Improved prime editing system enables gene size editing in human cells at therapeutic level

Scientists from the Broad Institute of MIT and Harvard have improved a gene-editing technology that is now capable of inserting or replacing entire genes in the genomes of human cells efficiently enough to be potentially useful for therapeutic applications.

The advance, from the laboratory of Broad Core Institute member David Liu, could one day help researchers develop a single gene therapy for diseases such as cystic fibrosis that are caused by one of hundreds or thousands of different mutations in a gene . Using this new approach, they would insert a healthy copy of the gene into the original location in the genome, rather than having to create another gene therapy to correct each mutation using other gene editing approaches that perform smaller edits.

The new method uses a combination of prime editing, which can instantly perform a wide range of edits up to about 100 or 200 base pairs, and newly developed recombinase enzymes that efficiently insert large stretches of DNA with a length of thousands of base pairs at specific sites . the genome. This system, called eePASSIGE, can perform gene size operations several times more efficiently than other comparable methods, and is reported in Nature Biomedical Technology.

“To our knowledge, this is one of the first examples of programmable targeted gene integration in mammalian cells that meets key criteria for potential therapeutic relevance,” said Liu, senior author of the study, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad, a professor at Harvard University and an investigator at the Howard Hughes Medical Institute.

“With this efficiency, we expect that many, if not most, genetic loss-of-function diseases could be ameliorated or rescued, if the efficiency we observe in cultured human cells can be translated into a clinical setting.”

Graduate student Smriti Pandey and postdoctoral researcher Daniel Gao, both in Liu’s group, were co-first authors of the study, which was also a collaboration with Mark Osborn’s group at the University of Minnesota and Elliot Chaikof’s group at Beth Israel Deaconess Medical Center.

“This system holds promise for cell therapies where it can be used to precisely insert genes into cells outside the body before administering them to patients to treat diseases, among other things,” Pandey said.

“It is exciting to see the high efficiency and versatility of eePASSIGE, which could enable a new category of genomic medicines,” Gao added. “We also hope that it will be a tool that scientists from across the research community can use to study fundamental biological questions.”

Main improvements

Many scientists have used prime editing to efficiently make changes to DNA that can be up to dozens of base pairs in length, enough to correct the vast majority of known pathogenic mutations. But introducing very healthy genes, often thousands of base pairs long, to their original locations in the genome has long been a goal in gene editing.

Not only would this potentially treat many patients, regardless of what mutation they have in a disease-causing gene, but it would also preserve surrounding DNA sequences, which would increase the chance that the newly installed gene is properly regulated, rather than over-regulated. to be expressed. , too little or at the wrong time.

In 2021, Liu’s lab reported a major step toward this goal, developing a prime editing approach called twinPE, which installed recombinase “landing sites” in the genome and then used natural recombinase enzymes such as Bxb1 to promote the insertion of new Catalyze DNA in the prime. edited target sites.

The biotech company Prime Medicine, co-founded by Liu, soon began using this technology, which they called PASSIGE (prime-editing-assisted site-specific integrase gene editing), to develop treatments for genetic diseases.

PASSIGE installs edits in only a modest fraction of cells, which is enough to treat some, but probably not most, genetic diseases that result from the loss of a functioning gene. Therefore, in the new work reported today, Liu’s team aimed to boost PASSIGE’s machining efficiency. They found that the recombinase enzyme Bxb1 was the culprit in limiting the efficiency of PASSIGE. They then used a tool previously developed by Liu’s group called PACE (phage-assisted continuous evolution) to quickly develop more efficient versions of Bxb1 in the lab.

The resulting newly developed and developed Bxb1 variant (eeBxb1) improved the eePASSIGE method to integrate an average of 30% of the gene size payload into mouse and human cells, four times more than the original technique and approximately 16 times more than another recently published method called PASTE.

“The eePASSIGE system provides a promising foundation for studies integrating healthy gene copies at locations of our choice into 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.”

To this end, Liu’s team is now working to combine eePASSIGE with delivery systems such as engineered virus-like particles (eVLPs) that can overcome barriers that have traditionally limited therapeutic delivery of gene editors into the body.