Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.
PLoS One. 2011;6(9):e24643. doi: 10.1371/journal.pone.0024643. Epub 2011 Sep 8.
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 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 FINDINGS: Here 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/SIGNIFICANCE: The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications.
通过衰减或插入治疗基因对痘病毒基因组进行遗传操作，已经产生了许多用于治疗各种人类疾病的载体候选物。重组痘病毒的开发通常涉及选择标记的基因组插入，以用于纯化和选择目的。然而，标记基因的使用不可避免地导致载体中含有无治疗价值的不需要的遗传信息。
方法/主要发现：在这里，我们描述了一种改进的策略，可用于创建任何物种的无标记重组痘病毒。可选择和可切除标记（SEM）系统包含一个独特的融合标记基因，用于有效选择痘病毒重组体，以及 Cre/loxP 系统，以方便随后去除标记。我们通过将外源基因插入痘苗病毒来定义和表征这个新的方法工具，随后去除选择标记。然后，我们分析了 Cre 重组过程中loxP 取向的重要性，并表明 SEM 系统可用于在病毒基因组中引入特异性缺失或反转。最后，我们证明 SEM 策略适用于其他痘病毒，如在这里创建的缺乏 EVM002 基因的疱疹病毒重组体所示。
结论/意义：因此，这里描述的系统为创建用于治疗基因治疗应用的临床就绪的重组痘病毒提供了更快、更简单和更有效的方法。
