Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2′-O-Et-2′,4′-bridged nucleic acid) gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA. We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin. Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-␣ antisense), have been characterized in mouse, rat, dog, monkey, and human. Plasma pharmacokinetics after i.v. administration exhibited relatively rapid distribution from plasma to tissues with a distribution half-life estimated from approximately 15 to 45 min in all species. Absorption after s.c. injection was high (80-100%), and absorption after intrajejunal administration in proprietary formulations was as high as 10% bioavailability compared with i.v. administration. Urinary excretion of the parent drug was low, with less than 1% of the administered dose excreted in urine after i.v. infusion in monkeys at clinically relevant doses (<5 mg/ kg). ISIS 104838 is highly bound to plasma proteins, likely preventing renal filtration. However, shortened oligonucleotide metabolites of ISIS 104838 lose their affinity to bind plasma proteins. Thus, excretion of radiolabel (mostly as metabolites) in urine (75%) and feces (5-10%) was nearly complete by 90 days. Elimination of ISIS 104838 from tissue was slow (multiple days) for all species, depending on the tissue or organ. The highest concentrations of ISIS 104838 in tissues were seen in kidney, liver, lymph nodes, bone marrow, and spleen. In general, concentrations of ISIS 104838 were higher in monkey tissues than in rodents at body weight-equivalent doses. Plasma pharmacokinetics scale well across species as a function of body weight alone. This favorable pharmacokinetic profile for ISIS 104838 provides guidance for clinical development and appears to support infrequent and convenient dose administration.