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

Self-amplifying RNA (saRNA) has the potential to provide durable, non-integrating transgene expression for transient gene therapy. However, its auto-replicative nature mimics viral infection, triggering innate immune responses that shut down 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, induces unintended changes in host gene expression, depletes transfected cells, and promotes inflammation—ultimately 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 signalling pathways. In mouse primary fibroblast-like synoviocytes—key cells in joint pathologies—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. Together, these findings support the development of reversible ‘immune-evasive saRNA’ constructs for transient gene therapy applications that avoid persistent immune activation and eliminate the need for external immune suppressants.