Single transcriptional unit gene-editing for inheritable lung disease

Enekwa, Ifeanyi (2022) Single transcriptional unit gene-editing for inheritable lung disease. Doctoral thesis, Northumbria University.

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Abstract

Genetic lung diseases such as Cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) are two of the most common examples of obstructive and restrictive lung diseases respectively, which pose an increasing challenge to health care systems. The Δ F508 mutation in cystic fibrosis transmembrane regulatory protein (CFTR) and rs35705950 single nucleotide polymorphism (SNP) in the Mucin 5 B (MUC5B) promoter region, are the two most common genetic conditions which are causative for CF and associated with IPF, respectively. Furthermore, a putative promoter associated non-coding (panc) RNA (AC061979.1) overlapping rs35705950 is predicted to be transcribed from this locus, positing a functional role in the development of IPF. Such mutations are prime candidates for gene-editing technologies aiming at disease treatment or prevention. Attempts to make this molecular tool more clinically relevant seek to enhance specificity, focus activity, or reduce the duration of action and thus risk of off-target gene editing events.

In this thesis, such an approach was taken by designing a single-transcriptional unit (STU) gene-editing cassette encoding the minimum components needed for genome editing. A set of functional CFTR-targeting gRNAs were designed, then tested in Cystic fibrosis bronchial epithelial (CFBE41o-), human alveolar basal epithelial (A549) and HeLa cells, at varying success rates as determined by T7 endonuclease 1 (T7E1) assay, with no statistically significant negative impact of cellular viability. The gRNA found to be most efficacious in CFBE41o- cells was then cloned into a STU gene-editing cassette which was found to express by RT-qPCR, but not translate in cellulo as determined by western blotting analysis. Troubleshooting eventually uncovered the lack of expression of the gene-editing endonuclease, present within the wild-type plasmid backbone used for cloning this STU gene-editing cassette.

After 3rd party next generation sequencing transcriptome data analysis, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was used to validate the expression of AC061979.1 pancRNA in lung epithelia, albeit at low copy number. Attempts to genome edit the rs35705950 locus proved unsuccessful, potentially because of the highly repetitive nature of this genomic locus.

Hence, this work validated the expression of a newly described pancRNA molecule in cellulo, which had only previously been predicted in silico to be expressed, and not explicitly reported in 3rd party next generation sequencing datasets. This thesis further contributes to the body of work attempting to improve production and design of clinically-relevant gene-editing approaches relevant to lung disease.

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