Cholesterol (Chol) conjugation to the 5′ or 3′ end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification.
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References
1.
CrookeST, eds. Antisense Drug Technologies, Second Edition. (2007). CRC Press, Boca Raton, FL.
2.
FreierSM and AltmannKH. (1997). The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res, 25:4429–4443.
3.
MartinP. (1995). Ein neuer Zugang zu 2′-O-Alkylribonucleosiden und Eigenschaften deren Oligonucleotide. Helv Chim Acta, 78:486–504.
4.
ObikaS, NanbuD, HariY, MorioK, InY, IshidaT and ImanishiT. (1997). Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3’-endo sugar puckering. Tetrahedron Lett, 38:8735–8738.
5.
ObikaS, NanbuD, HariY, AndohJ, MorioK, DoiT and ImanishiT. (1998). Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneribonucleosides. Tetrahedron Lett, 39:5401–5404.
6.
SinghSK, KoshkinAA, WengelJ and NielsenP. (1998). LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem Commun, 4:455–456.
7.
MoniaBP, LesnikEA, GonzalezC, LimaWF, McGeeD, GuinossoCJ, KawasakiAM, CookPD and FreierSM. (1993). Evaluation of 2′-modified oligonucleotides containing deoxy gaps as antisense inhibitors of gene expression. J Biol Chem, 268:14514–14522.
8.
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.
9.
PrakashTP, YuJ, MigawaMT, KinbergerGA, WanWB, ØstergaardME, 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.
10.
LetsingerRL, ZhangGR, SunDK, IkeuchiT and SarinPS. (1989). Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Proc Natl Acad Sci USA, 86:6553–6556.
11.
CrookeST, GrahamMJ, ZuckermanJE, BrooksD, ConklinBS, CumminsLL, GreigMJ, GuinossoCJ, KornbrustD, et al. (1996). Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J Pharmacol Exp Ther, 277:923–937.
12.
GaoWY, HanFS, StormC, EganW and ChengYC. (2000). Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. Mol Pharmacol, 41:223–229.
13.
WadaS, YasuharaH, WadaF, SawamuraM, WakiR, YamamotoT, Harada-ShibaM and ObikaS. (2016). Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides. J Control Release, 226:57–65.
14.
MatysiakS, FrankR and PfleidererW. (1997). Acetal oligonucleotide conjugates in antisense strategy. Nucleosides Nucleotides, 16:855–861.
15.
KurzA, BungeA, WindeckAK, RostM, FlascheW, ArbuzovaA, StrohbachD, MüllerS, LiebscherJ, HusterD and HerrmannA. (2006). Lipid-anchored oligonucleotides for stable double-helix formation in distinct membrane domains. Angew Chem Int Ed Engl, 45:4440–4444.
16.
TomkinsJM, BarnesKJ, BlackerAJ, WatkinsWJ and AbellC. (1997). Lipophilic modification of oligonucleotides. Tetrahedron Lett, 38:691–694.
17.
BryldT and LomholtC. (2007). Attachment of cholesterol to amino-LNA: synthesis and hybridization properties. Nucleosides Nucleotides Nucleic Acids, 26:1645–1647.
18.
ZhangZ, SmithJA, SmythAP, EisenbergW, PariGS and TangJY. (1996). Thiono triester modified antisence oligonucleotides for inhibition of human cytomegalovirus in vitro. Bioorg Med Chem Lett, 6:1911–1916.
19.
StraarupEM, FiskerN, HedtjärnM, LindholmMW, RosenbohmC, AarupV, HansenHF, ØrumH, HansenJB 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.
20.
ZhangZ, TangJX and TangJY. (1995). Syntheses and properties of novel thiono triester modified antisense oligodeoxynucleotide phosphorothioates. Bioorg Med Chem Lett, 5:1735–1740.
21.
ShimizuR, KitadeM, KobayashiT, HoriS and WatanabeA. (2015). Pharmacokinetic-pharmacodynamic modeling for reduction of hepatic apolipoprotein B mRNA and plasma total cholesterol after administration of antisense oligonucleotide in mice. J Pharmacokinet Pharmacodyn, 42:67–77.
22.
TurnpennyP, RawalJ, SchardtT, LamorattaS, MuellerH, WeberM and BradyK. (2011). Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid–liquid extraction LC–MS/MS analytical approach. Bioanalysis, 3:1911–1921.
23.
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.
24.
SaneyoshiH, KondoK, SagawaN and OnoA. (2016). Glutathione-triggered activation of the model of pro-oligonucleotide with benzyl protecting groups at the internucleotide linkage. Bioorg Med Chem Lett, 26:622–625.
25.
SaneyoshiH, IketaniK, KondoK, SaneyoshiT, OkamotoI and OnoA. (2016). Synthesis and characterization of cell-permeable oligonucleotides bearing reduction-activated protecting groups on the internucleotide linkages. Bioconjug Chem, 27:2149–2156.
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