Introduction to Antisense oligonucleotides (ASOs)
Antisense oligonucleotides (ASOs) modulate gene expression by targeting specific messenger RNA (mRNA) for degradation by ribonuclease H, translation arrest, or splicing inhibition. They offer therapeutic avenues for diseases that are currently unmet by alternative therapeutic modalities. However, the development of therapeutic ASOs is often hampered by hepatotoxicity. Here, we explore why 3D human liver microtissues are becoming an indispensable tool for de-risking ASO drug candidates and for reducing attrition in late-stage drug development.
The Challenge of DILI in Antisense Oligonucleotides Therapeutics
ASOs are short, synthetic strands of nucleic acids designed to bind to mRNAs of a target gene specifically. By binding to mRNA, ASOs can modulate gene expression through RNA degradation, inhibition of translation, or modulation of splicing. This targeted approach allows for the precise control of genetic pathways and offers potential treatments for various diseases, including genetic disorders, cancer, and inflammatory conditions.
However, their unique mechanisms of action and metabolic pathways pose distinct challenges in predicting hepatotoxicity. ASOs can induce liver injury through a variety of mechanisms, including mitochondrial toxicity, immune-mediated responses, or direct effects on hepatocyte RNA processes. The complex interplay of these mechanisms often goes undetected in conventional 2D in vitro assays or early animal studies, leading to unanticipated complications in later clinical phases.
The Advantages of 3D Liver Microtissues in DILI Prediction of ASOs
Scaffold-free co-culture spheroids consist of primary human liver cells (hepatocytes, Kupffer cells, LSECs) and represent a significant advancement over traditional testing paradigms. This platform offers several compelling advantages for Antisense Oligonucleotides testing due to the more liver-specific cellular composition and functionality: Unlike 2D cultures, 3D liver microtissues mimic the liver’s intricate architecture and cell-cell interactions, providing a more accurate physiological environment. This complexity is critical for observing how ASOs interact in a multi-cellular context, influencing metabolism and toxicity profiles.
The study of the biotransformation of antisense oligonucleotides in in vitro liver models is a critical area of research aimed at understanding the metabolic pathways and stability of these therapeutic agents. The metabolic competence of our 3D in vitro liver models provides a valuable platform for assessing the metabolic fate of ASOs. These models help to elucidate the enzymatic processes involved in the degradation and modification of antisense oligonucleotides, such as nuclease activity and phase I/II metabolic reactions.
At InSphero, we leverage several unique advantages of our 3D InSight™ Liver Microtissues to provide a tailored solution for reliable, in-depth, and fast-turnaround safety assessment of antisense oligonucleotides. The 28-day functional lifetime of the microtissues allows for long-term, multi-dose treatments, hence enabling the detection of ASOs that may cause a delayed toxic response. The miniaturization of the 3D InSight™ platform reduces the amount of ASO required for the safety evaluation: a 14-day cytotoxicity assay with seven concentrations and four biological replicates each requires less than two milligrams of ASO, significantly reducing compound costs. Our high degree of automation in microtissue production and assay execution results in excellent data reproducibility.
Finally, a key advantage of the 3D approach lies in the delivery method: 3D liver microtissues do not require a transfection agent to deliver antisense oligonucleotides into the liver spheroids. This avoids difficulties in attributing any potentially observed toxicity to its true cause.
Strategic Integration of 3D Liver Microtissues into ASO Development
The integration of 3D liver microtissues into the drug development pipeline for antisense oligonucleotides can be done strategically at several key stages:
Lead optimization
Early screening of multiple ASO candidates to identify and eliminate compounds with undesirable toxicity profiles before entering more costly stages. The affordable cost, low compound volume, and fast turnaround times of 3D liver microtissue assays are critical advantages at this stage.
Mechanistic studies
Detailed investigations into the pathways of observed toxicities that guide molecular modifications to improve safety. A wide range of physiologically relevant readouts and causality assays for better mechanistic understanding of ASO effects are an advantage of 3D liver models.
Regulatory submissions
Provide robust data on the hepatotoxic potential of ASOs to support regulatory submissions with comprehensive safety profiles generated using human in vitro models.
Assay Considerations for Success with Antisense Oligonucleotides Therapeutics
DILI assay protocols differ significantly between small molecules, for which 3D liver microtissues are considered the gold standard today, and biologics. Therefore, careful consideration of the assay conditions for ASOs is key to successful implementation.
Unlike small molecules, which are often dissolved in DMSO, ASOs are typically delivered in aqueous solutions and require careful consideration of the solvents used to ensure stability and compatibility. Common solvents such as water or buffer solutions are used, but the ionic strength and pH of these solutions can significantly affect ASO stability and hybridization efficiency.
In addition, ASO stability is highly dependent on nucleic acid integrity and resistance to enzymatic degradation, whereas chemical stability is critical for small molecules. Therefore, serum-containing cell-culture media can be detrimental to the DILI assessment of ASOs.
3D Liver Microtissues are a Fit-for-Purpose Solution to Assess ASO Toxicity
As the development of ASOs continues to expand, the adoption of 3D liver microtissues in preclinical testing is not only beneficial, but essential. These advanced models are a critical tool for innovative biotechnology companies to navigate the complex safety landscape of new therapeutics, to minimize DILI risks and to promote a smoother transition through clinical development. Strongly supported by our own data, we believe that the 3D liver microtissue approach, using state-of-the-art 3D cell culture technology, not only enhances the predictability of liver safety assessments, but also contributes significantly to the overall success and sustainability of ASO therapeutic development. The use of these innovative tools will propel us towards a future where drug development is both safer and more efficient.
