Optimization of oncolytic viruses for therapeutic applications requires the strategic removal or mutagenesis of virulence genes alongside the insertion of transgenes that enhance viral replication, spread and immunogenicity. However, the complexity of many viral genomes and the labour-intensive nature of methods for the generation and isolation of recombinant viruses have hindered the development of therapeutic oncolytic viruses. Here we report an iterative strategy that exploits the preferential susceptibility of viruses to certain antibiotics to accelerate the engineering of the genomes of oncolytic viruses for the insertion of immunomodulatory cytokine transgenes, and the identification of dispensable genes with regard to replication of the recombinant oncolytic viruses in tumour cells. We applied the strategy by leveraging insertional mutagenesis via the Sleeping Beauty transposon system, combined with long-read nanopore sequencing, to generate libraries of herpes simplex virus type 1 and vaccinia virus, identifying stable transgene insertion sites and gene deletions that enhance the safety and efficacy of the viruses.

优化溶瘤病毒的治疗应用需要战略性地去除或诱变毒力基因，同时插入增强病毒复制、传播和免疫原性的转基因。然而，许多病毒基因组的复杂性以及生成和分离重组病毒的方法的劳动密集型性质阻碍了治疗性溶瘤病毒的发展。在这里，我们报告了一种迭代策略，该策略利用病毒对某些抗生素的优先易感性来加速溶瘤病毒基因组的工程设计，以插入免疫调节细胞因子转基因，并鉴定与重组溶瘤病毒在肿瘤细胞中复制有关的可有可无的基因。我们应用该策略，利用睡美人转座子系统的插入诱变，结合长读长纳米孔测序，生成 1 型单纯疱疹病毒和牛痘病毒文库，鉴定稳定的转基因插入位点和基因缺失，从而提高病毒的安全性和有效性。