Small interfering RNA (siRNA) represents a transformative therapeutic class that enables precise gene silencing through RNA interference (RNAi). N-acetyl galactosamine (GalNAc)-conjugated siRNAs have achieved remarkable clinical success with six FDA-approved therapeutics targeting liver diseases. Following subcutaneous administration, GalNAc–siRNAs rapidly accumulate in hepatocytes via asialoglycoprotein receptor (ASGPR)-mediated endocytosis and are sequestered within endolysosomal compartments, creating an intracellular depot that sustains therapeutic effects for weeks to months. While RNAi-mediated degradation and nuclease metabolism contribute to siRNA clearance, the detection of full-length, active siRNA in circulation long after dosing suggests alternative clearance mechanisms exist. This review examines the pharmacokinetic properties and intracellular trafficking of GalNAc–siRNA, with particular focus on underexplored clearance pathways from hepatocytes. We discuss potential mechanisms including endosomal recycling, efflux via exosomes and lysosomal exocytosis that may facilitate siRNA redistribution from tissues to circulation. Understanding these clearance mechanisms could enable correlation of plasma and tissue drug concentrations, reduce preclinical animal use, inform clinical dosing strategies and guide the design of next-generation siRNA therapeutics with optimized tissue residence time and enhanced therapeutic durability.
ZhangJ, ChenB, GanC, et al.A comprehensive review of small interfering RNAs (siRNAs): Mechanism, therapeutic targets, and delivery strategies for cancer therapy. Int J Nanomedicine2023;18:7605–7635; doi: 10.2147/IJN.S436038
2.
AnG. Pharmacokinetics and pharmacodynamics of GalNAc-Conjugated siRNAs. J Clin Pharmacol2024;64:45–57; doi: 10.1002/jcph.2337
3.
SehgalI, EellsK, HudsonI. A comparison of currently approved small interfering RNA (siRNA) Medications to alternative treatments by costs, indications, and medicaid coverage. Pharmacy (Basel)2024;12:58; doi: 10.3390/pharmacy12020058
4.
ZhuY, ZhuL, WangX, et al.RNA-based therapeutics: An overview and prospectus. Cell Death Dis2022;13:644; doi: 10.1038/s41419-022-05075-2
5.
ChoiGW, KimJH, KangDW, et al.A journey into siRNA therapeutics development: A focus on Pharmacokinetics and Pharmacodynamics. Eur J Pharm Sci2025;205:106981; doi: 10.1016/j.ejps.2024.106981
6.
Ali ZaidiSS, FatimaF, Ali ZaidiSA, et al.Engineering siRNA therapeutics: Challenges and strategies. J Nanobiotechnology2023;21:381; doi: 10.1186/s12951-023-02147-z
7.
BrownCR, GuptaS, QinJ, et al.Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates. Nucleic Acids Res2020;48:11827–11844; doi: 10.1093/nar/gkaa670
8.
HuB, ZhongL, WengY, et al.Therapeutic siRNA: State of the art. Signal Transduct Target Ther2020;5:101; doi: 10.1038/s41392-020-0207-x
9.
SpiessM. The asialoglycoprotein receptor: A model for endocytic transport receptors. Biochemistry1990;29:10009–10018; doi: 10.1021/bi00495a001
10.
SpringerAD, DowdySF. GalNAc-siRNA conjugates: Leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther2018;28:109–118; doi: 10.1089/nat.2018.0736
11.
SchwartzAL, FridovichSE, LodishHF. Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem1982;257:4230–4237; doi: 10.1016/s0021-9258(18)34710-0
12.
BaenzigerJ, FieteD. Galactose and N-Acetylgalactosamine-Specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell1980;22:611–620; doi: 10.1016/0092-8674(80)90371-2
13.
SteerCJ, AshwellG. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem1980;255:3008–3013; doi: 10.1016/s0021-9258(19)85843-x
14.
