CoChat
Sign Up
Cited by 285RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryRNA and protein synthesis mechanisms

Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates

Open access
Brown, Christopher R, Gupta, Swati, Qin, June, Racie, Timothy, He, Guo, Lentini, Scott, Malone, Ryan, Yu, Mikyung +16 more·2020·Nucleic Acids Research
DOI 10.1093/nar/gkaa670PMC PMC7708070
Open this paper in CoChat — free accountOpen PDF

Abstract

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.

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 · 10.1093/nar/gkaa670

Cites

9 of 9 references verified0 citation errors · 0 not checkable

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?

Self-check

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

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

Cites

Flagged first, then by citations

Research works printed but not linked: 51

  1. [1]Tuschl T., Zamore P.D., Lehmann R., Bartel D.P., Sharp P.A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 1999; 13: 3191– 3197. 10617568 PMC317199
  2. [2]Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391: 806– 811. 9486653
  3. [3]Elbashir S.M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001; 411: 494– 498. 11373684
  4. [4]Akinc A., Maier M.A., Manoharan M., Fitzgerald K., Jayaraman M., Barros S., Ansell S., Du X., Hope M.J., Madden T.D. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 2019; 14: 1084– 1087. 31802031
  5. [5]Balwani M., Sardh E., Ventura P., Peiro P.A., Rees D.C., Stolzel U., Bissell D.M., Bonkovsky H.L., Windyga J., Anderson K.E. et al. Phase 3 trial of RNAi therapeutic givosiran for acute intermittent Porphyria. N. Engl. J. Med. 2020; 382: 2289– 2301. 32521132
  6. [6]Shen X., Corey D.R. Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res. 2018; 46: 1584– 1600. 29240946 PMC5829639
  7. [7]Nair J.K., Willoughby J.L.S., Chan A., Charisse K., Alam M.R., Wang Q., Hoekstra M., Kandasamy P., Kel’in A.V., Milstein S. et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 2014; 136: 16958– 16961. 25434769
  8. [8]Stockert R.J., Morell A.G., Scheinberg I.H. Mammalian hepatic lectin. Science. 1974; 186: 365– 366. 4472191
  9. [9]Nair J.K., Attarwala H., Sehgal A., Wang Q., Aluri K., Zhang X., Gao M., Liu J., Indrakanti R., Schofield S. et al. Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates. Nucleic Acids Res. 2017; 45: 10969– 10977. 28981809 PMC5737438
  10. [10]Matsuda S., Keiser K., Nair J.K., Charisse K., Manoharan R.M., Kretschmer P., Peng C.G., Kel’in A.V., Kandasamy P., Willoughby J.L.S. et al. 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. 25730476
  11. [11]Zimmermann T.S., Karsten V., Chan A., Chiesa J., Boyce M., Bettencourt B.R., Hutabarat R., Nochur S., Vaishnaw A., Gollob J. Clinical proof of concept for a novel hepatocyte-targeting GalNAc–siRNA conjugate. Mol. Ther. 2017; 25: 71– 78. 28129130 PMC5363199
  12. [13]Ray K.K., Landmesser U., Leiter L.A., Kallend D., Dufour R., Karakas M., Hall T., Troquay R.P., Turner T., Visseren F.L. et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 2017; 376: 1430– 1440. 28306389
  13. [15]Springer A.D., Dowdy S.F. GalNAc–siRNA conjugates: leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther. 2018; 28: 109– 118. 29792572 PMC5994659
  14. [19]Juliano R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016; 44: 6518– 6548. 27084936 PMC5001581
  15. [20]Hou K.K., Pan H., Schlesinger P.H., Wickline S.A. A role for peptides in overcoming endosomal entrapment in siRNA delivery - a focus on melittin. Biotechnol. Adv. 2015; 33: 931– 940. 26025036 PMC4540690
  16. [21]Jayaraman M., Ansell S.M., Mui B.L., Tam Y.K., Chen J., Du X., Butler D., Eltepu L., Matsuda S., Narayanannair J.K. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. Engl. 2012; 51: 8529– 8533. 22782619 PMC3470698
  17. [24]Gilleron J., Querbes W., Zeigerer A., Borodovsky A., Marsico G., Schubert U., Manygoats K., Seifert S., Andree C., Stoter M. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013; 31: 638– 646. 23792630
  18. [25]Wei J., Jones J., Kang J., Card A., Krimm M., Hancock P., Pei Y., Ason B., Payson E., Dubinina N. et al. RNA-induced silencing complex-bound small interfering RNA is a determinant of RNA interference-mediated gene silencing in mice. Mol. Pharmacol. 2011; 79: 953– 963. 21427169
  19. [26]Sahay G., Querbes W., Alabi C., Eltoukhy A., Sarkar S., Zurenko C., Karagiannis E., Love K., Chen D., Zoncu R. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 2013; 31: 653– 658. 23792629 PMC3814166
  20. [27]Dowdy S.F., Levy M. RNA therapeutics (almost) comes of age: targeting, delivery and endosomal escape. Nucleic Acid Ther. 2018; 28: 107– 108. 29782220
  21. [28]Pei Y., Hancock P.J., Zhang H., Bartz R., Cherrin C., Innocent N., Pomerantz C.J., Seitzer J., Koser M.L., Abrams M.T. et al. Quantitative evaluation of siRNA delivery in vivo. RNA. 2010; 16: 2553– 2563. 20940339 PMC2995415
  22. [29]Chen C., Ridzon D.A., Broomer A.J., Zhou Z., Lee D.H., Nguyen J.T., Barbisin M., Xu N.L., Mahuvakar V.R., Andersen M.R. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005; 33: e179. 16314309 PMC1292995
  23. [30]Parmar R.G., Brown C.R., Matsuda S., Willoughby J.L.S., Theile C.S., Charisse K., Foster D.J., Zlatev I., Jadhav V., Maier M.A. et al. Facile synthesis, geometry, and 2′-substituent-dependent in vivo activity of 5′-(E)- and 5′-(Z)-vinylphosphonate-modified siRNA conjugates. J. Med. Chem. 2018; 61: 734– 744. 29376650
