Chemical Modifications to Improve Uptake and Bioavailability of Antisense Oligonucleotides M. MANOHARAN: L. K. JOHNSON, D. P. C. McGEE, C. J. GUINOSSO, K. RAMASAMY, R. H. SPRINGER, C. F. BENNETT, D. J. ECKER, T. VICKERS, L. COWSERT, AND P. D. COOK Isis Pharmaceuticals 2280 Faraday Ave. Carlsbad, California 92008 The fate and function of antisense oligonucleotides are primarily controlled by their uptake and distribution in the cell.’ However, the efficiency of uptake is hampered by the negative charge on the backbone and also by the hydrophilic properties of oligonucleotides. Solutions to the uptake problem would be the modification of the antisense oligonucleotide to include: (1) hydrophobic moieties, (2) cationic modifications to overcome charge effects, (3) cell receptor binding molecules, and (4) amphipathic modifications having one or more of the foregoing properties. We have initiated chemical modification^^*^ aimed at improving uptake of antisense oligonucleotides using these guidelines, and our preliminary results are summarized here. To confer hydrophobicity to oligonucleotides, cholic acid was activated and conjugated to oligonucleotide DNA phosphodiesters, phosphodiester RNA mimics (2’-OMe analogs) and phosphorothioates at either the 5’ or the 3’ end using the appropriate aminolinker (FIG.1). In evaluating hybridization properties of cholic acid-conjugated oligonucleotides we observed that these conjugates did not affect the melting temperature of the parent oligomers against both DNA and RNA. Moreover, in the case of diesters, the 3’ conjugation offered significant nuclease stability in fetal calf serum (half-life > 24 hours). Thus, the conjugated diesters had a lifetime similar to that of unmodified thioates. Uptake was monitored either by fluorescent microscopy of the oligonucleotides in cells assessing subcellular distribution or by cellular activity in measuring protein synthesis. Fluorescent microscopy shows cellular localization of oligonucleotides, and protein synthesis assays with nonfluorescent conjugates showed the ultimate performance of these antisense oligonucleotides. Fluorescein was attached to the 5’ end of the oligonucleotide, whereas cholic acid was attached to the 3‘ end. Oligonucleotides targeted against human intercellular adhesion molecule-1 (ICAM-l), human immunodeficiency virus (HIV-l), and bovine papillomavirus (BPV-1) were used to study the effects of cholic acid conjugation on antisense activity. The ICAM-1 and BPV-1 oligos were 2‘-deoxy phosphorothioates, whereas the antisense HIV-1 oligos were 2’-O-methyl phosphodiesters. With ICAM-1, we observed localization of cholic acid-conjugated oligonucleotides in the cytoplasm by the fluorescent tag. In the protein synthesis assay, the conjugate did not change the potency of the parent oligonucleotide. However, in the ‘To whom correspondence should be addressed. 306

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presence of excess amounts of a cationic lipid, N-[ 1-(2,3-dioIeyloxy)propyl-N,N,Ntrimethylammoniumchloride (DOTMA),the conjugate was localized in the nucleus, and the cellular fluorescent intensity was enhanced. Also, protein synthesis was significantly reduced. Our control experiments prove that the antisense effects observed are sequence specific.

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The HIV-1 and BPV-1 oligonucleotides were evaluated in transactivation assays. Preliminary results indicate some uptake enhancement for the cholic acid conjugates. In BPV-1 assays, the 3‘ conjugate performed better than did the 5’ conjugates. In HIV-1 assays, there is approximately a threefold enhancement of activity for a 5’ conjugated oligomer and a twofold enhancement for a 3’ conjugated oligomer. These experiments are being repeated with increased pre-incubation time during the assays. In an alternate approach to chemically modify oligonucleotides, nucleosides were synthesized by incorporating alkyl groups and alkylamino groups either in the 2’ position of sugars or at the N2 position of purines. ICAM-1 oligonucleotides were synthesized, incorporating these modifications either in a site-specific manner in the sugar ring of adenosines (2’-O-nonyls and 2’-O-pentylamine~)~.~ and N2 position of guanosine (nonyls) or in a uniform fashion (2’-O-propyls and 2‘-0 -pentyls). (FIG.2). Evaluation of these modified oligonucleotides for uptake enhancement is in progress: TG*G GAG CCA TAG CGA GG*C (thioate, purine modification) TGG GA*G CCA* TA*G CGA* GGC (thioate, sugar modification in adenosine) UGG GAG CCA UAG CGA GG dC (thioate, uniform 2’-sugar modification).

REFERENCES 1. COOK,P. D. 1991. Anti-Cancer Drug Design 6 585 (see also references cited therein). 2. GUINOSSO, C. J., G. D. HOKE,S. FRIER, J. F. MARTIN, D. J. ECKER, C. K. MIRABELLI, S. T. CROOKE & P. D. COOK.1991. Nucleosides Nucleotides 1 0 259. 3. MANOHARAN, M., C. J. GUINOSSO & P. D. COOK.1991. Tetrahedron Lett. 32: 7171.