Antisense oligonucleotides are being evaluated in clinical trials as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have designed mixed-backbone oligonucleotides (MBOs) that contain phosphorothioate segments at the 3 and 5 ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligonucleotide. Some of these MBOs indicate improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H activation, and anti-HIV activity. In addition, more acceptable pharmacological, in vivo degradation and pharmacokinetic profiles were obtained with these MBOs.Synthetic oligonucleotides (oligos) complementary to messenger RNA have shown promise as therapeutic agents for various diseases (1). Phosphorothioate oligodeoxynucleotides (PSoligos) have been studied intensively in both in vitro and in vivo models, and clinical trials in humans are underway (2).Although PS-oligos continue to show promising results as first generation antisense oligos, they have certain limitations (2). Toxicity studies of PS-oligos in mice, rats, monkeys, and humans have shown some dose-dependent side effects (2). In mice and rats, these side effects include thrombocytopenia, elevation of liver transaminases, hyperplasia of reticuloendothelial cells in various organs, and renal tubular changes (2, 3). In monkeys, the side effects observed are activation of complement (4) and prolongation of activated partial thromboplastin time (aPTT; refs. 2 and 5). These side effects may be caused by the PS-oligos or by their in vivo metabolites. Because similar side effects have been observed after administration of dextran sulfate (2, 6), the inference is that these side effects are caused by the polyanionic nature of PS-oligos and are not nucleotide-sequence-specific. In addition, certain conditions (e.g., splenomegaly) may be caused by a sequence-dependent mitogenic response leading to cytokine induction (2, 7). The aim of the present study was to design less polyanionic oligos that would still be biologically active as antisense agents but less immune stimulatory than the original oligos. A related aim was to retain control of degradation in vivo and therefore generate a favorable pharmacological and safety profile.
MATERIALS AND METHODSSynthesis and Analysis of Oligonucleotides. The structures of oligos used in the present study are illustrated in Table 1. The synthesis of oligos was carried out on a 15-mol scale (Biosearch 8900 DNA Synthesizer) or on a 1-mmol scale (Pharmacia OligoPilot II Synthesizer) using appropriate nucleoside phosphoramidites (8). Oligos 1, 9, 10, and 11 were synthesized and purified as reported (8). Oligos 2, 4, 6, 8, and 15 were synthesized in a three-step reaction sequence. First, the 3Ј end was prepared using deoxynucleoside phosphoramidites. The central region was next synthesized using 2Ј-Omethylribonucleoside phosphoramidites, and finally, the 5Ј end was prepared from deoxyn...