  24. [31]Elkayam E., Parmar R., Brown C.R., Willoughby J.L., Theile C.S., Manoharan M., Joshua-Tor L. 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. 2017; 45: 3528– 3536. 27903888 PMC5389677
  25. [32]Hauptmann J., Meister G. Peptide-Based isolation of argonaute protein complexes using Ago-APP. Methods Mol. Biol. 2017; 1580: 107– 116. 28439830
  26. [33]Hauptmann J., Schraivogel D., Bruckmann A., Manickavel S., Jakob L., Eichner N., Pfaff J., Urban M., Sprunck S., Hafner M. et al. Biochemical isolation of Argonaute protein complexes by Ago-APP. Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 11841– 11845. 26351695 PMC4586862
  27. [34]Maier M.A., Jayaraman M., Matsuda S., Liu J., Barros S., Querbes W., Tam Y.K., Ansell S.M., Kumar V., Qin J. et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol. Ther. 2013; 21: 1570– 1578. 23799535 PMC3734658
  28. [35]Severgnini M., Sherman J., Sehgal A., Jayaprakash N.K., Aubin J., Wang G., Zhang L., Peng C.G., Yucius K., Butler J. et al. A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development. Cytotechnology. 2012; 64: 187– 195. 22105762 PMC3279583
  29. [36]Castellanos-Rizaldos E., Brown C.R., Dennin S., Kim J., Gupta S., Najarian D., Gu Y., Aluri K., Enders J., Brown K. et al. Reverse transcription quantitative polymerase chain reaction methods to support pharmacokinetics and drug mechanism of action to advance development of RNA interference therapeutics. Nucleic Acid Ther. 2020; 30: 133– 142. 32202961
  30. [37]Coelho T., Adams D., Silva A., Lozeron P., Hawkins P.N., Mant T., Perez J., Chiesa J., Warrington S., Tranter E. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 2013; 369: 819– 829. 23984729
  31. [38]Gantier M.P., McCoy C.E., Rusinova I., Saulep D., Wang D., Xu D., Irving A.T., Behlke M.A., Hertzog P.J., Mackay F. et al. Analysis of microRNA turnover in mammalian cells following Dicer1 ablation. Nucleic Acids Res. 2011; 39: 5692– 5703. 21447562 PMC3141258
  32. [40]Oliveira S., van Rooy I., Kranenburg O., Storm G., Schiffelers R.M. Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes. Int. J. Pharm. 2007; 331: 211– 214. 17187949
  33. [42]Stegmann T. Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion. Traffic. 2000; 1: 598– 604. 11208147
  34. [43]Bonnafous P., Stegmann T. Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion. J. Biol. Chem. 2000; 275: 6160– 6166. 10692407
  35. [44]Mastrobattista E., Koning G.A., van Bloois L., Filipe A.C., Jiskoot W., Storm G. Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins. J. Biol. Chem. 2002; 277: 27135– 27143. 12021269
  36. [46]Cummins L.L., Owens S.R., Risen L.M., Lesnik E.A., Freier S.M., McGee D., Guinosso C.J., Cook P.D. Characterization of fully 2′-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. 1995; 23: 2019– 2024. 7541132 PMC306978
  37. [47]Takahashi M., Minakawa N., Matsuda A. Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability. Nucleic Acids Res. 2009; 37: 1353– 1362. 19151085 PMC2651785
  38. [48]Gregoriadis G., Morell A.G., Sternlieb I., Scheinberg I.H. Catabolism of desialylated ceruloplasmin in the liver. J. Biol. Chem. 1970; 245: 5833– 5837. 5472375
  39. [49]Bridges K., Harford J., Ashwell G., Klausner R.D. Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 350– 354. 6281767 PMC345729
  40. [50]Wall D.A., Wilson G., Hubbard A.L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980; 21: 79– 93. 7407914
  41. [51]Geuze H.J., Slot J.W., Strous G.J., Lodish H.F., Schwartz A.L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983; 32: 277– 287. 6130851
  42. [52]Winter J., Diederichs S. Argonaute proteins regulate microRNA stability: Increased microRNA abundance by Argonaute proteins is due to microRNA stabilization. RNA Biol. 2011; 8: 1149– 1157. 21941127
  43. [53]Olejniczak S.H., La Rocca G., Gruber J.J., Thompson C.B. Long-lived microRNA-Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses. Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 157– 162. 23248281 PMC3538211
  44. [54]Erazo-Oliveras A., Najjar K., Truong D., Wang T.Y., Brock D.J., Prater A.R., Pellois J.P. The late endosome and its lipid BMP act as gateways for efficient cytosolic access of the delivery agent dfTAT and its macromolecular cargos. Cell Chem. Biol. 2016; 23: 598– 607. 27161484 PMC4874899
  45. [55]Juliano R.L. Intracellular trafficking and endosomal release of Oligonucleotides: What we know and what we Don’t. Nucleic Acid Ther. 2018; 28: 166– 177. 29708838 PMC5994677
  46. [56]Wagenaar T.R., Tolstykh T., Shi C., Jiang L., Zhang J., Li Z., Yu Q., Qu H., Sun F., Cao H. et al. Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells. Nucleic Acids Res. 2015; 43: 1204– 1215. 25550434 PMC4333411
  47. [57]Wang S., Sun H., Tanowitz M., Liang X.H., Crooke S.T. Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Res. 2017; 45: 5309– 5322. 28379543 PMC5605259
  48. [58]Siomi H., Siomi M.C. RISC hitches onto endosome trafficking. Nat. Cell Biol. 2009; 11: 1049– 1051. 19724258
  49. [59]Gibbings D.J., Ciaudo C., Erhardt M., Voinnet O. Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat. Cell Biol. 2009; 11: 1143– 1149. 19684575
  50. [60]Lee Y.S., Pressman S., Andress A.P., Kim K., White J.L., Cassidy J.J., Li X., Lubell K., Lim D.H., Cho I.S. et al. Silencing by small RNAs is linked to endosomal trafficking. Nat. Cell Biol. 2009; 11: 1150– 1156. 19684574 PMC2737091
  51. [61]Stalder L., Heusermann W., Sokol L., Trojer D., Wirz J., Hean J., Fritzsche A., Aeschimann F., Pfanzagl V., Basselet P. et al. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing. EMBO J. 2013; 32: 1115– 1127. 23511973 PMC3630355

