Abstract
Background/aims: A hexanucleotide repeat expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS-FTD). Toxicity arising from sense and antisense intranuclear RNA foci, and dipeptide repeat proteins generated from repeat-associated non-AUG dependent (RAN) translation of expanded RNA, are thought to be the primary drivers of disease in C9ALS-FTD. Consequently, a gene therapy strategy to knockdown mutant C9orf72 RNA using artificial miRNA encoded by an AAV9 vector may hold therapeutic potential and is explored in this project.Methods: A gene therapy cassette encoding artificial microRNA (miRNA) targeting intron 1 of C9orf72 was designed. Candidate miRNA sequences were screened in vitro using a dual-luciferase reporter (DLR) assay. The most potent candidates were packaged into self-complimentary AAV9 vectors. The potential of these viral vectors to knockdown endogenous C9orf72 was evaluated in human iPSC cortical neurons derived from C9ALS patients, and by intra-thalamic injection in a transgenic mouse model.
Results: A statistically significant reduction in intronic C9orf72 transcripts was observed in human iPSC cortical neurons transduced with AAV9 vectors and in transgenic mouse brains following bilateral intra-thalamic delivery. No mice developed any adverse reaction to the vector or the surgical procedure up until the point of sacrifice, suggesting the approach was well-tolerated.
Conclusion: A Viral vector-based gene silencing approach incorporating artificial miRNA targeting intron 1 of C9orf72 is a potentially viable therapeutic strategy for C9ALS-FTD. Further work is required to optimise vector dose, route of delivery, improve CNS transduction, and to evaluate the long-term safety and efficacy of the approach.
Date of Award | 1 May 2024 |
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Original language | English |
Awarding Institution |
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Supervisor | Younbok Lee (Supervisor), Jemeen Sreedharan (Supervisor) & Christopher Shaw (Supervisor) |