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MVV vector integration

ABSTRACT: MVV vector integration

PROVIDER: PRJNA803148 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR17871201.fastq.gz	Fastqsanger.gz
	SRR17871202.fastq.gz	Fastqsanger.gz
	SRR17871203.fastq.gz	Fastqsanger.gz
	SRR17871204.fastq.gz	Fastqsanger.gz
	SRR17871205.fastq.gz	Fastqsanger.gz

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The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice

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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MVV vector integration in human cells

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.

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Project description:The whole coding RNA of Actinoplanes sp. SE50/110 mutants containing two different integrative vectors (pSET152::acbB and pSETT4::acbB) were sequenced. Both vectors are integrated via a phiC31 integrase (Bierman et al. 1992) into the genetic locus ACSP50_6589 (former: acpl_6602) (Gren et al. 2016). The novel expression vector pSETT4 is excelled by an easy cloning mechanism allowing the integration of different promoters. By this, the system can be quickly adapted to further species of the order Actinomycetales. Additionally, T4-terminators were introduced before and after the expression cassette, since they are able to block the transcription efficiently and prevent antisense formation and read-through from the integrase gene into the gene of interest.

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