Nucleic Acids D. Conjugate Groups 4. Properties of Modified Oligonucleotides and Conjugates A. The Effect of Modification on Oligonucleotide Hybridization B. The Effect of Modification on Nuclease Resistance C. Cellular Uptake of Modified Oligonucleotides 1043-1802/90/2901-0165S02.50/0 D. Modified Antisense Oligonucleotides 5. Concluding Comments

The possibility ofusing oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-Ill) [the etiological agent of acquired imnunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P-labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynudeotides at 20 pM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 pM. Oligodeoynudeotides complementary to a region close to the tRNAL9 primer binding site on HTLV-M RNA and others complementary to HTLV-I mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynudeotides suggests that the antiviral effects were specific. Although these esults pertain to HTLV-I-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynudeotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex.

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus 1 (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The Fig. 1. All the modified oligonucleotides were found to be resistant to nucleases such as snake venom or spleen phosphodiesterases. amounts of material for toxicity studies were made by a manual solid-support method on an Omnifit assembly (13). Unmodified oligomers were made and purified by methods described previously (2). Phosphorothioates and phosphoramidates were synthesized by a modification of the Hphosphonate procedure (14,15). To generate phosphorothioate linkages, oxidation following the final coupling and detritylation was replaced by treatment with 0.1 M sulfur in carbon disulfide/triethylamine (9:1, vol/vol) at room temperature for up to 2 hr, depending on chain length (16). For the phosphoramidates, this step was replaced by treatment with a 10% solution of the appropriate amine in carbon tetrachloride for up to 1.5 hr, depending on chain length (16).After deblocking in concentrated ammonium hydroxide (550C for 51/2 hr or room temperature for >2 days), products were purified on 2-mm-thick preparative layer plates (Merck silica gel 60) in propanol/water/concentrated ammonium hydroxide (55:35:10, vol/vol). Phosphorothioates were further purified on DEAE-cellulose and C18 silica (details will be published elsewhere). Phosphoramidates from preparative layer plates were passed through Sephadex G-25 in aqueous 30% (vol/vol) ethanol and then through C18 silica and dialyzed. In all cases, small amounts of oligomers attached to the solid support were taken before the final treatment with sulfur or amine and were oxidized with 2% iodine in pyridine/water (98:2, vol/vol) (15) to phosphodiesters. These were used for determination of base composition by enzymatic degradation to nucleosides followed by HPLC. The phosphorothioates were also characterized by polyacrylamide gel electrophoresis, where they had similar mobility to the corresponding diesters.Phosphoramidates were further characterized by hydrolysis with formic acid to the phosphodiesters (16) for comparison with authentic samples on HPLC and degradation to nucleosides for determination of nucleoside composition and integrity by HPLC. A useful indicator of oligonucleotide purity and integrity is the melting curve of its duplex with a complementary DNA Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome. 7079The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

authors request that the following correction be noted. In both papers, the statement that toxicity studies in mice were performed using compound at a concentration of 640 ,ug/ml is incorrect. In all cases, various concentrations of oligodeoxynucleotides were in a volume of 1 ml of pyrogenfree distilled water.

Antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages were examined for their ability to inhibit human immunodeficiency virus (HIV)-induced syncytium formation and virus expression. HIV inhibitory activity was found to be dependent on both chain length and the number of phosphonate residues. Introduction of 18 phosphonate groups in an oligomer of chain length 20 significantly increased HIIV inhibitory activity relative to the parent oligonucleotide, whereas 5 such groups showed little or no increase in the HIV inhibition capacity. Methylphosphonate-linked oligomers are more stable to nuclease degradation and hence could be potentially useful in the treatment of acquired immunodeficiency syndrome.With the identification of human immunodeficiency virus (HIV) type 1 as the etiological agent of acquired immunodeficiency syndrome (AIDS), a number of approaches are being explored to develop therapeutic agents for the treatment of AIDS and AIDS-related complex (1). A molecular approach to inhibit HIV-1 replication involves the use of complementary antisense oligonucleotides that can selectively bind to defined sequences of viral RNA. Antiviral activity of specific antisense oligonucleotides was first observed in studies with Rous sarcoma virus (2, 3). The antisense oligomer approach has since been applied to inhibit a number of RNA and DNA viruses including HIV (4-6), vesicular stomatitis virus (7-9), herpes simplex virus type 1 (7, 10), and influenza virus (11). Attempts have been made to improve the activity ofantisense oligonucleotides by increasing their stability to nucleases, their cellular uptake, and their hybridization with complementary RNA. These studies have involved replacing the internucleoside phosphates by methylphosphonates (7,10), phosphorothioates (5), or phosphoramidates (22) or by modifying the ends of the molecule (2, 4, 6, 9, 11). In this paper, we show that methylphosphonate analogues are potent inhibitors of HIV-1 replication in cell culture and, with their added resistance to nucleases, could be potentially useful in the treatment of AIDS. MATERIALS AND METHODSChemical Synthesis. Oligonucleotides were synthesized by using an automated DNA synthesizer (Biosearch model 8600, San Rafael, CA). Phosphodiester bonds were constructed by using the standard phosphoramidite protocol of the manufacturer except that the oxidation step was performed before the capping reaction. Methylphosphonate linkages were assembled from nucleoside methylphosphonamidites by using the method previously reported (12). Oligonucleotides were cleaved from the support in concentrated ammonium hydroxide at room temperature for 2 hr. The supernatant was taken to dryness, and the acyl protecting groups were removed by treatment with ethylenediamine/ethanol, 1:1 (vol/vol) at room temperature for 4 hr. Solvent was evaporated under reduced pressure followed by coevaporation of two additional aliquots of ethanol.Oligonucleotides with mostly phosphodiester linkages were purified in the same way...