Location, Location, Location – GalNAc for targeted delivery to hepatocytes
In oligonucleotide therapeutics as in the property market, three things matter – location, location, location. If there are twisting roads with lots of compulsory diversions between your work place and your dream home (or the blood stream and your target tissue) it will likely be a No Go. Direct and reliable public transport might just change your mind, however.
In scientific terms, delivery of therapeutic oligonucleotides has long been considered a problem of such compulsory diversions. Distribution to intended target cells and tissues via systemic delivery is hampered because, along the way, therapeutic oligonucleotides are sequestered by the liver and kidney. Even within the liver, therapeutic oligonucleotides accumulate in the Kupffer and endothelial cells while the genes of therapeutic interest are often expressed only in hepatocytes.
Graham et al.1 showed in 1998 that phosphorothioate-modified antisense oligonucleotides (ASOs) are preferentially internalized by endothelial cells lining the liver sinusoids and by Kupffer cells (70-95% depending on dose) resulting in little ASO accumulation in hepatocytes. A year later, Biessen et al.2 showed that linking phosphorothioate oligonucleotides to a multivalent N-acetylgalactosamine cluster, a synthetic ligand for the asialoglycoprotein receptor (ASGPR), results in an ASO that accumulates mostly in hepatocytes (75 ± 4% of the total liver uptake). Unfortunately, this study did not assess the effect of GalNAc conjugation on ASO activity, and the benefits of GalNAc-targeted delivery remained unclear.
ASGPR is a highly expressed (~500 000 copies/cell compared to ~100 000 copies/cell for the insulin receptor) calcium-ion dependent lectin primarily found on mammalian hepatocytes. ASGPR is involved in clearing aged serum glycoproteins via clathrin-mediated endocytosis. The receptor has high avidity for multivalent GalNAc and consists of two subunits, a major 48 kDa subunit (ASGPR-1) and a minor 40 kDa subunit (ASGPR-2). These subunits form oligomers in various configurations with each subunit able to bind a monovalent GalNAc through bivalent calcium-ion chelation to form a tetravalent coordination complex. The low pH of endosomes causes disruption of the tetravalent calcium-chelation between the ligand and the receptor and release of the ligand into the digestive machinery of hepatocytes. After release of the ligand, the receptor complex recycles. A single receptor can cycle up to 200 times with a turnover time of around 15 minutes, allowing large amounts of ligand to be internalized into hepatocytes without saturation effects.
Trivalent Galactose cluster conjugated 2’-O-MOE “Gapmer” oligonucleotides were synthesized in 2003.3 In 2007 Rozema et al.4 used monovalent GalNAc ligands (NAG) to prepare a multi-component GalNAc-polymer that carried an siRNA. This complex mediated RNAi-based gene silencing in mice. Trivalent GalNAc-conjugated siRNAs were synthesized using solution-phase and solid-phase methods in 2010 and 2011 using a variety of linkers and scaffolds (Peng et al.5; Yamada et al.6). In 2014, Nair et al.7 reported reengineered bi- and triantennary GalNAc ligands that simplified covalent linking to siRNAs and are compatible with solid-phase oligonucleotide synthesis and deprotection. The synthesis yield was comparable to that of standard oligonucleotides. The authors also found that subcutaneous administration resulted in significantly higher levels of GalNAc-siRNA in the livers of C57BL/6 mice than intravenous injection, with 94% of the GalNAc-siRNA localized in hepatocytes. Further, these siRNA conjugates mediated efficient gene silencing.
In a clinical setting, subcutaneous administration offers distinct advantages over intravenous injection as the patient can self-administer the former while the latter requires a medical professional. Thus, GalNAc-mediated therapeutic oligonucleotide delivery by subcutaneous administration has advantages over lipid nanoparticle formulations delivered intravenously.
Also in 2014, Prakash et al.8 showed that second generation gapmer ASOs (fully phosphorothioated ASOs with 2′-O-methoxyethyl termini and a central gap region of 8–14 DNA nucleotides) linked to triantennary GalNAc were up to 10-fold more potent than the parent ASOs in mouse models. Further increases (up to 60-fold) were seen when the GalNAc was coupled to generation 2.5 gapmer ASOs containing the S-2-O-Et-2,4-bridged nucleic acid chemistry. The authors further demonstrated that the GalNAc-ASO conjugate was metabolized quickly once internalized, releasing the ASO from the complex and thus avoiding potential steric interference with the machinery that mediates gene silencing.
These results provided strong incentive to push GalNAc-linked ASOs and siRNAs through the preclinical stages into the clinic, even though the chemical synthesis of the GalNAc conjugates was quite intricate and time consuming. Therefore, the focus of GalNAc research in the last three years has been on optimizing the chemical structure and simplifying the synthesis of these compounds.
In 2015, Rajeev et al.9 synthesized and characterized a simplified serial trivalent GalNAc design and demonstrated that these GalNAc-siRNA conjugates mediated robust gene silencing in mice. Matsuda et al.10 subsequently showed that siRNAs with clusters of three monovalent GalNAcs in close proximity on the 3′-end of an siRNA had similar silencing efficiency to the triantennary GalNAc-siRNA in mice. Other notable papers include Østergaard et al.11, who delineated a simple solution-phase strategy to attach pentafluorophenyl ester triantennary GalNAc clusters to 5′-hexylamino modified ASOs and showed that attachment of the GalNAc clusters to the 5′-end increased in vivo efficiency. Migawa et al.12 describe a simplified synthesis method for producing triantennary GalNAc clusters in less than five steps, ready for linking to ASOs. This research culminated in data presented by Prakash et al.13 who systematically compared different linking scaffolds and tethers to clarify the structure-activity relationship of triantennary GalNAc moieties.
