RNA trans-splicing to rescue β-catenin: A novel approach for treating CTNNB1-Haploinsufficiency disorder

Loss-of-function mutations in the <i>CTNNB1</i> gene cause β-catenin deficiency, resulting in CTNNB1 syndrome, a rare neurodevelopmental disorder characterized by motor and cognitive impairments. Given the wide variety of mutations across <i>CTNNB1</i> and its dosage sensitivity, a mutation-independent therapeutic approach that preserves endogenous gene regulation is critically needed. This study introduces spliceosome-mediated RNA <i>trans</i>-splicing as a novel approach to restore β-catenin production. Pre-<i>trans</i>-splicing RNA molecules (PTMs) targeting <i>CTNNB1</i> introns 2, 5, and 6 were designed and evaluated using a split yellow fluorescent protein reporter system. Rationally designed short antisense RNAs, which mask splicing regulatory elements, significantly enhanced PTM-mediated <i>trans</i>-splicing at both mRNA and protein levels. Additionally, introducing a self-cleaving ribozyme at the PTM's 5' end further improved <i>trans</i>-splicing efficiency, likely due to increased nuclear retention. CMV promoter-driven PTM expression yielded the highest efficiency. Importantly, successful <i>trans</i>-splicing of the endogenous <i>CTNNB1</i> transcript confirmed the physiological relevance of this strategy. This study is the first to apply and optimize spliceosome-mediated RNA <i>trans</i>-splicing (SMaRT) for <i>CTNNB1</i> mRNA correction, providing a promising, mutation-agnostic approach for treating CTNNB1 syndrome.