Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8 (original) (raw)
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Journal of Medicinal Chemistry, 2009
We previously reported a novel class of stabilized immune-modulatory RNA (SIMRA) compounds that activates TLR8 or both TLR7 and TLR8 depending on the nucleotide composition and chemical modifications incorporated. In the present study, to identify TLR7-selective agonists, we designed and synthesized novel SIMRA compounds with varying sequence compositions substituting 7-deaza-G for natural guanosine and studied immune-stimulatory activity in cell-based assays and in vivo in mice. SIMRA compounds activated NF-κB in HEK293 cells expressing TLR7 and induced cytokine production in mouse spleen cells and human PBMCs and higher levels of IFN-R in human pDCs, which correlated with TLR7 activation. Subcutaneous administration of SIMRA compounds to mice increased serum cytokine levels. TLR knockout mouse studies showed that both TLR7 and MyD88 are required for activity of SIMRA compounds. The presence of a 5 0-AA/CN (A > C and N = U/C/7deaza-G) and/or C/AUU-3 0 (C > A) trinucleotide at the 5 0-and 3 0-ends of SIMRA compound along with a 5 0-AN 1 N 2 UG1A-3 0 (N 1 =A/C; N 2 =U/C/7-deaza-G) or UG1AZ 1 G1Z 2 UU (Z 1 =A < C; Z 2 =C < A) motif confers TLR7 selectivity over other sequence compositions. In conclusion, we have designed and synthesized novel SIMRA compounds that selectively act as agonists of TLR7.
Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery
Proceedings of the National Academy of Sciences, 2012
The specific activation of Toll-like receptors (TLRs) has potential utility for a variety of therapeutic indications including antiviral immunotherapy and as vaccine adjuvants. TLR7 and TLR 8 may be activated by their native ligands, single-stranded RNA, or by small molecules of the imidazoquinoline family. However the use of TLR7/8 agonists for in vivo therapy is limited by instability, in the case of RNA, or systemic biodistribution and toxicity in the case of small molecule agonists. We hypothesized that unique lipid-like materials, termed "lipidoids," could be designed to efficiently deliver immunostimulatory RNA (isRNA) to TLR-expressing cells to drive innate and adaptive immune responses. A library of lipidoids was synthesized and screened for the ability to induce type I IFN activation in human peripheral blood mononuclear cells when combined with isRNA oligonucleotides. Effective lipidoid-isRNA nanoparticles, when tested in mice, stimulated strong IFN-α responses following subcutaneous injection, had robust antiviral activity that suppressed influenza virus replication, and enhanced antiovalbumin humoral and cell-mediated responses when used as a vaccine adjuvant. Further, we demonstrate that whereas all immunological activity was MyD88-dependent, certain materials were found to engage both TLR7-dependent and TLR7-independent activity in the mouse suggestive of cell-specific delivery. These lipidoid formulations, which are materials designed specifically for delivery of isRNA to Toll-like receptors, were superior to the commonly used N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate-RNA delivery system and may provide new tools for the manipulation of TLR responses in vitro and in vivo.
Oligodeoxyribonucleotide-Based Antagonists for Toll-Like Receptors 7 and 9
Journal of Medicinal Chemistry, 2009
Oligodeoxyribonucleotides containing unmethylated CpG motifs act as TLR9 agonists. In this study, we evaluated oligonucleotides containing an unmethylated CpG motif in which two nucleotides adjacent to the CpG dinucleotide were substituted with 2′-O-methylribonucleotides, resulting in TLR7 and TLR9 antagonists. In mouse and human cell cultures, antagonists did not stimulate immune activation but inhibited TLR7 and TLR9 agonist-induced activity. In mice, antagonists inhibited immune responses induced by TLR9 agonists for up to several days, and the inhibition was dose-dependent. Antagonists also inhibited immune responses induced by an RNA-based TLR7/8 agonist but not TLRs 2, 3, 4, or 5 agonists in mice. Additionally, antagonist inhibited TLR9 agonist-induced IL-6 in lupus-prone MRL/lpr mouse spleen cell cultures. These results indicate that antagonists described herein can suppress immune responses induced by TLR7 and TLR9 agonists. Antagonists may be suitable candidates for treating inflammatory and autoimmune diseases where inappropriate or uncontrolled TLR activation has been implicated.
PLOS ONE, 2015
Activation of TLR7 and TLR9 by endogenous RNA-or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX 3-5 GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7-and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks Gmodification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.
Proceedings of the National Academy of Sciences, 2005
Bacterial DNA and synthetic oligomers containing CpG dinucleotides activate the immune system through Toll-like receptor (TLR) 9. Here, we compare the immunostimulatory activity of three immunomers with different nucleotide sequences containing a synthetic cytosine-phosphate-2′-deoxy-7-deazaguanosine dinucleotide (CpR), called immunomodulatory oligonucleotides (IMOs), in mouse, human, and monkey systems. IMOs induced IL-12 and IFN-γ secretion more than a control non-CpG IMO in mice. All three IMOs activated HEK293 cells expressing TLR9 but not TLR3, -7, or -8. IMOs induced human B-cell proliferation and enhanced expression of CD86 and CD69 surface markers on B cells. The three IMOs induced CD86 expression on human plasmacytoid dendritic cells, but only IMOs that contained a 5′-terminal TCR nucleotide sequence induced IFN-α secretion. A sequence that forms a duplex structure also was required for IFN-α induction in human peripheral blood mononuclear cell cultures. IMOs induced chemok...
Angewandte Chemie International Edition, 2020
As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses.T his study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site,f ormulated as nanoparticles with ac oordinative amphiphile,c ontaining az inc/dipicolylamine complex moiety as ac oordinative phosphate binder,a s as tabilizer for RNA-based adjuvants.T he nanoformulated ssRNAa djuvant was resistant to enzymatic degradation in vitro and in vivo,a nd that with ac oordinative amphiphile bearing an oleyl group (CA-O)w as approximately 100 nm, promoted effective recognition, and improved activation of antigen-presenting cells,leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants,p roving useful to meet the urgent need for vaccines during pathogen outbreaks.