BAC Mutagenesis-Driven Development of Oncolytic HSV for Preclinical Mouse Studies

The limited efficacy of cancer treatment has driven the development of novel therapeutic strategies, including oncolytic virotherapy. Herpes simplex virus type 1 (HSV-1) has emerged as a promising oncolytic platform due to its large genome, tumor-selective replication, and capacity to accommodate therapeutic transgenes. This study aimed to generate and validate oncolytic HSV-1 expressing the immunostimulatory transgenes interleukin-12 (IL-12) and class II transactivator (CIITA) using bacterial artificial chromosome (BAC) technology. Following the creation of the CIITA-ICP4 cassette, HSV-1 BAC mutagenesis was performed through λ Red-mediated homologous recombination in Escherichia coli, allowing for the incorporation of the cassette into the viral genome. These BAC constructs were verified by polymerase chain reaction (PCR) and agarose gel electrophoresis which then followed a BAC resolution process, allowing for the removal of a bacterial resistance gene, kanamycin. Successful excision of the kanamycin resistance cassette was confirmed by PCR analysis and the reconstituted virus was used to infect recombinant virus infection of CT2A murine glioma cells. Infection was evaluated by assessing the expression of using an HSV-1 BAC expressing the fluorescent reporter gene mCherry, that is present within the parental HSV-1 BAC.. These findings demonstrate that HSV-1 BAC technology provides an efficient platform for constructing recombinant oncolytic viruses and supports its application in the development of novel immunotherapeutic strategies for cancer.