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A team of researchers at the ӳ����ý, led by gene-editing pioneer David Liu, has developed a new genome-editing strategy that could potentially lead to a one-time treatment for multiple unrelated genetic diseases.

Gene-editing medicines are often made one at a time to treat a specific mutation, an approach that’s difficult to scale up to address the thousands of rare diseases affecting patients around the world. The new technology, called PERT (prime editing-mediated readthrough of premature termination codons), is designed to maximize the potential of gene editing by using just one editing agent to serve as many patients as possible.

“We're excited by the possibility that you could develop a single editing agent into a drug that may help many different types of patients, circumventing the need to invest multiple years and millions of dollars to develop each new genetic medicine for each individual,” said Liu, a core institute member, the Richard Merkin Professor, and director of the Merkin Institute for Transformative Technologies in Healthcare at ӳ����ý, professor at Harvard University, and Howard Hughes Medical Institute investigator.

PERT uses prime editing — a versatile and precise DNA editing system developed by Liu’s lab in 2019 — to rescue a type of mutation that can cause about a third of rare diseases. These “nonsense mutations” can appear in many different genes and cause cells to stop synthesizing their associated proteins too early, resulting in truncated, malfunctioning molecules that lead to disease. Among the 200,000 disease-causing mutations documented in the ClinVar database, 24 percent are nonsense mutations.

The PERT approach does not directly edit these nonsense mutations — a strategy that would require developing a different editing agent for each mutation — but instead makes another edit that equips cells with a tool to produce the normal, functional version of the protein, regardless of which gene is impacted.

In a paper published in , the team described how they tested PERT in human cell models of Batten disease, Tay-Sachs disease, and Niemann-Pick disease type C1, and in a mouse model of Hurler syndrome. The technology restored protein production and alleviated disease symptoms, with no detected off-target edits, changes in normal RNA or protein production, or toxicity to the cells.

The work was spearheaded by co-first authors Sarah Pierce and Steven Erwood, both postdoctoral associates in the Liu lab.

A creative solution

Liu and his lab have been developing new DNA-editing tools to address genetic diseases for many years. However, after seeing how challenging and resource-intensive it is for these technologies to reach patients, Liu began thinking about opportunities to streamline the process.

“In some cases, the bottlenecks in genetic medicine aren’t the science anymore,” he said. “They’re in meeting regulatory requirements, in the manufacturing costs associated with these treatments, and in the commercial challenges of drugs that treat very small numbers of patients. Witnessing gene-editing companies make the gut-wrenching decisions of which targets to pursue — synonymous with the gut-wrenching decisions of which patients are left behind — made it clear that we need creative scientific ways to help address some of these problems.”

The team found a potential solution by identifying a common cause of many different genetic diseases. Normally, when a cell needs to make a protein, it first transcribes DNA into mRNA. Other molecules called tRNAs then read the mRNA sequence and bring the corresponding amino acid building blocks together into a chain that becomes the final protein. A special three-letter sequence in the mRNA — UAA, UAG, or UGA — marks the end of the protein assembly instructions. This signal is called a termination codon.

But roughly 30 percent of genetic diseases are caused by DNA mutations that create an errant termination codon somewhere in the middle of the mRNA sequence, signaling the cell to halt protein production too early. Liu’s team sought to develop a universal way to permanently equip the cell to overcome these premature termination codons, allowing protein synthesis to continue as normal.

“Our hope is that this type of solution could provide a single, one-time gene-editing treatment that benefits patients with different diseases caused by nonsense mutations,” said Pierce.

Installing a new tRNA

The researchers came up with a creative application of prime editing to tackle this issue. They first turned to “suppressor” tRNAs. This type of tRNA adds an amino acid building block in response to a premature termination codon, allowing the cell to continue building the protein instead of halting protein synthesis midway. 

By testing tens of thousands of tRNA variants, Pierce, Erwood, and their colleagues engineered a new, highly efficient suppressor tRNA. They then optimized a prime editing system to install this tRNA directly into the genomes of cells, replacing an existing, redundant tRNA. 

“A lot of what made this possible was simply taking advantage of how versatile prime editing is,” said Erwood. “It let us make very complex changes to a tRNA in ways we couldn’t have done otherwise. We tested thousands of different prime edits until one tRNA design finally stood out.”

The resulting prime editor permanently equips cells with the new suppressor tRNA, which allows them to produce full-length protein regardless of which specific gene is carrying a nonsense mutation.

The researchers used prime editing to install this new suppressor tRNA in human cell models of Batten disease, Tay-Sachs disease, and Niemann-Pick disease type C1. Using the same prime editing system in these different cell models, they observed enzyme activity restored at approximately 20 to 70 percent of normal levels, a level theoretically high enough to alleviate disease symptoms.

The team also tested their strategy in a mouse model of Hurler syndrome, a lysosomal storage disorder. When the scientists analyzed tissue from the mouse brain, liver, and spleen — tissues normally impacted by the disorder — they determined that PERT had restored about 6 percent of normal enzyme activity, high enough to nearly eliminate all signs of disease.

The researchers found that PERT did not result in detected off-target edits, and did not affect the normal synthesis of other proteins. The team speculates that PERT minimally impacts normal protein production because mammalian cells have several additional ways of supporting proper protein synthesis, and because PERT leads to only low levels of the engineered suppressor tRNA in cells.

The team is now further optimizing PERT and testing it in a variety of animal models for different genetic diseases.

“We hope this research will eventually pave the way for a clinical trial of PERT, and will inspire other broadly applicable, disease-agnostic gene-editing strategies,” said Liu. “If you don’t have to target one mutation at a time, the size of the patient groups that could be treated with a single drug becomes much, much larger. We hope the result will be many more patients that benefit, as well as greater incentives to develop gene-editing drugs for rare diseases.”