Single-stranded siRNAs activate RNAi in animals.

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.

Mechanisms of palmitic acid-conjugated antisense oligonucleotide distribution in mice.

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-/-).

Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay.

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.

Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.

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.

Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.

The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.

Solid-phase synthesis of 5'-triantennary N-acetylgalactosamine conjugated antisense oligonucleotides using phosphoramidite chemistry.

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.

Targeted Delivery of Antisense Oligonucleotides Through Angiotensin Type 1 Receptor.

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.

Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins.

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H(2)O(2)), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl(-) secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H(2)O(2) inhibit Ca(2+)-dependent Cl(-) secretion across T(84) colonic epithelial cells by elevating cytosolic Ca(2+), which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca(2+)-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca(2+)-dependent stimuli is mediated in part by K(+) efflux through basolateral K(+) channels and Cl(-) uptake by the Na(+)-K(+)-2Cl(-) cotransporter, NKCC1. We demonstrate that H(2)O(2) inhibits Ca(2+)-dependent basolateral K(+) efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl(-) conductance. Thus, we have demonstrated that H(2)O(2) inhibits Ca(2+)-dependent Cl(-) secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.

5-Hydroxytryptamine contributes significantly to a reflex pathway by which the duodenal mucosa protects itself from gastric acid injury.

Although duodenal mucosal bicarbonate secretion (DMBS) is currently accepted as an important defense mechanism against acid-induced duodenal injury, the mechanism and the regulation of DMBS are largely unknown. 5-HT may regulate DMBS, but little is known about its physiological relevance in DMBS and the underlying mechanism(s). Thus, the aims of the present study were to demonstrate the role of 5-HT in acid-stimulated DMBS and to further elucidate the precise mechanisms involved in this process. Luminal acid stimulation significantly increased 5-HT release from the duodenal mucosa (P<0.01). SB204070, a selective 5-HT4 receptor antagonist, dose-dependently reduced luminal acid-stimulated HCO3(-) secretion of mice in vivo. In Ussing chamber studies, 5-HT-induced I(SC) and DMBS were abolished by removal of extracellular Ca2+, and significantly attenuated by pharmacological blockade of the Na+/Ca2+ exchanger (NCX), intermediate Ca2+-activated K+ channels (IK(Ca)), or cystic fibrosis transmembrane conductance regulator (CFTR). 5-HT increased cytoplasmic free calcium ([Ca2+]cyt) in SCBN cells, a duodenal epithelial cell line, and knockdown of NCX1 proteins with a specific siRNA greatly decreased this 5-HT-mediated Ca2+ signaling. Taken together, our data suggest that 5-HT plays a physiological role in acid-stimulated DMBS via a Ca2+ signaling pathway, in which the plasma membrane NCX transporter as well as IK(Ca) and CFTR channels may be involved.

Lipid nanoparticles improve activity of single-stranded siRNA and gapmer antisense oligonucleotides in animals.

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.

Addition of bromine chloride and iodine monochloride to carbonyl-conjugated, acetylenic ketones: synthesis and mechanisms.

The reactions of 3-butyn-2-one (1), 3-hexyn-2-one (2), and 4-phenyl-3-butyn-2-one (3) with bromine chloride (BrCl) and iodine monochloride (ICl) in CH(2)Cl(2), CH(2)Cl(2)/pyridine, and MeOH are described. The data show that the major products in CH(2)Cl(2) are (Z)-AM (anti-Markovnikov) regioisomers. With the exception of 3 and ICl, the (E)-AM regioisomers predominate when pyridine was added as an acid scavenger. Minor amounts of the M regioisomers were formed with 1 and 2 and BrCl. The percentage of M regioisomer increased significantly with 1 and BrCl in MeOH, but MeOH had little affect on the other reactions. Isolation and stability of the products are discussed. Detailed evidence for the structures of the products, involving a combination of MS, (1)H and (13)C NMR, and IR, is presented; HRMS analyses are provided as proofs for all of the products. The acid-catalyzed mechanism and the halonium ion mechanism are considered as possible pathways in the formation of the products.