DowdySF. Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem? RNA2023;29:396–401; doi: 10.1261/rna.079507.122
DowdySF, SettenRL, CuiXS, et al.Delivery of RNA therapeutics: The great endosomal escape!Nucleic Acid Ther2022;32:361–368; doi: 10.1089/nat.2022.0004
17.
SardhE, HarperP, BalwaniM, et al.Phase 1 trial of an RNA interference therapy for acute intermittent porphyria. N Engl J Med2019;380:549–558; doi: 10.1056/NEJMoa1807838
18.
FrishbergY, DeschênesG, GroothoffJW, study collaborators. et al.; Phase 1/2 study of lumasiran for treatment of primary hyperoxaluria type 1: A placebo-controlled randomized clinical trial. Clin J Am Soc Nephrol2021;16:1025–1036; doi: 10.2215/CJN.14730920
19.
LuoZ, HuangZ, SunF, et al.The clinical effects of inclisiran, a first-in-class LDL-C lowering siRNA therapy, on the LDL-C levels in Chinese patients with hypercholesterolemia. J Clin Lipidol2023;17:392–400; doi: 10.1016/j.jacl.2023.04.010
20.
HabtemariamBA, KarstenV, AttarwalaH, et al.Single-Dose pharmacokinetics and pharmacodynamics of transthyretin targeting N-acetylgalactosamine-Small interfering ribonucleic acid conjugate, Vutrisiran, in healthy subjects. Clin Pharmacol Ther2021;109:372–382; doi: 10.1002/cpt.1974
21.
HoppeB, KochA, CochatP, et al.Safety, pharmacodynamics, and exposure-response modeling results from a first-in-human phase 1 study of nedosiran (PHYOX1) in primary hyperoxaluria. Kidney Int2022;101:626–634; doi: 10.1016/j.kint.2021.08.015
22.
PasiKJ, LissitchkovT, MamonovV, et al.Targeting of antithrombin in hemophilia A or B with investigational siRNA therapeutic fitusiran-Results of the phase 1 inhibitor cohort. J Thromb Haemost2021;19:1436–1446; doi: 10.1111/jth.15270
McDougallR, RamsdenD, AgarwalS, et al.The nonclinical disposition and pharmacokinetic/pharmacodynamic properties of N-Acetylgalactosamine-Conjugated small interfering RNA are highly predictable and build confidence in translation to human. Drug Metab Dispos2022;50:781–797; doi: 10.1124/dmd.121.000428
30.
StenS, CardilinT, AntonssonM, et al.Plasma Pharmacokinetics of N-Acetylgalactosamine-Conjugated Small-Interfering Ribonucleic Acids (GalNAc-Conjugated siRNAs). Clin Pharmacokinet2023;62:1661–1672; doi: 10.1007/s40262-023-01314-7
31.
NairJK, AttarwalaH, SehgalA, et al.Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates. Nucleic Acids Res2017;45:10969–10977; doi: 10.1093/nar/gkx818
32.
BoianelliA, AokiY, IvanovM, et al.Cross-Species translation of biophase half-life and potency of GalNAc-Conjugated siRNAs. Nucleic Acid Ther2022;32:507–512; doi: 10.1089/nat.2022.0010
33.
Castellanos-RizaldosE, BrownCR, DenninS, et al.RT-qPCR methods to support pharmacokinetics and drug mechanism of action to advance development of RNAi therapeutics. Nucleic Acid Ther2020;30:133–142; doi: 10.1089/nat.2019.0840
CullenPJ, SteinbergF. To degrade or not to degrade: Mechanisms and significance of endocytic recycling. Nat Rev Mol Cell Biol2018;19:679–696; doi: 10.1038/s41580-018-0053-7
36.
Van de VyverT, De SmedtSC, RaemdonckK. Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics. Adv Drug Deliv Rev2022;181:114041; doi: 10.1016/j.addr.2021.114041
37.
