Antisense oligonucleotides (ASOs) are quickly becoming a leading tool in the treatment of various central nervous system (CNS) disorders, particularly neurodegenerative diseases like ALS. However, before advancing ASO discovery programs into clinical stages, there are many important considerations, particularly when transitioning from in vitro studies to animal models. Dr. Susanne Back, an expert in CNS pharmacology at Charles River Labs, provides insights on the challenges and best practices for this process.
Introduction to ASOs
ASOs are short, synthetic sequences of single-stranded DNA that can alter RNA expression and splicing. They are capable of modulating the production of proteins, often by either reducing, restoring, or modifying protein expression. Although ASOs have been used for gene therapy in certain applications, their development process more closely mirrors that of small molecule drugs than the traditional viral vector-based gene therapies. ASOs are proving particularly valuable in treating diseases with a strong genetic basis, such as ALS, Huntington’s disease, and rare genetic disorders, thanks to their ability to target specific RNA transcripts tied to disease-causing genes.
The Growing Interest in ASOs for ALS and Other CNS Disorders
ASOs have garnered increased attention in treating ALS due to their effectiveness in targeting the root causes of the disease. Genetic forms of ALS, particularly those related to mutations in genes like SOD1, C9orf72, and TDP-43, provide a clear foundation for ASO development. These ASOs can be specifically designed to target the mutated RNA transcripts, offering a new approach to disease management.
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FDA-approved ASOs, like those used in spinal muscular atrophy (SMA) treatment, have shown success in providing hope for patients with rare CNS disorders. This success has sparked further interest in the development of ASOs as a promising therapeutic modality for ALS and other neurodegenerative diseases. However, despite the optimism, there are several considerations that must be addressed before ASO programs move into animal models.
Key Considerations for Moving ASO Programs into Animal Models
When considering transitioning an ASO discovery program to animal models, particularly for diseases like ALS, it’s crucial to evaluate the genetic evidence supporting the involvement of the target gene. Dr. Back emphasizes the importance of understanding how genetic mutations influence disease pathology. Familial ALS models, where specific gene mutations have been identified, provide critical insights that guide the development of relevant preclinical models and pharmacological readouts.
Given the urgency of ALS, selecting the right preclinical models is essential to avoid unnecessary delays in the discovery process. ALS is a rapidly progressing disease, and drug discovery programs must prioritize studies that move efficiently from one stage to the next.
For ASO programs specifically, pharmacodynamic readouts are a key consideration. These may include measures of RNA degradation, splicing modulation, or other targeted effects. Early-stage safety assessments, such as evaluating bio-distribution and potential immunostimulatory effects, are also essential to ensure that the therapeutic approach is viable before moving to human clinical trials.
Limitations of Animal Models
While animal models play a vital role in drug discovery, there are inherent limitations when using them for complex human diseases like ALS. Even the most sophisticated animal models cannot replicate the full complexity of human ALS, which may lead to discrepancies between preclinical and clinical outcomes.
In response, Dr. Back highlights the growing interest in human-based in vitro models, such as human induced pluripotent stem cell (iPSC)-derived cell models. These models help bridge the gap between animal studies and human trials, providing a more accurate representation of the disease’s effects on human tissues. The challenge lies in balancing the use of animal models and human-based in vitro models to generate the most predictive and reliable data for advancing ASO therapies.
ASO Administration and Challenges in Neuroscience Disorders
A key difference between ASOs and traditional small molecule drugs is their inability to freely cross the blood-brain barrier (BBB). This presents a significant challenge when considering systemic administration of ASOs. Unlike small molecule drugs, which can be taken orally, ASOs must be directly delivered to the CNS.
The most common clinical method for delivering ASOs into the CNS is through intrathecal injection, a procedure where the drug is injected into the space around the spinal cord. This method has shown to be highly translatable to human trials, making it an essential technique in both preclinical animal models and human studies. Researchers are also investigating alternative methods for ASO administration, including conjugating ASOs to antibodies to facilitate BBB crossing and employing focused ultrasound to temporarily open the BBB.
Assessing Bioavailability and Distribution in vivo
Assessing the distribution and bioavailability of ASOs within the CNS is another critical component of preclinical studies. Several established methods are used to measure ASO levels in brain tissues. These include tissue dissection followed by liquid chromatography-mass spectrometry (LC-MS), hybridization ELISA, and quantitative PCR (qPCR). These techniques allow researchers to quantify ASO concentrations in specific regions of the brain.
Microdialysis is another useful technique, which involves microsampling to measure ASO concentrations in plasma and cerebrospinal fluid (CSF). This method can provide real-time measurements of ASO distribution, though it may not always be the most accurate due to the intracellular nature of ASO targets. For a more comprehensive view, researchers use PET imaging with radio-labeled ASOs, enabling them to track the distribution of the drug in living animals over time.
Half-life of ASOs in the CNS
The half-life of ASOs in the CNS can vary depending on their chemical structure and delivery method. For instance, in studies involving SOD1-targeting ASOs, effects have been observed lasting up to eight weeks following a single intrathecal injection. Similarly, in the C9orf72 mouse model of ALS, knockdown effects persisted for up to 24 weeks after two injections. These prolonged effects of ASOs are advantageous for patients, as they allow for extended therapeutic benefits compared to the shorter duration of action seen with small molecule drugs.
Conclusion
As the development of ASOs for CNS disorders like ALS progresses, the integration of preclinical in vivo models remains a cornerstone of research. The complexity of neurological diseases, however, calls for continuous refinement of animal models and alternative human-based in vitro systems to increase the accuracy and predictability of preclinical studies. While challenges persist, the potential of ASOs to transform the treatment landscape for rare and genetic CNS disorders is immense. Moving forward, advancements in ASO therapies may provide new hope for patients, but ensuring their safety and efficacy requires ongoing diligence and research.
