Chemically modified oligonucleotides exhibit decreased immune stimulation in mice

Phosphorothioate oligodeoxynucleotides produce splenomegaly and mononuclear cell infiltrates in multiple organs in mice after repeated i.v. administra tion. Several phosphorothioate oligodeoxynucleotides were studied to better understand the basis of immunostimulatory properties of these molecules in mice and to study the effects of chemically modified oligonucleotides. Che mical modifications examined included 5-methyl cytosine and 2'-methoxyethox y substituents. Male mice (six per group) were treated with oligonucleotide concentrations of 0, 2, 10, or 50 mg/kg by i.v. injection every other day for 14 days. Immune stimulation was assessed 24 h after the last dose by me asuring spleen weight, or histologic and immunohistochemical examination of liver and kidney. Immune stimulation was dose-dependent for the phosphorot hioate oligodeoxynucleotides studied, but potency varied as a function of s equence. Results from this study reveal that there is a close correlation b etween the extent of splenomegaly and other evidence of immune stimulation, such as the severity of cell infiltrates in liver and kidney in mice. Immu nohistochemical analysis indicated that cell infiltrates in liver and kidne y were primarily mononuclear cells associated with increased expression of the endothelial-leukocyte cellular adhesion molecule intracellular adhesion molecule-1 and the cytokine interleukin-6. Immune stimulation was markedly decreased with oligonucleotides containing the 5-methyl cytosine and furth er decreased by 2'-methoxyethoxy modifications. Administration of these mod ified oligonucleotides to mice did not produce splenomegaly even at the 50- mg/kg dose, and only produced minimal cell infiltrates despite the presence of comparable or greater tissue oligonucleotide concentrations. Thus, chem ical modifications appeared to increase the tolerability profile for these compounds that are representative of the second generation of antisense oli gonucleotides.