Gopal B. Inamati

Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays

The activity of synthetic antisense oligonucleotides (splicomers) designed to block pre‐mRNA splicing at specific exons has been demonstrated in a number of model systems, including constitutively spliced exons in mouse dystrophin RNA. Splicomer reagents directed to Duchenne muscular dystrophy (DMD) RNAs might thus circumvent nonsense or frame‐shifting mutations, leading to therapeutic expression of partially functional dystrophin, as occurs in the milder, allelic (Becker) form of the disease (BMD).

Electrochemical Detection of Surface Hybridization of Oligodeoxynucleotides Bearing Anthraquinone Tags at Gold Electrodes

Gold electrodes modified with a DNA 20-mer via a thiol linkage were shown to efficiently capture the complementary 20-mer that was tagged with anthraquinone (AQ) reporter groups. Treatment of the electrodes with 2-mercaptoethanol was required for both efficient surface hybridization and for reversible electron transfer between the gold electrode and the tethered anthraquinone tag. In addition, slow thermal annealing through the melting temperature of the duplex increased the efficiency of the process. Electrodes that had been modified with a ds-DNA bearing AQ tags gave slightly greater voltammetric signals, but after dissociation of the duplex were not as effective as the ss-DNA in capturing the complementary 20-mer from the buffer solutions.

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.

Improving antisense oligonucleotide binding to human serum albumin: Dramatic effect of ibuprofen conjugation

The interaction of antisense oligonucleotides with serum and cellular proteins determines their pharmacokinetic (transport to and distribution in target tissues) and pharmacodynamic (binding to the mRNA target) properties and hence their eventual pharmacology. In general, binding of drugs to serum albumin, 2-macroglobulin, immunoglobulins, and lipoproteins in the bloodstream governs their transport and tissue distribution. The first generation antisense compounds, 2 -deoxyphosphorothioate oligonucleotides (containing P S linkages, where R H and X S in Scheme 1), bind rapidly to serum and cellular

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.

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety1 has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oli...

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.