An RNA-targeting CRISPR–Cas13d system alleviates disease-related phenotypes in Huntington’s disease models

Abstract

Huntington’s disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR–Cas13d system (Cas13d–CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d–CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR–Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.

Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins

“…Because of their intrinsic RNase activity [36][37][38][39][40][41][42] , their high programmability, and their ability to be used in mammalian cells to knock down the expression of a target gene 36,41,[47][48][49]73 , RNAtargeting CRISPR effector proteins are a potentially powerful class of agents for gene silencing.…”

“…To target the mATXN2 mRNA, we used the Cas13d nuclease from Ruminococcus flavefaciens XPD3002 (RfxCas13d) 41 , a programmable RNA-targeting effector protein that we 47 and others 48,49 have demonstrated can silence target gene expression in the nervous system. To facilitate the identification of crRNAs for mATXN2, we created a reporter plasmid carrying the mATXN2 protein-coding sequence fused to an enhanced green fluorescence protein (EGFP) via a self-cleaving T2A peptide, linking mATXN2 expression to EGFP fluorescence.…”

Programmable macromolecule-based RNA-targeting therapies to treat human neurological disorders

“…AAV-mediated delivery of this mutant HTT mRNA targeting Cas13d system to the striatum of heterozygous zQ175 mice, an established mouse model of HD, resulted in allele-selective suppression of mutant HTT mRNA and protein aggregates while maintaining normal HTT mRNA and protein levels, significantly improved motor function, and attenuated striatal atrophy. These phenotypic improvements lasted for at least 8 months without adverse effects, and with minimal off-target transcriptomic effects 56 . This strategy also holds promise as a possible therapeutic approach for myotonic dystrophy type 1 (DM1) as we demonstrated Cas13d with CUG-targeting guide RNAs can eliminate toxic RNA foci in a cortical organoid of DM1 57 .…”

“…We recently demonstrated the proof-of-principle that a mutant allele-selective RNAtargeting CRISPR-Cas13d system eliminates toxic mutant HTT transcripts in HD patient-derived fibroblasts and iPSC-derived neurons with limited off-target effects on the human transcriptome 56 .…”

Massively Parallel Profiling of RNA-targeting CRISPR-Cas13d

“…For example, Cas13d-one of the most compact and biochemically active Cas13 enzymes-can efficiently knockdown RNA in mammalian cells and animal models 5,[18][19][20][21][22][23][24][25] . Moreover, Cas13d fusions are used for RNA tracking, editing, modification, and splicing regulation 5,7,26,27 .…”