Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates.

One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis.

BACKGROUND: Hereditary transthyretin amyloidosis is caused by pathogenic single-nucleotide variants in the gene encoding transthyretin ( TTR) that induce transthyretin misfolding and systemic deposition of amyloid. Progressive amyloid accumulation leads to multiorgan dysfunction and death. Inotersen, a 2'- O-methoxyethyl-modified antisense oligonucleotide, inhibits hepatic production of transthyretin. METHODS: We conducted an international, randomized, double-blind, placebo-controlled, 15-month, phase 3 trial of inotersen in adults with stage 1 (patient is ambulatory) or stage 2 (patient is ambulatory with assistance) hereditary transthyretin amyloidosis with polyneuropathy. Patients were randomly assigned, in a 2:1 ratio, to receive weekly subcutaneous injections of inotersen (300 mg) or placebo. The primary end points were the change in the modified Neuropathy Impairment Score+7 (mNIS+7; range, -22.3 to 346.3, with higher scores indicating poorer function; minimal clinically meaningful change, 2 points) and the change in the score on the patient-reported Norfolk Quality of Life-Diabetic Neuropathy (QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating poorer quality of life). A decrease in scores indicated improvement. RESULTS: A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. Both primary efficacy assessments favored inotersen: the difference in the least-squares mean change from baseline to week 66 between the two groups (inotersen minus placebo) was -19.7 points (95% confidence interval [CI], -26.4 to -13.0; P<0.001) for the mNIS+7 and -11.7 points (95% CI, -18.3 to -5.1; P<0.001) for the Norfolk QOL-DN score. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. There were five deaths in the inotersen group and none in the placebo group. The most frequent serious adverse events in the inotersen group were glomerulonephritis (in 3 patients [3%]) and thrombocytopenia (in 3 patients [3%]), with one death associated with one of the cases of grade 4 thrombocytopenia. Thereafter, all patients received enhanced monitoring. CONCLUSIONS: Inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis. Thrombocytopenia and glomerulonephritis were managed with enhanced monitoring. (Funded by Ionis Pharmaceuticals; NEURO-TTR ClinicalTrials.gov number, NCT01737398 .).

Tafamidis Treatment for Patients with Transthyretin Amyloid Cardiomyopathy.

BACKGROUND: Transthyretin amyloid cardiomyopathy is caused by the deposition of transthyretin amyloid fibrils in the myocardium. The deposition occurs when wild-type or variant transthyretin becomes unstable and misfolds. Tafamidis binds to transthyretin, preventing tetramer dissociation and amyloidogenesis. METHODS: In a multicenter, international, double-blind, placebo-controlled, phase 3 trial, we randomly assigned 441 patients with transthyretin amyloid cardiomyopathy in a 2:1:2 ratio to receive 80 mg of tafamidis, 20 mg of tafamidis, or placebo for 30 months. In the primary analysis, we hierarchically assessed all-cause mortality, followed by frequency of cardiovascular-related hospitalizations according to the Finkelstein-Schoenfeld method. Key secondary end points were the change from baseline to month 30 for the 6-minute walk test and the score on the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS), in which higher scores indicate better health status. RESULTS: In the primary analysis, all-cause mortality and rates of cardiovascular-related hospitalizations were lower among the 264 patients who received tafamidis than among the 177 patients who received placebo (P<0.001). Tafamidis was associated with lower all-cause mortality than placebo (78 of 264 [29.5%] vs. 76 of 177 [42.9%]; hazard ratio, 0.70; 95% confidence interval [CI], 0.51 to 0.96) and a lower rate of cardiovascular-related hospitalizations, with a relative risk ratio of 0.68 (0.48 per year vs. 0.70 per year; 95% CI, 0.56 to 0.81). At month 30, tafamidis was also associated with a lower rate of decline in distance for the 6-minute walk test (P<0.001) and a lower rate of decline in KCCQ-OS score (P<0.001). The incidence and types of adverse events were similar in the two groups. CONCLUSIONS: In patients with transthyretin amyloid cardiomyopathy, tafamidis was associated with reductions in all-cause mortality and cardiovascular-related hospitalizations and reduced the decline in functional capacity and quality of life as compared with placebo. (Funded by Pfizer; ATTR-ACT ClinicalTrials.gov number, NCT01994889 .).

