Dr. Xiaoxia Li, MD, Ph.D., DABT- Executive Technical Director of Toxicology, WuXi AppTec
Rare diseases (i.e., “orphan diseases”) represent a global health challenge that affects around 500 million people worldwide. Most rare disease sufferers face lifelong hurdles in diagnosis and obtaining effective medications. Despite the rapid pace of medical innovation, fewer than 6% of rare diseases have approved therapies,1 leaving many patients with few solutions and even less hope.
Rare diseases are often rooted in genetic mutations, highlighting the need for innovative precision medicines. One of the most promising classes of new therapeutics that can be tailored to unique molecular characteristics is antisense oligonucleotides (ASOs). ASOs are synthetic nucleotide sequences designed to bind disease-causing RNA and modify gene expression with great specificity. They are being considered with “special interest” for more than 8,000 rare diseases and have already been used to treat spinal muscular atrophy (SMA), homozygous familial hypercholesterolemia, and primary hyperoxaluria type 1.2
Despite incredible promise - and growing demand - an ASO’s journey from concept to candidate to market readiness includes rigorous preclinical efficacy and safety assessments. Scientists are specifically investigating the on/off-target effects and tissue-specific safety profiles associated with each ASO treatment. ASOs are poised to profoundly impact personalized medicine. They will continue playing an enormous role in fighting rare and ultra-rare diseases. But ASOs’ true potential depends on how well scientists can identify, evaluate, and select efficacious and relatively safe candidates.
ASO Design and Early Safety Considerations
Responsible ASO development must prioritize therapeutic efficacy and safety. Selecting the right target relies on advanced bioinformatics and in silico tools to minimize off-target interactions and unintended gene modulation. Scientists try to maximize sequence specificity to reduce the risk of unwanted off-target effects that can jeopardize safety during later testing.
Chemical modification is another important aspect of ASO design. Common strategies, including phosphorothioate backbone substitutions and 2’-O-methoxyethyl (2’-MOE) groups, improve molecular stability and enhance resistance to nucleases. Chemical modifications also impact toxicity profiles- certain chemicals show greater tolerability and less immunogenicity in preclinical models. Early-phase in silico screening also helps to identify sequence-related risk factors before laboratory work begins.
Delivery is also a significant hurdle in ASO therapy. Achieving meaningful tissue concentrations, whether systemically or in specific organs, requires consideration of pharmacokinetics (PK), biodistribution, and formulation science. For neurogenetic disorders, penetrating the blood-brain barrier (BBB) poses an additional challenge for oligonucleotides. Intrathecal administration and other novel delivery systems may eventually be used to optimize CNS targeting. Meanwhile, formulation advancements continue to shape ASO development, introducing carriers and chemical conjugates that can improve efficacy and safety.
These early-stage design considerations are the foundation of safe and effective ASO development. The rigor applied in target selection, chemical engineering, and delivery strategy reduces risks and allows for robust safety assessment as drug candidates move toward in vitro and in vivo studies.
Starting With In Vitro Safety Assessments
In vitro safety assessment plays a significant role in building design and engineering processes that can advance ASO candidates to clinical consideration. Identifying adverse effects at the cellular level helps refine drug candidates and filter out sequences most likely to present safety liabilities in later development.
Well-established in vitro assays using human cell lines or primary cells offer a critical first look at toxicity profile. As an example, standard cytotoxicity assays screen ASOs for their impact on vital tissues - i.e., liver, kidney, and heart - providing a clear picture of on- and off-target damage. These models also allow for immune response profiling through cytokine release assays that help uncover the potential for unwanted immune responses. Equally important is evaluating off target gene knockdown. Tools like qPCR and high-throughput RNA sequencing can map changes across the entire RNA profile.
Advanced technology, including patient-derived organoids and multiplexed micro-physiological platforms, help scientists model tissue-specific responses with greater accuracy. These three dimensional cultures mimic human tissue, allowing accurate predictions of efficacy and toxicity. Organoids can also be tailored to reflect the genetic backgrounds seen in rare disease populations, offering critical insight into safety risks unique to this population.
Layering cytotoxicity screening, immune profiling, and genome wide surveillance within cutting-edge tissue models lets researchers establish a robust framework for assessing ASO candidates before moving to in vivo safety studies. This integration sets the stage for greater predictability and reliability in subsequent in vivo assessments.
Moving On to In Vivo Safety Assessments
After in vitro data identify promising ASO candidates, a comprehensive battery of in vivo safety assessments helps demonstrate how these molecules behave in animal species, typically rodents (mice or rats) and non-rodents (e.g., nonhuman primates). These studies often employ identical or like clinical dosing regimens and are designed to comprehensively monitor the drug’s impact across biological systems, including deriving acute and chronic toxicity.
