Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease
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PROVIDER: PRJNA688203 | ENA |
REPOSITORIES: ENA
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Project description:The pre-clinical efficacy and safety data support the initiation of a phase I/II clinical trial for β-globin gene correction in patient-derived hematopoietic stem cells for the treatment of sickle cell disease.
2021-05-16 | GSE163912 | GEO
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.
2009-06-20 | E-GEOD-16728 | biostudies-arrayexpress
Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC
2009-06-20 | GSE16728 | GEO
Project description:We performed RNA-seq analysis on immortalized human erythroid cell line (HUDEP-2), that had been engineered to carry the sickle cell disease (SCD) mutation (sHUDEP-2), and on healthy donor mobilized CD34+ derived cells after transduction with a control GFP lentivirus and lentivirus expressing βT87Q-globin. Our results show activation of inflammation- and proliferation-related programs, suggesting minimal changes of background gene expression except for βT87Q-globin expression and endogenous β/βs-globin suppression.
2020-04-13 | GSE148529 | GEO
Project description:Human fetal γ-globin gene is developmentally silenced around the birth, and reactivation of γ-globin gene in adulthood sheds new light on ameliorating symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemias. However, the precise regulation process of γ-globin remains incompletely understood. Here, we found that a new protein directly interacted with SOX6 and exerted a significant repression effect on the expression of γ-globin gene in erythroid cells. Further studies have demonstrated that it bound directly to γ-globin gene promoter via octamer binding motif, which in turn suppressed the transcriptional activity of γ-globin gene promoter. Thus, these data indicate that this new protein acts as a novel transcriptional repressor in the regulation of γ-globin gene expression through direct promoter binding, implying a potential alternative therapeutic target for the treatment of SCD and β-thalassemias.
2022-02-16 | PXD024259 | Pride
Project description:Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle Cell Disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of ZFNs and CRISPR/Cas9 for editing and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODN), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLV) and adenovirus 5/35 serotype (Ad5/35) to transvert the base-pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base-pair transversions were significantly reduced when analyzing cells in vivo in immune-deficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODN. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high efficiency gene correction in HSPCs
2019-05-18 | GSE131387 | GEO
Project description:The benign condition Hereditary Persistence of Foetal Haemoglobin (HPFH) is known to ameliorate symptoms when co-inherited with beta-haemoglobinopathies, such as sickle cell disease and beta-thalassaemia. The condition is sometimes associated with point mutations in the foetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the beta-globin locus that all reside downstream of the foetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the foetal globin genes, including the deletion of silencer elements, of genes encoding non-coding RNA, and bringing downstream enhancer elements into proximity with the foetal globin gene promoters. Here we systematically analyse the deletions associated with both HPFH and a related condition known as beta-thalassaemia and propose a unifying mechanism. In all cases where foetal globin is up-regulated, the proximal adult beta-globin (HBB) promoter is deleted. We use CRISPR gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce promoter activity, result in elevated foetal globin expression. These results fit with previous models where the foetal and adult globin genes compete for the distal Locus Control Region and suggest that targeting the promoter might be explored to elevate foetal globin and reduce sickle globin expression as a treatment for beta-haemoglobinopathies.
2022-01-20 | GSE194064 | GEO
Project description:<p> <ol> <li>Implement an efficient, highly reproducible and 'scalable' system for the production of large numbers of sickle cell anemia-specific iPS cells (iPSC).</li> <li>Derive and characterize a novel, in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of 'clinical grade' transgene-free iPS cells; use this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes.</li> <li>Identify developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS.</li> <li>Determine the effects of the three known HbF major quantitative trait loci (QTL) on globin gene expression in disease-specific iPS cells during in vitro erythropoiesis.</li> <li>Search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment, by examining gene expression profiles and mRNA sequence of their iPSC-derived erythroid cells.</li> <li>Develop and use a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers, explore globin switching, and correct the HbS mutation in sickle iPSC lines.</li> </ol> </p>
| phs001212 | dbGaP
Project description:NextGen Consortium: Globin Gene Expression in Sickle Cell Genotype-Specific iPS cells
| PRJNA353151 | ENA
Project description:B-hemoglobinopathies such as Sickle Cell Disease (SCD) and b-thalassemia result from mutations in the adult b-globin gene. Reactivating the developmentally silenced fetal g-globin gene is a therapeutic goal for treating SCD and b-thalassemia1. Some forms of Hereditary Persistence of Fetal Hemoglobin (HPFH), a rare benign condition in which individuals express g‑globin throughout adulthood, are caused by point mutations in the g‑globin gene promoter at regions residing ~115 and 200 base pairs upstream of the transcription start site. Here we show that the major fetal globin repressors BCL11A and ZBTB7A/LRF directly bind to the -115 and ‑200 sites, respectively. Furthermore, introduction of naturally occurring HPFH mutations into erythroid cells by CRISPR/Cas9 disrupts repressor binding and raises g‑globin expression. These findings resolve the mystery surrounding how these HPFH mutations operate and demonstrate that BCL11A and ZBTB7A/LRF are major direct repressors of the fetal globin gene.
2018-01-29 | GSE103445 | GEO