Izboljšava trans-spajanja RNA za popravilo genetskih mutacij

CTNNB1 syndrome is a rare neurodevelopmental disorder with serious motor and cognitive impairments, caused by de novo loss-of-function mutations in the CTNNB1 gene, which encodes the ß-catenin protein. The diversity and distribution of pathogenic mutations across the CTNNB1 gene and its dosage sensitivity highlight the need for mutation-independent therapeutic strategies capable of restoring ß-catenin expression and function without disrupting endogenous gene regulation. This thesis primarily investigated an RNA-based therapeutic approach using spliceosome-mediated RNA trans-splicing (SMaRT) to restore ß-catenin production. A series of pre-trans-splicing molecules (PTMs) targeting introns 2, 3, 5, and 6 of CTNNB1 were designed and evaluated using a split YFP reporter system. Trans-splicing efficiency was significantly enhanced by rationally designed short antisense RNAs (asRNAs) that inhibit cis-splicing, as well as by incorporating a self-cleaving ribozyme at the 5' end of PTM to improve nuclear retention. Additionally, CMV promoter-driven PTM expression yielded the highest trans-splicing efficiency. Endogenous trans-splicing of CTNNB1 transcripts in HEK293T cells confirmed the physiological relevance of this strategy and demonstrated proof-of-concept for SMaRT-mediated RNA repair in CTNNB1 syndrome. In addition to the SMaRT strategy, splice-switching antisense oligonucleotides (SSOs) were investigated as a complementary RNA-based approach to modulate CTNNB1 RNA splicing. Specifically, SSOs were designed to induce skipping of exon 3, which contains phosphorylation motifs responsible for targeting ß-catenin for proteasomal degradation. Exon 3 skipping resulted in a stabilized and transcriptionally active form of ß-catenin. To identify effective candidates, multiple plasmid-encoded asRNAs were designed and screened, leading to the selection of a potent SSO that induced robust and specific exon 3 skipping. The most effective SSO was further validated in human induced neural stem cells (NSCs) carrying the CTNNB1 syndrome mutation, demonstrating its potential for therapeutic application in a disease-relevant context. Together, these studies establish two distinct, RNA-based strategies for restoring ß-catenin expression and activity in CTNNB1 syndrome. By targeting either mRNA repair through trans-splicing or transcript stabilization via exon skipping, these approaches offer a solid foundation for the development of potential RNA-based therapy for CTNNB1 syndrome.