Recombineering-based Manufacturing of Engineered Viral Vectors for Research and Therapy.

The increasing demand for engineered viral vectors in both basic and translational research has underscored the need for flexible, rapid, and scalable methods to generate recombinant DNA viruses. Strategies relying on restriction enzyme digestion and ligation are constrained by sequence-dependent limitations and time-consuming cloning steps. Here, we describe the recombination-mediated genetic engineering method (recombineering) that circumvents these limitations by enabling precise and seamless modifications of viral genomes in bacterial artificial chromosomes (BACs). This approach allows for efficient deletion, insertion, or substitution of coding or non-coding genetic elements, providing a powerful platform for high-throughput viral vector development. Recombineering is particularly valuable in basic research applications, such as the deletion or mutation of viral genes to investigate their function. More importantly, this methodology enables the generation of tens of recombinant viruses encoding distinct immunostimulatory or therapeutic payloads in parallel, making it exceptionally well-suited for the rapid preclinical evaluation of novel constructs. While this technology can be potentially implemented for any scientific purpose, this article focuses on the application of recombineering in two specific areas: the generation of oncolytic viruses based on herpes simplex virus, and the development of non-replicative adenoviral vectors for gene transfer. In conclusion, recombineering offers a versatile approach to viral genome engineering, significantly accelerating the pipeline from design to functional testing. Its relevance spans from fundamental virology to translational medicine to meet evolving research and clinical needs.