Ligand-targeted drug delivery (LTDD) has gained more attention in the field of nucleic acid therapeutics. To further elicit the potential of therapeutic oligonucleotides by means of LTDD, we newly developed (R)- and (S)-3-amino-1,2-propanediol (APD) manifold for ligand conjugation. N-acetylgalactosamine (GalNAc)/asialoglycoprotein receptor (ASGPr) system has been shown to be a powerful and robust paradigm of LTDD. Our novel APD-based GalNAc (GalNAcAPD) was shown to have intrinsic chemical instability that could play a role in better manipulation of active drug release. The APD manifold also enables facile production of conjugates through an on-support ligand cluster synthesis. We showed in a series of in vivo studies that while the knockdown activity of antisense oligonucleotides (ASOs) bearing 5′-GalNAcAPD was comparable to the conventional hydroxy-L-prolinol-linked GalNAc (GalNAcHP), 3′-GalNAcAPD elicited ASO activity by more than twice as much as the conventional 3′-GalNAcHP. This was ascribed partly to the GalNAcAPD's ideal susceptibility to nucleolytic digestion, which is expected to facilitate cytosolic internalization of ASO drugs. Moreover, an in vivo/ex vivo imaging study visualized the enhancement effect of monoantennary GalNAcAPD on liver localization of ASOs. This versatile manifold with chemical and biological instability would benefit therapeutic oligonucleotides that target both the liver and extrahepatic tissues.
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References
1.
DebackerAJ, VoutilaJ, CatleyM, BlakeyD and HabibN. (2020). Delivery of oligonucleotides to the liver with GalNAc: from research to registered therapeutic drug. Mol Ther, 28:1759–1771.
2.
HuangY. (2017). Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics. Mol Ther Nucleic Acids, 6:116–132.
3.
BenizriS, GissotA, MartinA, VialetB, GrinstaffMW and BarthélémyP. (2019). Bioconjugated oligonucleotides: recent developments and therapeutic applications. Bioconjug Chem, 30:366–383.
4.
ÄmmäläC, DruryWJ, KnerrL, AhlstedtI, Stillemark-BilltonP, Wennberg-HuldtC, AnderssonEM, ValeurE, Jansson-LöfmarkR, et al. (2018). Targeted delivery of antisense oligonucleotides to pancreatic β-cells. Sci Adv, 4:eaat3386.
5.
Pricer WEJ and AshwellG. (1971). The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem, 246:4825–4833.
6.
AshwellG and HarfordJ. (1982). Carbohydrate-Specific Receptors of the Liver. Annu Rev Biochem, 51:531–554.
7.
DrickamerK and TaylorME. (1993). Biology of animal lectins. Annu Rev Cell Biol, 9:237–264.
8.
Weigel PH. (1980). Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes. J Biol Chem, 255:6111–6120.
9.
SchwartzAL, FridovichSE, KnowlesBB and LodishHF. (1981). Characterization of the asialoglycoprotein receptor in a continuous hepatoma line. J Biol Chem, 256:8878–8881.
10.
SchwartzAL, FridovichSE and LodishHF. (1982). Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem, 257:4230–4237.
11.
SchwartzAL, RupD and LodishHF. (1980). Difficulties in the quantification of asialoglycoprotein receptors on the rat hepatocyte. J Biol Chem, 255:9033–9036.
12.
BischoffJ and LodishHF. (1987). Two asialoglycoprotein receptor polypeptides in human hepatoma cells. J Biol Chem, 262:11825–11832.
13.
SpiessM and LodishHF. (1985). Sequence of a second human asialoglycoprotein receptor: conservation of two receptor genes during evolution. Proc Natl Acad Sci U S A, 82:6465–6469.
14.
SmithEA, ThomasWD, KiesslingLL and CornRM. (2003). Surface plasmon resonance imaging studies of protein-carbohydrate interactions. J Am Chem Soc, 125:6140–6148.
15.
MamidyalaSK, DuttaS, ChrunykBA, PrévilleC, WangH, WithkaJM, McCollA, SubashiTA, HawrylikSJ, et al. (2012). Glycomimetic ligands for the human asialoglycoprotein receptor. J Am Chem Soc, 134:1978–1981.
16.
LeeYC, TownsendRR, HardyMR, LönngrenJ, ArnarpJ, HaraldssonM and LönnH. (1983). Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J Biol Chem, 258:199–202.
17.
ConnollyDT, TownsendRR, KawaguchiK, BellWR and LeeYC. (1982). Binding and endocytosis of cluster glycosides by rabbit hepatocytes. Evidence for a. short-circuit pathway that does not lead to degradation. J Biol Chem 257:939–945.
18.
SpringerAD and DowdySF. (2018). GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther, 28:109–118.
19.
HuangX, LerouxJC and CastagnerB. (2017). Well-defined multivalent ligands for hepatocytes targeting via asialoglycoprotein receptor. Bioconjug Chem, 28:283–295.
