DNA methylation response of hematopoietic stem cells to recombinant lentiviral vector infection
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE72867 | GEO | 2015/09/11
SECONDARY ACCESSION(S): PRJNA295266
REPOSITORIES: GEO
Similar Datasets
Project description:Human cord blood CD34+ cells respond to the infection with LV by modifying the methylation profile of a large number of CpG sites in their genome. Different vector preparations vary in their capacity to trigger a response; some induce sporadic modifications only while others trigger a strong response that systematically includes de novo methylation of the same 4126 CpG-s corresponding to 1059 genes Illumina Infinium 450K arrays were performed according to the manufacturer's directions on bisulfite converted gDNA extracted from CD34+ cells from cord blood. CpG methylation profiling on Illumina Infinium 450K arrays was performed.
2015-09-11 | E-GEOD-72867 | biostudies-arrayexpress
Project description:Human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.
2012-09-05 | GSE40631 | GEO
Project description:Human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols. 3 replicates of control unstimulated (Group 1), cytokine stimulated, cytokine strimulated and treated with polybren and cytokine stimulated and transduced with lentiviral vector coding for GFP in the presence of polybren were analysed
2012-09-05 | E-GEOD-40631 | biostudies-arrayexpress
Project description:Expression data from different expressed genes (DEGs) in human esophageal squamous cell carcinoma (ESCC) EC109 cells transfected with empty lentiviral vector (Lv-NC) or lentiviral vector carrying carrying KDM5C specific shRNA (Lv-shKDM5C) Lysine demethylase 5C (KDM5C), a member of lncRNAs, has tumor-suppressor properties and is downregulated in the majority of malignancies. KDM5C was found to suppress some sorts of tumors through regulating epithelial mesenchymal transformation,inducing cell cycle arrest,triggering apoptosis and so on. We used microarrays to investigate the possible regulatory mechanism of KDM5C in ESCC cell line.
2021-10-06 | GSE159102 | GEO
Project description:In the context of lentiviral vector (LV)-mediated gene correction of hematopoietic stem and progenitor cells (HSPC), the viral origin of LV could trigger cellular responses with potential functional consequences that have not been investigated to date. Here, a time-course genome-wide transcriptional profiling of transduced human HSPC reveals a remarkably limited impact of LV on cellular gene expression, supporting some stealth features of the vector. In particular, LV efficiently escaped innate immune sensing that instead led to robust type I IFN signaling upon transduction with a gamma-retroviral vector. However, recognition of LV DNA prior to integration did trigger DNA damage responses that were activated also by non-integrating Adeno-associated vector DNA. As a consequence of this transient p53 activation, transduced HSPC showed increased apoptosis in vitro and reduced engraftment in vivo at limiting cell doses, but no long-term impact or competitive disadvantage were detected. Pharmacological inhibition of the LV signaling partially rescued both apoptosis and engraftment, potentially providing a novel strategy to further dampen the impact of ex vivo gene therapy on HSPC. Overall, our results shed light on the molecular mechanisms of viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.
2017-06-28 | GSE92652 | GEO
Project description:Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e. the conventional VSV-LV and the CD8-targeted CD8-LV. LV exposed human donor PBMC were evaluated for a panel of 400 immune-response related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A demanding for a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells resulting in a target cell selectivity of above 99%. Beyond that, the differential gene expression analysis revealed the upregulation of restriction factors in CAR-negative cells thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-Seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation.
2021-10-01 | GSE184895 | GEO
Project description:A major challenge in Down syndrome (DS) is to understand how the extra-dose of functional chromosome 21 (HSA21) genetic elements can impact on the tissue-specific transcriptome to contribute to phenotypic alterations. MiRNAs are post-transcriptional modulators with genome-wide regulatory effects. Five microRNAs have been identified in HSA21 that are present in triple copy in DS individuals. Interestingly, in the Ts65Dn mouse model of DS two of these miRNAs, miR-155 and miR-802, are also triplicated resulting in its overexpression. In the current work, we have developed a lentiviral miRNA-sponge genetic strategy for miR-155 and miR-802 (Lv-miR155-802T) to identify novel mRNA targets involved in hippocampal function. Hippocampal injection of the lentiviral sponge in Ts65Dn mice reduced miR-155 and miR-802 overexpression. Noticeable lentiviral sponge rescued the expression of the miRNA predicted targets showing the potential of the strategy to identify miRNA dosage-sensitive genes with potential involvement in DS-hippocampal phenotypes. Euploid and trisomic adult mice were bilaterally injected at the level of the ventral hippocampus at selected coordinates (AP=-3.3mm, L=+/- 3mm, DV=-3.3mm and -2.3mm relative to bregma). Up to 108 transducing units (3µl of viral suspensions of Lv-Contol or Lv-miR155-802T) were injected into each hemisphere at a rate of 0.2 µl/min, under the precise control of an infusion pump (Ultramicropump, World Precision Instruments). Mice were euthanized for hippocampus collection at day 23 after administration. Transcriptome of hippocampus of euploid and trisomic mice treated with Lv-Control or Lv-miR155-802T was analysed using an Agilent SurePrint G3 Mouse gene expression 8x60K Microarray (ID 028005). A total RNA 100 ng, obtained using miRNeasy Mini Ki (QIAGEN), were labeled using LowInputQuick Amp Labeling kit (Agilent 5190-2305) following manufacturer instructions. Briefly, mRNA was reverse transcribed in the presence of T7-oligo-dT primer to produce cDNA. cDNA was then in vitro transcribed with T7 RNA polymerase in the presence of Cy3-CTP to produce labeled cRNA. The labeled cRNA was hybridized to the Agilent SurePrint G3 Mouse gene expression 8x60K Microarray (ID 028005) according to the manufacturer's protocol. The arrays were washed, and scanned on an Agilent G2565CA microarray scanner at 100% PMT and 3µm resolution. Intensity data was extracted using the Feature Extraction software (Agilent). Replicates from each genotypes and treatment group were distributed as follows: EU+Lv-Contol n=4, EU+Lv-miR155-802T n=4, TS+Lv-Contol n=5, TS+Lv-miR155-802T n=3.
2015-05-16 | E-GEOD-68074 | biostudies-arrayexpress
Project description:Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.
| S-EPMC3034847 | biostudies-other
Project description:Lentiviral vectors (LV) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPC), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete towards the 3’ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPC. By combining three modifications to vector design and production (shortening the vector length to 5.3-kb; expressing TAT protein during packaging; and packaging in PKR-/- cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPC. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.
2020-09-21 | GSE158252 | GEO
Project description:Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 out of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear sub-compartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures, identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may impact the efficacy of LV-based cellular therapies.
2023-10-06 | GSE163082 | GEO