Gene editing uses a prime editor and sophisticated enzymes called recombinases, which could lead to universal gene therapies that are effective for conditions such as cystic fibrosis.
Researchers at the Broad Institute of MIT and Harvard have enhanced a gene-editing technique that can efficiently insert or replace entire genes in the genomes of human cells, making it suitable for therapeutic use.
This advance, from the lab of Broad Core Institute member David Liu, could one day help develop single gene therapies for diseases such as cystic fibrosis that are caused by one of hundreds or thousands of different mutations in genes. This new approach allows a healthy copy of the gene to be inserted in its natural place in the genome, eliminating the need to create a separate gene therapy to correct each mutation using other gene editing approaches that make smaller edits.
This new method combines prime editing, which can directly make large edits of as little as 100 or 200 base pairs, with newly developed recombinase enzymes that efficiently insert larger fragments. DNA The system, called eePASSIGE, can make gene-sized edits several times more efficiently than other similar methods, making edits that are thousands of base pairs long at specific sites in the genome. Nature Biomedical Engineering.
“To our knowledge, this is one of the first examples of programmable targeted gene integration in mammalian cells that meets the main criteria for potential therapeutic relevance,” said Liu, senior author of the study, the Richard Mirkin Professor and director of the Mirkin Institute for Transformative Technologies in Medicine at the Broad Institute, a professor at Harvard University, and a Howard Hughes Medical Institute Investigator. “These efficiencies give us hope that if the efficiencies observed in cultured human cells could be translated into the clinical setting, they could ameliorate or rescue many or most loss-of-function genetic diseases.”
Smriti Pandey, a graduate student in Liu’s group, and Daniel Gao, a postdoctoral researcher, were co-first authors of the study, which also collaborated with Mark Osborn’s group at the University of Minnesota and Elliot Chaikoff’s group at Beth Israel Deaconess Medical Center.
“This system offers promising opportunities for cell therapy, for example, to precisely insert genes into cells outside the body and then administer them to patients to treat diseases,” Pandey said.
“We are excited by the efficiency and versatility of eePASSIGE, which may lead to the realization of a new category of genomic medicines,” Gao added, “and hope that this tool can be used by scientists across the research community to study fundamental biological questions.”
Prime Improvements
Many scientists have used prime editing to efficiently introduce changes to DNA up to tens of base pairs in length, enough to correct the majority of known pathogenic mutations. But introducing entire healthy genes, often thousands of base pairs in length, into their native location in the genome has been a long-standing goal of the gene editing field. Not only would this potentially cure many patients of whatever disease-causing gene mutation they have, it would also preserve the surrounding DNA sequence, increasing the chances that the newly introduced gene will be properly regulated, rather than being expressed too much, too little, or at the wrong time.
In 2021, Liu’s lab reported an important step towards this goal, developing a prime-editing approach called twinPE, which places a recombinase “landing site” in the genome and uses a natural recombinase enzyme, such as Bxb1, to catalyze the insertion of new DNA into the prime-edited target site.
Prime Medicine, a biotech company Liu co-founded, quickly began using the technique, which it calls PASSIGE (PrimeEditing-Assisted Site-Specific Integrase Gene Editing), to develop treatments for genetic diseases.
PASSIGE only edits a small percentage of cells, enough to treat some genetic diseases caused by loss of functional genes, but probably not enough to cure most. So in a new study published today, Liu’s team set out to improve the editing efficiency of PASSIGE. They found that the recombinase enzyme Bxb1 was what limited the efficiency of PASSIGE, so they used a tool previously developed by Liu’s group. pace (phage-assisted continuous evolution) allows us to rapidly evolve more efficient versions of Bxb1 in the lab.
As a result, the newly evolved and engineered Bxb1 mutant (eeBxb1) improved on the eePASSIGE method, enabling an average of 30 percent integration of gene-sized cargo into mouse and human cells, four times faster than the original technique and about 16 times faster than another recently published method, PASTE.
“The eePASSIGE system provides a promising platform for research into the integration of healthy gene copies at any site in cellular and animal models of genetic disease for the treatment of loss-of-function diseases,” Liu said. “We hope this system will be an important step toward realizing the benefits of targeted gene integration for patients.”
With this goal in mind, Liu’s team is currently working on pairing eePASSIGE with delivery systems such as: Artificial virus-like particles (eVLP) hurdle Traditionally, the therapeutic delivery of gene editors into the body has been limited.
Reference: “Efficient Site-Specific Integration of Large Genes by Serially Evolving Recombinases and Prime Editing in Mammalian Cells” by Smriti Pandey, Xin D. Gao, Nicholas A. Krasnow, Amber McElroy, Y. Allen Tao, Jordyn E. Duby, Benjamin J. Steinbeck, Julia McCreary, Sarah E. Pierce, Jakub Tolar, Torsten B. Meissner, Elliot L. Chaikof, Mark J. Osborn, David R. Liu, June 10, 2024, Nature Biomedical Engineering.
Publication date: 10.1038/s41551-024-01227-1
This study National Institutes of Healththe Bill & Melinda Gates Foundation, and the Howard Hughes Medical Institute.