In a new study, researchers at the University of Oregon’s Phil and Penny Night Campus used start codon disruption with CRISPR-Cas9 gene editing to prevent Fuchs corneal dystrophy in mice. This is the first proven use of a technique called start codon disruption to treat genetic disorders in post-mitotic tissue, revolutionizing the treatment of Fuchs’ dystrophy by replacing the need for corneal transplantation. May cause. It can also lead to new treatments for other genetic diseases, even disorders that affect non-germ cells.
A new paper, “Start Codon Destruction by CRISPR / Cas9 Prevents Endothelial Corneal Dystrophy in Mouse Fuchs,” was published in a journal led by Baramurarian Bati, a research professor at Night Campus. eLife..
This treatise details the results of an eight-year study addressing a disease that affects approximately one in 2,000 people worldwide.Fuchs’ corneal dystrophy causes the corneal endothelium cell Death can cause swelling and lead to poor vision, pain, visual impairment and blindness.
“Many people are working to prevent these cells from dying, but in the end, the challenge is that they are post-mitotic cells. You have the cells you have. It’s a problem when they were born with them and they started dying. “
Currently, the only treatment for Fuchs’ dystrophy is corneal transplantation. This is a major surgery with associated risks and many potential complications such as infection, rejection and glaucoma. The disease is the leading cause of corneal transplants in the United States, and while corneal tissue is readily available in this country, it is deficient in many parts of the world.
Researchers have focused on an early-onset subset of diseases that usually affect patients in their late 30s or early 40s. They focused on single-point mutations in collagen proteins known as COL8A2 or collagen type VIII alpha double chains.
“Previously it was shown that if you knock out this gene (COL8A2), your cornea will be fine,” Ambati said. “It is this variant of this protein that is particularly problematic.”
Researchers sought to test whether protein knockdown could provide a new treatment strategy for the disease. They turned to CRISPR-Cas9 gene editing to target pathogenic proteins in adult mutant mice, but faced the challenge of using this technique in postmitotic cells.
“In post-mitotic cells, it is very difficult or impossible to induce homologous recombination. Therefore, we must consider other ways to achieve our goals.” Hironori Uehara, a researcher in the laboratory and the lead author of the paper, said.
Uehara has developed an innovative means of blocking the expression of the COL8A2 gene by targeting the start codon. The start codon is the starting site for protein synthesis. Destruction of the start codon can result in termination of protein expression. Targeting other sites can also terminate protein expression by frameshifting, but it may induce other unwanted protein expression. Targeting downstream of the start codon increases the risk of missense mutations, resulting in viable mutant proteins with unknown activity.
“We decided that we could destroy the start codon and thereby knock it down. protein It is selectively expressed by delivering the gene therapy only to the cornea. “
The researchers treated it via an adenovirus that encodes SpCas9 and induced RNA by injecting it into the anterior chamber of mice directly facing the corneal endothelial cells. In a study examining the safety of treatment, they determined that the surrounding tissues were not affected by gene therapy. They studied other off-target genes to confirm that they were unaffected and determined that their maximum tolerated dose was safe for the retina, iris, and other parts of the eye.
The researchers have shown that they can not only maintain the density and structure of corneal endothelial cells, but also save their function. During a swelling-induced functional rescue trial, they made some surprising secondary discoveries about the cornea. As the researchers expected, applying water to the cornea did not induce swelling. Instead, they induce swelling by the invasion of aqueous humor into the cornea through the corneal endothelium (the backside of the cornea), so the challenge of hyperosmolar solutions on the surface of the cornea after epithelial removal is most of the cornea. Determined to have caused swelling. ..
The research team used mice bred by Dr. Albert Jun of the Wilmer Eye Institute at Johns Hopkins University in Baltimore, Maryland. The mutation is the same as the cause of Fuchs’ dystrophy in humans. Jun was the contributor to this treatise.
This study lays the foundation for future studies investigating the feasibility of using COL8A2 gene knockdown as a treatment for Fuchs’ dystrophy in clinical trials involving animals, non-human primates, and ultimately humans. .. Reducing the need for corneal transplants in patients with Fuchs’ dystrophy can indirectly help other patients in need of corneal tissue, Ambati said. In future studies, Cas9-mediated gene knockdown targets other genetic disorders of postmitotic cells with single-point mutations, including neurological disorders, immune disorders, and specific disorders affecting joints. You can also investigate the impact of mitosis.
“It has the potential to expand the therapeutic target pool of the CRISPR-Cas system to tissues that are unable to divide,” Ambati said.
Hironori Uehara and others use CRISPR / Cas9 to disrupt the start codon to prevent endothelial corneal dystrophy in mouse hooks. eLife (2021). DOI: 10.7554 / eLife.55637
University of Oregon
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Scientists use CRISPR-Cas9 to prevent Fuchs corneal dystrophy in mice
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