Designing self-amplifying replicons for transient gene expression of interleukin-18

Abstract

RNA-based therapeutics are on the rise as a re-emerging technology bolstered by the SARS-CoV-2 pandemic. However, issues such as storage, protein expression, and delivery are major challenges for the broad applicability. To address this issue, non-viral RNA replicon delivery systems can induce transient gene expression of immunomodulators and open the door to numerous potential targets that were once not viable due to dose-limiting toxicities affecting safety and efficacy. To that end, this work targets IL-18, a pleiotropic proinflammatory cytokine, which has been primarily viewed as a prognostic marker of various diseases like atherosclerosis, arthritis, diabetes, and cancer. In combination with another cytokine (IL-12), IL-18 is capable of enhancing the production of IFN-γ critical for anti-tumor immune responses. First, the synthesis of RNA replicons through in vitro transcription, characterization, and validation of IL-18 RNA replicons is described. Next, IL-18 RNA replicons are encapsulated in lipid nanoparticles and their function is analyzed using a melanoma cell line. Lastly, evidence of synergy between RNA replicons encoding for both IL-18 and IL-12 is shown through the production of robust IFN-γ responses in B16F10 co-cultures with naïve splenocytes. These studies provide a fundamental understanding to observe transient gene expression and synergy of dual expressing cytokines. Lastly, this system has the potential to be a promising tool for cancer immunotherapy.