Broad Institute researchers have improved gene editing to efficiently insert entire genes into human cells, offering the potential for single-gene therapies for diseases such as cystic fibrosis. This method combines state-of-the-art editing with novel enzymes to increase editing efficiency, which may revolutionize gene therapy.
The gene editing technique uses primary editors along with advanced enzymes known as recombinases. This method has the potential to lead to universal gene therapies that are effective for conditions such as cystic fibrosis.
Researchers at the Broad Institute of MIT and Harvard have improved gene-editing technology that can now efficiently insert or replace entire genes in the genomes of human cells, potentially making it suitable for therapeutic use.
Advances from the lab of Broad core institute member David Liu could one day help researchers develop a single gene therapy for diseases like 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 its native place in the genome, rather than having to create a different gene therapy to correct each mutation using other gene editing approaches that make smaller adjustments.
The new method uses a combination of primary editing, which can directly perform a wide range of modifications up to 100 or 200 base pairs, and newly developed recombinase enzymes that efficiently insert large DNA thousands of base pairs in length at specific locations in the genome. This system, called eePASSIGE, can perform gene size adjustments several times more efficiently than other similar methods and is presented in Nature Biomedical Engineering.
“To our knowledge, this is one of the first examples of programmable targeted gene integration in mammalian cells that meets major criteria for potential therapeutic relevance,” said Liu, lead 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 efficacy, we expect that many, if not most, loss-of-function genetic diseases could be alleviated or rescued if the efficacy we see in cultured human cells can be translated into the clinical setting.”
Postdoctoral fellow Smriti Pandey and postdoctoral fellow Daniel Gao, both in Liu’s group, were first authors on the study, which was also a collaboration with Mark Osborne’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 administering them to patients to treat diseases, among other things,” Pandey said.
“It is exciting to see the high efficacy and versatility of eePASSIGE, which could enable a new category of genomic medicine,” added Gao. “We also hope it will be a tool that scientists across the research community can use to study fundamental biological questions.”
A first class upgrade
Many scientists have used primary editing to efficiently install changes in DNA that are up to tens of base pairs long, sufficient to correct the vast majority of known pathogenic mutations. But introducing entire healthy genes, often thousands of base pairs long, into their natural 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 what mutation they have in the disease-causing gene, but it would also preserve the surrounding DNA sequences, making it more likely that the newly installed gene is properly regulated rather than overexpressed. , too little or at the wrong time.
In 2021, Liu’s lab announced a key step toward this goal, developing a major editing approach called twinPE that installed recombinase “landing sites” into the genome and then used natural recombinase enzymes such as Bxb1 to catalyze the insertion of new DNA into the primary edited target site.
The biotech company Prime Medicine, co-founded by Liu, soon began using this technology, which they called PASSIGE (primary editing-assisted site-specific integrase gene editing), to develop treatments for genetic diseases.
PASSIGE installs modifications 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 functional gene. So, in new work published today, Liu’s team set out to increase the efficiency of PASSIGE’s editing. They found that the recombinase enzyme Bxb1 was the culprit in limiting the effectiveness of PASSIGE. They then used a tool previously developed by Liu’s group called PACE (phage-assisted continuous evolution) to rapidly develop more efficient versions of Bxb1 in the lab.
The resulting newly developed and engineered variant of Bxb1 (eeBxb1) improved the eePASSIGE method to integrate an average of 30 percent of gene-sized cargo in mouse and human cells, four times more than the original technique and about 16 times more than another recently published method. called PASTE.
“The eePASSIGE system provides a promising foundation for studies integrating healthy gene copies at sites of our choice in cell and animal models of genetic disease to treat loss-of-function disorders,” said Liu. “We hope 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 now working to combine eePASSIGE with delivery systems such as engineered virus-like particles (eVLPs), which can overcome 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 Evolved Recombinases and Primary 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, 10 Jun 2024, Nature Biomedical Engineering.
DOI: 10.1038/s41551-024-01227-1
This work was partially supported National Institute of HealthThe Bill and Melinda Gates Foundation and the Howard Hughes Medical Institute.