New modality drugs, or advanced oligonucleotide therapeutics, refer to innovative approaches that go beyond traditional small molecule approaches to target diseases more precisely and effectively.
They are designed to address complex diseases and conditions that are difficult to treat with conventional therapies and may have been classed as “undruggable”.
According to The 2024 New Drug Modalities Report | BCG, new modalities represent $168 billion in Pharma/Biotech projected pipeline value, up 14% from 2023. By 2029, analysts project that 9 of the top 10 drugs will be new modalities. Although antibodies make up most of the later-stage preclinical pipeline, a plethora of other modalities can be found in the earlier phases including oligonucleotides which are more amenable to intracellular targets.
However, oligonucleotides have unique development challenges that require adaptations to preclinical testing workflows. A major challenge relates to their in vivo testing. Due to the high degree of conservation of nucleic acid base sequences between humans and non-human primates (NHPs), their use is common but, expensive. However, even NHPs can incorrectly predict human responses – as exemplified in the phase 1 clinical trial of TGN1412, a monoclonal antibody, in 2006. Six healthy volunteers were left with life-threatening morbidities due to the lack of CD28 expression on CD4+ effector memory T cells in macaques (Eastwood et al., 2010). Consequently, ensuring you have as much confidence in oligonucleotide delivery, safety and efficacy ahead of in vivo studies is advantageous to minimize risk. To achieve this, the pre-clinical toolbox requires modernization to become more human-centric. New Approach Methodologies (NAMs), including Organ-on-a-Chip (OOC), provide a viable path forward to address many development challenges. And, following the passing of the FDA’s Modernization Act 2.0 in 2022, their use is supported by the largest global regulator for evaluating drug safety and effectiveness in place of animal testing – where appropriate.
In this blog, we explore why oligonucleotide-based therapeutics are increasing in popularity, their ADMET discovery and development challenges, and how in vitro human OOC models help to circumvent these by providing data-rich insights that better inform/ justify the expense of in vivo NHP or human studies.
Why are Oligonucleotide-based Therapies Rising in Popularity?
Oligonucleotide-based therapeutics, or RNA-based therapeutics, are short oligonucleotide sequences that interfere with a specific RNA. They include antisense oligonucleotides (ASOs), RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), and aptamers. Interest in oligonucleotide therapeutics arose from our understanding of the human genome and the genetic link to many diseases, combined with their relatively fast drug discovery process compared to traditional small molecules and accessibility to organizations of any size.
Around 18 oligonucleotide-based therapeutics have received market approval from 1998-2024 (Table 2). Significant advances over more recent years are due to the use of delivery systems, such as lipid nanoparticles (LNPs) and the N-acetylgalactosamine (GalNAc)-conjugations, that enable drug targeting to the liver for the treatment of hepatic disorders. For example, in 2016 Patisiran (marketed as Onpattro) for transthyretin amyloidosis (ATTR) uses LNPs to deliver siRNA to the liver. In 2019, Givosiran (marketed as Givlaari), which uses GalNAc-conjugation to target the liver was approved for acute hepatic porphyria.
Liver targeting using the sugar molecule GalNAc is made possible because of the asialoglycoprotein receptor (ASGPR), which is found primarily on the cell surface of hepatocytes. GalNAc-conjugated oligonucleotides bind with high affinity to ASGPRs and are rapidly removed from the systemic circulation via endocytosis. Once inside the cell, the therapeutic oligonucleotide is released from the receptor to exert its therapeutic effect by targeting specific messenger RNA (mRNA) sequences. This approach improves potency and effectiveness at lower doses, reduces off-target effects, and potentially enables longer dosing intervals with lower immunogenicity. With continued research and development, GalNAc-based therapeutics hold immense future potential for treating a wide range of diseases, including genetic disorders, metabolic diseases, and infectious diseases.
