Modified Antisense Oligonucleotides Directed against Tumor Necrosis Factor Receptor Type I Inhibit Tumor Necrosis Factor α-Mediated Functions

Abstract

Tumor necrosis factor alpha (TNF alpha), a polypeptide produced by activated macrophages, is a highly pleiotropic cytokine which elicits inflammatory and immunological reactions. The binding of TNF alpha to tumor necrosis factor receptor type I (TNFRI) is considered the initial step responsible for some of the multiple biological functions mediated by TNF alpha. The role of TNF alpha as an inflammatory mediator through human TNFRI makes TNFRI an attractive target for intervention in both acute and chronic inflammatory diseases. In this study, we have identified partial phosphorothioate oligodeoxyribonucleotides (ODNs) containing C-5 propynyl or hexynyl derivatives of 2'-deoxyuridine which specifically inhibited TNFRI and subsequently inhibited the functions of TNF alpha mediated through TNFRI. The most active ODNs were directed against the 3'-poly adenylation signal site on the TNFRI mRNA, and in a cellular assay, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations, in the presence of Cellfectin. The inhibition of gene expression correlated with the binding affinity of the ODN for the target mRNA. The ODNs lowered TNFRI protein levels and TNF alpha-mediated functions by specifically reducing levels of TNFRI mRNA. These anti-TNFRI ODNs offer a novel approach for controlling biological functions of TNF alpha and may be useful as human therapeutic agents for treating diseases in which TNF alpha has been implicated.

TNFα as Therapeutic Target: New Drugs, More Applications

“…The latter approach has been utilized by the study of modified antisense oligonucleotides to TNFα RI that inhibit the receptor and therefore reduce signaling mediated by TNFα [73]. The compounds were partial phosphorothioate oligodeoxyribonucleotides containing C-5 propynyl or hexynyl derivatives of 2'-deoxyuridine, and the most active ones targeted the 3'-polyadenylation signal on the TNFαRI mRNA.…”

Oligodeoxynucleotide Targeted to the αv Gene Inhibits αv Integrin Synthesis, Impairs Osteoclast Function, and Activates Intracellular Signals to Apoptosis

“…After cleavage from the solid support and final deprotection by treatment with concentrated ammonia, ODNs were purified by polyacrylamide gel electrophoresis. The C‐ 5 propynyl pyrimidine–modified ODNs were prepared as described previously (29) . All ODNs were analyzed by negative ion electrospray mass spectroscopy (Bio‐Q; Fisons, Danvers, MA, U.S.A.), which in all cases confirmed the calculated mass.…”

Inhibition of gene expression by anti-sense C-5 propyne oligonucleotides detected by a reporter enzyme

“…The resulting C-5 propyne oligophosphorothioate, 17AS PS.P, bound to the 19T PO with greater affinity than the 17AS PO oligophosphodiester ( Table 2). These melting experiments showed that the C-5-propyne modification stabilized the binding of phosphorothioates to the DNA target, probably by increasing the stacking interactions of the bases [13,42]. Therefore the C-5propyne modification should stabilize the binding of phosphorothioates to the IGF-1 RNA target.…”

“…Short oligophosphorothioates containing C-5 propyne pyrimidines might represent a good alternative to oligophosphorothioates. Phosphorothioate oligonucleotides containing C-5-propynyl-2h-deoxyuridine (pdU) and C-5-propynyl-2h-deoxycytidine (pdC) have been shown to be potent and specific antisense inhibitors of protein expression such as simian virus 40 (SV40) TAg [5][6][7], p34(cdc2) kinase [8], cyclin B1 [8], p27kipl [9,10], HIV en [11], HIV re [12], tumour necrosis factor α [13], Escherichia coli β-galactosidase [5] and firefly (Photinus pyralis) luciferase [14,15] in various cellular systems. In most of these studies, C-5 propyne oligophosphorothioates were introduced together with their plasmid targets in cells by nuclear microinjection to compensate for their poor cellular uptake.…”