Synthetic Oligoribonucleotides Research Articles

Analysis of RNA and Its Modifications.

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins, and modulation of many biochemical processes. They are also often the key components of viruses. Synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics. In many cases, RNAs will be chemically modified, either naturally via enzymatic systems within a cell or intentionally during their synthesis. Analytical methods to detect, sequence, identify, and quantify RNA and its modifications have demands that far exceed requirements found in the DNA realm. Two complementary platforms have demonstrated their value and utility for the characterization of RNA and its modifications: mass spectrometry and next-generation sequencing. This review highlights recent advances in both platforms, examines their relative strengths and weaknesses, and explores some alternative approaches that lie at the horizon.

RNA-Polymer Hybrids via Direct and Site-Selective Acylation with the ATRP Initiator and Photoinduced Polymerization.

Combining synthetic polymers with RNA paves the way for creating RNA-based materials with non-canonical functions. We have developed an acylation reagent that allows for direct incorporation of the atom transfer radical polymerization (ATRP) initiator into both short synthetic oligoribonucleotides and natural biomass RNA extracted from torula yeast. The acylation was performed in a quantitative yield. The resulting initiator-functionalized RNAs were used for grafting polymer chains from the RNA by photoinduced ATRP, resulting in RNA-polymer hybrids with narrow molecular weight distributions. The RNA initiator was used for the polymerization of oligo(ethylene oxide) methyl ether methacrylate, poly(ethylene glycol) dimethacrylate, and N-isopropylacrylamide monomers, resulting in RNA bottlebrushes, hydrogels, and stimuli-responsive materials. This approach, readily applicable to both post-synthetic and nature-derived RNA, can be used to engineer the properties of a variety of RNA-based macromolecular hybrids and assemblies providing access to a wide variety of RNA-polymer hybrids.

RNA-Cholesterol Nanoparticles Function as Potent Immune Activators via TLR7 and TLR8.

The innate immune system senses viral and bacterial ribonucleic acid (RNA) via pattern recognition receptors (PRR) leading to subsequent activation of the immune system. One group of RNA sensors is formed by endosomal/lysosomal Toll-like receptors (TLR) such as TLR7 and TLR8. During viral or bacterial infection, immunostimulatory RNA is part of the pathogen reaching the endosomal/lysosomal compartment after cellular uptake. Synthetic single-stranded or double-stranded oligoribonucleotides (ORN) can mimic RNA from pathogens and are widely used as activating ligands for TLR7 and TLR8. However, one limitation in the use of synthetic ORN driven immune stimulation is the need for transfection reagents for RNA delivery into cells. Here we demonstrate that the conjugation of cholesterol to a double-stranded version of immunostimulatory RNA40 strongly enhanced RNA uptake into monocytes and plasmacytoid dendritic cells when compared to naked RNA. Cholesterol-conjugated RNA (RNA-chol) formed nanoparticles that were superior to RNA-liposomes complexes in regard to induction of type I interferon from human and murine plasmacytoid dendritic cells as well as proinflammatory cytokine production (e.g. TNF-α, IL12p70 or IL-6) in human monocytes. Furthermore, the RNA40-chol induced cytokines in human monocyte cultures supported TH1 and TFH cell differentiation underscoring a strong adjuvant function of RNA-chol nanoparticles for adaptive immune responses. In summary, cholesterol-conjugated immunostimulatory RNA forms nanoparticles and functions as a potent immune adjuvant in human and murine immune cells. It further simplifies the use of immunostimulatory RNA by avoiding the need for liposomal transfection reagents.

Urban air PM modifies differently immune defense responses against bacterial and viral infections in vitro

