Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptides in vivo

Cabrera, Gabriela Toro; Meijboom, Katharina E.; Abdallah, Abbas; Tran, Helen; Foster, Zachariah W.; Weiss, Alexandra; Wightman, Nicholas; Stock, Rachel; Gendron, Tania F.; Gruntman, Alisha M.; Giampetruzzi, Anthony; Petrucelli, Leonard; Brown, Robert C.; Mueller, Christian

doi:10.21203/rs.3.rs-2081412/v1

Cited by 2 publications

(2 citation statements)

References 44 publications

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“…To date, therapeutic strategies to target C9 ALS/FTD repeat RNAs have focused on targeting the sense G4C2 repeat transcripts [12][13][14][15][16] , however, there is a considerable and growing body of evidence that supports the contribution of the antisense strand to C9 ALS/FTD disease pathogenesis [17][18][19][20][21] . Additionally, recent failures in clinical trial of two antisense oligonucleotides (ASOs) targeting C9orf72 sense repeat-containing transcripts (BIIB078; clinicaltrials.gov: NCT03626012 and WVE-004; clinicaltrials.gov: NCT04931862), despite showing target engagement and lowering of the sense transcript, highlight the importance of developing therapeutic approaches that can also target C9orf72 antisense repeat RNA transcripts.…”

Section: Introductionmentioning

confidence: 99%

Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

Kempthorne,

Vaizoglu,

Cammack

et al. 2024

Preprint

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The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

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Section: Introductionmentioning

confidence: 99%

Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

Kempthorne,

Vaizoglu,

Cammack

et al. 2024

Preprint

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“…Given the evidence in support of a role for the hexanucleotide repeat-containing RNAs in C9ORF72-linked ALS and FTD -hereafter referred to as C9-ALS/FTD -strategies for silencing their production hold potential for the disorder [25][26][27][28][29][30] , as they can inhibit the formation of abnormal RNA foci 25,27 and DPR protein deposits 31,32 . One emerging technology with the capabilities to enable this is Cas13 [33][34][35] , a class 2 type VI CRISPR effector protein that, when complexed with a CRISPR RNA (crRNA) guide molecule carrying complementarity to a target transcript, can cleave it via its intrinsic ribonuclease (RNase) activity.…”

Section: Introductionmentioning

confidence: 99%

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

McCallister,

Lim,

Terpstra

et al. 2023

Preprint

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An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

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Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptides in vivo

Cited by 2 publications

References 44 publications

Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

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