A new paper from the laboratory of David Liu, PhD, at the Broad Institute describes a genome-editing strategy that could result in a one-time treatment for multiple unrelated genetic diseases. The new technique dubbed prime editing-mediated readthrough of premature termination codons or PERT is detailed in Nature in a new paper titled “Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.” The work is spearheaded by co-first authors Sarah Pierce, PhD, and Steven Erwood, PhD, both of whom are postdoctoral associates in the Liu lab.
According to its developers, PERT is designed to maximize the potential of gene editing by using a single agent to target multiple disorders. Specifically, it uses prime editing, also developed by the Liu lab, to rescue nonsense mutations, which, when they appear, cause cells to stop protein synthesis early, resulting in malfunctional forms of proteins that are linked to various rare diseases.
Importantly, PERT does not directly edit nonsense mutations, which account for 24 percent of pathogenic alleles in the ClinVar database. It works by “permanently converting a dispensable endogenous tRNA into an optimized [suppressor]-RNA.” This conversion equips edited cells to produce functional forms of the necessary protein, regardless of which gene has the mutation.
In the paper, the team describes 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. In each case, 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, according to the scientists.
For Liu, a core institute member and director of the Broad’s Merkin Institute for Transformative Technologies in Healthcare, the possibility of developing “a single editing agent into a drug that may help many different types of patients” while “circumventing the need to invest multiple years and millions of dollars to develop each new genetic medicine for each individual” is an attractive one. Earlier this year, he shared some insights into his lab’s work in this regard on an episode of Behind the Breakthroughs, an Inside Precision Medicine, podcast.
Liu’s lab is well-known for its work in developing multiple gene editors. They are also familiar with the challenges and resource-intensive nature of translating these technologies into treatments for patients. “In some cases, the bottlenecks in genetic medicine aren’t the science anymore,” said Liu, who is also a professor at Harvard University and a Howard Hughes Medical Institute investigator. These days, “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.”
PERT is one such creative solution. About 30 percent of genetic diseases are caused by mutations that introduce a termination codon in the middle of an mRNA sequence, which signals the cell to halt protein production before it’s done. To address this issue, Liu’s team focused on suppressor tRNAs or sup-tRNAs, which add an amino acid building block in response to a premature termination codon. This lets the cell continue building the protein in question instead of halting synthesis midway.
Sup-tRNAs are promising as a potential therapeutic strategy but “lipid nanoparticle (LNP)-based or adeno-associated virus (AAV)-mediated delivery of sup-tRNAs face the challenge of supporting sup-tRNA production throughout the lifetime of the patient,” the scientists wrote in Nature. AAV-based methods, for example “may be limited to a single dose, owing to the generation of high levels of neutralizing antibodies following dosing” and that “may not be sufficient to treat genetic diseases that require restoration of protein expression for the lifetime of the patient.” For this and other reasons, “recent efforts to rescue nonsense mutations have relied on the overexpression or delivery of high levels of sup-tRNAs with suboptimal suppression efficiency” and “possible toxicity.”
This led Liu, Pierce, Erwood, and their colleagues to focus on using prime editing to “enable permanent expression of endogenous levels of sup-tRNA optimize for suppression efficiency without apparent toxicity.” After testing thousands of variants of all 418 human tRNAs, the team engineered what they describe as a highly efficient suppressor tRNA. “We then used the versatility of prime editing to permanently convert a redundant endogenous human tRNA to these optimized sup-tRNAs,” they wrote. Once edited, cells are able to produce full-length protein regardless of which gene carries the nonsense mutation.
When tested in human cell models of Batten disease, Tay-Sachs disease, and Niemann-Pick disease type C1, the scientists observed enzyme activity restored at approximately 20 to 70 percent of normal levels, a level which is theoretically high enough to alleviate disease symptoms. Turning to the mouse model of Hurler syndrome, when the scientists analyzed tissue from the mouse brain, liver, and spleen, they determined that PERT restored about six percent of normal enzyme activity, which was high enough to nearly eliminate all signs of disease.
PERT did not appear to cause any off-target effects, and analysis of transcriptomic and proteomic data found no significant effects on either process. The researchers speculate that this is likely because mammalian cells have additional methods of supporting proper protein synthesis, and because PERT leads to only low levels of the engineered suppressor tRNA in cells.
Though additional studies will be necessary to ensure the safety and efficacy of the technique, PERT has the potential to treat a wide range of patients and conditions. “For example, approximately 8,000 people with cystic fibrosis, 252,000 people with Stargardt disease, 31,000 people with phenylketonuria, and 43,500 people with Duchenne muscular dystrophy have nonsense mutations in CFTR, ABCA4, PAH, or DMD, respectively, that could in principle be treated with common PERT prime editing agents after further optimization,” the researchers wrote.
Those are part of the team’s next steps. They are working on optimizing PERT and testing it in various animal models for different genetic diseases. Hopefully, the findings reported here “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.”