Epidemiological evidence has shown the association between exposure to ambient fine particulate matter (PM) and increased susceptibility to bacterial and viral respiratory infections. However, to date, the underlying mechanisms of immunomodulatory effects of PM remain unclear. Our objective was to explore how exposure to relatively low doses of urban air PM alters innate responses to bacterial and viral stimuli in vitro. We used secondary alveolar epithelial cell line along with monocyte-derived macrophages to replicate innate lung barrier in vitro. Co-cultured cells were first exposed for 24 h to PM2.5-1 (particle aerodynamic diameter between 1 and 2.5 μm) and subsequently for an additional 24 h to lipopolysaccharide (TLR4), polyinosinic-polycytidylic acid (TLR3), and synthetic single-stranded RNA oligoribonucleotides (TLR7/8) to mimic bacterial or viral stimulation. Toxicological endpoints included pro-inflammatory cytokines (IL-8, IL-6, and TNF-α), cellular metabolic activity, and cell cycle phase distribution. We show that cells exposed to PM2.5-1 produced higher levels of pro-inflammatory cytokines following stimulation with bacterial TLR4 ligand than cells exposed to PM2.5-1 or bacterial ligand alone. On the contrary, PM2.5-1 exposure reduced pro-inflammatory responses to viral ligands TLR3 and TLR7/8. Cell cycle analysis indicated that viral ligands induced cell cycle arrest at the G2-M phase. In PM-primed co-cultures, however, they failed to induce the G2-M phase arrest. Contrarily, bacterial stimulation caused a slight increase in cells in the sub-G1 phase but in PM2.5-1 primed co-cultures the effect of bacterial stimulation was masked by PM2.5-1. These findings indicate that PM2.5-1 may alter responses of immune defense differently against bacterial and viral infections. Further studies are required to explain the mechanism of immune modulation caused by PM in altering the susceptibility to respiratory infections.

Synthesis of Peptido RNAs from Unprotected Peptides and Oligoribonucleotides via Coupling in Aqueous Solution

Peptido RNAs are hybrid molecules with a phosphoramidate link between the N‐terminus of a peptide and the 5'‐phosphate of an oligoribonucleotide. Such species are formed in spontaneous co‐oligomerizations of amino acids and ribonucleotides in aqueous condensation buffer. To shed light on the properties of these fascinating molecules, a synthetic method for their preparation in monodisperse form is needed. Herein, we report how peptido RNAs can be prepared via solution‐phase coupling of unprotected peptides and oligoribonucleotides in aqueous solution. The preferred protocol uses pre‐activation of the 5'‐phosphate of the RNA as an imidazolide at pH 6.5, followed by precipitation and coupling to the peptide at pH 8 with an organocatalyst. The procedure gave peptido RNAs from water‐soluble peptides and synthetic oligoribonucleotides in up to 68 % yield. The method is convenient and inexpensive and can produce NMR quantities, opening the door to the systematic exploration of the chemistry of peptido RNAs.

23S rRNA from Vibrio parahaemolyticus regulates the innate immune response via recognition by TLR13 in orange-spotted grouper (Epinephelus coioides)

Toll-like receptors (TLRs) are major pattern recognition receptors (PRRs) that recognize multiple pathogen-associated molecular patterns (PAMPs) through the leucine-rich repeat (LRR) domain and mount effective immune responses. Vibrio parahaemolyticus is the main pathogen that causes vibriosis in aquatic animals, yet the mechanisms of its recognition by innate immune system in teleost fish remain unknown. Here, the results reveal that TLR13 in orange-spotted grouper (Epinephelus coioides) (EcTLR13) recognizes a conserved 23S ribosomal RNA (23S rRNA) sequence in V. parahaemolyticus, and the 13-nucleotide motif near the 23S rRNA ribozyme activation site (VP13) acts as a PAMP. After challenge with RNA and 23S rRNA from V. parahaemolyticus and with the synthetic oligoribonucleotide VP13, the expression of EcTLR13 in grouper spleen cells (GS cells) was significantly increased. EcTLR13-knockdowned GS cells were stimulated with the same stimulants as listed above, the expression of IL-6, IL-12, IL-1β and TNFα was significantly reduced. RNA-protein immunoprecipitation revealed that VP13 could directly bind to EcTLR13. The dual-luciferase reporter assay also showed that EcTLR13 enhanced the fluorescence activity of IFNβ rather than that of NF-κB when the cells were challenged with RNA from V. parahaemolyticus or with synthetic VP13. Our study established the mechanism of fish TLR13-mediated recognition of microbial products during V. parahaemolyticus infection.

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P.falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Automated Chemical Solid-Phase Synthesis and Deprotection of 5-Hydroxymethylcytosine-Containing RNA.

