Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Mediator cyclin-dependent kinases upregulate transcription of inflammatory genes in cooperation with NF-κB and C/EBPß on stimulation of Toll-like receptor 9.

In eukaryotes, the Mediator complex has important roles in regulation of transcription by RNA polymerase II. Mediator is a large complex with more than 20 subunits that form head, middle, tail and CDK/cyclin modules. Among them, CDK8 and/or CDK19 (CDK8/19), and their counterpart cyclin C, form the CDK/cyclin module together with Mediator subunits MED12 and MED13. Despite evidences of both activation and repression, the precise functional roles of CDK8/19 in transcription are still elusive. Our previous results indicate that CDK8/19 recruits epigenetic regulators to repress immunoresponse genes. Here, this study focused on Toll-like receptors (TLRs), which exert innate immune responses through recognition of pathogen-associated molecular patterns and examined the functional roles of CDK8/19. As a result, CDK8/19 regulated transcription of inflammatory genes on stimulation of TLR9 in myeloma-derived RPMI8226 cells, which led to expression of inflammation-associated genes such as IL8, IL10, PTX3 and CCL2. Mediator subunits CDK8/19 and MED1, inflammation-related transcriptional activator NF-κB and C/EBPß, and general transcription factors TFIIE and TFIIB colocalized at the promoter regions of these genes under this condition. Our results show that CDK8/19 positively regulates inflammatory gene transcription in cooperation with NF-κB and C/EBPß on stimulation of TLR9.