The Ultrastructure of Tissue Damage by Amyloid Fibrils.

Amyloidosis is a group of diseases that includes Alzheimer's disease, prion diseases, transthyretin (ATTR) amyloidosis, and immunoglobulin light chain (AL) amyloidosis. The mechanism of organ dysfunction resulting from amyloidosis has been a topic of debate. This review focuses on the ultrastructure of tissue damage resulting from amyloid deposition and therapeutic insights based on the pathophysiology of amyloidosis. Studies of nerve biopsy or cardiac autopsy specimens from patients with ATTR and AL amyloidoses show atrophy of cells near amyloid fibril aggregates. In addition to the stress or toxicity attributable to amyloid fibrils themselves, the toxicity of non-fibrillar states of amyloidogenic proteins, particularly oligomers, may also participate in the mechanisms of tissue damage. The obscuration of the basement and cytoplasmic membranes of cells near amyloid fibrils attributable to an affinity of components constituting these membranes to those of amyloid fibrils may also play an important role in tissue damage. Possible major therapeutic strategies based on pathophysiology of amyloidosis consist of the following: (1) reducing or preventing the production of causative proteins; (2) preventing the causative proteins from participating in the process of amyloid fibril formation; and/or (3) eliminating already-deposited amyloid fibrils. As the development of novel disease-modifying therapies such as short interfering RNA, antisense oligonucleotide, and monoclonal antibodies is remarkable, early diagnosis and appropriate selection of treatment is becoming more and more important for patients with amyloidosis.

A pair of peptides inhibits seeding of the hormone transporter transthyretin into amyloid fibrils.

The tetrameric protein transthyretin is a transporter of retinol and thyroxine in blood, cerebrospinal fluid, and the eye, and is secreted by the liver, choroid plexus, and retinal epithelium, respectively. Systemic amyloid deposition of aggregated transthyretin causes hereditary and sporadic amyloidoses. A common treatment of patients with hereditary transthyretin amyloidosis is liver transplantation. However, this procedure, which replaces the patient's variant transthyretin with the WT protein, can fail to stop subsequent cardiac deposition, ultimately requiring heart transplantation. We recently showed that preformed amyloid fibrils present in the heart at the time of surgery can template or seed further amyloid aggregation of native transthyretin. Here we assess possible interventions to halt this seeding, using biochemical and EM assays. We found that chemical or mutational stabilization of the transthyretin tetramer does not hinder amyloid seeding. In contrast, binding of the peptide inhibitor TabFH2 to ex vivo fibrils efficiently inhibits amyloid seeding by impeding self-association of the amyloid-driving strands F and H in a tissue-independent manner. Our findings point to inhibition of amyloid seeding by peptide inhibitors as a potential therapeutic approach.

Optimization of kinetic stabilizers of tetrameric transthyretin: A prospective ligand efficiency-guided approach.

In the past few years, attempts have been made to use decision criteria beyond Lipinski's guidelines (Rule of five) to guide drug discovery projects more effectively. Several variables and formulations have been proposed and investigated within the framework of multiparameter optimization methods to guide drug discovery. In this context, the combination of Ligand Efficiency Indices (LEI) has been predominantly used to map and monitor the drug discovery process in a retrospective fashion. Here we provide an example of the use of a novel application of the LEI methodology for prospective lead optimization by using the transthyretin (TTR) fibrillogenesis inhibitor iododiflunisal (IDIF) as example. Using this approach, a number of compounds with theoretical efficiencies higher than the reference compound IDIF were identified. From this group, ten compounds were selected, synthesized and biologically tested. Half of the compounds (5, 6, 7, 8 and 10) showed potencies in terms of IC50 inhibition of TTR aggregation equal or higher than the lead compound. These optimized compounds mapped within the region of more efficient candidates in the corresponding experimental nBEI-NSEI plot, matching their position in the theoretical optimization plane that was used for the prediction. Due to their upstream (North-Eastern) position in the progression lines of NPOL = 3 or 4 of the nBEI-NSEI plot, three of them (5, 6 and 8) are more interesting candidates than iododiflunisal because they have been optimized in the three crucial LEI variables of potency, size and polarity at the same time. This is the first example of the effectiveness of using the combined LEIs within the decision process to validate the application of the LEI formulation for the prospective optimization of lead compounds.

