Identification and evaluation of novel intergenic sites in the vaccinia virus genome for transgene insertion

Vaccinia virus (VACV) is widely used as a vaccine and oncolytic vector due to its safety profile and its capacity to accommodate exogenous DNA. However, its compact genome offers limited loci for transgene insertion without disrupting functional viral sequences. To develop VACV vectors capable of delivering multiple therapeutic genes, additional insertion sites that support stable transgene expression are needed. In this study, we validated the suitability of two new intergenic sites for this purpose. Using in silico analysis, we identified candidate sites based on genomic features and the conservation of flanking genes across VACV strains. Using a reporter transgene expression cassette, we assessed the potential of these newly identified sites to support transgene expression. Our results demonstrated that the D10R-D11L and E8R-E9L intergenic sites enable stable and robust transgene expression without diminishing the expression of adjacent viral genes. The modified viruses retained their ability to replicate and kill cancer cells and exhibited similar pathogenicity in mouse models compared to control viruses. These findings identify the D10R-D11L and E8R-E9L intergenic regions as promising insertion sites for developing VACV-based clinical candidates carrying multiple therapeutic transgenes.