Journal Article

A Long‐Term Human Liver Spheroid Model for Assessing Silencing and Durability of GalNAc‐Conjugated siRNAs

Nordling, Åsa, Sadhasivam, Lingheswar, Scholten, Gijs‐Jan, Grundmann, Clara, Coskun, Caroline, Einfalt, Tomaž, Riet, Sander, Engelhardt, Volker, Ingelman‐Sundberg, Magnus

2026

Overview

Advances in RNA interference technology have established it as a powerful therapeutic tool with important future potential. The design and the chemical modifications of the siRNA nucleotide backbone have greatly enhanced stability, durability, and pharmacokinetics while minimizing tolerability risks. The optimal combination of these modifications depends on the target gene, tissue, and RNA sequence, necessitating an iterative, experimental approach that currently relies heavily on animal models. To reduce the reliance and number of (humanized) animals required, we developed a human long‐term liver 3D spheroid model designed for screening GalNAc‐conjugated siRNAs which captures the process of uptake, potency, and durability for early in vitro screening. These liver spheroids remain viable in culture for at least 5 weeks while maintaining expression of the asialoglycoprotein receptor to facilitate GalNAc mediated uptake. siRNA was efficiently internalized by the spheroids without the need for transfection reagents, and its durable silencing efficiency was assessed by monitoring AHSA1 target gene expression over time. Target gene silencing in the spheroid model persisted up to 5 weeks post‐treatment. Fluorescently labeled siRNA enabled visualization of uptake and distribution within the spheroid, revealing somewhat reduced siRNA accumulation in pericentral CYP3A4+ hepatocytes accompanied with somewhat reduced ASGR1 expression. No signs of hepatotoxicity were observed under the conditions used. By varying the number of phosphorothioate modifications in the siRNA backbone, distinct differences in silencing efficiency and durability were observed which were principally similar as obtained in vivo in mice. We propose that this long‐term human liver spheroid model provides a valuable preclinical platform for evaluating siRNA‐based therapeutics with respect to uptake, durability, and silencing efficiency, and could refine early in vitro screening and accelerate drug development. Study Highlights What is the current knowledge on the topic? ○Chemical modifications to the siRNA backbone are essential for enhancing molecular stability, reducing immunogenicity, increasing resistance to enzymatic degradation, and minimizing toxicity. ○Such modifications are pivotal for optimizing siRNA performance in therapeutic settings. ○However, despite growing interest in siRNA‐based therapeutics, no long‐term in vitro model exists to evaluate silencing efficiency and durability. ○As a result, preclinical development still relies heavily on humanized mouse models. What question did this study address? ○Can the human liver spheroids be used as an in vitro model to evaluate the effect of backbone modifications of the GalNAc‐conjugated siRNA on the silencing efficiency and durability? What does this study add to our knowledge? ○We present a new human spheroid model for assignment of the properties of structurally different GalNAc‐conjugated siRNA molecules. ○The spheroids were maintained in culture for up to 5 weeks while retaining expression of the asialoglycoprotein receptor. ○These spheroids can be directly treated with GalNAc‐conjugated siRNA without the need for transfection reagents, enabling sustained target gene silencing to be monitored over the entire five‐week period. ○Using fluorescently labeled siRNA, intracellular trafficking was visualized, revealing heterogeneity in hepatocyte uptake capacity. ○Moreover, the model enabled clear discrimination of silencing performance across five distinct siRNA backbones. How might this change clinical pharmacology or translational science? ○This study shows that human liver spheroids provide a robust, scalable platform for evaluating GalNAc‐conjugated siRNA therapeutics. ○The model offers a valuable tool for early‐stage compound selection, potentially accelerating screening workflows and reducing reliance on animal models in preclinical development.

• Publisher: John Wiley & Sons, Inc,Wiley
• Subject: Acetylgalactosamine - chemistry / Animal models / Animals / asialoglycoprotein receptor / Asialoglycoprotein Receptor - genetics / Asialoglycoprotein Receptor - metabolism / Cell culture / Drug development / Gene expression / Gene Silencing / Genetic engineering / Glycoproteins / Health aspects / Hepatocytes / Hepatocytes - metabolism / Hepatotoxicity / Humans / Liver / Liver - cytology / Liver - metabolism / liver spheroids / Nucleotide sequence / Pharmacokinetics / Phosphorothioate / Regulatory approval / RNA Interference / RNA, Small Interfering - chemistry / RNA, Small Interfering - genetics / RNA-mediated interference / silencing efficiency / siRNA / siRNA stability / Spheroids / Spheroids, Cellular - metabolism / Transfection / White people