Engineering RNA-Targeting CRISPR/Cas13 for the Treatment of Spinocerebellar Ataxia Type 3

Overview

Project Summary

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disorder, integrating the group of polyglutamine (polyQ) diseases as the most prevalent form of spinocerebellar ataxia. SCA3 is caused by the overexpansion of a cytosine-adenine-guanosine (CAG) trinucleotide repeat in the ATXN3 gene encoding the Ataxin-3 deubiquitinase. As a result, the formation of an expanded polyQ segment within Ataxin-3 confers a toxic gain-of function to this mutant isoform manifested by the generation of protein microaggregates and toxic species that trigger neuronal cell death by disrupting vital cellular mechanisms. There is no available treatment for SCA3.

Major efforts to treat SCA3 have focused on preventing the production of Ataxin-3. Chief among these is the use of RNA interference (RNAi) to silence ATXN3 expression at the post-transcriptional level. The connection of Ataxin-3 to numerous cellular mechanisms suggests that shutting down ATXN3 expression could be deleterious to patients in the long run. This lays the call for novel and innovative methods that can target the ATXN3 transcriptome and precisely remove transcripts encoding the the SCA3-causing polyQ segment from the Ataxin-3 while keeping its endogenous physiological activity.

One promising approach that has emerged in recent years is the discovery of CRISPR-based Cas13 for RNA targeting. Cas13 is a member of the CRISPR-associated family of enzymes that can target specific RNA sequences and induce cleavage. The discovery of Cas13 has opened new opportunities for the targeted manipulation of RNA, including the capability to precisely silence or influence the splicing outcome of predetermined genes without requiring the involvement of internal pathways for processing the target transcripts. Utilizing Cas13 for RNA targeting holds the potential to address some of the drawbacks associated with conventional RNAi-mediated gene silencing, primarily, the global transcriptomic off-target effects or the toxic imbalance of RNAi pathways. The proposed research aims to use the CRISPR/ Cas13 system to treat SCA3 by removing the toxic gain-of-function from Ataxin-3 while preserving its endogenous physiological activity. Two different approaches will be used to harness the CRISPR/Cas13 system to target the ATXN3 transcript. The first approach is allele-specific knockdown of mutant ATXN3 transcription by an RNA-targeting CRISPR/Cas13 RNase. The second approach is non-allele-specific modulation of ATXN3 pre-mRNA splicing by an RNA-targeting deactivated CRISPR/Cas13. Both strategies will increase the chances of effectively correcting the genetic defect and improving patient outcomes. We will evaluate the efficacy and safety of ATXN3-targeting CRISPR/Cas13 sequences in a human in vitro model of SCA3 neuropathology based on patient-derived neurons. A rapid and established method will be used to generate homogenous mature neurons within a short period by bypassing progenitor states and directly driving neuronal induction. The study will then treat the SCA3 neurons with adeno-associated virus that will deliver the ATXN3-targeting CRISPR/Cas13 sequences to evaluate the effects on the ATXN3 transcriptome and the alleviation of the disease phenotype.

The research conducted with support from this grant could provide a new avenue for the development of novel therapies for SCA3 and other polyglutamine diseases. We expect that the outcome of this study will support CRISPR/Cas13 as a next-generation treatment towards a viable treatment for SCA3 and potentially other neurodegenerative disorders.

Project Details