Abstract
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.
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.
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► Chemically modified single-stranded siRNAs silence gene expression in animals ► ss-siRNAs require 5′ phosphate and association with AGO2 for gene silencing ► Passenger strand is dispensable for potent RNAi-mediated gene silencing ► Single-stranded siRNAs provide an alternate option for RNAi therapeutics
Chemically modified RNA oligonucleotides load into Ago2-containing RISC complexes to effect RNA silencing without the need for a passenger strand. These single-stranded siRNAs can be applied directly in animals and thus offer new alternatives for developing therapeutics and diagnostics.
Abstract
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.
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-/-).
Abstract
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.
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.
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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.
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.
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 Isc 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 (IKCa), 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 IKCa and CFTR channels may be involved.--Smith, A. J., Chappell, A. E., Buret, A. G., Barrett, K. E., Dong, H. 5-Hydroxytryptamine (5-HT) contributes significantly to a reflex pathway by which the duodenal mucosa protects itself from gastric acid injury.
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