Manufacturing of LVV and AAV can be hindered by expression of certain therapeutic genes, and the byproducts of production cell death can increase downstream process burden. Methods of silencing the cargo gene are needed to reliably produce certain hard-to-manufacture viral vectors carrying cytotoxic genetic cargos with high therapeutic potential.
For LVV, we developed a shRNA-based cargo gene silencing system, achieving up to 95% gene silencing during manufacturing and up to a four-fold increase in LVV production titre, demonstrated in XOFLX® packaging cell line. The silencing system also enabled otherwise unrecoverable stable producer cell lines to recover as a more scalable and cost-effective LVV manufacturing platform.
AAV presents specific challenges for traditional shRNA-mediated cargo-silencing strategies, in particular issues around PKR activation and translational shutdown. Our novel silencing technology has shown near-complete silencing of reporters and up to a 16-fold improvement in yields using known toxic cargo genes.
