Universal strategy to generate lentiviral vectors encoding lethal proteins

Lentiviral vectors have emerged as a versatile and robust platform for gene therapy. However, their use for delivering toxic genes has been hindered by challenges related to packaging lentiviral genomes that encode inherently lethal transgenes. In this study, we introduce a novel multi-layered approach to overcome this obstacle. Our results show that combining a regulatable promoter, an inverted orientation of the lethal transgene expression cassette, and CRISPR-based inhibition to repress vector transgene transcription effectively reduces toxicity in producer cells, enabling the efficient production of high-titer lentiviral vectors expressing lethal proteins. Notably, these lentiviral vectors were able to deliver transgenes encoding pro-apoptotic proteins or toxins that induced cell death in cancer cells, providing a translational proof of concept for therapeutic payload delivery. Overall, this work establishes an innovative framework that expands the utility of the lentiviral platform for the controlled delivery of cytotoxic factors in gene therapy applications.