Engineering multiple levels of specificity in an RNA viral vector.

Engineered molecular circuits encoded in RNA can act as programmable therapeutics that sense cellular states and elicit precise responses within diseased cells. However, their application depends critically on systems for delivering circuits into cells. Here, we engineer a model delivery system based on the rabies virus that incorporates multiple levels of control over the viral life cycle and cargo. We demonstrate controlled release of viral vectors from sender cells, conditional entry into target cells based on cell-surface proteins, restricted viral replication governed by intracellular protein content, and an escaper-resistant mechanism for viral elimination with drugs. In parallel, we integrate RNA-sensing and protease-controlled circuits to regulate cargo expression and activity at post-transcriptional and post-translational levels. Together, these strategies illustrate how viral and protein engineering can establish multi-level control at both the viral and cargo levels to facilitate specificity in future therapeutic RNA delivery systems.