5-Hydroxymethylcytosine is an epigenetic base modification that is part of the demethylation pathway of 5-methylcytosine in DNA. 5-Methylcytosine is iteratively oxidized to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine by enzymes of the TET protein family. Since the discovery of 5-hydroxymethylcytosine also in RNA its role in regulatory processes and metabolism remains elusive. To gain more insight into the function of RNA containing 5-hydroxymethylcytidine, innovative and interdisciplinary approaches are required. In this context, synthetic oligoribonucleotides containing 5-hyroxymethylcytidine are an inevitable tool. Therefore, in this chapter, we present the efficient synthesis of RNA oligonucleotides containing 5-hydroxymethylcytosine by solid-phase synthesis. The incorporation of the modified cytosine derivative into RNA is compatible with standard phosphoramidite-based synthesis procedures of oligoribonucleotides.

Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation

In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel. Chemical probing with BzCN revealed that h16 in the 48S PICs containing eIF3j or scanning factor DHX29 is strongly shielded; the effect was observed with all the mRNAs used, and h16 remained protected as well in 80S post-initiation complexes lacking these factors. Altogether, the obtained results allowed us to suggest that eIF3j bound at the 48S PICs makes the rp uS3 inaccessible for binding of RNAs and this factor subunit is responsible for the decrease of h16 conformational flexibility; the latter is manifested as reduced accessibility of h16 to BzCN. Thus, our findings provide new insights into how eIF3j is implicated in ensuring the proper conformation of the mRNA entry channel, thereby facilitating mRNA loading.

Optimal antisense target reducingINSintron 1 retention is adjacent to a parallel G quadruplex

Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression.

RNA and imidazoquinolines are sensed by distinct TLR7/8 ectodomain sites resulting in functionally disparate signaling events.

TLRs 7 and 8 are pattern recognition receptors controlling antiviral host defense or autoimmune diseases. Apart from foreign and host RNA, synthetic RNA oligoribonucleotides (ORN) or small molecules of the imidazoquinoline family activate TLR7 and 8 and are being developed as therapeutic agonists. The structure-function relationships for RNA ORN and imidazoquinoline sensing and consequent downstream signaling by human TLR7 and TLR8 are unknown. Proteome- and genome-wide analyses in primary human monocyte-derived dendritic cells here showed that TLR8 sensing of RNA ORN versus imidazoquinoline translates to ligand-specific differential phosphorylation and transcriptional events. In addition, TLR7 and 8 ectodomains were found to discriminate between RNA ORN and imidazoquinolines by overlapping and nonoverlapping recognition sites to which murine loss-of-function mutations and human naturally occurring hyporesponsive polymorphisms map. Our data suggest TLR7 and TLR8 can signal in two different "modes" depending on the class of ligand. Considering RNA ORN and imidazoquinolines have been regarded as functionally interchangeable, our study highlights important functional incongruities whose understanding will be important for developing TLR7 or 8 therapeutics with desirable effector and safety profiles for in vivo application.

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA(Leu(UUR)) and tRNA(Lys).

5-Taurinomethyluridine (τm(5)U) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) are located at the wobble position of human mitochondrial (hmt) tRNA(Leu(UUR)) and tRNA(Lys), respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys) are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm(5)U and τm(5)s(2)U into 17-mers related to the sequence of the anticodon arms hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys), respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm(5)s(2)U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.

Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2′-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1β and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.

Abstract P5-10-16: miRNAs as novel therapeutic adjuvants for the prognosis and treatment of triple negative breast cancers

