New Gene Editing Tool for Viral Vectors

The SHOT 2.0 system leverages a baculovirus, a type of insect virus, to engineer bacmids within *E. coli*. This edited bacmid is then used to produce recombinant baculoviruses in insect cells, which can efficiently transduce a wide variety of mammalian cell types, including hard-to-transfect iPSCs and liver cancer cells. This approach expands the baculovirus engineering toolbox, providing a more flexible platform for gene delivery. A key advantage of SHOT 2.0 is its ability to integrate large DNA cargos of at least 14 kb. This capacity is a significant leap over older recombineering methods, whose efficiency drops sharply with DNA fragments larger than 3-4 kb. This enables the delivery of larger, more complex genetic payloads, a critical need for developing therapies for diseases caused by mutations in large genes. The system demonstrates high efficiency, achieving up to 85.6% in HEK293T cells and, notably, up to 37.1% in iPSCs. This level of efficiency in iPSCs is particularly relevant for developing cell-based therapies and creating more accurate disease models, a process that has been historically challenging. The technology builds on CRISPR-associated transposon (CAST) systems, which allow for RNA-guided DNA insertion without inducing the double-strand breaks that can lead to errors from cellular repair pathways. From a biomanufacturing perspective, SHOT 2.0's design offers enhanced stability. By integrating transgenes at specific loci like ODVe56, it markedly improves transgene stability during the serial passaging of the virus. This addresses a common bottleneck in viral vector production, where transgene loss can limit scalability and lead to batch-to-batch inconsistency, a major challenge for CDMOs aiming for standardized, GMP-compliant processes. This advancement in programmable viral vector engineering directly impacts the process development and manufacturing workflows within a CDMO. The compatibility of SHOT 2.0 with the established Bac-to-Bac® workflow allows for dual-gene insertion, streamlining the production of more complex vectors. This improved efficiency and flexibility can accelerate timelines for creating stable producer cell lines, reducing reliance on transient transfection methods and their inherent variability. The development of more capable viral vectors directly influences the competitive landscape for gene therapy CDMOs. Platforms that can efficiently produce stable, high-titer vectors for complex payloads provide a distinct advantage. Such technologies are critical for enabling the next generation of therapies and can help partners move from early-stage development to commercial-scale manufacturing more predictably.