In 2021 Novo Nordisk acquired Dicerna to capitalize on this potential. A subsequent report in Bioprocess International, in 2023, citing Marcus Schindler, executive vice president of research and early development at Novo Nordisk, stated “a significant portion of my pipeline now is oligonucleotide”. The reason, he explained, “it’s really to do with building a toolbox to accelerate everything that we do in drug discovery”. Regarding how Novo Nordisk is supporting oligo-development, he said, “so first and foremost, we are taking what we call a human-centric approach to understanding complex biology. We start with datasets and experimental models that mimic the one species that we’re interested in. And that is humans.”
So, why is it that this type of drug requires a more human-centric approach, and what are the challenges in its discovery and development that require a rethinking of the preclinical toolbox?
The ADMET Challenges of Oligonucleotide Therapeutic Discovery & Development for Liver Diseases
The Absorption, Distribution, Metabolism, Excretion and Toxicology (ADMET) challenges relate to the point above, oligonucleotides aren’t traditional small molecule compounds and animals aren’t humans. Oligonucleotides have multiple modes of activity by which they can achieve their intended pharmacological effect, however, most commonly, they contain complementary nucleotide base sequences to a targeted sequence of an intracellular nucleic acid. Some may be highly specific to humans and consequently, this either forces the use of NHPs, or in some instances negates the use of non-human species. Test species-active surrogate oligonucleotides are an option but this doesn’t get around the fact that animals are not humans. In silico and in vitro methodologies help to identify potential off-target hybridization effects, however, in vitro models are limited by their physiological relevance (explored in more detail below) and in silico models are only as good as the input data.
Many challenges remain in the development of oligonucleotide-based therapeutics for liver disease and their preclinical ADMET profiling, the most pertinent of which are summarized below (Table 1).
Table 1
The key challenges in ADMET profiling of oligonucleotide therapeutics.
| Specificity/Delivery & Cellular Uptake | While GalNAc-conjugations target the liver and facilitate cellular uptake, there’s a need to improve their specificity to minimize off-target effects in other tissues. Challenges remain around ensuring oligonucleotides reach their intended intracellular targets without being degraded or losing efficacy. |
| Stability and Bioavailability | Enhancing the stability and bioavailability of oligonucleotides is crucial, requiring chemical modifications to improve the stability in the bloodstream and their ability to penetrate cell membranes |
| Complex ADME/PK Profiles | Oligonucleotides can exhibit complex pharmacokinetic (PK) behaviors, including rapid distribution to tissues, short plasma life, long pharmacodynamic half-life, and variable clearance rates. |
| Off-target Effects | Minimizing off-target effects is another challenge. Oligonucleotides can sometimes bind to unintended RNA sequences, leading to unwanted side effects and reduced specificity. |
| Immunogenicity | Oligonucleotides can engage and activate components of the innate immune system leading to potential side effects. Certain chemical modifications help mitigate this, but it remains a challenge. |
| Model Limitations | Simplistic in vitro cell culture models fail to replicate the complexity of human biology, plus culture viability is relatively short-lived. Interspecies differences between in vivo animal models and humans can limit their suitability for oligonucleotides that bind human-specific targets and change the way that they are processed. |
| Data Integration | Combining data from in vitro, in vivo, and in silico models to create a comprehensive understanding of drug ADMET is complex. Each model has its limitations, integrating these data to make reliable predictions for human trials is a significant challenge. |
| Ethical Constraints | In vivo testing is subject to strict regulations. Drugs with human-specific targets and pathways may require NHP models that are not readily available and costly. |
How OOC Models Support Advancements in the ADMET Profiling of Oligonucleotide-therapeutics for Liver Disease
These challenges underscore the need for innovative new approaches, such as OOC, to improve the predictive power of preclinical oligonucleotide testing. OOC platforms, also known as microphysiological systems (MPS), condense all the technologies needed to build functional tissues that model specific organ-level responses into a miniaturized format. To replicate the liver, primary human hepatocytes (PHHs) and the liver’s resident immune cell population (Kupffer cells, as necessary) are cultured to form 3D microtissues in bespoke engineered scaffolds. Microtissues are continuously perfused by media to mimic blood flow, providing gas exchange and nutrients. This dynamic 3D microenvironment promotes the viability and functionality of liver cells (quantified by albumin and lactate dehydrogenase) over multiple weeks, compared to just a few days for 2D in vitro hepatocyte cultures. They aim to help circumvent traditional preclinical model limitations by improving human and physiological relevance. Their insights support the responsible use of animal models, by justifying the progression of only the most promising candidates, and better-informed decision-making ahead of clinical trials where no pharmacologically relevant model exists.
