Cap-independent co-expression of dsRNA-sensing and NF-κB pathway inhibitors enables controllable self-amplifying RNA expression with reduced immunotoxicity.

Self-amplifying RNA (saRNA) holds promise for durable therapeutic gene expression, but its broader utility beyond vaccines is limited by potent innate immune responses triggered during replication. These responses shut down translation, induce cytotoxicity, degrade host mRNAs, and drive cytokine production. While exogenous immunosuppressants can blunt these effects, they complicate treatment and risk systemic side effects. To address this, we engineered 'immune-evasive saRNA' that intrinsically suppresses the innate immune pathways triggered by its own replication. This strategy leverages cap-independent translation to co-express a suite of inhibitors from a single saRNA transcript, targeting key innate immune pathways, including protein kinase R (PKR), oligoadenylate synthase (OAS)/RNase L, and nuclear factor-κB (NF-κB). In primary mouse fibroblast-like synoviocytes, a cell type central to the pathology of joint diseases, immune-evasive saRNA enables sustained transgene expression without external immunosuppressants, substantially reducing cytotoxicity and antiviral cytokine secretion. Crucially, this system offers both concentration-dependent control of expression and on-demand termination via a small-molecule antiviral. Together, these findings establish a framework for developing saRNA therapeutics with an improved tolerability profile that can be switched off once therapeutic outcomes are met, offering a path toward a controllable gene expression platform that fills the therapeutic gap between the transience of mRNA and the permanence of viral vectors.