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.

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

“…2) was performed as previously reported (20,22). ss/dsRNAs and purified viral RNAs were transfected with dioleoyl trimethylammonium propane (DOTAP; Roche) and pure RPMI in biological triplicate as previously described (19)(20)(21)(22). The ratios of DOTAP to ssRNA (at 80 M) were 3.74 g/l of ssRNA (see Fig.…”

“…The cells were incubated for 4 h at 37°C in 5% CO 2 atmosphere before stimulation by ssRNAs and dsRNAs. Immortalized Tlr7 Ϫ/Ϫ bone marrow macrophages (BMMs) and wildtype (wt) BMMs were generated using the J2 retrovirus encoding v-raf and v-myc as previously described (19) and were maintained in 80% Dulbecco's modified Eagle medium (DMEM) supplemented with 1ϫ antibiotic/ antimycotic and 10% FBS (referred to as complete DMEM), with 20% L-929 cell-conditioned medium. Human acute monocytic leukemia (THP-l) cells were grown in suspension in complete RPMI.…”

“…In mice, however, both TNF-␣ and IFN-␣ production relate to Tlr7 activation (13). We and others have previously established that TLR7/8 sensing of ssRNAs is strongly dependent on the propensity of ssRNAs to form inter-and intramolecular secondary structures, with the limitation that perfect dsRNA molecules are less stimulatory than imperfect ones (13,18,19). Whether or not inosine incorporation in RNA can modulate TLR7/8 sensing has not been characterized to date.…”

siRNA and RNAi optimization

“…Even though it was noted that some secondary structures stimulate the activation of TLRs, their activation is mostly sequence-dependent. Indeed, polyuridine tracts and GU-rich sequences, are strong RNA immunostimulatory motifs that should be avoided in tailoreddesigned siRNA 85,86 . .…”

“…Although the necessity of neutralizing the immunostimulatory potential of siRNA molecules is fundamental goal to achieve for reliable therapeutic purposes, the siRNA adjuvant effect has been exploited to activate the cellular immunity. A promising approach for cancer and viral therapy came from the development of immunostimulatory siRNAs (is-siRNAs) 86,94 . These bifunctional molecules, linking potent gene silencing properties to suitable production of IFNs, can effectively control chronic viral diseases such as HIV-AIDS, HBV and HCV infections [95][96][97] .…”