Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1074750 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27916000.fastq.gz | Fastqsanger.gz | |||
| SRR27916001.fastq.gz | Fastqsanger.gz | |||
| SRR27916002.fastq.gz | Fastqsanger.gz | |||
| SRR27916003.fastq.gz | Fastqsanger.gz | |||
| SRR27916004.fastq.gz | Fastqsanger.gz |
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Project description:Recombinant adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes can integrate into the host genome, but the theoretical risk of tumorigenesis may depend on vector regulatory features. A mouse model was used to investigate long-term kinetics and integration profiles of an AAV serotype 5 (AAV5) vector that mimics key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads identified by target enrichment sequencing were derived from episomes, and mean integration frequency was 2.70 (standard deviation, 1.24) integrations per 1000 cells. Longitudinal integration analysis suggested AAV5 vector integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal, and only 5.46% of integrations had a common integration site order ≥5, suggesting random distributions when looking at a 50-kb genomic window. No integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver (P = 1x10−4) and less enriched for genes with low or no liver expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.
2024-02-01 | GSE254503 | 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 development of neutralizing FVIII antibodies is the most serious complication of hemophilia A treatment. The currently known patient- and treatment-related risk factors for inhibitor development do not accurately predict this adverse event in all patients. The composition of the gut microbiota has been shown to influence immune mediated diseases at distant anatomical sites (e.g. lungs, brain and joints). We demonstrate that a disrupted gut microbiome can be created in a mouse model of hemophilia A using a broad-spectrum antibiotic. Under controlled conditions, this sustained dysbiosis was associated with an enhanced anti-FVIII immune response as evidenced by increased splenic B cells and the development of higher titre FVIII specific IgG antibodies following FVIII challenge. Splenic and mesenteric lymph node cytokines, T cells and dendritic cells were unaffected prior to FVIII administration. However, the immune transcriptome of both aforementioned secondary lymphoid organs was significantly modified. Short chain fatty acids (SCFA), which are microbial metabolites, were depleted in cecal contents of the dysbiotic mice. Furthermore, supplementation of the drinking water with the most immunologically active SCFA, butyrate, successfully achieved attenuation of the FVIII immune response. Collectively, our data suggest that the composition of the gut microbiome alters the FVIII immune response via the action of specific microbial metabolites on the immune cell transcriptome and that oral butyrate supplementation effectively reduces the FVIII immune response. Mice treated with one week of ampicillin at 3 weeks of age have a dysbiotic gut microbiome at 6 weeks of age and at the study endpoint. The immune response to FVII was compared between the two cohorts. The dysbiotic cohort had a greater immune response to FVIII
2019-10-04 | GSE138330 | GEO
Project description:The development of neutralizing FVIII antibodies is the most serious complication of hemophilia A treatment. The currently known patient- and treatment-related risk factors for inhibitor development do not accurately predict this adverse event in all patients. The composition of the gut microbiota has been shown to influence immune mediated diseases at distant anatomical sites (e.g. lungs, brain and joints). We demonstrate that a disrupted gut microbiome can be created in a mouse model of hemophilia A using a broad-spectrum antibiotic. Under controlled conditions, this sustained dysbiosis was associated with an enhanced anti-FVIII immune response as evidenced by increased splenic B cells and the development of higher titre FVIII specific IgG antibodies following FVIII challenge. Splenic and mesenteric lymph node cytokines, T cells and dendritic cells were unaffected prior to FVIII administration. However, the immune transcriptome of both aforementioned secondary lymphoid organs was significantly modified. Short chain fatty acids (SCFA), which are microbial metabolites, were depleted in cecal contents of the dysbiotic mice. Furthermore, supplementation of the drinking water with the most immunologically active SCFA, butyrate, successfully achieved attenuation of the FVIII immune response. Collectively, our data suggest that the composition of the gut microbiome alters the FVIII immune response via the action of specific microbial metabolites on the immune cell transcriptome and that oral butyrate supplementation effectively reduces the FVIII immune response. Mice treated with one week of ampicillin at 3 weeks of age have a dysbiotic gut microbiome throughout the study and an enhanced immune response to FVIII which was administered at 6 weeks of age. Here we assess the immune transcriptome in the mice spleen and Mesenteric lymph nodes at 6 weeks of age, just prior to exposure to FVIII challenge.