So, how do these in vitro models address ADMET challenges? Firstly, the longevity of OOC cultures versus static PHH cultures enables the concentration effects of oligonucleotides to be measured. This is achieved by dosing liver microtissues and subsequently measuring oligonucleotide uptake and gene knockdown – the latter of which takes time to manifest. Additionally, their longevity permits repeat oligonucleotide dosing to inform the most effective strategy before in vivo animal, or first-in-human (FIH) studies. Furthermore, the health of Liver-on-a-chip cultures can be monitored throughout the experiment to flag any potential adverse effects, including activation of innate immune cells. Finally, their human-relevant data can be used to “feed” in silico models to improve the performance of predictions.
Liver-on-a-chip use for small molecule drug metabolism, accumulation and toxicity testing has already been recognized by the US FDA as superior to traditional approaches. Now, their ability to improve the ADMET profiling of oligonucleotide therapies is being explored by Pharma and Biotech via PhysioMimix® OOC platform adoption, or our contract research services. The following case studies highlight recent findings.
Case studies
Case Study 1: Human-Relevant ASO Delivery and Uptake
In a recent publication by GSK, Majer et al., (2024), explored OOC’s potential for addressing one of the major hurdles in therapeutic ASOs, delivering oligonucleotides in sufficient concentration to the target tissue and the cells of interest. Their targeted delivery is important for reducing off-target effects and improving the specificity of gene silencing, both of which are essential for safe and effective therapeutics.
Their study evaluated the cellular uptake and localization of Alexa488 (A488)-labelled non-conjugated ASO and GalNAc-conjugated ASO by a PhysioMimix Liver-on-a-chip model using label/label-free imaging techniques.
The authors cite “by replicating key aspects of the liver function, such as drug metabolism, bile production and immune responses in a controlled in vitro environment, these models provide a cost-efficient, robust, and human biology-relevant ethical alternative to traditional in vivo animal testing that can accelerate drug discovery and development”.
An interesting observation made by the publication is that Liver-on-a-chip narrows the gap between in vitro assay and in vivo human liver environment because most PHHs in the model were cuboidal. Cuboidal PHHs were found to exhibit different chemical composition, morphology, and ASO distribution compared to circular hepatocytes, which are more common/less physiologically relevant in 2D culture. For this reason, the authors discounted data from circular hepatocytes These results call into question the usefulness of 2D, or 3D spheroid models for this purpose.
The study observed that uptake of non-conjugated ASOs by Liver-on-a-chip PHHs was slower compared to GalNAc-conjugated ASOs at early time points, although a greater amount of non-conjugated ASOs was taken up over time. This is probably caused by the high binding affinity of GalNAc and insufficient receptor recycling.
Case Study 2: ASO Uptake vs. Knockdown
In an independent CN Bio study, using a PHH-only model, similar results to the Majer et al., study were observed i.e., greater hepatocyte uptake (signal intensity) over time by the non-GalNAc conjugated ASOs versus GalNAc-conjugated – likely due to different processing of the ASOs by PHHs. However, the gene knockdown data tells a different story. This hint into the efficacy of ASOs highlights why it is important to measure knockdown and uptake. Register for our next webinar to find out more!
