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We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1−/−) and FcRn knockout (FcRn−/−).

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

Interactions of chemically modified nucleic acid therapeutics with plasma proteins play an important role in facilitating distribution from the injection site to peripheral tissues by reducing renal clearance. Despite the importance of these interactions, analytical methods that can characterize binding constants with individual plasma proteins in a reliable and high throughput manner are not easily available. We developed a fluorescence polarization (FP) based assay and measured binding constants for the 25 most abundant human plasma proteins with phosphorothioate (PS) modified antisense oligonucleotides (ASOs). We evaluated the influence of sequence, sugar modifications, and PS content on ASO interactions with several abundant human plasma proteins and determined the effect of salt and pH on these interactions. PS ASOs were found to associate predominantly with albumin and histidine-rich glycoprotein (HRG) in mouse and human plasma by size-exclusion chromatography. In contrast, PS ASOs associate predominantly with HRG in monkey plasma because of higher concentrations of this protein in monkeys. Finally, plasma proteins capable of binding PS ASOs in human plasma were confirmed by employing affinity chromatography and proteomics. Our results indicate distinct differences in contributions from the PS backbone, nucleobase composition and oligonucleotide flexibility to protein binding.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

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.

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

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.

A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5'-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5'-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5'-triantennary GalNAc cluster conjugated ASOs and helps understand the structure-activity relationship for targeting hepatocytes with oligonucleotide therapeutics.

The pharmacokinetics of a 2'-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-alpha 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 (< or = 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.

We evaluated the abilities of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), and a single-stranded siRNA (ss-siRNA) to inhibit expression from the PTEN gene in mice when formulated identically with lipid nanoparticles (LNPs). Significantly greater reductions in levels of PTEN mRNA were observed for LNP-formulated agents compared to unformulated drugs when gene silencing was evaluated after a single dose in the livers of mice. An unformulated ss-siRNA modified with a metabolically stable phosphate mimic 5'-(E)-vinylphosphonate showed dose-dependent reduction of PTEN mRNA in mice, albeit at doses significantly higher than those observed for formulated ss-siRNA. These results demonstrate that LNPs can be used to deliver functional antisense and ss-siRNA therapeutics to the liver, indicating that progress in the field of siRNA delivery is transferable to other classes of nucleic acid-based drugs.