Cited by

Sorted by citations

Authors
Brown, C. R., Gupta, S., Qin, J., Racie, T., He, G., Lentini, S., Malone, R., Yu, M., Matsuda, S., Shulga-Morskaya, S., Nair, A. V., Theile, C. S., Schmidt, K., Shahraz, A., Goel, V., Parmar, R. G., Zlatev, I., Schlegel, M. K., Nair, J. K., Jayaraman, M., Manoharan, M., Brown, D., Maier, M. A., & Jadhav, V.
Affiliations
  • Alnylam Pharmaceuticals (United States) (US)
  • Alnylam Pharmaceuticals, Inc., Cambridge, MA 02142, USA
  • Harvard University (US)
Corresponding author
Jadhav, Vasant
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2020-08-18
Volume
48
Issue
21
Pages
11827-11844
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkaa670
PMC
PMC7708070
OpenAlex
W3063613132
Cited by
285
References
9
Citations by year
2026: 37 · 2025: 64 · 2024: 62 · 2023: 50 · 2022: 44 · 2021: 25 · 2020: 3
FWCI
9.90
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Alnylam Pharmaceuticals
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
siRNA delivery, RNA interference, Argonaute 2, lipid nanoparticles, siRNA stability
MeSH terms
Acetylgalactosamine, Animals, Biological Transport, Drug Carriers, Drug Stability, Female, Glycoconjugates, Humans, Hydrogen-Ion Concentration, Liver, Mice, Inbred C57BL, Prealbumin, Time Factors, Gene Silencing, Hepatocytes, RNA, Small Interfering, Asialoglycoprotein Receptor, Mice, Nanoparticles, Argonaute Proteins

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