Long-read RNA-Seq analysis of the full-length F8 transcript in iPSCs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE229621 | GEO | 2023/04/24
REPOSITORIES: GEO
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Project description:Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for hemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralize F8 activity. Taking advantage of split inteins-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV split-inteins vectors whose co-administration to HemA mice results in F8 protein reconstitution and expression of therapeutic levels of F8 over time without anti-F8 antibodies development.
2022-03-31 | PXD031884 | Pride
Project description:Coagulation Factor VIII (FVIII) plays a pivotal role within the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types. Our current investigation delves into small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes. The outcome of this exploration underscores the discernible microRNA expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver. Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset - miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p - whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above miRNA emerged as potential targets for innovative therapies in Hemophilia A patients.
2024-02-19 | GSE242926 | GEO
Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.
2015-02-04 | E-GEOD-65581 | biostudies-arrayexpress
Project description:The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 (∼14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 (∼36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3′ end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3′ splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3’ splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.
2024-01-17 | GSE230495 | GEO
Project description:In human F8 deficiency leads to hemophilia A and is largely synthesized and secreted by the sinusoidal endothelial cells of the liver (LSECs). However, the specificity and characteristics of these cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human LSECs together with other fetal endothelial cells, from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal plenty of expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differential regulated pathways and functions in fetal LSECs. We used targeted bisulfite re-sequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in house iPSs generated vascular endothelial cells to fetal and adult LSECs; a higher similarity was found to the fetal than to adult LSECs. Our molecular profiling study provides a guide to test the effectiveness of production of in vitro differentiated LSECs that could be used in cellular therapies. In-silico analysis to identify molecular signature of liver sinusoidal endothelial cells in comparison to other endohtelial cells
2020-10-25 | GSE140078 | GEO
Project description:In human F8 deficiency leads to hemophilia A and is largely synthesized and secreted by the sinusoidal endothelial cells of the liver (LSECs). However, the specificity and characteristics of these cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human LSECs together with other fetal endothelial cells, from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal plenty of expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differential regulated pathways and functions in fetal LSECs. We used targeted bisulfite re-sequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in house iPSs generated vascular endothelial cells to fetal and adult LSECs; a higher similarity was found to the fetal than to adult LSECs. Our molecular profiling study provides a guide to test the effectiveness of production of in vitro differentiated LSECs that could be used in cellular therapies. In-silico analysis to identify molecular signature of liver sinusoidal endothelial cells in comparison to other endohtelial cells
2020-10-25 | GSE139993 | GEO
Project description:In human F8 deficiency leads to hemophilia A and is largely synthesized and secreted by the sinusoidal endothelial cells of the liver (LSECs). However, the specificity and characteristics of these cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human LSECs together with other fetal endothelial cells, from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal plenty of expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differential regulated pathways and functions in fetal LSECs. We used targeted bisulfite re-sequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in house iPSs generated vascular endothelial cells to fetal and adult LSECs; a higher similarity was found to the fetal than to adult LSECs. Our molecular profiling study provides a guide to test the effectiveness of production of in vitro differentiated LSECs that could be used in cellular therapies. In-silico analysis to identify molecular signature of liver sinusoidal endothelial cells in comparison to other endohtelial cells
2020-10-25 | GSE140295 | GEO
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