Researchers at the University of California San Diego School of Medicine have used CRISPR-Cas13d technology to target and eliminate the RNA transcript that encodes the toxic protein that causes Huntington's disease (HD). This approach, which targets RNA rather than DNA, may be safer and more reversible than traditional CRISPR methods that target DNA, according to a study published in Nature Neuroscience. The developed technology was shown to ameliorate HD signs in mouse prototypes.
The study discussed the potential benefits of using an RNA-targeting CRISPR enzyme over a DNA-targeting enzyme. Specifically, the researchers noted that, if an RNA-targeting enzyme were to cause negative side effects, it could be halted and the effects would likely be reversible. In contrast, a DNA-targeting enzyme could potentially cause irreversible, unintended changes to the patient's genome. This is one of the reasons why the researchers believe that RNA-targeting CRISPR technology may be safer than DNA-targeting CRISPR technology, as reported by Genetic Engineering & Biotechnology News.
The San Diego team used CRISPR-Cas13d technology to target the mutant RNA that is produced when a mutant version of the huntingtin (HTT) gene is expressed. This mutant gene contains an unusually large number of repetitions of a specific DNA segment (CAG), which leads to the production of an unusually large huntingtin protein. This protein can be harmful to neurons, and the researchers used CRISPR-Cas13d technology to eliminate the RNA that encodes this protein to alleviate the symptoms of Huntington's disease in mouse models.
Cas13d-CAGEX
Huntington's disease is caused by the accumulation of toxic proteins in the brain, which leads to the death of neurons in certain areas of the brain. This causes the symptoms of HD, such as loss of movement and cognitive function. More than 200,000 people worldwide are affected by HD, including around 30,000 in the United States. In the US, more than 250,000 people are at risk of inheriting HD from an affected parent. Currently, HD is considered as an untreatable disease.
Repeated sequences of DNA are difficult for cells to copy accurately, and these copying errors can cause the repetitive sequences to become longer over time, according to Gene Yeo, Ph.D., the senior author of a Nature Neuroscience article and a professor of cellular and molecular medicine at UC San Diego School of Medicine. In the case of the Huntingtin gene, these repeats can sometimes become much longer than normal, leading to the formation of toxic clumps of the repeat-expanded protein in a part of the brain called the striatum. The striatum is important for controlling movement, and the loss of functional neurons in this region of the brain leads to the symptoms of HD.
Yeo and his team, along with colleagues at UC Irvine and Johns Hopkins University, investigated whether a recently developed CRISPR technology that targets RNA could be used to treat HD. They developed a system called Cas13d-CAGEX, which uses CRISPR-Cas13d to eliminate the toxic RNA associated with HD in cells derived from HD patients and in neurons derived from induced pluripotent stem cells. They also tested whether delivering Cas13d-CAGEX directly into the striatum of the brain using a viral vector would reduce the levels of mutant Huntingtin protein in a mouse model of HD.
In Huntington’s disease, toxic accumulations of the huntingtin protein produced by the mutated HTT gene results in progressive destruction of neurons in a part of the brain responsible for regulating movement.
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Developing Treatment for Huntington
The team published their results in an article titled "An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models." The researchers found that delivering Cas13d-CAGEX directly into the striatum improved motor coordination, reduced brain atrophy, and reduced the levels of mutant Huntingtin protein aggregates in a mouse model of HD. These improvements lasted for at least eight months without any adverse effects and with minimal off-target effects on other genes. As stated by the university's press release, the researchers conclude that their RNA-targeting CRISPR-Cas13d system shows promise as a potential therapeutic approach for HD, and could also have implications for the treatment of other genetic disorders.
The investigators found that their approach was effective at targeting and destroying the mutant RNA molecules that cause HD, as well as clearing out the toxic protein buildup associated with the disease. They also demonstrated that the therapy did not disrupt the expression of most other human genes. The goal of the research was to develop a therapy that would only target the toxic RNA in HD without affecting the rest of the genome, according to co-first writer Kathryn Morelli, Ph.D., a research associate in Yeo's laboratory. The team specifically tested their top therapeutic constructs in cell lines derived from HD patients to ensure that they did not have any off-target effects on other genes.
Developing effective therapies for HD has been difficult, and several clinical trials for promising gene therapies have been halted due to disappointing results. In 2021, for example, two clinical trials for potential HD treatments were stopped after the drugs did not perform as well as expected. Currently, there are no treatments that can change the course of HD, although medications can help manage some of the symptoms. The failure of the clinical trials was a major setback for the HD community, according to Yeo, but it has also renewed the scientific community's efforts to find alternative strategies for treating the disease.
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