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Huntingtin CAG repeat expansions in induced pluripotent stem cell models of Huntington’s Disease

Donaldson, Jasmine Jo ORCID: 2019. Huntingtin CAG repeat expansions in induced pluripotent stem cell models of Huntington’s Disease. PhD Thesis, Cardiff University.
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the huntingtin gene, HTT. The length of the CAG repeat is inversely correlated with age at motor onset, but other factors influence onset including genetic variation elsewhere in the genome. Recent genome-wide association studies have identified genetic variants in the vicinity of FAN1, a nuclease involved in DNA interstrand cross-link repair, as modifiers of age at onset of HD. It is thought that FAN1 might modify age at onset through direct modulation of the expanded HTT CAG repeat. Further expansions of the CAG repeat in post-mitotic neurons are thought to accelerate disease-onset and therefore factors that license or inhibit expansions represent therapeutic targets. This thesis demonstrates that HD-iPSC lines with expanded CAG repeat tracts of >100 CAG repeats (Q109) exhibit repeat instability in culture, with the repeat tract undergoing further expansions in pluripotent cells and upon neuronal differentiation. These cell lines therefore represent a cellular model of repeat expansion which can be utilised to characterise how DNA repair genes affect cells harbouring expanded CAG repeats. Employing CRISPR-Cas9 and a piggyBac transposon-based homologous recombination approach the expanded HTT CAG repeat tract was genetically corrected to a wild-type repeat length of 22 HTT CAGs (Q22). Corrected HD-iPSCs retained pluripotency and differentiation potential. Additionally, metabolic deficits in Q109 neural progenitor cells were rescued in Q22 neural progenitor cells. This thesis also aimed to establish the effect of FAN1 on HTT CAG repeat expansions. Using CRISPRCas9, FAN1 was knocked-out of an isogenic pair of HD-iPSCs containing either 22 or >100 HTT CAG repeats. FAN1-/- clones were significantly more susceptible to interstrand cross-linking agent MMC, indicative of defective interstrand cross-link repair. Increased CAG repeat expansion was observed in FAN1-/- iPSCs and iPSC-derived neurons, suggestive of a protective role of FAN1 against CAG repeat expansions. These novel model systems provide a platform for investigating the cellular phenotypes associated with expanded CAG repeats, and the effects of DNA-repair associated genetic modifiers.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 24 February 2020
Last Modified: 19 May 2023 01:45

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