Charlotte Bon (1), Erich Koller (2), Martina Mueller (2), Wouter Driessen (3), Kevin Brady (3), Franz Schuler (2), Ben-Fillippo Krippendorff (1)
(1) Quantitative and Systems Pharmacology, Pharmaceutical Sciences, pRED, Roche Innovation Center Basel, (2) Drug Disposition and Safety, Pharmaceutical Sciences, pRED, Roche Innovation Center Basel, (3) DMPK and Bioanalytical R&D, Pharmaceutical Sciences, pRED, Roche Innovation Center Basel
Objectives: GalNAc, triantennary N-acetyl galactosamine, is a high-affinity ligand to the asialoglycoproteins receptor (ASGPR) which is highly expressed at the hepatocyte cell surface [1, 2]. Therefore, ASGPR is thought to be a promising target for enhancing uptake into hepatocyte. We aim to develop an in-vitro target mediated uptake model of GalNAc conjugated antisense oligonucleotides. The model is intended to help understand the different cellular processes influencing cellular uptake of GalNAc-LNA gapmers and quantify the capacity of ASGPR as a shuttle.
Methods: We used literature information on the asialoglycoprotein receptor kinetics and ligand affinity to develop the target mediated uptake model [3, 4]. The ASGPR has been extensively scrutinized since its discovery in the mid-1970s [5] which allowed us to implement a detailed mechanistic model structure [6] in Simbiology®/Matlab®. In-vitro uptake data were obtained using primary rat hepatocytes and exposing them to different GalNAc-LNA gapmers concentrations. The data was then fitted to the uptake model and parameters were then compared to the ones found in the literature. Also a sensitivity analysis was performed allowing the identification of crucial parameters influencing the ASGPR shuttle capacity.
Results: We find that the developed uptake model allowed to quantitatively predict the dose proportional cellular uptake at low GalNAc-LNA concentrations. The ASGPR density and the ratio of internalization vs recycling rates appear to be crucial parameters. The model suggested that the uptake data can be explained when including a loss of ASGPR expression on hepatocytes over time, probably due to the in-vitro conditions.
Conclusions: Modeling the target mediated uptake due to ASGPR allowed to characterize this shuttle quantitatively and helped to understand the dynamics influencing the in-vitro cellular uptake data.
References:
[1] Weigel, P.H. and J.H. Yik, Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors. Biochim Biophys Acta, 2002. 1572(2-3): p. 341-63.
[2] Lee, Y.C., et al., Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J Biol Chem, 1983. 258(1): p. 199-202.
[3] Schwartz, A.L., S.E. Fridovich, and H.F. Lodish, Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line. J Biol Chem, 1982. 257(8): p. 4230-7.
Reference: PAGE 25 () Abstr 5849 [www.page-meeting.org/?abstract=5849]
Poster: Drug/Disease modeling - Other topics