Li, Yiman; (2025) Development of In Vitro Transcribed mRNA Therapeutics for Cystic Fibrosis. Doctoral thesis (Ph.D), UCL (University College London).

Text Yiman Li PhD Thesis.pdf - Accepted Version Access restricted to UCL open access staff until 1 October 2026. Download (45MB)

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The dysfunctional CFTR protein results in thick mucus buildup, chronic bacterial infection, and lung function decline. Although modulator drugs benefit many patients, approximately 10% still lack effective treatment. We propose to develop in vitro transcribed CFTR messenger ribonucleic acid (mRNA) as a universal therapy for all CF patients. The project aimed to formulating a non-viral nanoparticle delivery system and optimize mRNA structural element to enhance translation in epithelial cell models. We have adapted a previously established lipopolyplex system and refined it for delivery of mRNA to human bronchial epithelial cells. Among the tested cationic lipids, ditetradecyl trimethyl ammonium propane (DTDTMA) demonstrated superior transfection efficiency compared to the commonly used dioctadecenyl trimethyl ammonium propane (DOTMA). An optimal peptide was identified from a library of five candidates; however, further studies are required to validate its targeting efficacy. Delivery in air-liquid interface culture failed to achieve successful transfection, indicating the need for further optimisation. In vitro transcription and mRNA elements were optimised to enhance synthetic mRNA expression. Modulation of the poly(A) tail length in synthetic mRNA revealed its critical role in regulating reporter mRNA translation in bronchial epithelial cells. A range of non-native untranslated regions (UTRs), including two novel UTRs, were demonstrated to enhance reporter mRNA translation in bronchial epithelial cells. In vitro transcribed (IVT) luciferase mRNA outperformed commercial mRNA in epithelial cell transfection, and IVT CFTR mRNA showed high translation in CFBE cells. Overall, this work lays the foundation for developing mRNA therapies for cystic fibrosis.

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