Undiscovered Roles for Transthyretin: From a Transporter Protein to a New Therapeutic Target for Alzheimer's Disease.

Transthyretin (TTR), an homotetrameric protein mainly synthesized by the liver and the choroid plexus, and secreted into the blood and the cerebrospinal fluid, respectively, has been specially acknowledged for its functions as a transporter protein of thyroxine and retinol (the latter through binding to the retinol-binding protein), in these fluids. Still, this protein has managed to stay in the spotlight as it has been assigned new and varied functions. In this review, we cover knowledge on novel TTR functions and the cellular pathways involved, spanning from neuroprotection to vascular events, while emphasizing its involvement in Alzheimer's disease (AD). We describe details of TTR as an amyloid binding protein and discuss its interaction with the amyloid Aß peptides, and the proposed mechanisms underlying TTR neuroprotection in AD. We also present the importance of translating advances in the knowledge of the TTR neuroprotective role into drug discovery strategies focused on TTR as a new target in AD therapeutics.

Uncovering the Mechanism of Aggregation of Human Transthyretin.

The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify ß-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy.

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.

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.

Native State Stabilization of Amyloidogenic Proteins by Kinetic Stabilizers: Inhibition of Protein Aggregation and Clinical Relevance.

Proteinopathies or amyloidoses are a group of life-threatening disorders that result from misfolding of proteins and aggregation into toxic insoluble amyloid aggregates. Amyloid aggregates have low clearance from the body due to the insoluble nature, leading to their deposition in various organs and consequent organ dysfunction. While amyloid deposition in the central nervous system leads to neurodegenerative diseases that mostly cause dementia and difficulty in movement, several other organs, including heart, liver and kidney are also affected by systemic amyloidoses. Regardless of the site of amyloid deposition, misfolding and structural alteration of the precursor proteins play the central role in amyloid formation. Kinetic stabilizers are an emerging class of drugs, which act like pharmacological chaperones to stabilize the native state structure of amyloidogenic proteins and to increase the activation energy barrier that is required for adopting a misfolded structure or conformation, ultimately leading to the inhibition of protein aggregation. In this review, we discuss the kinetic stabilizers that stabilize the native quaternary structure of transthyretin, immunoglobulin light chain and superoxide dismutase 1 that cause transthyretin amyloidoses, light chain amyloidosis and familial amyotrophic lateral sclerosis, respectively.

Anionic phenolic compounds bind stronger with transthyretin than their neutral forms: nonnegligible mechanisms in virtual screening of endocrine disrupting chemicals.

The molecular structures of many endocrine-disrupting chemicals (EDCs) contain groups that ionize under physiological pH conditions. It is unclear whether the neutral and ionic forms have different binding mechanisms with the macromolecular targets. We selected phenolic compounds and human transthyretin (hTTR) as a model system and employed molecular docking with quantum mechanics/molecular mechanics optimizations to probe the mechanisms. The binding patterns of ionizable ligands in hTTR crystal structures were also analyzed. We found that the anionic forms of the phenolic compounds bind stronger than the corresponding neutral forms with hTTR. Electrostatic and van de Waals interactions are the dominant forces for most of the anionic and neutral forms, respectively. Because of the dominant and orientational electrostatic interactions, the -O(-) groups point toward the entry port of the binding site. The aromatic rings of the compounds also form cation-π interactions with the -NH3(+) group of Lys 15 residues in hTTR. Molecular descriptors were selected to characterize the interactions and construct a quantitative structure-activity relationship model on the relative competing potency of chemicals with T4 binding to hTTR. It is concluded that the effects of ionization should not be neglected when constructing in silico models for screening of potential EDCs.

Specific disruption of transthyretin(105-115) fibrilization using "stabilizing" inhibitors of transthyretin amyloidogenesis.

