Lex M. Cowsert

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

Cholic acid-oligonucleotide conjugates for antisense applications

Abstract To improve the pharmacokinetics of antisense oligonucleotides, cholic acid was conjugated to several biologically active sequences. Physical and biological properties of the conjugates were examined. While the cholic acid conjugation changes the lipophilicity of the oligonucleotides, the in vitro biological activity observed was dependent on the disease target, cell lines and the method of oligonucleotide treatment.

5′ Phospholipid Phosphatase SHIP-2 Causes Protein Kinase B Inactivation and Cell Cycle Arrest in Glioblastoma Cells

ABSTRACT The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)–protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G1 in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27KIP1. Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

Bovine Serological Response to a Recombinant BPV-1 Major Capsid Protein Vaccine

Four of five groups of Holestine by Angus calves (5 calves/group) were immunized with different formulations of a recombinant BPV-1 DNA vaccine using a BPV-1 major capsid:B-galactosidase fusion protein as the immunogen. Group 5 was not vaccinated. Vaccinated calves received the vaccine on days 0 and 21 of the trial, and calves from all five groups were challenged intradermally with 10(10) BPV-1 particles at each of two different sites on day 56. All calves were bled on days 3, 24, 55, 77, and 104 of the trial, and the sera were tested for reactivity with intact and disrupted BPV-1 particles by ELISA. At the time of challenge with BPV-1 virions (day 56), 19 of 20 vaccinated calves were seropositive for disrupted BPV-1 particles; sera from 3 of 20 calves reacted with intact BPV-1 virions. By day 77, 11 of 19 vaccinated calves had developed antibody titers to intact BPV-1 virions; only 1 calf in group 5 developed antibodies (transiently) against BPV-1 capsid epitopes. After challenge, 24 of 25 calves from the five groups developed intradermal fibromas, the biological end point of this study. Fibromas appeared to increase in size in group 5 (unvaccinated, inoculated controls), whereas most tumors from the four vaccinated groups (1-4) stabilized or decreased in size. Although the calves developed fibromas, 90% of calves (in groups 1-4) developed antibodies against disrupted BPV-1 capsid proteins whereas 58% developed antibodies that reacted with intact virions. The immunologic response of vaccinated calves to intact and disrupted BPV-1 particles appeared to be determined in large part by the various formulations of the vaccine, particularly the adjuvant.

A novel drug screening assay for papillomavirus specific antiviral activity

Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.

Identification of linear epitopes of the BPV-1 L1 protein recognized by sera of infected or immunized animals

Sera from cattle that had been inoculated with BPV-1 virions or with recombinant L1 proteins and serum from a rabbit that had been immunized with SDS-denatured virions were evaluated for their reactivity with 466 overlapping synthetic peptides corresponding to 95% of the BPV-1 L1 protein. The late serological response of cattle to both intact virions and recombinant L1 proteins exhibited a similar profile of reactivity with approximately 70% (7 of 10) of L1 antigenic sites. However, the L1 serological response of the rabbit to SDS-denatured virions exhibited a significant difference from bovine serum antibodies in the profile of epitopes recognized, including a relative lack of response to major bovine epitopes located between L1 amino acids (AAs) 300-400. Importantly, only the sera from animals inoculated/immunized with intact virions was capable of neutralizing BPV-1 infectivity of murine C127 cells, suggesting that nonlinear epitopes are important for papillomavirus neutralization.

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...