Linda M. van der Graaf

Antisense oligonucleotides (AONs) are small pieces of chemically modified DNA or RNA that bind to RNA in a sequence-specific manner based on Watson-Crick base-pairing. Splice-switching AONs are designed to modulate pre-mRNA splicing, thereby for instance restoring protein expression or modifying the eventual protein to restore its function or reduce its toxicity. Given the current lack of in silico methods that adequately predict off-target splicing events, assessment of off-target effects of AONs in human cells using RNAseq could be a promising approach. The identification and prioritization of potential off-target effects for validation and further investigation into the biological relevance would contribute to the development of safe and effective AONs. In this study, we used three different splice-switching AONs targeting three different human transcripts to study their transcriptome-wide, hybridization-dependent off-target effects with short read RNAseq. Using the computational tools rMATS and Whippet, we identified differential splicing events of which only a minority could be explained by hybridization, illustrating the difficulty of predicting off-target effects based on sequence homology. The main splicing events could all be validated with RT-PCR. Furthermore, from the three AONs studied, one AON induced considerably more changes in gene expression and splicing compared to the two other AONs assessed, which was confirmed in a validation experiment. Our study demonstrates that the interpretation of short read RNAseq data to determine off-target effects is challenging. Nonetheless, we showed that valuable results can be obtained as it allows the comparison of toxicity between different AONs within an experiment and identification of AON-specific off-target profiles.

Background: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin‐1 protein resulting in neuropathology including mutant ataxin‐1 protein aggregation, aberrant neurodevelopment, and mitochondrial dysfunction.Objectives: Identify SCA1‐relevant phenotypes in patient‐specific fibroblasts and SCA1 induced pluripotent stem cells (iPSCs) neuronal cultures.Methods: SCA1 iPSCs were generated and differentiated into neuronal cultures. Protein aggregation and neuronal morphology were evaluated using fluorescent microscopy. Mitochondrial respiration was measured using the Seahorse Analyzer. The multi‐electrode array (MEA) was used to identify network activity. Finally, gene expression changes were studied using RNA‐seq to identify disease‐specific mechanisms.Results: Bioenergetics deficits in patient‐derived fibroblasts and SCA1 neuronal cultures showed altered oxygen consumption rate, suggesting involvement of mitochondrial dysfunction in SCA1. In SCA1 hiPSC‐derived neuronal cells, nuclear and cytoplasmic aggregates were identified similar in localization as aggregates in SCA1 postmortem brain tissue. SCA1 hiPSC‐derived neuronal cells showed reduced dendrite length and number of branching points while MEA recordings identified delayed development in network activity in SCA1 hiPSC‐derived neuronal cells. Transcriptome analysis identified 1050 differentially expressed genes in SCA1 hiPSC‐derived neuronal cells associated with synapse organization and neuron projection guidance, where a subgroup of 151 genes was highly associated with SCA1 phenotypes and linked to SCA1 relevant signaling pathways.Conclusions: Patient‐derived cells recapitulate key pathological features of SCA1 pathogenesis providing a valuable tool for the identification of novel disease‐specific processes. This model can be used for high throughput screenings to identify compounds, which may prevent or rescue neurodegeneration in this devastating disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.