Abstract Alternative splicing of messenger RNA (mRNA) is a regulatory mechanism that controls transcript localization, translation, and stability, and enables the expression of multiple protein isoforms from a single gene. In neurons, splicing is finely controlled and is critical for regulation of neurogenesis, neuronal migration and structure, synaptogenesis, and synaptic function. Growing evidence highlights a role for splicing alterations in neurological diseases, emphasizing the need to better understand the mechanisms of RNA processing in order to develop splicing-targeted therapies. Recent studies have shown that neurons can control gene expression during critical developmental stages by coupling alternative splicing with nonsense mediated decay (NMD). This mechanism relies on inclusion of cassette exons, known as poison exons, to create in-frame premature termination codons that trigger transcript NMD and reduce protein expression. Importantly, genetic variants promoting constitutive inclusion of poison exons have been associated with neurodevelopmental and, more recently, neurodegenerative diseases. However, the field lacks rigorous methods to identify and annotate poison exons and variants affecting their splicing. A well-established example in which increased exon inclusion can lead to neurological diseases is the aberrant splicing of the microtubule associated protein tau (MAPT). Pathogenic splicing mutations that promote MAPT exon 10 inclusion lead to increased 4R-tau isoform expression and aberrant tau accumulation, whereas missense and deletion gain-of-function mutations in exon 10 are associated with mutant 4R-tau pathology. In both contexts, an approach that promotes MAPT exon 10 exclusion would be therapeutically beneficial. Given the increase relevance of misplicing in disease, the goal of the proposed work is to advance the understanding of the role of splicing alterations in neurological diseases with the objective of developing splicing-targeted therapeutics. In Aim 1, we will develop a transcriptomic approach to map poison exons relevant for neuronal development and survival, and by intersecting the identified poison exons with ClinVar pathogenic variants, we will catalog mutations that are likely to affect their splicing and contribute to disease. Then, generation of CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC)-derived neuronal models for a selective number of these mutations will allow us to evaluate the impact of aberrant poison exon inclusion on neuronal phenotypes. In Aim 2, we will use a mRNA-targeted strategy that promotes MAPT exon 10 skipping as proof-of-principle for the therapeutic potential of splicing modulator compounds, by showing rescue of neuronal disease phenotypes in patient-derived neuronal models of frontotemporal dementia (FTD). The development of novel mRNA-targeted therapies that specifically correct the molecular defects leading to disease will be a major advance for the treatment of incurable neurological diseases caused by splicing alterations.

Narrative The goal of our proposed research is to advance the knowledge regarding the role played by splicing alterations in neurological diseases with the objective of developing novel mRNA-targeted therapeutics. Our first aim is to use a computational approach to identify new neuron-specific poison exons for which mis-splicing is associated with neurodevelopmental and neurodegenerative diseases. We will then use patient-derived cellular models of frontotemporal dementia associated with MAPT mis-splicing as proof-of-principle to demonstrate the therapeutic potential of a novel class of splicing modulator compounds.