Abstract Approximately 15–20% of all breast cancers account for triple negative breast cancers (TNBCs) that exhibit an aggressive, distinct metastatic pattern and poor prognosis resulting in a disproportionate number of breast cancer deaths. More than 50% of patients with TNBC develop chemoresistance and do not respond to chemotherapeutic drugs, leading to early relapse and shorter survival. Understanding the mechanisms underlying such resistance is therefore crucial for the development of new, efficacious cancer drugs. In spite of extensive inquiry in this field, little is known about the key molecules/signaling pathways that regulate this phenomenon. Recently, we have discovered that small non-coding RNAs-miRNAs play critical roles in mediating drug sensitivity/resistance in TNBCs. We have identified miRNAs that are differentially expressed between chemo-resistant and sensitive breast cancer cells. In particular, through high-throughput miRNA inhibitor library screens, we have identified miRNA inhibitors that sensitize resistant TNBC cells to paclitaxel, a drug commonly used to treat triple negative breast cancers. Interestingly, our studies revealed that specific miRNAs including miR-185 (patent pending) that sensitize resistant TNBCs to paclitaxel are expressed at significantly lower levels in relapsed metastatic TNBC patient sera compared to sera from their healthy siblings. Furthermore, using liposome- or biocompatible PLGA nanoparticle-based approaches, we show that systemic delivery of one of the sensitizer miRNAs suppresses breast cancer lung metastasis without any hepatotoxicity in preclinical mouse tumor models. Importantly, we show that one of the mechanisms by which sensitizer miRNAs regulate paclitaxel sensitivity is by targeting Stathmin, a microtubule destabilizer protein that can bind to tubulin dimers and stimulate microtubule destabilization. These findings suggest that tumor-specific miRNAs render selective cell cytotoxicity in a drug-specific manner, that these miRNAs may serve as detection markers for identifying patients who might benefit most from specific drug treatment, and that these miRNAs may represent novel therapeutic tools for the treatment of the TNBCs. Since miRNAs are endogenously expressed and can be easily manipulated using synthetic oligoribonucleotides, we believe that they represent more attractive targets than the single gene or gene product targeted by conventional cancer treatments that are typically prone to drug resistance. Since paclitaxel is used for the treatment of many cancers, the long-term implications of this study are likely not limited to breast cancer alone but may apply to other tumor types. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-16.

Design of synthetic oligoribonucleotide-based agonists of Toll-like receptor 3 and their immune response profiles in vitro and in vivo

Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-β, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.

Improving the efficacy of chemotherapy drugs for the treatment of triple-negative breast cancers.

107 Background: Approximately 15-20% of all breast cancers account for triple-negative breast cancers that exhibit aggressive, distinct metastatic pattern and poor prognosis. More than 50% of patients with triple negative breast cancers develop chemoresistance and do not respond to chemotherapeutic drugs, leading to early relapse and shorter survival. Understanding the mechanisms underlying such resistance is therefore crucial for the development of new, efficacious cancer drugs. Methods: Through high-throughput miRNA inhibitor library screens, we have identified miRNA inhibitors that sensitize resistant triple negative breast cancer cells to paclitaxel, a drug commonly used to treat triple negative breast cancers. Results: Through high-throughput miRNA inhibitor library screens, we have identified miRNA inhibitors that sensitize resistant triple negative breast cancer cells to paclitaxel, a drug commonly used to treat triple negative breast cancers. Since miRNAs are endogenously expressed and can be easily manipulated using synthetic oligoribonucleotides, we believe that they represent more attractive targets than the single gene or gene product that is the target of conventional cancer treatments that are typically prone to drug resistance. Supporting this, we have recently demonstrated that miRNAs can be systemically delivered to treat breast cancer lung metastasis without any hepatotoxicity. In addition to being a potent therapeutic regimen, our preliminary analyses reveal that miRNAs can be bonafide early prognostic markers to monitor treatment response to specific drugs in triple-negative breast cancers. Conclusions: Taken together, these findings suggest that miRNA can serve as potent therapeutic adjuvants and although the data content of miRNA profiles is far less than that of gene expression profiles, by virtue of their ability to modulate entire spectrum of genes and pathways miRNAs have potential to be better classifiers for the prognosis and response to treatment of cancers. We believe that the identification of miRNAs that mediate chemoresistance could lead to more efficient treatment selection at the patient level and an improved response rates at the population level.

Novel Mechanism of RNA Repair by RtcB via Sequential 2′,3′-Cyclic Phosphodiesterase and 3′-Phosphate/5′-Hydroxyl Ligation Reactions

RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Immunostimulatory Potential of Silencing RNAs Can Be Mediated by a Non-Uridine-Rich Toll-Like Receptor 7 Motif

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.

Synthesis and immunological activities of novel Toll-like receptor 7 and 8 agonists

Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.