title: Immunostimulatory potential of silencing RNAs can be mediated by a non-uridine-rich toll-like receptor 7 motif.
abstract: Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.
authors.affiliation: Pfizer Oligonucleotide Therapeutics Unit, Coley Pharmaceutical GmbH, Merowingerplatz 1a, Düsseldorf, Germany. marion.jurk@pfizer.com
authors.forename: Marion
authors.initials: M
authors.lastname: Jurk
authors.forename: Ghania
authors.initials: G
authors.lastname: Chikh
authors.forename: Bettina
authors.initials: B
authors.lastname: Schulte
authors.forename: Andrea
authors.initials: A
authors.lastname: Kritzler
authors.forename: Denise
authors.initials: D
authors.lastname: Richardt-Pargmann
authors.forename: Catherine
authors.initials: C
authors.lastname: Lampron
authors.forename: Rachel
authors.initials: R
authors.lastname: Luu
authors.forename: Arthur M
authors.initials: AM
authors.lastname: Krieg
authors.forename: Alain P
authors.initials: AP
authors.lastname: Vicari
authors.forename: Jörg
authors.initials: J
authors.lastname: Vollmer
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D058621:Blood Buffy Coat; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D007114:Immunization / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D051199:Toll-Like Receptor 7 / Q000737:chemistry* / Q000378:metabolism*; D014529:Uridine / Q000737:chemistry
chemical_list: D016207:Cytokines; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D009843:Oligoribonucleotides; D054735:Phosphorothioate Oligonucleotides; D034741:RNA, Small Interfering; D051199:Toll-Like Receptor 7; D014529:Uridine