Scientists in the laboratory of David Liu, PhD, at the Broad Institute of MIT, Harvard University, and Howard Hughes Medical Institute (HHMI) have designed an improved version of prime editing technology that can efficiently insert or substitute entire genes (or gene-sized DNA segments) in human cells.
Details of the method are published in Nature Biomedical Engineering in a report titled, “Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.” Liu previewed highlights of the work in a plenary lecture last month at the American Society of Gene and Cell Therapy conference in Baltimore, as well as last week’s “The State of CRISPR and Gene Editing” virtual summit hosted by GEN.
The new method, which is called eePASSIGE, combines prime editing and new recombinase enzymes that efficiently insert DNA sequences that are thousands of base pairs long at specific sites in the genome. According to the Liu lab, the system makes gene-sized edits several times more efficiently than similar methods. The technology has important implications for therapeutic applications of prime editing. Conceivably, it could be used to design a single gene therapy for a disease like cystic fibrosis or Stargardt’s disease, which can result from one of hundreds of different mutations in the same gene, by simply replacing the mutated gene with a healthy copy.
“To our knowledge, this is one of the first examples of programmable targeted gene integration in mammalian cells that satisfies the main criteria for potential therapeutic relevance,” said Liu, who is senior author of the study, director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad, a professor at Harvard University, and an HHMI investigator. “At these efficiencies, we expect that many if not most loss-of-function genetic diseases could be ameliorated or rescued, if the efficiency we observe in cultured human cells can be translated into a clinical setting.”
Liu’s lab pioneered prime editing with a landmark report in 2019. It next reported the 2021 development of twinPE, an editing approach that installed recombinase “landing sites” in the genome and then used natural recombinase enzymes like Bxb1 to catalyze the insertion of new DNA into the target sites. The technology has been commercialized by Prime Medicine, a company co-founded by Liu, under the moniker prime-editing-assisted site-specific integrase gene editing, or PASSIGE. Its ability to edit a fraction of cells is enough to treat some genetic diseases but probably not most genetic diseases that are linked to loss of function genes.
The improved version, eePASSIGE, builds on the capabilities of its predecessor by using a more efficient version of the recombinase enzyme Bxb1, which turns out to be the limiting factor of the PASSIGE system. So Liu and his colleagues turned to a tool called phage-assisted continuous evolution or PACE—developed in the Liu lab more than a decade ago—to engineer more efficient Bxb1 variants in the lab.
Using one of those variants, eeBxb1, eePASSIGE was able to integrate an average of 30% of gene-sized cargo in mouse and human cells. That amounts to four times more cargo than PASSIGE and 16 times more than a similar method called PASTE, developed by Omar Abudayyeh, PhD, Jonathan Gootenberg, PhD, and colleagues (Tome has detailed advances of its own in the PASTE platform in a commentary published in GEN).
“It’s exciting to see the high efficiency and versatility of eePASSIGE, which could enable a new category of genomic medicines,” said David Gao, PhD, a postdoctoral researcher in the Liu lab and a co-author on the new paper. “We also hope that it will be a tool that scientists from across the research community can use to study basic biological questions.”
In fact, Liu’s team is already taking steps toward therapeutic applications. For example, the team is trying to combine eePASSIGE with delivery systems such as engineered virus-like particles. “The eePASSIGE system provides a promising foundation for studies integrating healthy gene copies at sites of our choosing in cell and animal models of genetic diseases to treat loss-of-function disorders,” Liu said. “We hope this system will prove to be an important step toward realizing the benefits of targeted gene integration for patients.”