JulianoRL. Intracellular trafficking and endosomal release of oligonucleotides: What we know and what we don’t. Nucleic Acid Ther2018;28:166–177; doi: 10.1089/nat.2018.0727
38.
GeuzeH, SlotJ, StrousG, et al.Intracellular site of asialoglycoprotein receptor-ligand uncoupling: Double-Label lmmunoelectron microscopy during receptor-mediated endocytosis. Cell1983;32:277–287; doi: 10.1016/0092-8674(83)90518-4
39.
HumphreysSC, ThayerMB, CampbellJ, et al.Emerging siRNA design principles and consequences for biotransformation and disposition in drug development. J Med Chem2020;63:6407–6422; doi: 10.1021/acs.jmedchem.9b01839
40.
PrakashTP, GrahamMJ, YuJ, et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res2014;42:8796–8807; doi: 10.1093/nar/gku531
41.
KollerE, VincentTM, ChappellA, et al.Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res2011;39:4795–4807; doi: 10.1093/nar/gkr089
42.
GearyRS, WancewiczE, MatsonJ, et al.Effect of dose and plasma concentration on liver uptake and pharmacologic activity of a 2′-methoxyethyl modified chimeric antisense oligonucleotide targeting PTEN. Biochem Pharmacol2009;78:284–291; doi: 10.1016/j.bcp.2009.04.013
43.
DepreyK, BatistatouN, KritzerJA. A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics. Nucleic Acids Res2020;48:7623–7639; doi: 10.1093/nar/gkaa576
44.
BrownDW, WeeP, BhandariP, et al.Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles. Cell2024;187:5357–5375.e24; doi: 10.1016/j.cell.2024.07.023
45.
LiX, KheirabadiM, DoughertyPG, et al.The endosomal escape vehicle platform enhances delivery of oligonucleotides in preclinical models of neuromuscular disorders. Mol Ther Nucleic Acids2023;33:273–285; doi: 10.1016/j.omtn.2023.06.022
46.
ZhangH, et al.Comparative Metabolism of a GalNAc-Conjugated siRNA, inclisiran, among various in vitro systems and correlations with in vivo metabolism in rats. Drug Metabolism and Disposition2025; doi: 10.1016/j.dmd.2025.100089
47.
BasiriB, XieF, WuB, et al.Introducing an in vitro liver stability assay capable of predicting the in vivo pharmacodynamic efficacy of siRNAs for IVIVC. Mol Ther Nucleic Acids2020;21:725–736; doi: 10.1016/j.omtn.2020.07.012
48.
HouseleyJ, TollerveyD. The many pathways of RNA degradation. Cell2009;136:763–776; doi: 10.1016/j.cell.2009.01.019
49.
ZhaoYG, ZhangH. Autophagosome maturation: An epic journey from the ER to lysosomes. J Cell Biol2019;218:757–770; doi: 10.1083/jcb.201810099
50.
AmaralO, MartinsM, OliveiraAR, et al.The biology of lysosomes: From order to disorder. Biomedicines2023;11:213; doi: 10.3390/biomedicines11010213
KuSH, JoSD, LeeYK, et al.Chemical and structural modifications of RNAi therapeutics. Adv Drug Deliv Rev2016;104:16–28; doi: 10.1016/j.addr.2015.10.015
54.
CaoX, SunY, LuP, et al.Fluorescence imaging of intracellular nucleases-A review. Anal Chim Acta2020;1137:225–237; doi: 10.1016/j.aca.2020.08.013
55.
KhvorovaA, WattsJK. The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol2017;35:238–248; doi: 10.1038/nbt.3765
56.
LiuJ, CarmellMA, RivasFV, et al.Argonaute2 is the catalytic engine of mammalian RNAi. Science2004;305:1437–1441; doi: 10.1126/science.1102513
57.
NakanishiK. Anatomy of RISC: How do small RNAs and chaperones activate Argonaute proteins? Wiley Interdiscip Rev RNA2016;7:637–660; doi: 10.1002/wrna.1356
OlejniczakSH, La RoccaG, GruberJJ, et al.Long-lived microRNA-Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses. Proc Natl Acad Sci U S A2013;110:157–162; doi: 10.1073/pnas.1219958110
60.
