Love, K. S.; Lende-Dorn, B. A.; Galloway, K. E.

Lentiviral vectors enable efficient delivery of genetic cargoes for gene and cell therapies. With their ~10-kb packaging limit, lentiviral vectors can encode multiple transcription units, supporting delivery of compact gene circuits. RNA-based devices offer highly compact control including ligand-responsive induction and closed-loop regulation. However, RNA devices such as ribozymes and splicing switches may interfere with vector production via activity on the single-stranded RNA genome. Here, we examine the impact of gene syntax and genetic parts to define design strategies for two-gene vectors encoding RNA devices. We find that titer decreases with genetic parts that interfere with transcription or processing of the viral transcript during production. Compared to initial vectors, our best-performing design boosts titer more than 30-fold, enabling fine-scale tuning of expression to optimize cell-fate conversion within a non-monotonic landscape. Together, this work illuminates principles for constructing two-gene lentiviral vectors with both high titer and robust expression, enhancing efficacy for downstream applications.