Improvement of RNA trans-splicing for the repair of genetic mutations

Maruna, Matea (author), Jerala, Roman (mentor)

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.

• Language: English
• Keywords: trans-splicing, exon skipping, antisense oligonucleotides, CTNNB1, β-catenin, CTNNB1 syndrome, rare disease, RNA therapy
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2025
• PID: 20.500.12556/RUL-175870
• Publication date in RUL: 12.11.2025

Secondary language

• Language: Slovenian
• Title: Izboljšava trans-spajanja RNA za popravilo genetskih mutacij​

Abstract

Sindrom CTNNB1 je redka nevrološko-razvojna motnja, za katero so značilne hude motorične in kognitivne prizadetosti. Povzročajo jo naključne de novo točkovne mutacije v genu CTNNB1, ki kodira protein β-katenin. Zaradi raznolikosti in razpršenosti omenjenih mutacij ter vloge gena pri nastanku raka ob njegovem prekomernem izražanju, je razvoj terapevtskega pristopa, ki omogoča popravljanje mutacij ob hkratnem ohranjanju naravne regulacije izražanja gena, ključnega pomena. V doktorski nalogi smo se osredotočili na RNA-terapevtski pristop, natančneje na trans-spajanje RNA, posredovano s spajalnim telescem (angl. splicesome mediated RNA trans-splicing, SMaRT). Razvili smo nabor trans-spajalnih RNA molekul (angl. pre-trans RNA molecule, PTM), usmerjenih na introne 2, 5 in 6 gena CTNNB1, ter njihovo učinkovitost ovrednotili z uporabo poročevalskega sistema, zasnovanega na cepljenem rumenem fluorescenčnem proteinu. Ugotovili smo, da se učinkovitost trans-spajanja znatno poveča z uporabo racionalno zasnovanih kratkih protismiselnih RNA (angl. antisense RNA, asRNA), ki zavirajo cis-spajanje, kot tudi z vključitvijo samoizrezujočega ribocima na 5′ koncu PTM, ki izboljša zadrževanje molekule v jedru. Najvišjo učinkovitost trans-spajanja smo dosegli z izražanjem PTM pod promotorjem CMV. Potrdili smo tudi endogeno trans-spajanje v celicah HEK293T, kar potrjuje uporabnost te strategije za popravljanje mutacij, povezanih s sindromom CTNNB1. Poleg tega smo raziskali terapevtski potencial kratkih protismiselnih oligonukleotidov (angl. Splice-switching oligonucleotides, SSO) za modulacijo spajanja RNA. Ti so bili načrtovani za induciranje preskoka eksona 3 v genu CTNNB1, ki vsebuje fosforilacijske motive, ključne za proteasomalno razgradnjo β-katenina. Pokazali smo, da preskok eksona 3 vodi do stabilne in transkripcijsko aktivne oblike β-katenina. Načrtovali smo nabor kratkih asRNA, ki smo jih ovrednotili z uporabo poročevalskega sistema, ter identificirali učinkovit SSO, ki je sprožil močno in specifično preskakovanje eksona 3. Učinkovitost SSO smo potrdili tudi v induciranih človeških nevralnih matičnih celicah (angl. neuronal stem cells, NSC) z mutacijo v genu CTNNB1, ki povzroča sindrom CTNNB1, kar dodatno potrjuje njegov potencial kot terapevtsko orodje. V doktorski nalogi sta predstavljeni dve obetavni RNA strategiji za obnovo izražanja β-katenina pri sindromu CTNNB1, ki temeljita na trans-spajanju in preskakovanju eksona 3. Obe strategiji ponujata obetavno platformo za nadaljnji razvoj RNA-terapije za zdravljenje sindroma CTNNB1.

• Keywords: trans-spajanje, preskakovanje eksonov, protismiselni oligonukleotidi, CTNNB1, β-katenin, sindrom CTNNB1, redka bolezen, RNA terapija