Charles John Guinosso

Chemical Modifications to Improve Uptake and Bioavailability of Antisense Oligonucleotides

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

Novel functionalization of the sugar moiety of nucleic acids for multiple labeling in the minor groove

Abstract The sugar ring of adenosine was alkylated at the 2′-O- position using N-(5-bromopentyl)phthalimide and the modified nucleoside was chemically incorporated into oligonucleotide diesters, thioates and RNA analogs. The free amino group, available after oligonucleotide deprotection, is useful for site-specific introduction of reporter groups and therapeutic agents at the 2′-position. This novel methodology permits multiple labeling of nucleic acids in the minor groove.