A Modular Multilocus Engineering Platform for Iterative Prototyping of Oncolytic Adenoviruses

The development of oncolytic adenovirus type 5 (Ad5) has been constrained by inefficient genome-engineering workflows and a lack of reusable backbones for iterative vector optimization. To address these limitations, we established a modular and reusable engineering workflow for constructing tumor-selective oncolytic Ad5 genomes by integrating BJ5183-mediated homologous recombination with in vitro homology-based DNA assembly. First, the BJ5183-based system enables targeted modification of multiple loci within adenoviral genomes, including the installation of locus-compatible restriction sites such as BstBI, ClaI, and SwaI. Second, an in vitro homology-based DNA assembly module enables insertion of target gene expression cassettes. Third, PacI-linearized genomes are rescued in HEK-293A cells. The resulting backbone retained viral rescue competence and replication selectivity. This platform supports engineering of therapeutic and structural loci within a retained full-length plasmid backbone while allowing the final recombinant plasmid to be sequence-verified before virus rescue. Thus, it provides a practical framework for iterative and parallelized prototyping of armed, capsid-modified, and combinatorial oncolytic Ad5 vectors.