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)XXX3-5GGG 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 G-modification. 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 TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.
Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor ...delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic ...acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5
mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50
cm neutrally coated capillary at 60
°C together with a 7
M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions ≥1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.
Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl‐deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving ...Toll‐like receptor 9 (TLR9). We have investigated the structure–activity relationship (SAR) of base‐modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20‐mer oligonucleotides having just a single human recognition motif (5′‐GTCGTT‐3′) in functional cell‐based TLR9 assays. Substitution of guanine by hypoxanthine and 6‐thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2‐aminopurine, 2,6‐diaminopurine, and 8‐oxo‐7,8‐dihydroguanine substitution resulted in approximately 40–60 % reduction in activity, and 7‐deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species‐specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.
Activation of Toll‐like receptors (TLRs) results in stimulation of the innate immune response, including secretion of proinflammatory cytokines, up‐regulation of co‐stimulatory molecules, and secretion of cytokines and chemokines. In our SAR model, the CpG dinucleotide motif of immune stimulatory oligodeoxynucleotides is recognized by TLR9 by the Hoogsteen site of guanine and the C5 corner of cytosine resulting in potent stimulation of the innate immune response.
Small noncoding RNAs (ncRNAs) have been shown to guide epigenetic silencing complexes to target loci in human cells. When targeted to gene promoters, these small RNAs can lead to long-term stable ...epigenetic silencing of gene transcription. To date, small RNAs have been shown to modulate transcriptional gene silencing (TGS) of human immunodeficiency virus type 1 (HIV-1) as well as several other disease-related genes, but it has remained unknown as to what extent particular chemistries can be used to generate single-stranded backbone-modified oligonucleotides that are amenable to this form of gene targeting and regulation. Here, we present data indicating that specific combinations of backbone modifications can be used to generate single-stranded antisense oligonucleotides that can functionally direct TGS of HIV-1 in a manner that is however, independent of epigenetic changes at the target loci. Furthermore, this functionality appears contingent on the absence of a 5′ phosphate in the oligonucleotide. These data suggest that chemically modified oligonucleotide based approaches could be implemented as a means to regulate gene transcription in an epigenetically independent manner.
The preparation of novel monomethoxytrityl (Mmt) protected monomers for the synthesis of polyamide nucleic acids (PNAs) is described. The use of base-labile acyl-type nucleobase protecting groups and ...of a succinyl-linked solid-support offers a synthetic strategy similar to standard oligonucleotide synthesis conditions. This strategy has been successfully applied for the synthesis of PNAs of mixed base sequence.
The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other ...oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.
The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', ...4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl cET). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.
Oligonucleotide analogs are of major interest as tools in molecular biology, as diagnostics, and as potential pharmaceuticals which bind in a predictable way to certain nucleic acid target sequences, ...aiming at the inhibition of expression of disease-causing genes. One of the most promising nucleic acid mimetics are the peptide - or polyamide- nucleic acids (PNA) which bind with higher affinity to DNA and RNA than natural oligonucleotides. In these non-ionic PNAs, the entire sugar-phosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure. A unique property of PNA is its ability to displace one strand of a DNA double-helix. This strand displacement process, which is inefficient with DNA, is supported by the formation of an unusually stable internal (PNA)2 x DNA triple helix. The combination of PNA and DNA in one molecule results in PNA/DNA chimeras with new properties. They show improved aqueous solubility compared to pure PNAs due to their partially negatively charged structure. Furthermore, the cellular uptake of the chimeras is better than of pure PNAs. In contrast to PNA, the chimeras bind exclusively in the antiparallel orientation under physiological conditions. The binding affinity is generally stronger when the PNA/DNA chimeras are hybridized to RNAthan to DNA, whereby the strength of binding strongly depends on the PNA: DNA ratio. Most interestingly, PNA/DNA chimeras are recognized as substrates by various nucleic acid processing enzymes, and consequently can also assume biological functions, such as a primer function for DNA polymerases. Pure PNA cannot induce RNase H cleavage of target RNA, which is believed to support the biological efficacy of antisense agents. However, DNA-PNA chimeras are able to stimulate cleavage of the target RNA by RNase H upon formation of an RNA x chimera duplex.
Inhibitory TLR7 and/or TLR9 oligonucleotides (inhibitory oligonucleotide INH-ODN) are characterized by a phosphorothioate backbone and a CC(T)XXX₃₋₅GGG motif, respectively. INH-ODN 2088 is a ...prototypic member of this class of INH-ODN and acts as a TLR7 and TLR9 antagonist. It contains a G quadruple that leads to higher order structures by the formation of G tetrads. These structures are unfavorable for the prediction of their pharmacological and immunological behavior. We show in this study that modification of Gs within the G quadruple by 7-deaza-guanine or 7-deaza-2'-O-methyl-guanine avoids higher order structures and improves their inhibitory potential. Whereas TLR9-induced TNF-α secretion of bone marrow-derived macrophages and conventional dendritic cells was equally inhibited by INH-ODN 2088 and G-modified INH-ODNs such as INH-ODN 24888, TLR7-induced TNF-α release and TLR7- and TLR9-induced IL-12p40 release were significantly more impaired by G-modified INH-ODNs. Similarly, the IL-6 release of B cells from wild-type and autoimmune MRL/Mp-lpr/lpr mice was more efficiently impaired by G-modified INH-ODNs. Surprisingly, INH-ODN 2088 stimulated B cells to proliferate when used in higher doses. Finally, in vivo, in wild-type and autoimmune MRL/Mp-lpr/lpr mice, G-modified INH-ODN 24888 was significantly more efficient than unmodified INH-ODN 2088. In summary, G modification allows the development of INH-ODNs with superior inhibitory potency for inflammatory diseases with high medical need such as systemic lupus erythematosus.
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