Article Text
Abstract
Background Toll-like receptors (TLRs) are an integral part of innate immune cell activation and play an essential role in mounting effective antigen-specific adaptive immune responses. TLR7 and TLR8 are highly homologous TLR family members that recognize ssRNA derived from viruses and induce distinct cytokine profiles due to differential expression in immune cells. Recent studies demonstrate that TLR7 and TLR8 recognize degraded ssRNA products generated by endosomal RNases. We hypothesized that site-specific modifications to foundational ssRNA sequences containing both TLR7 and TLR8 binding motifs could slow or inhibit RNase-mediated degradation of TLR8 binding site motifs. Here we designed oligoribonucleotides (ORNs) that reduce TLR8 activity while maintaining TLR7 agonism and developed a high throughput automated in vitro assay to identify TLR7 selective ORNs.
Methods We engineered 20–25 base pair ssRNA foundation sequences containing both TLR7 and TLR8 binding motifs with 2’ modifications placed on select nucleotides to slow or stop RNase-mediated processing of ORNs into ligands activating TLR8. This allowed for the abrogation of TLR8 and preservation of TLR7 activity. We established an automated screening platform to assemble and deliver lipid complexes containing a TLR-selective ORN and mRNA encoding GFP, and assessed their immunostimulatory activity in vitro. Cytokine responses from human PBMCs were evaluated to identify TLR7 selective agonists by Luminex, and monocyte maturation and antigen expression were assessed by flow cytometry. Top performing TLR7 specific ORNs were administered systemically to mice. DC maturation and serum cytokines were measured ex vivo by flow cytometry and Luminex.
Results Introduction of 2’ sugar-modified bases into ORNs limited or blocked RNase-mediated cleavage 3’ to the modified nucleotide at specific phosphodiester bonds. The nucleotide modifications of ssRNAs modulate endosomal RNase processing of these sequences, preventing degradation into monomeric uridine required for TLR8 activation, while preserving TLR7 activation. To mimic the effects of a co-delivered mRNA-based therapeutic, ssRNA ORN and mRNA were assembled into lipid complexes with DOTAP and cultured with human PBMCs. We identified top performing TLR7 specific oligos, classified as S2-PS-F.F and S5-F, that induced a robust IFNα response and weak TNFα response, accompanied by monocyte maturation and mRNA encoded GFP expression. These modified ORNs induced splenic DC maturation and cytokine release in vivo.
Conclusions Using a rationale design we created ORNs that have diminished TLR8 and preserved TLR7 activity. This design strategy and screening platform can be applied and expanded to identify additional novel TLR7 selective agonists for potential use with mRNA-based therapeutics.
Ethics Approval Obtained informed consent from healthy adult volunteer apheresis donors who have passed the standard blood donor health history questionnaire. All animal experiments were performed under protocols approved by the Genentech Institutional Animal Care and Use Committee.
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