20.
HangelandJJ, LevisJT, LeeYC and Ts'oPO. (1995). Cell-type specific and ligand specific enhancement of cellular uptake of oligodeoxynucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3. Bioconjug Chem, 6:695–701.
21.
HangelandJJ, FlesherJE, DeamondSF, LeeYC, Ts'OPO and FrostJJ. (1997). Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse. Antisense Nucleic Acid Drug Dev, 7:141–149.
22.
BiessenEA, VietschH, RumpET, FluiterK, KuiperJ, BijsterboschMK and van BerkelTJ. (1999). Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo. Biochem J, 340:783–792.
23.
PrakashTP, GrahamMJ, YuJ, CartyR, LowA, ChappellA, SchmidtK, ZhaoC, AghajanM, et al. (2014). Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res, 42:8796–8807.
24.
NairJK, WilloughbyJLS, ChanA, CharisseK, AlamMR, WangQ, HoekstraM, KandasamyP, KelinAV, et al. (2014). Multivalent N -acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc, 136:16958–16961.
25.
NairJK, AttarwalaH, SehgalA, WangQ, AluriK, ZhangX, GaoM, LiuJ, IndrakantiR, et al. (2017). Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates. Nucleic Acids Res, 45:10969–10977.
26.
FitzgeraldK, WhiteS, BorodovskyA, BettencourtBR, StrahsA, ClausenV, WijngaardP, HortonJD, TaubelJ, et al. (2017). A highly durable RNAi therapeutic inhibitor of PCSK9. N Engl J Med, 376:41–51.
27.
KhvorovaA and WattsJK. (2017). The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol, 35:238–248.
28.
MaierMA, YannopoulosCG, MohamedN, RolandA, FritzH, MohanV, JustG and ManoharanM. (2003). Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting. Bioconjug Chem, 14:18–29.
29.
PrakashTP, YuJ, MigawaMT, KinbergerGA, WanWB, ME Østergaard, CartyRL, VasquezG, LowA, et al. (2016). Comprehensive structure-activity relationship of triantennary N-acetylgalactosamine conjugated antisense oligonucleotides for targeted delivery to hepatocytes. J Med Chem, 59:2718–2733.
30.
ØstergaardME, YuJ, KinbergerGA, WanWB, MigawaMT, VasquezG, SchmidtK, GausHJ, MurrayHM, et al. (2015). Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides. Bioconjug Chem, 26:1451–1455.
31.
MigawaMT, PrakashTP, VasquezG, WanWB, YuJ, KinbergerGA, ME Østergaard, SwayzeEE and SethPP. (2016). A convenient synthesis of 5’-triantennary N-acetyl-galactosamine clusters based on nitromethanetrispropionic acid. Bioorg Med Chem Lett, 26:2194–2197.
32.
MeadeBR, GogoiK, HamilAS, Palm-ApergiC, Van Den BergA, HagopianJC, SpringerAD, EguchiA, KacsintaAD, et al. (2014). Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat Biotechnol, 32:1256–1261.
33.
WatanabeA, NakajimaM, KasuyaT, OnishiR, KitadeN, MayumiK, IkeharaT and KugimiyaA. (2016). Comparative characterization of hepatic distribution and mRNA reduction of antisense oligonucleotides conjugated with triantennary N-acetyl galactosamine and lipophilic ligands targeting apolipoprotein B. J Pharmacol Exp Ther, 357:320–330.
34.
MatsudaS, KeiserK, NairJK, CharisseK, ManoharanRM, KretschmerP, PengCG, Kel'inAV, KandasamyP, et al. (2015). siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chem Biol, 10:1181–1187.
35.
YamamotoT, SawamuraM, WadaF, Harada-ShibaM and ObikaS. (2016). Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides. Bioorganic Med Chem, 24:26–32.
36.
SharmaVK, OsbornMF, HasslerMR, EcheverriaD, LyS, UlashchikEA, Martynenko-MakaevYV, ShmanaiVV, ZatsepinTS, KhvorovaA and WattsJK. (2018). Novel cluster and monomer-based GalNAc structures induce effective uptake of siRNAs in vitro and in vivo. Bioconjug Chem, 29:2478–2488.
37.
FarzanVM, UlashchikEA, Martynenko-MakaevYV, KvachMV, AparinIO, BrylevVA, PrikazchikovaTA, MaklakovaSY, MajougaAG, et al. (2017). Automated solid-phase click synthesis of oligonucleotide conjugates: from small molecules to diverse N-acetylgalactosamine clusters. Bioconjug Chem, 28:2599–2607.
38.
AviñóA, OcampoSM, LucasR, ReinaJJ, MoralesJC, PeralesJC and EritjaR. (2011). Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations. Mol Divers, 15:751–757.
39.
