Phillip Dan Cook

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

MAKING DRUGS OUT OF OLIGONUCLEOTIDES : A BRIEF REVIEW AND PERSPECTIVE

I provide a brief review and perspective thoughts concerning the antisense oligonucleotide, drug discovery paradigm.

Synthesis and Biological Evaluation of Antisense Oligonucleotides Containing Modified Pyrimidines

Abstract Antisense oligodeoxynucleotides (ODNs) containing certain pyrimidines modified at the 5 and/or 6-position(s) of the heterocycle were synthesized. The ODNs were evaluated for their resistance to nuclease degradation in serum, hybridization properties, and ability to activate RNase H.

Purine Based Combinatorial Chemistry: Solution Phase Simultaneous Addition of Functionalities. Iterative Deconvolution by Orthogonal Protection to a Single Compound with Potent Antibacterial Activity

Abstract We have recently described a method to prepare combinatorial chemistry libraries by solution phase simultaneous addition of functionalities (SPSAF).1–2 SPSAF has been used to create libraries based on the purine heterocycle. The nucleophilic sites (secondary nitrogens) in the planer heteroaromatic purine scaffold were built in via linkers. Thus, to continue the use of electophilic functionalities, as in previous libraries, a bifunctional nucleophilic linker was required. Piperazines readily served this purpose. Nucleophilic displacement of the chloro groups on 2,6-dichloropurine with piperazines provides reactive, constrained secondary amines for combinatorialization (Figure 1, 1). An additional piperazine was placed in the 9-position by alkylation of 2,6-dipiperazinylpurine. In this manner, the functionality that differentiates each pool (sublibrary) could be placed last in the synthetic scheme (fix last concept).1

MMI linkage modification increases potency and stability of H-ras antisense oligonucleotides.

Abstract Phosphorothioate antisense oligodeoxyribonucleotides (PS-ASOs) have proven to be useful first generation antisense tools for in vitro and in vivo uses and now show great promise as human therapeutic agents. However, there are two characteristics of PS-ASOs that make it desirable to continue to attempt to improve their biophysical characteristics through chemical modification. First, PS-ASOs have been reported, at very high concentrations, to have some nonspecific activities, both in vitro and in vivo, usually attributed to their protein binding properties. Second, while significantly more stable than their phosphodiester analogues, the in vivo stability of phosphorothioate oligonucleotides can still be improved. This instability is primarily due to 3′ exonucleases, 5′ exonucleases, and to a lesser degree, endonucleases. There is a strong rationale for exploring backbone modifications that can reduce the P=S content and maintain or increase nuclease resistance of antisense oligonucleotides. One su...