CoChat
Sign Up
Cited by 279RNA Interference and Gene DeliveryDrug-Induced Hepatotoxicity and ProtectionToxin Mechanisms and Immunotoxins

Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity

Open access
Janas, Maja M., Schlegel, Mark K., Harbison, Carole E., Yilmaz, Vedat O., Jiang, Yongfeng, Parmar, Rubina, Zlatev, Ivan, Castoreno, Adam +7 more·2018·Nature Communications
DOI 10.1038/s41467-018-02989-4PMC PMC5818625
Open this paper in CoChat — free accountOpen PDF

Abstract

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.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41467-018-02989-4

Cites

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The naturally occurring RNAi pathway can be harnessed in extrahepatic organs by building on nearly two decades of liver siRNA design optimization, but novel delivery approaches are needed.

    “The naturally occurring RNAi pathway can also be harnessed in extrahepatic organs by building on nearly two decades of efforts on liver siRNA design optimization [9][10][11][12] ; however, novel delivery approaches are needed.”

    From Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates (2022) · cites this paper as [10]

    The Introduction (page unavailable) says exogenous siRNAs can harness the natural RNAi pathway and describes chemical modifications and GalNAc-mediated delivery to hepatocytes. It does not address harnessing RNAi in extrahepatic organs, establish nearly two decades of liver siRNA design optimization, or say that novel delivery approaches are needed for those organs. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Off-target mitigation strategies involving placing a thermally destabilizing modification such as glycol nucleic acid in the antisense strand seed region also performed well, thus allowing for translation of safety improvements with the ESC+ platform beyond liver.

    “Additionally, off-target mitigation strategies involving placing a thermally destabilizing modi cation such as GNA in the antisense strand seed region 10 also performed well, thus allowing for translation of safety improvements with the ESC+ platform beyond liver.”

    From Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates (2022) · cites this paper as [10]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • ESC+ chemistry employing glycol nucleic acid in the antisense seed region for increased specificity retained activity in rat two weeks after a single 0.9 mg intrathecal dose.

    “Furthermore, the ESC+ chemistry employing glycol nucleic acid (GNA) in the antisense seed region for increased speci city 10,30 retained activity in rat two weeks after a single 0.9 mg IT dose (Fig. 1d).”

    From Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates (2022) · cites this paper as [10]

  • The off target effects observed in this study are minor in comparison to a previous study evaluating RNA-seq data in siRNA-treated rat liver.

    “The off target effects observed in this study are minor in comparison to a previous study evaluating RNA-seq data in siRNA-treated rat liver38.”

    From A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system (2019) · cites this paper as [38]

  • siRNA off-target effects are a major concern in development of siRNA-based therapeutics.

    “siRNA off-target effects are a major concern in development of siRNA-based therapeutics38.”

    From A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system (2019) · cites this paper as [38]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

