Broad Institute researchers have improved gene editing to efficiently insert entire genes into human cells, providing potential for single-gene therapies for diseases like cystic fibrosis. This method combines master editing with new enzymes to improve editing efficiency, potentially revolutionizing gene therapy.
The gene editing technique uses top-notch editors along with advanced enzymes called recombinases. This method has the potential to lead to effective universal gene therapies for diseases like cystic fibrosis.
Researchers at the Broad Institute of MIT and Harvard have improved a gene-editing technology that can now efficiently insert or replace entire genes into the genome of human cells, making it potentially suitable for therapeutic uses.
This advance, made by the lab of David Liu, a member of the Broad Core Institute, could one day help researchers develop a unique gene therapy for diseases such as cystic fibrosis, caused by any of hundreds or even thousands of different mutations of a gene. Using this new approach, they would insert a healthy copy of the gene into its original location in the genome, rather than having to create a different gene therapy to correct each mutation using other gene editing approaches that make smaller changes.
The new method uses a combination of master editing, which can directly perform a wide range of edits of up to about 100 or 200 base pairs, and newly developed recombinase enzymes that efficiently insert large pieces of
” data-gt-translate-attributes=”({“attribute”:”data-cmtooltip”, “format”:”html”})” tabindex=”0″ role=”link”>DNA thousands of base pairs in length at specific sites in the genome. This system, called eePASSIGE, can make gene size changes several times more efficiently than other similar methods, and is reported in Natural biomedical engineering.
“To our knowledge, this is one of the first examples of targeted and programmable gene integration in mammalian cells that meets key criteria for potential therapeutic relevance,” said Liu, lead author of the study. , Professor Richard Merkin and director of the study. Merkin Institute of Transformative Technologies in Healthcare at the Broad, professor at Harvard University and researcher at the Howard Hughes Medical Institute. “With these efficiencies, we hope that many, if not most, loss-of-function genetic diseases could be ameliorated or rescued, if the efficiencies we observe in cultured human cells can be translated to the clinical setting.”
Graduate student Smriti Pandey and postdoctoral researcher Daniel Gao, both members of 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 offers promising opportunities for cell therapies where it can be used to precisely insert genes into cells outside the body before delivering them to patients to treat disease, among other applications,” 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 across the research community can use to study fundamental biological questions.” »
Major improvements
Many scientists have used prime editing to efficiently install changes in DNA of up to tens of base pairs, which is enough to correct the vast majority of known disease-causing mutations. But introducing entire healthy genes, often thousands of base pairs long, back to their original location in the genome has been a long-standing goal in the field of gene editing. Not only could this potentially treat many patients, regardless of which mutation they carry in a disease-causing gene, but it would also preserve surrounding DNA sequences, making it more likely that the newly installed gene will be properly regulated, rather that too expressed. , too little or at the wrong time.
In 2021, Liu’s lab reported a key step toward this goal and developed a prime editing approach called twinPE that installed recombinase “landing sites” in the genome and then used natural recombinase enzymes such as Bxb1 to catalyze the insertion of new DNA into the main genome. modified target sites.
The biotechnology company Prime Medicine, co-founded by Liu, quickly began using the technology, which it called PASSIGE (prime-editing-assisted site-special integrase gene edition), to develop treatments for genetic diseases.
PASSIGE installs changes in only a modest fraction of cells, which is enough to treat some, but probably not most, genetic diseases resulting from the loss of a functional gene. So Liu’s team, in the new work reported today, set out to improve the efficiency of PASSIGE editing. They discovered that the recombinase enzyme Bxb1 was responsible for limiting the effectiveness of PASSIGE. They then used a tool previously developed by Liu’s group called PACE (phage-assisted continuous evolution) to rapidly evolve more efficient versions of Bxb1 in the lab.
The newly developed and engineered Bxb1 variant (eeBxb1) improved the eePASSIGE method to fit an average of 30% of the size of a gene into mouse and human cells, four times larger than the original technique and approximately 16 times larger than ‘another recently published method. called PASTE.
“The eePASSIGE system provides a promising basis for studies integrating copies of healthy genes at sites of our choosing in cellular and animal models of genetic diseases to treat loss-of-function disorders,” Liu said. “We hope that this system will prove to 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 to combine eePASSIGE with delivery systems such as virus-like particles (eVLPs) that could overcome the obstacles that have traditionally limited the therapeutic delivery of gene editors. in the body.
Reference: “Efficient site-specific integration of large genes in mammalian cells via continuously evolving recombinases and master editing” 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 and David R. Liu, June 10, 2024, Natural biomedical engineering.
DOI: 10.1038/s41551-024-01227-1
This work was supported in part by the
” data-gt-translate-attributes=”({“attribute”:”data-cmtooltip”, “format”:”html”})” tabindex=”0″ role=”link”>National Institutes of Healththe Bill and Melinda Gates Foundation and the Howard Hughes Medical Institute.