Abstract
BackgroundGenetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.Methodology/Principal FindingsHere we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.Conclusion/SignificanceThe system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.
Highlights
Poxviruses comprise a large family of double-stranded DNA viruses that infect a wide range of hosts
Our results suggest that the Selectable and Excisable Marker (SEM) vector development system will be useful for the creation of novel poxvirus therapeutics, and for basic virological studies
The cellular distribution of Cre from Cre recombinaseexpressing cell lines was analyzed by immunofluorescence from parental U2OS cells, nuclear Cre cells (Nuc-Cre), and cytoplasmic Cre (Cyto-Cre) cells illustrating that the absence of the nuclear localization sequence in the Nuc-Cre cells leads to accumulation of the enzyme in the cytoplasm (Fig. 1C)
Summary
Poxviruses comprise a large family of double-stranded DNA viruses that infect a wide range of hosts. The poxvirus genome is genetically modified and can accommodate inserts exceeding 25 kb [4] using strategies that are dependent upon virusencoded homologous recombination [5,6] Using these approaches, recombinant VV has since proven to be valuable as a vector for gene therapy in a number of therapeutic applications [4,7,8,9,10,11,12,13,14,15]. Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The use of marker genes inevitably results in a vector that contains unwanted genetic information of no therapeutic value
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