The citation lists haven’t been fetched yet

References 41 · Cited by 279

Research works printed but not linked: 40

  1. [1]Carthew RW Sontheimer EJ Origins and mechanisms of miRNAs and siRNAs Cell 2009 136 642 655 10.1016/j.cell.2009.01.035 19239886 PMC2675692 · DOI
  2. [2]Nakanishi K Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins? Wiley Interdiscip. Rev. RNA 2016 7 637 660 10.1002/wrna.1356 27184117 PMC5084781 · DOI
  3. [3]Swarts DC The evolutionary journey of Argonaute proteins Nat. Struct. Mol. Biol. 2014 21 743 753 10.1038/nsmb.2879 25192263 PMC4691850 · DOI
  4. [4]Liu J Argonaute2 is the catalytic engine of mammalian RNAi Science 2004 305 1437 1441 10.1126/science.1102513 15284456 · DOI · Paper page
  5. [5]Wittrup A Lieberman J Knocking down disease: a progress report on siRNA therapeutics Nat. Rev. Genet. 2015 16 543 552 10.1038/nrg3978 26281785 PMC4756474 · DOI
  6. [6]Matsuda S siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes ACS Chem. Biol. 2015 10 1181 1187 10.1021/cb501028c 25730476 · DOI
  7. [7]Rajeev KG Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo Chembiochem 2015 16 903 908 10.1002/cbic.201500023 25786782 · DOI
  8. [8]Nair JK Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing J. Am. Chem. Soc. 2014 136 16958 16961 10.1021/ja505986a 25434769 · DOI · Paper page
  9. [9]Manoharan M RNA interference and chemically modified small interfering RNAs Curr. Opin. Chem. Biol. 2004 8 570 579 10.1016/j.cbpa.2004.10.007 15556399 · DOI
  10. [10]Bumcrot D Manoharan M Koteliansky V Sah DW RNAi therapeutics: a potential new class of pharmaceutical drugs Nat. Chem. Biol. 2006 2 711 719 10.1038/nchembio839 17108989 PMC7097247 · DOI
  11. [12]Janas MM Impact of oligonucleotide structure, chemistry, and delivery method on in vitro cytotoxicity Nucleic Acids Ther. 2016 27 11 22 10.1089/nat.2016.0639 27923110 · DOI
  12. [13]Janas MM Exposure to siRNA-GalNAc conjugates in systems of the standard test battery for genotoxicity Nucleic Acids Ther. 2016 26 363 371 10.1089/nat.2016.0622 27387336 · DOI
  13. [14]Liang XH Sun H Shen W Crooke ST Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages Nucleic Acids Res. 2015 43 2927 2945 10.1093/nar/gkv143 25712094 PMC4357732 · DOI
  14. [15]Shen W Liang XH Sun H Crooke ST 2’-fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF Nucleic Acids Res. 2015 43 4569 4578 10.1093/nar/gkv298 25855809 PMC4482069 · DOI
  15. [16]Crooke ST Wang S Vickers TA Shen W Liang XH Cellular uptake and trafficking of antisense oligonucleotides Nat. Biotechnol. 2017 35 230 237 10.1038/nbt.3779 28244996 · DOI · Paper page
  16. [17]Frazier KS Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist’s perspective Toxicol. Pathol. 2015 43 78 89 10.1177/0192623314551840 25385330 · DOI
  17. [18]Valdmanis PN RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice Nat. Med. 2016 22 557 562 10.1038/nm.4079 27064447 PMC4860119 · DOI
  18. [19]Grimm D Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways Nature 2006 441 537 541 10.1038/nature04791 16724069 · DOI · Paper page
  19. [20]Jackson AL Expression profiling reveals off-target gene regulation by RNAi Nat. Biotechnol. 2003 21 635 637 10.1038/nbt831 12754523 · DOI · Paper page
  20. [21]Jackson AL Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity RNA 2006 12 1179 1187 10.1261/rna.25706 16682560 PMC1484447 · DOI
  21. [22]Birmingham A 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets Nat. Methods 2006 3 199 204 10.1038/nmeth854 16489337 · DOI · Paper page
  22. [23]Burel SA Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts Nucleic Acids Res. 2016 44 2093 2109 10.1093/nar/gkv1210 26553810 PMC4797265 · DOI
  23. [24]Kamola PJ In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization Nucleic Acids Res. 2015 43 8638 8650 10.1093/nar/gkv857 26338776 PMC4605310 · DOI
  24. [25]Wilczynska A Bushell M The complexity of miRNA-mediated repression Cell Death Differ. 2015 22 22 33 10.1038/cdd.2014.112 25190144 PMC4262769 · DOI
  25. [26]Zlatev, I. et al. REVERSIR™ platform for the tailored control of GalNAc-siRNA conjugate pharmacology. Nat. Biotechnol. in press (2018).
  26. [27]John M Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway Nature 2007 449 745 747 10.1038/nature06179 17898712 PMC3019095 · DOI
  27. [28]Schirle NT MacRae IJ The crystal structure of human Argonaute2 Science 2012 336 1037 1040 10.1126/science.1221551 22539551 PMC3521581 · DOI
  28. [29]Wang Y Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes Nature 2009 461 754 761 10.1038/nature08434 19812667 PMC2880917 · DOI
  29. [30]Parmar R 5’-(E)-vinylphosphonate: a stable phosphate mimic can improve the RNAi activity of siRNA-GalNAc conjugates Chembiochem 2016 17 985 989 10.1002/cbic.201600130 27121751 · DOI
  30. [31]Weitzer S Martinez J The human RNA kinase hClp1 is active on 3’ transfer RNA exons and short interfering RNAs Nature 2007 447 222 226 10.1038/nature05777 17495927 · DOI
  31. [32]Haringsma HJ mRNA knockdown by single strand RNA is improved by chemical modifications Nucleic Acids Res. 2012 40 4125 4136 10.1093/nar/gkr1301 22253019 PMC3351186 · DOI
  32. [34]Schlegel MK Chirality dependent potency enhancement and structural impact of glycol nucleic acid modification on siRNA J. Am. Chem. Soc. 2017 139 8537 8546 10.1021/jacs.7b02694 28570818 · DOI
  33. [35]Bramsen JB A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects Nucleic Acids Res. 2010 38 5761 5773 10.1093/nar/gkq341 20453030 PMC2943616 · DOI
  34. [36]Mook O In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts Artif. DNA PNA XNA 2010 1 36 44 10.4161/adna.1.1.12204 21687525 PMC3109444 · DOI
  35. [37]Vaish N Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs Nucleic Acids Res. 2011 39 1823 1832 10.1093/nar/gkq961 21047800 PMC3061082 · DOI
  36. [38]Lee HS Abasic pivot substitution harnesses target specificity of RNA interference Nat. Commun. 2015 6 10154 10.1038/ncomms10154 26679372 PMC4703836 · DOI
  37. [39]Seok H Jang ES Chi SW Rationally designed siRNAs without miRNA-like off-target repression BMB Rep. 2016 49 135 136 10.5483/BMBRep.2016.49.3.019 26839153 PMC4915226 · DOI
  38. [40]Elkayam, E. et al. siRNA carrying an (E)-vinylphosphonate moiety at the 5’ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2. Nucleic Acids Res.45, 3528–3536 (2016). PMC5389677 27903888
  39. [41]Liao Y Smyth GK Shi W featureCounts: an efficient general purpose program for assigning sequence reads to genomic features Bioinformatics 2014 30 923 930 10.1093/bioinformatics/btt656 24227677 · DOI · Paper page
  40. [42]Love MI Huber W Anders S Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biol. 2014 15 550 10.1186/s13059-014-0550-8 25516281 PMC4302049 · DOI · Paper page

