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CRISPR-based therapy alleviates Huntington’s disease symptoms in mouse models

Researchers have investigated the application of RNA-targeting CRISPR technology in the treatment of Huntington’s disease (HD). The study, published in Nature Neuroscience, details the use of the therapy in patient-derived cells and a mouse model of HD. The RNA targeting strategy reduced toxic protein build-up and had a long-lasting effect without altering other parts of the genome.

The pathophysiology of Huntington’s disease

HD is a neurodegenerative disorder. It is characterised by a progressive loss of cognitive function (dementia), psychiatric symptoms (depression), involuntary spasmodic movement, and impaired coordination and balance. Symptoms usually develop between the ages of 30 and 50. The disease is fatal and incurable. HD has an autosomal dominant pattern of inheritance. Only one copy of the mutated gene is needed for a person to develop the disorder. Therefore, if one parent has the disease, their child has a 50% chance of developing HD too.

HD is caused by a mutation in the huntingtin gene (HTT). HTT contains a repeat of three nucleotides: cytosine, adenine and guanine (CAG). A mutation in the gene causes excessive CAG repeats. The pathogenic HTT mRNA is translated to an abnormal form of the huntingtin protein. The abnormal proteins build up and form clusters in neurons within the striatum (the area of the brain that regulates movement). These clusters are difficult to break down. The build-up of the toxic proteins is associated with neurodegeneration.

CRISPR-Cas based gene editing approaches are able to alter DNA at very specific locations. However, current CRISPR technologies still have off-target effects. Therefore, they run the risk of introducing permanent alterations to the DNA in unintended paces in the genome. Recent research has focused on developing CRISPR technology that can target RNA molecules (the intermediate molecule between DNA transcription and protein production). This will mean that the genome remains unaltered as the intermediate RNA molecules are targeted rather than the DNA itself.

Researchers at the University of California San Diego, University of California Irvine and Johns Hopkins University developed a therapeutic strategy based on RNA-targeting CRISPR/Cas13d technology. The therapy specifically targets the toxic RNA that builds up in neurons and causes HD. 

Kathryn Morelli, co-first author and Postdoctoral Fellow in the Yeo lab at UC San Diego said, “Our goal was to engineer a type of therapy that would only target the toxic RNA that causes HD and could keep the rest of the human genome and transcriptome intact. We specifically screened our top therapeutic constructs in HD patient cell lines to make sure of it.”

The hunt for an effective Huntington’s disease therapy

First, the researchers developed a CRISPR/Cas13d system that targets the toxic CAG repeats in the RNA. They packaged Cas13d, which an RNA-targeting enzyme, and a guide RNA, which targets CAG expansions in HTT RNA, within a viral delivery vehicle (figure 1). The therapy was delivered to patient-derived fibroblasts and patient-derived stem cells, which were differentiated to striatal neurons. They found that the mutant RNA molecules were destroyed and there was a reduction in the build-up of the toxic protein.

The researchers then injected the therapy into the striatum of mouse models of HD. This reduced mutant HTT mRNA and toxic protein levels in the striatum of mice. Mice also showed improved motor coordination and reduced neuronal death in the striatum. The improvements lasted approximately eight months.

Figure 1: Treatment schematic of RNA-targeting CRISPR/Cas13d therapy. Cas13d (green) and the guide RNA (red) form a complex. The guide RNA molecule targets CAG expansions in the HTT RNA molecule (blue). This was tested in neuronal cultures derived from stem cells that were obtained from patients with HD and a mouse model of HD.  Source: Published in Nature Neuroscience.

As CRISPR technology is not always precise, the researchers analysed the expression of other human genes. There were minimal off-target transcriptomic effects, which showed that other human genes were not disrupted by the therapy.

The development of effective therapies is challenging   

The study outlines a new approach that has shown effectiveness in mouse models. The CRISPR-based strategy could also be used to treat other neurodegenerative diseases.

Unfortunately, the development of effective and prognosis-altering treatments for HD has proven difficult. For example, clinical trials for two game-changing gene therapy treatments were stopped in 2021 due to unsatisfactory results.

Gene Yeo, senior author of the study and Professor or Cellular and Molecular Medicine at UC San Diego said, “The Huntington’s community was devastated when the clinical trials failed, primarily due to target specificity and toxic effects. But their termination has only re-energized the scientific community to find alternative strategies.”