Like any other novel drug safety assessment, for ASO, general toxicity assessments consist of (but are not limited to) continuously monitoring clinical observations, body weights and food intake, periodically blood sampling for hematology, biochemistry and immune reactions, as well as microscopic examination of tissues collected from study animals. ASOs have their own unique characteristics. ASOs tend to accumulate in certain specific organs such as liver, kidney and spleen, leading to those organ specific toxicities. ASOs, particularly those with unmodified phosphodiester backbones, can activate the immune system, leading to immunogenicity or inflammation.
Hepatotoxicity
- ALT/AST: Elevated serum levels signal liver cell injury or impaired liver function, making these enzymes primary markers for hepatic safety.
- Liver histology: Direct microscopic examination allows detection of cell degeneration, infiltration, and structural abnormalities that may not present in bloodwork alone.
Nephrotoxicity
- Blood urea nitrogen (BUN), creatinine, urinary biomarkers (e.g., KIM-1, NGAL, clusterin, cystatin C): Reveal impaired renal clearance or acute kidney injury, with specialized urinary panels offering earlier and more sensitive detection than traditional serum markers.
- Renal histology: Assessment for tubular degeneration/ regeneration and structural changes confirms and contextualizes biomarker findings.
Immunotoxicity
- Cytokines (e.g., IL-6, TNF-α) and complement split products (e.g., C3a, Bb, C5a): Reveal activation of innate immunity, inflammation, or complement-driven adverse effects.
- Immuno-stimulation assays: Antidrug antibody production indicates potential for proinflammatory or hypersensitivity reactions.
Hematologic Endpoints
- Platelet counts: Used to monitor for thrombocytopenia, which has been observed with certain ASO chemistries; decreased levels can signal bone marrow suppression or peripheral destruction.
- Coagulation parameters (e.g., PT, aPTT): Assessed to detect changes in clotting function and risk of bleeding or thrombosis.
CNS Toxicity (for neuro-targeted ASOs)
- Neurobehavioral assessments: Tests of locomotor activity, cognition, and sensorimotor function identify overt and subtle deficits in nervous system function.
- Neuronal cell assays and histology: Cellular and tissue analysis post-administration reveals direct neurotoxicity, axonal injury, or demyelination.
In addition, although ASOs generally have a lower risk of genotoxicity and carcinogenicity compared to small-molecule drugs, these potential risks must still be evaluated, particularly for long-term therapies.
A Final Word on Preclinical Toxicology and ASOs
Preclinical toxicology is the frontline for ASO development targeting rare genetic disorders. It ensures that only the most efficacious and (relatively) safe drug candidates advance to patient trials. A tiered strategy of in silico, in vitro, and in vivo evaluations helps drug developers and sponsors anticipate, mitigate, and address adverse effects long before clinical risks arise.
Continued innovation in predictive models and high-value biomarkers could be essential to meet the evolving challenges of ASO therapies. For organizations without in-house expertise, collaborating with a trusted lab testing partner is the surest way to uphold the highest standards in ASO development - especially safety assessment - and accelerate the next wave of life-changing therapies.
References
- McDowall S, Aung-Htut M, Wilton S, Li D. “Antisense oligonucleotides and their applications in rare neurological diseases.” Front Neurosci. 2024 Sep 23;18:1414658. doi: 10.3389/ fnins.2024.1414658. PMID: 39376536; PMCID: PMC11456401.
- Lauffer, M. C., van Roon-Mom, W., & Aartsma-Rus, A., N=1 Collaborative. “Possibilities and limitations of antisense oligonucleotide therapies for the treatment of monogenic disorders.” Communications Medicine. 2024, January 5.
Author Biography
Dr. Xiaoxia Li, MD, Ph.D., DABT, is Executive Technical Director of Toxicology at WuXi AppTec with more than 22 years of experience in preclinical drug development. A board-certified toxicologist, she specializes in designing and managing nonclinical programs across various modalities, including toxicology, pharmacokinetics, ADME, and safety pharmacology. Her expertise spans CRO oversight, regulatory strategy, and CTD-compliant submissions for INDs and NDAs. Prior to WuXi AppTec, Dr. Li held scientific leadership roles in CROs and pharmaceutical companies in Canada and Japan. She holds a Ph.D. in Pharmacology from Japan, and an MD and MSc from China.
Subscribe to our e-Newsletters.
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!