Authors
Janas, M. M., Schlegel, M. K., Harbison, C. E., Yilmaz, V. O., Jiang, Y., Parmar, R., Zlatev, I., Castoreno, A., Xu, H., Shulga-Morskaya, S., Rajeev, K. G., Manoharan, M., Keirstead, N. D., Maier, M. A., & Jadhav, V.
Affiliations
Alnylam Pharmaceuticals (United States) (US)
Corresponding author
Jadhav, Vasant
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-02-19
Volume
9
Issue
1
Pages
723-723
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-02989-4
PMC
PMC5818625
OpenAlex
W2802881274
Cited by
279
References
41
Citations by year
2026: 32 · 2025: 43 · 2024: 40 · 2023: 38 · 2022: 32 · 2021: 26 · 2020: 34 · 2019: 27 · 2018: 6 · 2013: 1
FWCI
8.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Alnylam Pharmaceuticals
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Life Sciences › Immunology and Microbiology › Immunology › Toxin Mechanisms and Immunotoxins
Keywords
siRNA conjugates, hepatotoxicity, off-target effects, RNA interference, rat toxicity, interspecies extrapolation
MeSH terms
Acetylgalactosamine, Animals, Liver, Male, Rats, Sprague-Dawley, RNA Interference, RNA, Small Interfering, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review