Inhibition of dsRNA-sensing pathway enhances mRNA-LNP expression efficiency 2531

Description mRNA-LNPs are a platform for therapeutic applications such as protein replacement therapies. However, optimizing mRNA translation efficiency is challenging due to their immunogenicity, which can repress mRNA translation via RNA degradation or interferon release. This study explored whether modulating several RNA-sensing pathways can improve EGFP mRNA-LNP translation in PBMCs and THP-1 cell line. Inhibiting Toll-like receptors 7 and 8 (TLR7/8) that recognize single-stranded RNA did not improve translation efficiency. Surprisingly, genes related to double-stranded RNA (dsRNA) sensing were significantly upregulated after mRNA-LNPs treatment. These findings suggest that mRNA may assume double-stranded secondary structure, making dsRNA sensors critical in mRNA translation inhibition. Therefore, small molecules that inhibit dsRNA regulation pathways were tested. Inhibiting mitochondrial antiviral signaling protein (MAVS) only modestly enhanced protein expression (< 2-fold increase), while targeting retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3) had no effect. However, inhibiting RNase L with compound 17a led to a five-fold increase in mRNA translation in human monocytes. Targeting RNase L prevents RNA degradation, allowing sustained translation and enhanced protein expression. This novel finding on RNase L’s role in regulating mRNA-LNP translation offers important insights in optimizing mRNA-LNPs to minimize immune responses and maximize protein expression. Funding Sources Financial support for this work was provided by Pfizer Inc. The authors would like to acknowledge that HMN received financial support from Pfizer as a RNA Accelerator Postdoctoral Fellow.