Transthyretin (TTR) is a cerebrospinal fluid and serum protein that undergoes ordered aggregation (amyloidogenesis) in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). It is now widely accepted that dissociation of the native TTR tetramer is a precondition for amyloidogenesis; thus, molecules that stabilize the tetramer have received much attention as potential TTR amyloidosis inhibitors. Many of these inhibitors bind to the thyroxine (T(4)) binding pocket and interact specifically with a section of the TTR sequence, corresponding to residues 105-115, that is implicated in amyloidogenic propensity. In this work, we study the effects of "stabilizing" inhibitors on ordered aggregation of TTR(105-115) peptide. We show that molecules known to bind full-length TTR at the T(4) site are potent, specific inhibitors of ordered aggregation, while molecules that do not interact with TTR exhibit milder, nonspecific disruption through a "hyperbundling" effect. Our results suggest that, in addition to annealing the native tetramer, "stabilizing" inhibitors may also directly disrupt amyloidogenic aggregation of TTR monomers through specific interactions with the exposed TTR(105-115) sequence.

Cyclodextrin, a novel therapeutic tool for suppressing amyloidogenic transthyretin misfolding in transthyretin-related amyloidosis.

TTR (transthyretin), a ß-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched ß-CyDs, GUG-ß-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-ß-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-ß-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-ß-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-ß-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-ß-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.

Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs.

The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.

Rigorous treatment of multispecies multimode ligand-receptor interactions in 3D-QSAR: CoMFA analysis of thyroxine analogs binding to transthyretin.

For a rigorous analysis of the receptor-ligand binding, speciation of the ligands caused by ionization, tautomerism, covalent hydration, and dynamic stereoisomerism needs to be considered. Each species may bind in several orientations or conformations (modes), especially for flexible ligands and receptors. A thermodynamic description of the multispecies (MS), multimode (MM) binding events shows that the overall association constant is equal to the weighted sum of the sums of microscopic association constants of individual modes for each species, with the weights given by the unbound fractions of individual species. This expression is a prerequisite for a precise quantitative characterization of the ligand-receptor interactions in both structure-based and ligand-based structure-activity analyses. We have implemented the MS-MM correlation expression into the comparative molecular field analysis (CoMFA), which deduces a map of the binding site from structures and binding affinities of a ligand set, in the absence of experimental structural information on the receptor. The MS-MM CoMFA approach was applied to published data for binding to transthyretin of 28 thyroxine analogs, each forming up to four ionization species under physiological conditions. The published X-ray structures of several analogs, exhibiting multiple binding modes, served as templates for the MS-MM superposition of thyroxine analogs. Additional modes were generated for compounds with flexible alkyl substituents, to identify bound conformations. The results demonstrate that the MS-MM modification improved predictive abilities of the CoMFA models, even for the standard procedure with MS-MM selected species and modes. The predicted prevalences of individual modes and the generated receptor site model are in reasonable agreement with the available X-ray data. The calibrated model can help in the design of inhibitors of transthyretin amyloid fibril formation.

The discovery and development of transthyretin amyloidogenesis inhibitors: what are the lessons?

Transthyretin (TTR) is associated with several human amyloid diseases. Various kinetic stabilizers have been developed to inhibit the dissociation of TTR tetramer and the formation of amyloid fibrils. Most of them are bisaryl derivatives, natural flavonoids, crown ethers and carborans. In this review article, we focus on TTR tetramer stabilizers, genetic therapeutic approaches and fibril remodelers. The binding modes of typical bisaryl derivatives, natural flavonoids, crown ethers and carborans are discussed. Based on knowledge of the binding of thyroxine to TTR tetramer, many stabilizers have been screened to dock into the thyroxine binding sites, leading to TTR tetramer stabilization. Particularly, those stabilizers with unique binding profiles have shown great potential in developing the therapeutic management of TTR amyloidogenesis.

Targeting transthyretin in Alzheimer's disease: Drug discovery of small-molecule chaperones as disease-modifying drug candidates for Alzheimer's disease.

Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aß) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.

Syntheses of Morpholine-Based Nucleotide Analogs for Hepatic siRNA Targeting and Stabilization.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.