anemia caused by mutations in the HBB (β-globin) gene. We show that the severity of β-thalassemia in the frequently studied Hbbth3/+ mouse model is influenced by ancestral β-globin gene (Hbb) haplotypes that differ in common strains.
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Project description:The advent of human induced pluripotent stem (iPS) cells enables for the first time the derivation of unlimited numbers of patient-specific stem cells and holds great promise for regenerative medicine. However, realizing the full potential of iPS cells requires robust, precise and safe strategies for their genetic modification. Safe human iPS cell engineering is especially needed for therapeutic applications, as stem cell-based therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify iPS cells from patients with beta-thalassemia in a potentially clinically relevant manner. Our approach is based on the identification and selection of âsafe harborâ sites for transgene expression in the human genome. We show that thalassemia patient iPS cell clones harboring a transgene can be isolated and screened according to chromosomal position. We next demonstrate that iPS cell clones that meet our âsafe harborâ criteria resist silencing and allow for therapeutic levels of beta-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. Combined bioinformatics and functional analyses thus provide a robust and dependable approach for achieving desirable levels of transgene expression from selected chromosomal loci. This approach may be broadly applicable to introducing therapeutic or suicide genes into patient specific iPS cells for use in cell therapy. iPS cell clones were derived from beta-thalassemia patients. A single copy of beta-globin transgene cis-linked to locus control region (LCR) elements and an excisable Neo-eGFP transcription unit were inserted into these cell clones. beta-globin expression was induced by erythroid differentiation.
2010-10-26 | E-GEOD-24901 | biostudies-arrayexpress
Project description:A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic βA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and βA-T87Q-globin gene expression identical to the parent vector. Compared with the first βA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype.
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Project description:Haemoglobin, the oxygen-carrying molecule of red blood cells, at all stages of development comprises a tetramer of two α-like and two β-like globin chains. In humans and mice the highly conserved α- and β-globin loci contain genes encoding globin chains specifically expressed only during the first trimester of gestation (termed embryonic globins) in addition to the genes encoding globin chains expressed throughout adult life. In humans the β-globin locus also encodes a β-like chain that is expressed throughout the second and third trimesters of development; this is termed γ-globin and complexes with α-globin to form fetal haemoglobin (HbF). The sequential activation and repression of the genes at each of these loci throughout development to maturity is termed haemoglobin switching and is a poorly understood process. α-thalassemia, which results from insufficient production of α-globin, is a major global health problem with several hundred thousand sufferers world-wide. The embryonically expressed ζ-globin, encoded by the gene HBZ (Hba-x in mice), can functionally substitute for α-globin in adult erythroid cells. Reactivation of this gene is consequently likely to ameliorate the symptoms of α-thalassemia in individuals with a severe phenotype. Little is currently known about the regulation of ζ-globin expression in terms of both the trans- and cis-acting regulatory elements responsible for high-levels of expression of this gene during embryonic erythropoiesis and its maintenance in a transcriptionally inactive state throughout adult life. Using ATACseq and NG Capture C and mutant mouse lines this work aims to identify and define the cis-acting regulatory network underlying zeta-globin expression, and define the contribution of each regulatory element.
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Project description:Haemoglobin, the oxygen-carrying molecule of red blood cells, at all stages of development comprises a tetramer of two α-like and two β-like globin chains. In humans and mice the highly conserved α- and β-globin loci contain genes encoding globin chains specifically expressed only during the first trimester of gestation (termed embryonic globins) in addition to the genes encoding globin chains expressed throughout adult life. In humans the β-globin locus also encodes a β-like chain that is expressed throughout the second and third trimesters of development; this is termed γ-globin and complexes with α-globin to form fetal haemoglobin (HbF). The sequential activation and repression of the genes at each of these loci throughout development to maturity is termed haemoglobin switching and is a poorly understood process. α-thalassemia, which results from insufficient production of α-globin, is a major global health problem with several hundred thousand sufferers world-wide. The embryonically expressed ζ-globin, encoded by the gene HBZ (Hba-x in mice), can functionally substitute for α-globin in adult erythroid cells. Reactivation of this gene is consequently likely to ameliorate the symptoms of α-thalassemia in individuals with a severe phenotype. Little is currently known about the regulation of ζ-globin expression in terms of both the trans- and cis-acting regulatory elements responsible for high-levels of expression of this gene during embryonic erythropoiesis and its maintenance in a transcriptionally inactive state throughout adult life. Using ATACseq and NG Capture C and mutant mouse lines this work aims to identify and define the cis-acting regulatory network underlying zeta-globin expression, and define the contribution of each regulatory element.
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Project description:To determine the heterogeneity of g-globin expression in the context of deletions that induce fetal hemoglobin, single-cell RNA-seq was performed on primary reticulocytes and undifferentiated HUDEP-2 cells. Single-cell RNA-sequencing (10x Genomics) showed participants and cell lines with the Agdb-thalassemia deletion had overall lower g-globin expression and maintained a lower %HBG expression (HBG/(HBG+HBB)) compared to participants and cell lines with deletional HPFH.
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