Covalent Polymer-RNA Conjugates for Potent Activation of the RIG-I Pathway.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutic with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here we address this challenge by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, we found that covalent conjugation of 3pRNA improved loading efficiency, enhanced immunostimulatory activity, protected against nuclease degradation, and improved serum stability. Additionally, we found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter was only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increased type-I interferon levels relative to analogous carriers that used electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I. This article is protected by copyright. All rights reserved.