Development and Validation of SIMOA-based total and mutant Ataxin-2 Immunoassay for biomarker studies in SCA2 and SCA3 

Principal researchers: Dr Stefan Hauser, Prof Schöls Ludger and Jacob Helm, German Center for Neurodegenerative Diseases (Germany).

Scientific summary 

This project aims to identify single nucleotide polymorphisms (SNPs) associated with the cytosine-adenine-guanine (CAG) trinucleotide repeat expansion that causes spinocerebellar ataxia type-3 (SCA3) to develop an allele-specific therapeutic approach using antisense oligonucleotides (ASOs). SCA3, also known as Machado-Joseph disease (MJD), is an autosomal dominant neurodegenerative disorder caused by the expansion of a cytosine-adenine-guanine (CAG) trinucleotide repeat in exon 10 of the ATXN3gene. The expanded CAG repeat is translated into an abnormally long stretch of polyglutamine (polyQ), which drives the pathogenic process by inducing a toxic gain of function.

We aim to develop antisense oligonucleotides (ASOs) that selectively reduce the polyQ-expanded ataxin-3 while minimising the impact on the wildtype protein. The aim of our project is to develop a cost effective, high-throughput method for allele-specific sequencing of ataxin-3 to identify novel SNPs associated with the CAG repeat expansion.

We designed primers for a 2 kb amplicon flanking the CAG repeat. Using patient DNA, the mutant and wild-type allele could be separated electrophoretically and Sanger sequenced. This allowed us to confirm linkage of rs12895357 to the expanded CAG repeat, and identify the association of previously unknown variants with the mutant ATXN3. To confirm the strategy of allele-specific SNP identification across the ATXN3gene, we have already performed nanopore sequencing of selected individual patients, made possible by a grant from Dr Holger Hengel. For high-throughput sequencing, we have gained access to DNA samples from the European SCA3/MJD Initiative (ESMi). We carefully selected 25 patients from each of the three cohorts located in London (UK), Tübingen (Germany) and Coimbra and Azores (Portugal) to ensure representation of different haplotypes.

Lay summary  

Ataxia UK has awarded funding to Dr Stefan Hauser and colleagues at the German Center for Neurodegenerative Diseases, Tübingen, Germany, which paves the way for a technique that can switch off faulty ataxin-3 genes in Spinocerebellar Ataxia Type-3 (SCA3) without turning off healthy versions. This is known as allele-specific gene silencing. The method involves the use of synthetic gene sequences called antisense oligonucleotides (ASOs), which bind to instructions for the sequence of genes including the faulty ataxin-3, and degrade the damaged ataxin-3 genes, keeping only healthy ataxin-3 genes.

For the ASOs to bind predominantly to the mutant form, it is necessary to identify differences between the mutant and the healthy copy. These differences can be called single nucleotide polymorphisms (SNPs). Long-read sequencing is required and supported by Ataxia UK to identify SNPs that are on the same gene as the disease-causing mutation. Identifying these SNPs in SCA patient cohorts is essential for the development of ASOs that can target the damaged ataxin-3. The team will use human brain cells differentiated from induced pluripotent stem cells. These stem cells, reprogrammed from patients’ skin cells, can be turned into any type of cell in the body. The advantage of using cells taken from the skin of someone living with SCA3 is that they contain the identical gene sequences of the patient.

Previous studies by the team have shown that 80% of the faulty ataxin-3 was removed from the brain cells by 10 days following gene silencing compared to untreated cells. By identifying novel targets, the team hopes to increase the likelihood of safe and selective degradation of damaged ataxin-3. Future projects will also investigate the safety and tolerability of the method, as well as the long-term effects on brain cells. Once established, this method could help to slow the progression of SCA3.

 

For more support or information please contact:  
Ataxia UK, 12 Broadbent Close, London, N6 5JW 
Website: www.ataxia.org.uk.   
Helpline: 0800 995 6037 Tel: +44 (0)20 7582 1444   
Email: helpline@ataxia.org.uk.