title: Rational design of immunostimulatory siRNAs.
abstract: Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.
authors.affiliation: Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Victoria, Australia.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Gantier
authors.forename: Stephen
authors.initials: S
authors.lastname: Tong
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Irving
authors.forename: Martha
authors.initials: M
authors.lastname: Lappas
authors.forename: Ulrika W
authors.initials: UW
authors.lastname: Nilsson
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.forename: Nigel A J
authors.initials: NA
authors.lastname: McMillan
authors.forename: Bryan R G
authors.initials: BR
authors.lastname: Williams
mesh_terms: D000818:Animals; D006801:Humans; D007114:Immunization; D007249:Inflammation / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D009000:Monocytes / Q000276:immunology; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology*; D051199:Toll-Like Receptor 7 / Q000276:immunology; D051200:Toll-Like Receptor 8 / Q000276:immunology; D014529:Uridine / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014777:Virus Diseases / Q000628:therapy*
chemical_list: D018950:Lipopolysaccharide Receptors; D035683:MicroRNAs; D034741:RNA, Small Interfering; C495348:TLR7 protein, human; C495349:TLR8 protein, human; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D014529:Uridine