A Liver Microphysiological System for Evaluating Targeted Delivery and Efficacy of Oligonucleotide Therapeutics

New modalities can be defined as a type of therapeutic used to treat diseases, previously inaccessible by traditional drugs. Small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) are examples of oligonucleotide-based modalities.

Early adopters of oligonucleotide-based therapeutics made use of N-acetylgalactosamine (GalNAc) binding the asialoglycoprotein receptor (ASGPR), found primarily on the cell surface of liver hepatocytes. GalNAc-conjugated oligonucleotides bind ASGPR and are rapidly removed from the systemic circulation by endocytosis. Since these therapeutics have human-specific receptor and gene targets, use of physiologically relevant Organ-on-a-chip (OOC) technology was necessary to understand drug delivery and its efficacy.

Here we investigate the uptake of GalNAc-conjugated oligonucleotides by primary human hepatocytes (PHHs) using our PhysioMimix® Liver microphysiological system (MPS) or Liver-on-a-chip. Using our Liver-12 multi-chip consumable plate, PHHs were seeded onto collagen-coated 3D scaffolds and cultured for 11-14 days, under fluidic flow. ASOs or siRNA targeting human GAPDH or albumin gene (ALB) expression were introduced on Day 4. Readouts of cell health (albumin) and cell damage (lactate dehydrogenase) were measured by media sampling throughout the experiment. Analysis of siRNA/ASO uptake (immunocytochemistry) and gene knockdown (qPCR) was carried for each fluorescently labelled modality.

For each modality tested, we observed healthy liver tissue over the entire experimental duration. With siRNA targeting GAPDH (siGAPDH), we observed a reduction in gene expression. This knockdown was greater in the GalNAc-conjugated siGAPDH compared with non-conjugated (naked; Nkd) siGAPDH; despite greater fluorescence observed in the Nkd-siGAPDH condition compared with GalNAc-siGAPDH.

ASOs targeting ALB also caused a reduction in gene expression. With a greater knockdown caused by the GalNAc-conjugated targeting ASO, despite greater fluorescence observed in the Nkd-targeting ASO condition; as found by Majer and colleagues. Both modalities showed a dose-dependent response, with greater gene knockdown (efficacy), and fluorescence intensity (delivery) caused by increasing concentration, or by repeating dose.

With increasing numbers of new modalities entering clinical trials, there is greater demand for long-term culture of human-relevant in vitro models. Here we demonstrate the suitability of our PhysioMimix OOC to study the delivery and efficacy of oligonucleotide-based therapeutics targeted to the liver, in a sensitive and longitudinal manner.