Viral and synthetic single-stranded RNAs are the ligands for Tolllike receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications. The present study describes one pool of RNA compounds, referred to as stabilized immune modulatory RNA (SIMRA) compounds, in which two RNA segments are attached through their 3 ends. SIMRA compounds showed greater stability in human serum compared with linear RNA and activated human TLR8, but not TLR7, in HEK293 cells without using lipid carriers. Interestingly, another set of SIMRA compounds containing 7-deazaguanosine substituted for natural guanosine activated human TLR7 and TLR8. Additionally, TLR7-and TLR8-activating compounds, but not the compounds that activated only TLR8, stimulated mouse immune cells in vitro and in vivo and produced dose-dependent T helper 1-type cytokines. Both types of compounds activated human peripheral blood mononuclear cells, but only TLR7-and TLR8-activating compounds activated plasmacytoid dendritic cells and produced high levels of IFN-␣. In monkeys, s.c. administration of both types of SIMRA compounds induced transient changes in peripheral blood monocytes and neutrophils, and activated T lymphocytes, monocytes, and NK cells. Both types of compounds induced IFN-␥-inducible protein 10, but only the 7-deazaguanosine-containing compound that activated both TLR7 and TLR8 induced IFN-␣ in monkeys. This is a comprehensive study of RNA-based compounds containing structures and synthetic stimulatory motifs in mouse, monkey, and human systems without using lipid carriers. oligoribonucleotides T oll-like receptors (TLRs) recognize specific molecular signatures called pathogen-associated molecular patterns present within pathogens (1). Eleven TLRs (TLR1-TLR11) have been identified in mammals that recognize different pathogen-associated molecular patterns present in bacteria and viruses. Among the 11 TLRs, TLRs 3, 7, 8, and 9 are present on the membranes of endosomes in the cells and detect nucleic acid molecular patterns of intracellular DNA and RNA pathogens (2-7). The other TLRs are present on the cell surface and recognize molecular patterns associated with extracellular pathogens. Synthetic and bacterial DNA containing unmethylated CpG motifs are the ligands for TLR9 (7). Viral and synthetic double-stranded RNAs are the ligands for TLR3 (2). Viral and synthetic single-stranded RNAs are the ligands for TLR7 and TLR8 (4-6). Imidazoquinoline-based small molecules and certain guanosine-based nucleosides also have been shown to act as ligands for TLR7 and TLR8 (3).In addition to the differences in the cellular localization of TLRs, different immune cell subtypes express different TLRs (8). For example, TLRs 7 and 9 are expressed in human plasmacytoid dendritic cells (pDCs) and B cells, and TLR8 is expressed in human myeloid dendritic...

Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem. 2002, 13, 966-974). In the present study, we conjugated a model peptide, a 28-amino-acid-long beta-amyloid peptide, to either the 5'- or the 3'-end of an ODN via C3 and C6 alkyl linkers. We compared the immune stimulatory activity of the resulting conjugates with that of a parent ODN without conjugation in TLR9-transfected cells, mouse spleen cell cultures, and in vivo in mice. ODN with the peptide conjugated at the 3'-end via C3 and C6 linkers had immune stimulatory activity similar to that of the parent ODN in both in vitro and in vivo in mice. On the contrary, conjugation of peptide at the 5'-end of the ODN significantly abrogated immune stimulatory activity. In conclusion, the results presented here demonstrate that peptide/protein conjugation to ODN is optimal at the 3'-end with either C3 or C6 linker and conjugation at the 5'-end leads to significant loss of TLR9-mediated immune stimulation.

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Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation.

Oligodeoxynucleotides containing a CpG motif and double-or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-␣) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-␣-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonist's ability to induce immune responses, including IFN-␣ induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes. A third agonist showed a biphasic melting transition in the heating cycle and a monophasic melting transition with lower hyperchromicity during the cooling cycle, suggesting the formation of both intra-and intermolecular duplexes. All three agonists induced the production of Th1-type cytokines and chemokines, including high levels of IFN-␣, in human peripheral blood mononuclear cell and plasmacytoid dendritic cell cultures. Subcutaneous administration of the two intermolecular duplex-forming agonists, but not the intramolecular duplex-forming agonist, induced cytokine secretion in mice. In nonhuman primates, the two agonists that formed intermolecular duplexes induced IFN-␣ and IP-10 secretion. On the contrary, the agonist that formed an intramolecular duplex induced only low levels of cytokines in nonhuman primates, suggesting that this type of structure formation is less immunostimulatory in vivo than the other structure. Taken together, the present results suggest that oligonucleotide-based agonists of TLR9 that form intermolecular duplexes induce potent immune responses in vivo.The vertebrate immune system recognizes highly conserved molecular patterns that are present in pathogens through a number of pattern recognition receptors. Toll-like receptors (TLRs) are among the well-characterized pattern recognition receptors. At least 10 TLRs have been identified in humans, and one of them, TLR9, is the receptor for bacterial and synthetic DNA containing unmethylated CpG motifs (4). TLR9 is expressed predominantly in B cells and plasmacytoid dendritic cells (pDCs) in humans. Activation of these two cell types by synthetic oligonucleotides containing unmethylated CpG motifs via TLR9 results in a Th1-type immune response which includes the secretion of interferon alpha (IFN-␣), IFN-␥, interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-␣), and IL-6 with an increase in the levels of costimulatory surface molecules (1,11,12,17,19,28). A number of TLR9 agonists are currently being evaluated in clinical trials as therapies for various diseases, including cancers, infectious diseases, allergy, and asthma, and as vaccine adjuvants (1, 10).The immune response profiles induced via TLR9 stimulation depend on the stimulatory motif and secondary structure present in the oligonucleotides (...

Oligonucleotides are being employed for gene-silencing activity by a variety of mechanisms, including antisense, ribozyme, and siRNA. In the present studies, we designed novel oligonucleotides complementary to targeted mRNAs and studied the effect of 3'-end exposure and oligonucleotide length on gene-silencing activity. We synthesized both oligoribonucleotides (RNAs) and oligodeoxynucleotides (DNAs) with phosphorothioate backbones, consisting of two identical segments complementary to the targeted mRNA attached through their 5'-ends, thereby containing two accessible 3'-ends; these compounds are referred to as gene-silencing oligonucleotides (GSOs). RNA and/or DNA GSOs targeted to MyD88, VEGF, and TLR9 mRNAs had more potent gene-silencing activity than did antisense phosphorothioate oligonucleotides (PS-oligos) in cell-based assays and in vivo. Of the different lengths of GSOs evaluated, 19-mer long RNA and DNA GSOs had the best gene-silencing activity both in vitro and in vivo. These results suggest that GSOs are novel agents for gene silencing that can be delivered systemically with broader applicability.

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