Broad-spectrum immune suppression encoded in self-amplifying RNA enables non-cytotoxic, non-immunostimulatory, externally controllable transgene expression

Self-amplifying RNA (saRNA) has the potential to provide durable, non-integrative transgene expression for transient gene therapy. However, its auto-replicative nature mimics viral infection, triggering innate immune responses that shutdown cap-dependent translation, degrade cellular mRNA, induce cell death, and release cytokines. In non-immunotherapy applications, this immune activation is undesirable as it limits transgene expression, depletes transfected cells, and induces inflammation, undermining therapeutic outcomes. Moreover, the use of exogenous immune suppressants to mitigate these effects often increases treatment complexity and the risk of unintended systemic side effects. To address these challenges, we developed a strategy to encode broad-spectrum innate immune suppression directly within saRNA. This approach leverages cap-independent translation to bypass saRNA-triggered translation shutdown, enabling the expression of multiple inhibitors targeting diverse double-stranded RNA-sensing and inflammatory signaling pathways. In mouse primary fibroblast-like synoviocytes—a cell type relevant to inflammatory joint diseases—this strategy eliminates the need for external immune inhibitors, reduces cytotoxicity and antiviral cytokine secretion, and enables sustained transgene expression that can be controlled with a small-molecule antiviral. These findings support the development of saRNA therapeutics that offer durable, non-integrative, externally controllable transgene expression without persistent immune activation or reliance on exogenous immune suppressants.