MVV vector integration in sheep cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA803152 | ENA |
REPOSITORIES: ENA
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Project description:To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected sheep CPT3, LKO1 (PSIP1-null), LHKO1 and LHKO2 (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to sheep genome.
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Project description:MVV vector integration in human cells
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Project description:To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected human HEK293T, LKO (PSIP1-null), and LHKO (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to human genome.
2022-02-06 | GSE196040 | GEO
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.
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Project description:Transcriptomic profiling of sheep skin lymph DC subsets after 16h in vitro culture in medium or stimulated by non replicative Canine Adenovirus serotype 2 Reference design: total enriched lymph dendritic cells- Biological replicates: 4 sheep (70958, 50274, 30066, 11261)- Samples: FACS sorted CD103 -type and CD11b type cultured in medium or with CAV2
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Project description:Transcriptomic profiling of sheep skin lymph DC subsets after 16h in vitro culture in medium or stimulated by non replicative Canine Adenovirus serotype 2
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Project description:Transcriptomic profiling of ovine skin lymph dendritic cell subsets CD26+ and CD26- compared to total enriched lymph dendritic cells. CD26+ and CD26- dendritic cells (DC) from sheep skin lymph were sorted by flow cytometry and were analysed for their transcriptomic profile. We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8a+ and human BDCA3+ DC. This allowed the identification of a common set of phenotypic characteristics for CD8a-like DC in the 3 mammalian species, i.e. SIRPlo, CADM1hi, CLEC9Ahi, DEC205hi, XCR1hi. Compared to CD26- DC, the sheep CD26+ DC show (1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifn-gamma and Il22 genes; (2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble antigen; (3) selective expression of functional pathways associated to high capacity for antigen cross-presentation. Reference design (total enriched lymph dendritic cells) - Biological replicates: 2 sheep #10081 and #30066
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Project description:Transcriptomic profiling of ovine skin lymph dendritic cell subsets CD26+ and CD26- compared to total enriched lymph dendritic cells. CD26+ and CD26- dendritic cells (DC) from sheep skin lymph were sorted by flow cytometry and were analysed for their transcriptomic profile. We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8a+ and human BDCA3+ DC. This allowed the identification of a common set of phenotypic characteristics for CD8a-like DC in the 3 mammalian species, i.e. SIRPlo, CADM1hi, CLEC9Ahi, DEC205hi, XCR1hi. Compared to CD26- DC, the sheep CD26+ DC show (1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifn-gamma and Il22 genes; (2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble antigen; (3) selective expression of functional pathways associated to high capacity for antigen cross-presentation.
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Project description:Recent studies demonstrated that fibroblasts could be converted into induced neural stem cells (iNSCs). However, the insertional mutation caused by random integration of viral vectors has been a major limitation of iNSCs for the future clinical translation. Here we show that non-viral transfection of episomal vectors encoding Brn4/Pou3f4, Sox2, Klf4, and c-Myc sufficiently generates iNSCs. The episomal vector mediated iNSCs closely resemble brain-derived NSCs as well as iNSCs generated by retrovirus in morphology, gene expression profile, epigenetic status, self-renewal capacity and both in vitro and in vivo differentiation capacity. The novel conversion protocol defined in the current study offers a method for generating integration-free iNSCs for the clinical research we developed a novel method for generating integration-free iNSCs. We demonstrated that oriP/EBNA1-based episomal vectors could generate iNSCs by single transfection
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