Genome editing to rescue Machado-Joseph disease by alternative splicing

Overview

Project Summary

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder caused by an unstable over-repetition of a CAG tract located in the exon 10 of the coding region of ATXN3 gene, which translates into a polyglutamine (polyQ) repeat expansion that confers a toxic gain of function to the resultant Ataxin-3 protein. There is no therapy for this disorder.

Silencing ATXN3 expression by RNA interference (RNAi) sequences became the most widely used therapeutic approach to treat MJD, showing promising results in pre-clinical mouse models. However, RNAi methods can only target gene expression at the post-transcriptional level, demanding repetitive and long-term administration of inhibitory sequences which may trigger cellular pathway oversaturation and toxicity. Moreover, Ataxin-3 role in numerous cellular functions may be indicative that fully silencing its activity could be deleterious to patients.

An ideal approach to treat MJD would alleviate the toxicity caused by the CAG expansion of ATXN3 gene while preserving the Ataxin-3 physiological activity. Relevant to this, previous studies using anti-sense oligonucleotides (ASO) to modulate ATXN3 splicing support that the polyQ region is not required for Ataxin-3 physiological function, laying the basis for novel therapeutic approaches to correct MJD in a precise and safe manner. Nevertheless, similar to RNAi sequences, ASO can only modulate gene expression at the RNA level and only provide a transient effect. Therefore, there is a need to increase the effectiveness and reduce potential issues of this approach by designing platforms that can mediate a permanent effect by targeting ATXN3 at the DNA level.

The development of CRISPR-based platforms for genome engineering opened novel perspectives for permanent correction of genetic defects in a targeted and efficient manner. In particular, CRISPR-base editors (CRISPR-BE) promote the targeted mutation of genomic sequences without causing deleterious DNA double-strand breaks or requirement of endogenous cell repair pathways that are generally inactive in post-mitotic cells. Genomic DNA recognition is mediated by a small guide RNA (sgRNA) that directs an inactive Cas9 nuclease domain fused to a nucleobase deaminase that induces a specific base-pair conversion. We reasoned that we could take advantage of this innovative platform to modulate ATXN3 splicing at the DNA level to promote the efficacious exon-skipping of CAG-coding region in a permanent manner.

In this proposal, we will develop CRISPR-base editor sequences to promote ATXN3 alternative splicing as a novel approach to rescue MJD neurodegeneration. We will test the CRISPR-BE capacity to promote ATXN3 alternative splicing and rescue neurodegeneration in the context of MJD by treating patient cells or animal models. Delivery of CRISPR-BE sequences will be mediated by adeno-associated virus (AAV) which will enable high levels of CRISPR-BE in the cerebellum, the most affected region in MJD patients. We will also generate ATXN3 edited lines to precisely evaluate the impact of alternative splicing within the context of human brain organoids. We anticipate that ATXN3 alternative splicing by CRISPR-BE platforms will pave the way for a novel gene therapy approach to treat MJD patients and which could be expanded to other neurodegenerative disorders.

Project Details