Disrupting Splicing Regulation to Rescue β-Catenin: A Novel Approach for Treating CTNNB1-Haploinsufficiency Disorder

Abstract

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