Developing antisense oligonucleotide therapies using iPSC-based cell types

The working group “Advanced Cellular Models of Neurodegeneration” at the DZNE Tübingen, led by Dr. Stefan Hauser, focuses on the development of cutting-edge cellular models for the study of monogenetic neurodegenerative diseases.

These models can be used to develop precise antisense oligonucleotides (ASOs) that specifically reduce the levels of the mutant protein with preserving the wild type. For this purpose, an optimized and standardized workflow from target identification up to multimodal validation of ASOs in complex iPSC-derived cellular models has been developed.

ASOs are short RNA-based sequences that bind to complementary (pre-)mRNA in the cytosol or nucleus and alter the processing of the target gene by regulating splicing or initiating degradation through RNase H recruitment. Our research has demonstrated the remarkable specificity of gapmer ASOs, enabling them to selectively target single nucleotide polymorphisms (SNPs) that are exclusively present on the mutant allele. As a result, we achieved a specific reduction of mutant protein levels with a single application of the ASO in our in vitro 2D culture of cortical neurons and 3D cerebellar organoids from patient-derived induced pluripotent stem cells (iPSCs).

Although animal studies are essential for clinical approval, the possibilities offered by the evaluation of ASOs in patient-derived iPSCs are outstanding. The differentiation into different cell types with an identical genetic background to the patient and the versatility of the screening methods can more accurately simulate potential off-targets and adverse effects more accurately in the future.

A platform that can analyze ASOs in a human disease-relevant context in a scalable approach is urgently needed. In addition to off-target effects, acute and long-term toxicity as well as efficacy should be assessed in a simple and scalable manner. Brain organoids, a state-of-the-art model for various diseases, can effectively serve this purpose. Brain organoids can be generated from patient-derived iPSCs, providing a platform that mimics the increased complexity and maturity of an organ while maintaining the genetic background of the patient.

This model is ideal for replicating the pathogenic phenotype in vitro. In addition, acute and long-term toxicity of ASOs can be monitored using various readouts, including calcium imaging, multielectrode array recordings or the evaluation of specific biomarkers such as NfL. Omics technologies (RNA-Seq, proteomics) allow precise and in-depth analysis of on- and off-target effects.

Once established, this platform is adaptable to many diseases affecting different parts of the brain, such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia (HSP), or trinucleotide repeat disorders such as spinocerebellar ataxias and Huntington’s disease (HD). This platform can be used not only as a read out for ASOs, but also for the evaluation of therapeutics such as mRNA vaccines or siRNA in general, where the assessment of toxicity and adverse side effects is essential.