Guanine modification of inhibitory oligonucleotides potentiates their suppressive function

J Immunol. 2013 Sep 15;191(6):3240-53. doi: 10.4049/jimmunol.1300706. Epub 2013 Aug 21.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / drug effects
  • B-Lymphocytes / immunology
  • Cell Proliferation / drug effects
  • Cytokines / biosynthesis
  • Dendritic Cells / drug effects
  • Dendritic Cells / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Guanine / chemistry*
  • Immune Tolerance / drug effects*
  • Immune Tolerance / immunology
  • Macrophages / drug effects
  • Macrophages / immunology
  • Membrane Glycoproteins / antagonists & inhibitors
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Inbred MRL lpr
  • Oligonucleotides / chemistry*
  • Oligonucleotides / pharmacology*
  • Toll-Like Receptor 7 / antagonists & inhibitors
  • Toll-Like Receptor 9 / antagonists & inhibitors

Substances

  • Cytokines
  • Membrane Glycoproteins
  • Oligonucleotides
  • Tlr7 protein, mouse
  • Tlr9 protein, mouse
  • Toll-Like Receptor 7
  • Toll-Like Receptor 9
  • Guanine