A5009680488- RNA Interference and Gene Delivery
- MicroRNA in disease regulation
- Advanced biosensing and bioanalysis techniques
- RNA modifications and cancer
- RNA Research and Splicing
- DNA and Nucleic Acid Chemistry
- Cancer-related molecular mechanisms research
- RNA regulation and disease
- Circular RNAs in diseases
- Hepatitis Viruses Studies and Epidemiology
- Extracellular vesicles in disease
- RNA and protein synthesis mechanisms
- Hepatitis C virus research
- Porphyrin Metabolism and Disorders
- Hepatitis B Virus Studies
Alnylam Pharmaceuticals (United States)
2015-2025
Harvard University
2008-2013
Dana-Farber Cancer Institute
2009-2013
Broad Institute
2009-2013
Harvard Stem Cell Institute
2008
The developmentally regulated RNA-binding protein Lin28 blocks processing of let-7 family microRNAs (miRNAs) in embryonic cells. molecular basis for this selective miRNA block is unknown. Here we find that selectively binds the terminal loop region precursors vitro and mediates inhibition vivo. Additionally, identify domains required inhibition. These findings establish a regulatory role maturation provide insight into mechanism by which negatively regulates processing.
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for variety of indications. The typical development candidate selection process includes evaluation the most active compounds toxicity rats at pharmacologically exaggerated doses. subset GalNAc-siRNAs that show rat hepatotoxicity is not advanced development. Potential mechanisms can be associated with intracellular accumulation...
Background & Aims Current treatment for chronic hepatitis B virus (cHBV) infection requires lifelong treatment. New therapy aimed towards HBV functional cure would represent a clinically meaningful advancement. ALN-HBV and VIR-2218 (modified from by Enhanced Stabilization Chemistry Plus technology reducing off-target, seed-mediated binding while maintaining on-target antiviral activity) are investigational RNAi therapeutics that target all major transcripts. Methods We report the safety of...
MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to consensus view all miRNAs associated with Argonaute (Ago) proteins, we determine expressed in a 13-fold excess relative Agos HeLa cells and bound mRNAs sevenfold Agos, implying existence miRNA–mRNA duplexes not stoichiometrically by Agos. We show four can repress duplexes, but only Ago2 cleave interfering RNA–mRNA vitro. visualize direct Ago binding...
Abstract Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc–siRNA conjugates. Here, we demonstrate that single glycol nucleic acid or 2′–5′-RNA modification can substantially reduce small interfering (siRNA) seed-mediated binding to transcripts while maintaining on-target activity. In siRNAs with established driven by effects, these novel designs seed-pairing destabilization, termed enhanced...
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides not known occur naturally, their safety profile was assessed when in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), same sequence but different modification pattern metabolic stability, conjugated an N-acetylgalactosamine (GalNAc) ligand for targeted delivery hepatocytes. Exposure...
Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than natural DNA RNA, in context small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine...
The majority of mammalian microRNA (miRNA) genes reside within introns protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA spliced mRNA are poorly understood. Analysis melanoma invasion suppressor miR-211 expressed from intron 6 melastatin revealed that microprocessing promotes splicing exon 6–exon 7 junction by a mechanism requiring RNase III activity Drosha. Additionally, mutations in 5′ splice site (5′SS), but not...
Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures repeat-dose regimen. We have previously shown that these suprapharmacological doses, hepatotoxicity observed with subset GalNAc–siRNAs largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar microRNA-like regulation. However, the RISC...
Single-stranded (ss) 2'-fluoro (2'-F)-modified oligonucleotides (ONs) with a full phosphorothioate (PS) backbone have been reported to be cytotoxic and cause DNA double-strand breaks (DSBs) when transfected into HeLa cells. However, the molecular determinants of these effects not fully explored. In this study, we investigated impact ON structure, chemistry, delivery method, cell type on in vitro cytotoxicity DSBs. We found that ss PS-ONs were more than double-stranded (ds) PS-ONs,...
In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose modifications. The siRNAs designed to silence transthyretin (Ttr) gene conjugated a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery hepatocytes. Sense antisense strands of parent duplex synthesized single ANA residues at each position on...
Although 2′-deoxy-2′-α-F-2′-β-C-methyl (2′-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis 2′-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into Despite C3′-endo preorganization parent nucleoside, single RNA or DNA resulted significant thermal destabilization duplex due to unfavorable enthalpy, likely resulting from steric...
Registration of pharmaceuticals requires an assessment their genotoxic potential using in vitro and vivo tests outlined the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages various combinations 2'-fluoro 2'-O-methyl ribose modifications multiple nucleotides ICH battery assays, all which uniformly yielded negative results. To verify these genotoxicity results, this...
Serum protein interactions are evaluated during the drug development process since they determine free concentration in blood and thereby can influence drug's pharmacokinetic pharmacodynamic properties. While impact of serum proteins on disposition small molecules is well understood, it not yet characterized for a new modality, RNA interference therapeutics. When administered systemically, interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligand bind present circulation....
Abstract We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that single incorporation 2′-F-NMC within guide or passenger strand tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were into at positions 1 2, resulted loss vitro activity. Activity...
To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at 5'-end sense inhibits its interaction with RISC ensuring selection desired strand. improve this antagonizing binding property even further, new set morpholino-based analogues, Mo2 and Mo3, piperidine analogue, Pip, were designed based on known structure Argonaute2, slicer enzyme...