JohannesL, WunderC. Retrograde transport: Two (or more) roads diverged in an endosomal tree? Traffic2011;12:956–962; doi: 10.1111/j.1600-0854.2011.01200.x
61.
LiuXM, HalushkaMK. Beyond the bubble: A debate on microRNA sorting into extracellular vesicles. Lab Invest2025;105:102206; doi: 10.1016/j.labinv.2024.102206
62.
PayandehZ, TangruksaB, SynnergrenJ, et al.Extracellular vesicles transport RNA between cells: Unraveling their dual role in diagnostics and therapeutics. Mol Aspects Med2024;99:101302; doi: 10.1016/j.mam.2024.101302
63.
VojtechL, WooS, HughesS, et al.Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res2014;42:7290–7304; doi: 10.1093/nar/gku347
64.
WeiZ, BatagovAO, SchinelliS, et al.Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nat Commun2017;8:1145; doi: 10.1038/s41467-017-01196-x
65.
SantangeloL, GiuratoG, CicchiniC, et al.The RNA-Binding protein SYNCRIP Is a component of the hepatocyte exosomal machinery controlling MicroRNA sorting. Cell Rep2016;17:799–808; doi: 10.1016/j.celrep.2016.09.031
66.
HoborF, DallmannA, BallNJ, et al.A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets. Nat Commun2018;9:831; doi: 10.1038/s41467-018-03182-3
67.
BurattaS, TanciniB, SaginiK, et al.Lysosomal exocytosis, exosome release and secretory autophagy: The autophagic- and endo-lysosomal systems go extracellular. Int J Mol Sci2020;21:2576; doi: 10.3390/ijms21072576
68.
VermeulenLMP, BransT, De SmedtSC, et al.Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy. Nano Today2018;21:74–90; doi: 10.1016/j.nantod.2018.06.007
69.
ChowFW-N, KoutsovoulosG, Ovando-VázquezC, et al.Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides. Nucleic Acids Res2019;47:3594–3606; doi: 10.1093/nar/gkz142
70.
MeloSA, SugimotoH, O’ConnellJT, et al.Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell2014;26:707–721; doi: 10.1016/j.ccell.2014.09.005
71.
ArroyoJD, ChevilletJR, KrohEM, et al.Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A2011;108:5003–5008; doi: 10.1073/pnas.1019055108
72.
TurchinovichA, WeizL, LangheinzA, et al.Characterization of extracellular circulating microRNA. Nucleic Acids Res2011;39:7223–7233; doi: 10.1093/nar/gkr254
73.
LiQ, PengG, LiuH, et al.Molecular mechanisms of secretory autophagy and its potential role in diseases. Life Sci2024;347:122653; doi: 10.1016/j.lfs.2024.122653
74.
LiX, ZhaoH. Targeting secretory autophagy in solid cancers: Mechanisms, immune regulation and clinical insights. Exp Hematol Oncol2025;14:12; doi: 10.1186/s40164-025-00603-0
75.
LandesmanY, SvrzikapaN, CognettaA, et al.In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR). Silence2010;1:16; doi: 10.1186/1758-907X-1-16
76.
JeonJY, AyyarVS, MitraA. Pharmacokinetic and pharmacodynamic modeling of siRNA therapeutics - a minireview. Pharm Res2022;39:1749–1759; doi: 10.1007/s11095-022-03333-8
77.
PeiD, BuyanovaM. Overcoming endosomal entrapment in drug delivery. Bioconjug Chem2019;30:273–283; doi: 10.1021/acs.bioconjchem.8b00778
78.
EstupiñánHY, BaladiT, RoudiS, et al.Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotides in vitro. Mol Ther Nucleic Acids2025;36:102522; doi: 10.1016/j.omtn.2025.102522