WadaF, YamamotoT, UedaT, SawamuraM, WadaS, Harada-ShibaM and ObikaS. (2018). Cholesterol-GalNAc dual conjugation strategy for reducing renal distribution of antisense oligonucleotides. Nucleic Acid Ther, 28:50–57.
40.
YamamotoT, SawamuraM, TeradaC, KashiwadaK, WadaF, YamayoshiA, ObikaS and Harada-ShibaM. (2020). Effect of modular conjugation strategy for N-acetylgalactosamine-targeted antisense oligonucleotides. Nucleosides Nucleotides Nucleic Acids, 39:109–118.
41.
WeingärtnerA, BethgeL, WeissL, SternbergerM and LindholmMW. (2020). Less is more: novel hepatocyte-targeted siRNA conjugates for treatment of liver-related disorders. Mol Ther Nucleic Acids, 21:242–250.
42.
RajeevKG, NairJK, JayaramanM, CharisseK, TanejaN, O'SheaJ, WilloughbyJLS, YuciusK, NguyenT, et al. (2015). Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo. ChemBioChem, 16:903–908.
43.
YamamotoT, TeradaC, KashiwadaK, YamayoshiA, Harada-ShibaM and Obika.S (2019). Synthesis of monovalent N-acetylgalactosamine phosphoramidite for liver-targeting oligonucleotides. Curr Protoc Nucleic Acid Chem, 78:e99.
44.
KinbergerGA, PrakashTP, YuJ, VasquezG, LowA, ChappellA, SchmidtK, MurrayHM, GausH, SwayzeEE and SethPP. (2016). Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes. Bioorganic Med Chem Lett, 26:3690–3693.
45.
SchmidtK, PrakashTP, DonnerAJ, KinbergerGA, GausHJ, LowA, ME Østergaard, BellM, SwayzeEE and SethPP. (2017). Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor. Nucleic Acids Res, 45:2294–2306.
46.
AzhayevAV and AntopolskyML. (2001). Amide group assisted 3′-dephosphorylation of oligonucleotides synthesized on universal A-supports. Tetrahedron, 57:4977–4986.
47.
GuzaevAP and ManoharanM. (2003). A conformationally preorganized universal solid support for efficient oligonucleotide synthesis. J Am Chem Soc, 125:2380–2381.
48.
SchroederGK, LadC, WymanP, WilliamsNH and WolfendenR. (2006). The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA. Proc Natl Acad Sci U S A, 103:4052–4055.
49.
SoutschekJ, AkincA, BramlageB, CharisseK, ConstienR, DonoghueM, ElbashirS, GelckA, HadwigerP, et al. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature, 432:173–178.
50.
CrookeRM, GrahamMJ, LemonidisKM, WhippleCP, KooS and PereraRJ. (2005). An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J Lipid Res, 46:872–884.
51.
StraarupEM, FiskerN, HedtjarnM, LindholmMW, RosenbohmC, AarupV, HansenHF, OrumH, HansenJBR and KochT. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res, 38:7100–7111.
52.
KasteleinJJP, WedelMK, BakerBF, SuJ, BradleyJD, YuRZ, ChuangE, GrahamMJ and CrookeRM. (2006). Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation, 114:1729–1735.
53.
ShawJP, KentK, BirdJ, FishbackJ and FroehlerB. (1991). Modified deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Res, 19:747–750.
54.
PostN, YuR, GreenleeS, GausH, HurhE, MatsonJ and WangY. (2019). Metabolism and disposition of volanesorsen, a 2’-O-(2 methoxyethyl) antisense oligonucleotide, across species. Drug Metab Dispos, 47:1164–1173.
55.
YuRZ, GrahamMJ, PostN, RineyS, ZanardiT, HallS, BurkeyJ, ShemeshCS, PrakashTP, et al. (2016). Disposition and pharmacology of a GalNAc3-conjugated ASO targeting human lipoprotein (a) in mice. Mol Ther Nucleic Acids, 5:e317.
56.
ShemeshCS, YuRZ, GausHJ, GreenleeS, PostN, SchmidtK, MigawaMT, SethPP, ZanardiTA, et al. (2016). Elucidation of the biotransformation pathways of a Galnac3-conjugated antisense oligonucleotide in rats and monkeys. Mol Ther Nucleic Acids, 5:e319.
57.
BonC, HoferT, Bousquet-MélouA, DaviesMR and KrippendorffBF. (2017). Capacity limits of asialoglycoprotein receptor-mediated liver targeting. MAbs, 9:1360–1369.
58.
LendvaiG, VelikyanI, BergströmM, EstradaS, LaryeaD, VäliläM, SalomäkiS, LångströmB and RoivainenA. (2005). Biodistribution of 68Ga-labelled phosphodiester, phosphorothioate, and 2′-O-methyl phosphodiester oligonucleotides in normal rats. Eur J Pharm Sci, 26:26–38.
59.
GearyRS, NorrisD, YuR and BennettCF. (2015). Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev, 87:46–51.
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