May 14, 2024  |  Neurogenomics

New hope for neurodegenerative diseases

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PacBio PureTarget amplification-free targeted sequencing makes cutting edge advancements in dementia research a possibility.

Four years ago neurologist Claire Clelland, MD, PhD, and her Laboratory at the University of California San Francisco Weill Institute for Neurosciences launched a pioneering project investigating dementia. As a physician-scientist, Dr. Clelland not only treats patients with dementia but is also at the forefront of researching and developing new therapies aimed at treating or potentially curing the condition. Her research focuses on using CRISPR gene editing to target genetic forms of neurodegenerative diseases. She is dedicated to meticulously quantifying the outcomes of these edits, establishing best practices for gene therapy in neurological diseases, and creating a tool intended for use in future clinical trials.

It is estimated that more than 55 million people worldwide have dementia, and this number is expected to rise with the aging population. Dr. Clelland’s investigations into dementia have led her and her team to focus on the C9orf72 gene. The C9orf72 gene is the leading genetic cause of both frontotemporal dementia and ALS (Amyotrophic Lateral Sclerosis), sometimes referred to as Lou Gehrig’s disease.

“We became very interested in this gene because C9orf72 is amenable to CRISPR therapeutics and the path to a clinical trial is more straight forward in ALS than dementia. Our goal is to develop a CRISPR therapy to remove the mutation from the genome while also preserving normal gene function,” says Dr. Clelland. “The genetics are well known, and the biology correlates with the gene mutation, but the hard part is the very large repeat expansion which traditional sequencing tools do not work on. You cannot PCR amplify large repeats.”

Tandem repeats are one of the most abundant types of variation in the human genome, and due to their repetitive nature, they have the highest mutation rate in the genome. This genomic instability is a major driver of diseases like ALS. Furthermore, traditional sequencing methods, which rely on some form of amplification, have consistently struggled with genomic regions containing repetitive sequences.

“We were stuck even in figuring out sizing of the repeat expansions in the cell lines we had – trying to figure out what mutations our cell lines had and if we had corrected them with CRISPR. It was very hard for us to tell,” recalls Dr. Clelland.

That’s where the PureTarget repeat expansion panel came in. This no-amplification solution was transformative for Dr. Clelland’s research. The PureTarget repeat expansion panel offers a scalable and more comprehensive solution for profiling repeat expansions compared to genotyping and next-generation sequencing methods. With PureTarget and the precursor method, No-amp, Dr. Clelland and her team could quantify what was going on in cell lines and were able to size different lines accurately up to thousands of repeats – something they hadn’t been able to quantify with traditional sequencing techniques. “We could quantify it and then we could see the changes that occurred after gene editing, opening up a whole avenue of inquiry for us that was not possible before,” said Dr. Clelland.

“PureTarget has been more reliable and scalable, bringing down the cost so we can do more testing.”

By scalable, Claire means that before PureTarget, her team could do around five to ten samples per no amplification run. With PureTarget, they were able to start with 16 samples per run and still have capacity left, helping to reduce their overall costs.

The team is now testing the capacity to use this protocol to read out CRISPR editing outcomes in an unbiased fashion. “Because there is no amplification bias since it is not PCR-based, we are now using this technology to look at editing outcomes in pools of cells rather than clonal cell lines,” explains Dr. Clelland. “PCR amplification bias probably does impact results and we would like to see what the amplification bias really is.”

Interested in learning more?

To learn more about PureTarget, sign up to attend the upcoming webinar, “Characterizing C9ORF72 repeat expansions and transcripts using HiFi sequencing” on May 22 and 23.

We look forward to seeing you there!

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