Kempthorne, Liam; (2023) Utilising CRISPR-Cas13 systems to target C9orf72 repeat expansion-containing RNA. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The most common genetic cause of both frontotemporal dementia and amyotrophic lateral sclerosis is a G4C2 hexanucleotide repeat expansion (HRE) in intron 1 of the C9orf72 gene. The C9orf72 HRE has been shown to undergo bidirectional transcription and repeat-associated non-AUG translation producing sense and antisense RNA foci and five dipeptide repeat proteins, with poly-GR and poly-PR shown to be the most toxic. Recent discoveries of RNA-targeting CRISPR-Cas13 systems open promising avenues for novel therapeutics. We designed guide RNAs to target CRISPR-Cas13s upstream of the C9orf72 G4C2 HRE in intron 1 to target the sense repeat-containing transcripts, and upstream of the C4C2 repeat expansion in the antisense strand to target the antisense repeat-containing transcripts. We tested two CRISPR-Cas13 orthologs, Cas13b (originating from Prevotella sp. P5-125) and Cas13d (CasRx; originating from Ruminococcus flavifaciens) in HEK293T cells overexpressing either 92 sense G4C2 repeats or 55 antisense C4G2 repeats. We show that CRISPR-Cas13s can successfully target both the sense and antisense transcripts of the C9orf72 HRE resulting in a reduction of sense and antisense RNA foci, and poly-GR and poly-PR to background levels in transient repeat-expressing models. We find CRISPR-CasRx to be the most efficient ortholog at targeting the C9orf72 HRE and show it has the ability to mature its own guide array to allow targeting of multiple transcripts. We developed lentiviruses expressing both CRISPR-CasRx and our most efficacious guide RNAs determined in our transient expression models. Using these lentiviruses we show that CRISPR-CasRx can effectively target and reduce both the endogenous sense and antisense repeat-containing transcripts in three independent C9orf72 patient-derived neuronal lines and reduce DPRs by >50% after only 5 day treatments. This was achieved without any overt toxicity or significant off target transcriptional changes. We then packaged both CRISPR-CasRx and our guide RNA array targeting the sense and antisense C9orf72 transcripts into a single AAV-PHP.eB vector to produce AAVs to treat two mouse models of the C9orf72 HRE. We first use a mouse model overexpressing 149 pure G4C2 repeats and 120 nucleotides of the human C9orf72 sequence upstream and downstream of the repeats delivered by an AAV (149R AAV). By administering our CasRx-expressing C9orf72-targeting PHP.eB-AAV and the 149R AAV to post-natal day 0 mice we show we can reduce repeat-containing transcripts by >50% after 3 weeks. Furthermore, we show a >50% reduction in repeat-containing RNA when treating 12 month old adult C9orf72 BAC mice expressing the full human C9orf72 sequence and 500 G4C2 repeats after 3 months. Additionally, we show that CasRx can target repeat-containing RNA in a zebrafish model of the C9orf72 HRE expressing 50 G4C2 repeats to a therapeutically relevant level and rescue a larval behavioural phenotype. These data illustrate CRISPR-CasRx can effectively target the C9orf72 HRE in vivo. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD. Future work is focused on optimising this strategy for the clinic and further testing in larger animal cohorts. Additionally, further analysis into off target effects and immunogenicity is warranted and planned in future in vivo studies.

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