Kathleen L. Tivel

Oligonucleotide Conjugates: Alteration of the Pharmacokinetic Properties of Antisense Agents

Abstract Cholic acid, cholesterol, several polyamines and polyethylene glycols were conjugated to antisense oligonucleotides targeted to human or murine intercellular adhesion molecule-1 (ICAM-1) mRNA to study their effects on cellular absorption.

Introduction of a lipophilic thioether tether in the minor groove of nucleic acids for antisense applications

Abstract A 2′- O -hexylthiotrityl adenosine phosphoramidite has been synthesized and incorporated into oligonucleotide phosphodiesters and phosphorothioates. This tether has potential antisense applications, offers a convenient nucleophile for conjugation of various moieties that would reside in the minor groove, and may form novel tertiary structures.

Lipidic nucleic acids

Abstract Lipophilic nucleosides were synthesized from uridine via either a 2′- O -hexylamino tether or the corresponding 3′-linker. These synthons allowed site-specific incorporation of the lipophilic moieties into antisense oligonucleotides. A spectrum of lipophilicity was observed in the oligonucleotides, depending on the pendent group.

2′-O- and 3′-O- pyrimidine aminotether-containing oligonucleotides: Synthesis and conjugation chemistry

Abstract Nucleoside synthons carrying an aminohexyl moiety tethered at the 2′- O - or 3′- O - positions of uridine have been synthesized and incorporated into oligonucleotides. The aminohexyl tether was used to conjugate functionalities such as intercalators, nucleic acid alkylators and absorption-modifying agents. The 2′-modification allows conjugations in the minor groove in the normal 3′-5′-linked oligomers; the 3′-modification allows synthesis of 2′-5′-connected oligonucleotides with a built-in nucleophile. This structural motif has the potential to build novel lariat RNAs and ribozymes.

Conjugated Antisense Oligonucleotides

Abstract We have employed chemical modification strategies to improve cellular ahsorption of oligonucleotides. These include the conjugation of various pendant moieties to the oligonucleotide to affect its overall physical properties such as hydrophobicity, charge, and amphipathicity as well as pendants that may mediate absorption by binding to certain cellular receptors which internalize specific ligands. Our laboratory has prepared polyamines, polyethylene glycols and lipidic constituents conjugated to oligonucleotidcs in order to study their effects in enhancing absorption of antisense agents. These conjugates were targeted against human or murine Intercellular Adhesion Molecule- 1 (ICAM-1) mRNA.

2′- and 3′- Cholesterol-Conjugated Adenosine and Cytosine Nucleoside Building Blocks: Synthesis of Lipidic Nucleic Acids

Abstract Recently our laboratory introduced1 chemistries to synthesize 2′- and 3′- cholesteroluridine conjugates which were incorporated into several antisense oligonucleotides. We have now extended this chemistry to other nucleosides (adenosine and cytosine) and synthesized antisense oligonucleotide conjugates for several disease targets. Synthesis of these cholesterol nucleosides was carried out hy condensing choleskrol chloroformate with 2′-O-alkylamine or 3′-O-alkylamine of the appropriate nucleoside. The 2′-O-alkylamines were deiived from direct alkylation procedure.

2′- and 3′- Biotin Conjugated Nucleoside Building Blocks: Synthesis of Biotinylated Oligonucleotides

Abstract The vitamin biotin plays a significant role in biological assays based on its unusually high affinity [KD=10−15M] to streptavidin and avidin. This assay can be used for monitoring cellular trafficking of antisense oligonucleotides using hiotin conjugation. In addition to the above diagnostic application, biotin conjugation to macromolecules could be used as a vitamin-mediated delivery system for macromolecules into cells. Complexation of avidin to hiotin-oligonucleotides (phosphodiesters or PNA) have been used to enhance the uptake of oligonucleotides1. Appropiiate placement of biotin in oligonucleotides could also provide increased nuclease resistance.

Cholesterol Conjugated Uniform and Gapmer Phosphorothioate Oligonucleotides Targeted Against PKC-α and C-raf Gene Expression

Abstract In vitra and in vivo antitumor activity of phosphorothioate antisense oligonucleotides targeted against two protein kinases within the mitogen-activated protein (MAP) kinase signaling cascade has been well documented by ISIS 3521/CGP 6412XA (targeted against PKC-α protein) and ISIS 5132KGP69846A (targeted against C-rwfl kinase). For both of these compounds, cationic lipid formulations are necessary to observe any pharmacological activity in cell culture. In contrast, in vivo functional delivery of phosphorothioate oligonucleotides to cells in tissues does not appear to be a prohlem. These oligonucleotides have demonstrated reduction in either PKC-α or C-raf gene expression in tissues or human tumor xenografts following systemic administration.

Modification of a Phosphorothioate Oligonucleotide with Cholesterol Induces Association of the Oligonucleotide to Serum Lipoproteins and Affects Its Biological Fate

Abstract A cholesterol-conjugated phosphorothioate ICAM-1 antisense oligo-nucleotide was evaluated for its binding to lipoproteins and its biodistribution. Our study indicates that the conjugate behaves differently from the parent compound.