2019-10-04 | GSE138331 | GEO
Project description:The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) remains the most serious complication in the treatment of hemophilia A. While immune tolerance induction (ITI) is the standard strategy to eliminate these antibodies, it fails in approximately 30% of patients with severe hemophilia A, underscoring the need for innovative approaches to promote FVIII-specific tolerance. To address this challenge, we generated fusion proteins composed of A2, A3-C1-C2 (light chain, LCh), and C2 domains of FVIII linked to Annexin A5 (AnxA5), a protein that binds phosphatidylserine (PS), a hallmark of apoptotic cells. ELISA confirmed high-affinity binding of all fusion proteins to immobilized PS. To model PS exposure in vitro, red blood cells (RBCs) were treated with phorbol 12-myristate 13-acetate (PMA), leading to the release of PS-exposing microvesicles. Flow cytometry showed that FVIII-AnxA5 fusion proteins selectively bound to PS-exposing microvesicles but not to intact RBCs. Using mass spectrometry-based immunopeptidomics, we demonstrated that macrophages pulsed with FVIII-AnxA5 fusion proteins efficiently processed and presented FVIII-derived peptides on HLA-DR molecules. These findings suggest that FVIII-AnxA5 fusion proteins can engage apoptotic cell clearance pathways to facilitate antigen presentation in a potentially tolerogenic context. This strategy may offer a novel means of inducing immune tolerance to FVIII in hemophilia A.
2025-10-20 | PXD068424 | Pride
Project description:Background: The main complication in hemophilia A treatment is the development of inhibitory antibodies against factor (F)VIII. Immune tolerance induction, the gold standard for eradicating anti-FVIII antibodies, is efficient in only 60% to 80% of cases. This underscores the need for more efficient induction of tolerance in patients with hemophilia A with FVIII inhibitors. Objectives: In this study, we explored whether red blood cells (RBCs) can be utilized as antigen delivery system to modulate the immune response against FVIII. Methods: Two promiscuously HLA-DR–presented peptides derived from the A2 and C1 domains of FVIII were fused to the TAT cell-penetrating peptide and incubated with RBCs. Results: Biotinylated TAT-A2 and TAT-C1 peptides were found to interact with RBCs as shown by flow cytometry and imaging flow cytometry. Moreover, macrophages efficiently phagocytosed TAT-FVIII peptide–treated RBCs. Using mass spectrometry– based immunopeptidomics we established that TAT-FVIII peptides were presented on major histocompatibility complex class II of macrophages that phagocytosed TAT peptide–pulsed RBCs. Specifically, the TAT-A2 peptide exhibited efficient processing and presentation on HLA-DR molecules. Importantly, incubation of TAT-C1 peptide– treated RBCs-loaded macrophages with a FVIII-specific T-cell hybridoma led to a significant increase in IL-2 production, suggesting functional presentation of TAT-C1– derived peptides by macrophages. Conclusion: Our findings indicate that RBCs can serve as effective vehicle for the delivery of FVIII-derived peptides to antigen-presenting cells. The successful display of T-cell epitopes on antigen-presenting cell using ex vivo–loaded RBC may be potentially utilized to modulate pathogenic immune responses such as observed in a subset of patients with hemophilia A.
2025-10-02 | PXD069058 | Pride
Project description:Immune responses in hemophilia A patients to replacement factor VIII can be either tolerogenic or immunogenic, the latter resulting in induction of non-neutralizing anti-factor VIII antibodies or neutralizing antibodies called inhibitors. Since these inhibitors render replacement FVIII treatment essentially ineffective, preventing or eliminating them are of top priority in disease management. The extended half-life recombinant factor VIII Fc fusion protein (rFVIIIFc) is an approved therapy for hemophilia A patients. In addition, it has been reported that rFVIIIFc can induce tolerance to FVIII in hemophilia A patients that have developed inhibitors. Given that the IgG1 Fc region has the potential to interact with immune cells expressing Fc receptors and thereby affect the immune response to rFVIII, we investigated how human macrophages, expressing both Fc receptors and receptors reported to bind FVIII, respond to rFVIIIFc. We show herein that rFVIIIFc, but not rFVIII, uniquely skews macrophages towards an alternatively activated regulatory phenotype. rFVIIIFc initiates signaling events that result in morphological changes, as well as a specific gene expression and metabolic profile that is characteristic of the regulatory type Mox/M2-like macrophages. Further, these changes are dependent on rFVIIIFc-Fc receptor interactions. Our findings elucidate mechanisms of potential immunomodulatory properties of rFVIIIFc.
2018-11-20 | GSE118157 | GEO
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:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
2015-01-01 | E-GEOD-57597 | biostudies-arrayexpress