Overcoming the innate sensing of self-amplifying RNA for improved vaccines & therapeutics
Embargo Date
2027-09-02
OA Version
Citation
Abstract
Messenger RNA (mRNA) therapeutics have transformed modern medicine, enabling the rapid and flexible development of treatments for infectious diseases, cancer, genetic disorders, and more. A landmark demonstration of this technology’s potential was the swift development and global deployment of mRNA vaccines in response to the COVID-19 pandemic, showcasing the scalability, efficacy, and safety of protein-encoding RNA. Despite these successes, mRNA therapeutics face notable limitations including transient protein expression lasting only a few days and the need for large and repeated doses to achieve therapeutic efficacy. To address these challenges, self-amplifying RNA (saRNA) has emerged as a promising alternative. saRNA encodes an RNA-dependent RNA polymerase (RdRp), typically derived from an alphavirus, which enables intracellular replication and amplification of the RNA. This process results in robust and prolonged protein expression from small doses. However, the clinical translation of saRNA has been hindered by a poor safety profile. saRNA strongly activates innate immune responses, resulting in the production of interferons and the induction of systemic side effects at doses as low as 10 micrograms in humans. Unlike mRNA, prior studies have suggested that saRNA is incompatible with modified nucleotides—the Nobel prize winning solution that underpins the improved safety and performance of mRNA medicines—due to suspected interference with the RdRp function. As a result, saRNA development has remained limited in scope and application. This dissertation addresses the challenges of saRNA translation through multiple strategies: 1) development of modified saRNA, 2) evolution of the RdRp to enhance function, and 3) engineering saRNA vector tropism for targeted expression. Using a functional screening approach, we identified for the first time several modified nucleotides that are compatible with saRNA. These modifications significantly enhanced protein expression across diverse cell types, suppressed innate immune activation in primary human immune cells, and improved the in vivo tolerability by reducing systemic inflammatory cytokine expression. As a vaccine platform, modified saRNA demonstrated potent efficacy, achieving protection against a lethal SARS-CoV-2 challenge in mice after a 10 ng dose. Through long-term evolution, we identified an adaptive mutation in the viral RdRp sequence that significantly improved protein expression in multiple cell types in vitro and after intramuscular administration in vivo. We further explored strategies to engineer saRNA vector tropism, enhancing localized protein expression while minimizing off-target effects. Together, this work establishes modified saRNA as a powerful and versatile technology, overcoming key limitations of conventional mRNA and paving the way to develop the next generation of vaccines and RNA therapeutics.
Description
2025
License
Attribution-NonCommercial-NoDerivatives 4.0 International