Case Study 3: Resolving ASO Toxicity Data Discrepancies
A further study in collaboration with AstraZeneca utilized our PhysioMimix Liver-on-a-chip and DILI assay to resolve conflicting ASO toxicology data between animal models and 2D hepatocytes. In their publication, Sewing et al., (2016), cite “A series of reports describe changes observed in rodent and non-rodent studies related to SSO (single-stranded oligo)-induced toxicity, but as the safest preclinical candidates progress into the clinic, the human relevance of the observed effects remained unclear so far.” CN Bio was asked to test two ASOs to ascertain if a human system can provide more insight. Human-relevant markers including alanine aminotransferase (ALT) and CYP3A4 activity were able to clearly show and predict human outcomes between ASO-1 (toxic) and ASO-2 (safe). This gave the authors further clarity regarding which would be safest for humans.
Going forward, recent advancements in OOC technology enable the linking of individual organs together into systems. As more models become available to fluidically interconnect, these systems can be utilized to explore off-target effects and potentially explore renal excretion.
Summary
The rise of oligonucleotide therapeutics represents a significant advancement for the treatment of complex diseases, offering targeted and effective solutions where traditional therapies fall short. Despite their potential, oligonucleotides face unique challenges that necessitate a shift towards more human-centric preclinical testing approaches.
Use of human OOC models offers a solution to many of these challenges. These models replicate human organ functions in a controlled in vitro environment, providing more relevant and predictive data compared to animal models. By improving the physiological relevance of preclinical testing, OOC platforms help to better predict human responses, justify the use of expensive NHP studies or provide a path forward where in vivo models aren’t suited, and ultimately accelerate the development of safe and effective oligonucleotide therapies.
Published case studies from AstraZeneca and GSK, highlight the effectiveness of OOC models in addressing key hurdles in oligonucleotide development. These studies demonstrate how OOCs provide clearer insights into human-specific responses.
Furthermore, as OOC technology continues to advance, it holds the potential to further revolutionize the field by enabling the integration of multiple organ systems, thereby offering a comprehensive understanding of drug behavior and safety.
To conclude, the adoption of human-centric approaches, such as OOC, is crucial for overcoming the development challenges of oligonucleotide therapeutics and unlocking their full potential.
Find out more about the possibilities offered by PhysioMimix® Liver-on-a-chip models in our next webinar: Register here or contact us for more information.
Table 2
FDA-approved oligonucleotide therapeutics from 1998 to 2024
| Fomivirsen | Approved for cytomegalovirus retinitis in immunocompromised patients | 1998 |
| Mipomersen | Approved for familial hypercholesterolemia | 2013 |
| Nusinersen | Approved for spinal muscular atrophy | 2016 |
| Eteplirsen | Approved for Duchenne muscular dystrophy | 2016 |
| Defibrotide | Approved for veno-occlusive disease | 2016 |
| Inotersen | Approved for hereditary transthyretin-mediated amyloidosis | 2018 |
| Patisiran | Approved for hereditary transthyretin-mediated amyloidosis | 2018 |
| Golodirsen | Approved for Duchenne muscular dystrophy | 2019 |
| Volanesorsen | Approved for familial chylomicronaemia syndrome | 2019 |
| Givosiran | Approved for acute hepatic porphyria | 2019 |
| Viltolarsen | Approved for Duchenne muscular dystrophy | 2020 |
| Lumasiran | Approved for primary hyperoxaluria type 1 | 2020 |
| Inclisiran | Approved for primary hypercholesterolemia | 2021 |
| Casimersen | Approved for Duchenne muscular dystrophy | 2021 |
| Vutrisiran | Approved for hereditary transthyretin-mediated amyloidosis | 2022 |
| Nedosiran | Approved for primary hyperoxaluria | 2023 |
| Eplontersen | Approved for hereditary transthyretin amyloidosis | 2023 |
| Tryngolza | Approved for familial chylomicronemia syndrome | 2024 |
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Advancing oligonucleotide therapies with liver-on-a-chip models.
April 1st, 2025. 3 pm BST. 10:00 am ET