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

## TL;DR

Researchers found new spots in the vaccinia virus genome to insert multiple genes safely, which could improve its use in vaccines and cancer treatments.

## Contribution

The study identifies and validates two novel intergenic sites in the vaccinia virus genome suitable for transgene insertion.

## Key findings

- The D10R-D11L and E8R-E9L intergenic sites support stable and robust transgene expression.
- Modified viruses retain replication ability and cancer cell-killing properties.
- The identified sites do not disrupt adjacent viral gene expression.

## Abstract

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.

Bueno-Merino and colleagues identified and validated novel intergenic regions in the vaccinia virus genome as insertion sites for multitransgene vector development, demonstrating that two of the three tested candidates support stable transgene expression without compromising viral function.