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Nature Communications Feb 2018Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of...
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
Topics: Acetylgalactosamine; Animals; Liver; Male; RNA Interference; RNA, Small Interfering; Rats; Rats, Sprague-Dawley
PubMed: 29459660
DOI: 10.1038/s41467-018-02989-4 -
Molecular Therapy : the Journal of the... Mar 2018Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the...
Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.
Topics: Acetylgalactosamine; Animals; Argonaute Proteins; Gene Expression Regulation; Gene Silencing; Hepatocytes; Liver; Male; Mice; Mice, Transgenic; RNA Interference; RNA, Messenger; RNA, Small Interfering
PubMed: 29456020
DOI: 10.1016/j.ymthe.2017.12.021 -
The New England Journal of Medicine Jan 2017
Topics: Acetylgalactosamine; Anticholesteremic Agents; Drug Discovery; Hepatocytes; Humans; RNA, Small Interfering; RNAi Therapeutics
PubMed: 28052224
DOI: 10.1056/NEJMp1614154 -
Nature Jan 2024Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established, little is known about how host genetics regulates... (Meta-Analysis)
Meta-Analysis
Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.
Topics: Humans; Acetylgalactosamine; Bacteria; Cohort Studies; Computer Simulation; Faecalibacterium prausnitzii; Gastrointestinal Microbiome; Genome, Human; Genotype; Host Microbial Interactions; In Vitro Techniques; Metagenome; Multigene Family; Netherlands; Tanzania
PubMed: 38172637
DOI: 10.1038/s41586-023-06893-w -
Drugs Feb 2020Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the... (Review)
Review
Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. Givosiran is being developed by Alnylam Pharmaceuticals for the treatment of acute hepatic porphyria (AHP). In November 2019, givosiran was approved in the USA for the treatment of adults with AHP based on the positive results from the multinational, phase III ENVISION trial. In the EU, givosiran received a positive opinion in January 2020 for the treatment of AHP in adults and adolescents aged 12 years and older. This article summarizes the milestones in the development of givosiran leading to this first approval for the treatment of adults with AHP.
Topics: 5-Aminolevulinate Synthetase; Acetylgalactosamine; Drug Approval; Enzyme Inhibitors; Humans; Porphobilinogen Synthase; Porphyrias, Hepatic; Pyrrolidines; RNA, Messenger
PubMed: 32034693
DOI: 10.1007/s40265-020-01269-0 -
Nature Oct 2021Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates...
Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease.
Topics: Acetylgalactosamine; Animals; Bacteroides; Colon; Crystallography, X-Ray; Female; Galactose; Gastrointestinal Microbiome; Humans; Male; Mice; Models, Molecular; Mucins; Substrate Specificity; Sulfatases
PubMed: 34616040
DOI: 10.1038/s41586-021-03967-5 -
Nucleic Acids Research Nov 2017Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated...
Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.
Topics: Acetylgalactosamine; Animals; Area Under Curve; Drug Delivery Systems; Humans; Kidney; Liver; Male; Metabolic Clearance Rate; Mice, Inbred C57BL; RNA Interference; RNA, Small Interfering
PubMed: 28981809
DOI: 10.1093/nar/gkx818 -
Molecules (Basel, Switzerland) Aug 2019Galactosaminoglycans (GalAGs) are sulfated glycans composed of alternating -acetylgalactosamine and uronic acid units. Uronic acid epimerization, sulfation patterns and... (Review)
Review
Galactosaminoglycans (GalAGs) are sulfated glycans composed of alternating -acetylgalactosamine and uronic acid units. Uronic acid epimerization, sulfation patterns and fucosylation are modifications observed on these molecules. GalAGs have been extensively studied and exploited because of their multiple biomedical functions. Chondroitin sulfates (CSs), the main representative family of GalAGs, have been used in alternative therapy of joint pain/inflammation and osteoarthritis. The relatively novel fucosylated chondroitin sulfate (FCS), commonly found in sea cucumbers, has been screened in multiple systems in addition to its widely studied anticoagulant action. Biomedical properties of GalAGs are directly dependent on the sugar composition, presence or lack of fucose branches, as well as sulfation patterns. Although research interest in GalAGs has increased considerably over the three last decades, perhaps motivated by the parallel progress of glycomics, serious questions concerning the effectiveness and potential side effects of GalAGs have recently been raised. Doubts have centered particularly on the beneficial functions of CS-based therapeutic supplements and the potential harmful effects of FCS as similarly observed for oversulfated chondroitin sulfate, as a contaminant of heparin. Unexpected components were also detected in CS-based pharmaceutical preparations. This review therefore aims to offer a discussion on (1) the current and potential therapeutic applications of GalAGs, including those of unique features extracted from marine sources, and (2) the potential drawbacks of this class of molecules when applied to medicine.
Topics: Acetylgalactosamine; Arthralgia; Chondroitin Sulfates; Humans; Osteoarthritis; Polysaccharides; Uronic Acids
PubMed: 31374852
DOI: 10.3390/molecules24152803 -
Chembiochem : a European Journal of... Jun 2016Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be...
Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
Topics: Acetylgalactosamine; Animals; Apolipoproteins B; Argonaute Proteins; Cells, Cultured; Factor IX; Hepatocytes; Humans; Lipoproteins, LDL; Mice; Mice, Inbred C57BL; Organophosphonates; RNA Interference; RNA, Small Interfering; RNA-Binding Proteins; RNA-Induced Silencing Complex; Transcription Factors; Vinyl Compounds
PubMed: 27121751
DOI: 10.1002/cbic.201600130 -
Applied Microbiology and Biotechnology Oct 2019β-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though... (Review)
Review
β-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though hydrolytic enzymes per se, their good stability, easy recombinant production, absolute stereoselectivity, and a broad substrate specificity predestine these enzymes for challenging applications in carbohydrate synthesis. This mini-review aims to demonstrate the catalytic potential of β-N-acetylhexosaminidases in a range of unusual reactions, processing of unnatural substrates, formation of unexpected products, and demanding reaction designs. The use of unconventional media can considerably alter the progress of transglycosylation reactions. By means of site-directed mutagenesis, novel catalytic machineries can be constructed. Glycosylation of difficult substrates such as sugar nucleotides was accomplished, and the range of afforded glycosidic bonds comprises unique non-reducing sugars. Specific functional groups may be tolerated in the substrate molecule, which makes β-N-acetylhexosaminidases invaluable allies in difficult synthetic problems.
Topics: Biocatalysis; Glycosylation; Mutant Proteins; beta-N-Acetylhexosaminidases
PubMed: 31401752
DOI: 10.1007/s00253-019-10065-0