Journal
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
Volume 2, Issue -, Pages -Publisher
CELL PRESS
DOI: 10.1038/mtm.2015.35
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Funding
- UK charity Pancreatic Cancer Research Fund
- Ministry of Sciences and Technology, China [2013DFG32080]
- QMUL Innovation Ltd.
- MRC [MR/M015696/1] Funding Source: UKRI
- Cancer Research UK [12008] Funding Source: researchfish
- Medical Research Council [MR/M015696/1] Funding Source: researchfish
- Pancreatic Cancer UK [2010 Grant - Wang] Funding Source: researchfish
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The current method for creation of vaccinia virus (VACV) vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK) region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (similar to 90%) in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP) could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP) that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.
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