Alnylam recently reported results from a phase II open label extension study of Revusiran (ALN-TTRsc), a GalNAc-conjugated siRNA designed to treat transthyretin amyloidosis with cardiomyopathy.14 The company reported downregulation of transthyretin with indication of clinical benefit in treated patients. Data from a phase I study of fitusiran (ALN-AT3), a GalNAc-conjugated siRNA that targets the mRNA encoding antithrombin (AT) for the treatment of hemophilia also showed good down-regulation of AT expression and a reduced number of bleeding episodes in treated patients.15
Phase I results are also available from Ionis Pharmaceuticals for APO(a)-LRx, a -triantennary GalNAc-ASO, designed to reduce elevated Lp(a) for the treatment of cardiovascular disease and aortic stenosis.16 There was a 32-fold improvement in efficiency of the linked ASO compared to the parent ASO in the study participants. No injection site reactions, flu-like symptoms, or laboratory abnormalities were reported in the 16 volunteers with elevated Lp(a) who participated in the study.
From these data, it looks like GalNAc conjugates do achieve the dream hepatocyte location for therapeutic oligonucleotides in the human liver without annoying diversions into endothelial or Kupffer cells.
If your project too would benefit from targeted hepatocyte-specific delivery using GalNAc and you lack the chemistry expertise necessary to produce these compounds, please contact either Punit Seth at Ionis Pharmaceuticals (for single-strand oligonucleotides) or Muthiah Manoharan at Alnylam (for siRNA) to discuss a MTA or collaboration with them.
- Graham MJ, Crooke ST, Monteith DK, Cooper SR, Lemonidis KM, Stecker KK, Martin MJ, and Crooke RM. In vivo distribution and metabolism of a phosphorothioate oligonucleotide within rat liver after intravenous administration. J. Pharmacol. Exp. Ther. 1998, 286, 447-58.
- Biessen EA, Vietsch H, Rump ET, Fluiter K, Kuiper J, Bijsterbosch MK, and van Berkel TJ. Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo. Biochem. J. 1999, 340, 783-92
- Maier MA, Yannopoulos CG, Mohamed N, Roland A, Fritz H, Mohan V, Just G, Manoharan M. Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting. Bioconjugate Chem. 2003, 14, 18-29.
- Rozema DB, Lewis DL, Wakefield DH, Wong SC, Klein JJ, Roesch PL, Bertin SL, Reppen TW, Chu Q, Blokhin AV, Hagstrom JE, and Wolff JA. Dynamic polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12982-12987.
- Peng CG, Butler D, Varghese JP, Maier MA, Rajeev KG, and Manoharan M, Non-nucleoside building blocks for copper assisted and copper free click chemistry for the efficient synthesis of RNA Conjugates. Org. Lett. 2010, 12, 5410–5413.
- Yamada T, Peng, CG, Matsuda S, Addepalli H, Jayaprakash KN, Alam R, Mills K, Maier MA, Charisse K, Sekine M, Manoharan M, and Rajeev KG. Versatile site-specific conjugation of small molecules to siRNA using click chemistry. J. Org. Chem. 2011, 76, 1198-1211.
- Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel’in AV, Milstein S, Taneja N, O’Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME, Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier MA, Rajeev KG, and Manoharan M. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 2014, 136, 16958-16961.
- Prakash TP, Graham MJ, Yu J, Carty R, Low A, Chappell A, Schmidt K, Zhao C, Aghajan M, Murray HF, Riney S, Booten SL, Murray SF, Gaus H, Crosby J, Lima WF, Guo S, Monia BP, Swayze EE, and Seth PP. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 2014, 42, 8796-8807.
- Rajeev KG, Nair JK, Jayaraman M, Charisse K, Taneja N, O’Shea J, Willoughby JLS, Yucius K, Nguyen T, Shulga-Morskaya S, Milstein S, Liebow A, Querbes W, Borodovsky A, Fitzgerald K, Maier MA, and Manoharan M. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo. ChemBioChem 2015, 16, 903-908.
- Matsuda S, Keiser K, Nair JK, Charisse K, Manoharan RM, Kretschmer P, Peng CG, V Kel’in A, Kandasamy P, Willoughby JL, Liebow A, Querbes W, Yucius K, Nguyen T, Milstein S, Maier MA, Rajeev KG, and Manoharan M. 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.
- Østergaard ME, Yu J, Kinberger GA, Wan WB, Migawa MT, Vasquez G, Schmidt K, Gaus HJ, Murray HM, Low A, Swayze EE, Prakash TP, and Seth PP. Efficient synthesis and biological evaluation of 5′-GalNAc conjugated antisense oligonucleotides. Bioconjug. Chem. 2015, 26, 1451-1455.
- Migawa MT, Prakash TP, Vasquez G, Wan WB, Yu J, Kinberger GA, Østergaard ME, Swayze EE, and Seth PP. A convenient synthesis of 5′-triantennary N-acetyl-galactosamine clusters based on nitromethanetrispropionic acid. Bioorg. Med. Chem. Lett. 2016, 26, 2194-2197.
- Prakash TP, Yu J, Migawa MT, Kinberger GA, Wan WB, Østergaard ME, Carty RL, Vasquez G, Low A, Chappell A, Schmidt K, Aghajan M, Crosby J, Murray HM, Booten SL, Hsiao J, Soriano A, Machemer T, Cauntay P, Burel SA, Murray SF, Gaus H, Graham MJ, Swayze EE, and Seth PP. Comprehensive structure-activity relationship of triantennary N-acetylgalactosamine conjugated antisense oligonucleotides for targeted delivery to hepatocytes. J. Med. Chem. 2016, 59, 2718-2733.