Complete gene insertion is now possible into human cells

Researchers at the Broad Institute have improved gene editing to efficiently insert entire genes into human cells, offering the possibility of single-gene therapies for diseases such as cystic fibrosis. This method combines prime editing with novel enzymes to enhance editing efficiency, which could revolutionize gene therapy.

Gene editing technology uses primer editors along with advanced enzymes known as recombinases. This approach 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 University have advanced gene-editing technology that can now efficiently insert or replace entire genes into the genomes of human cells, making them suitable for therapeutic uses.

This advance, made from the lab of Broadcore 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 the gene. Using this new approach, they will be able to insert a healthy copy of a 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 modifications.

The new method uses a combination of prime editing, which can directly make a wide range of modifications of up to about 100 or 200 base pairs, and newly developed recombination enzymes that efficiently insert large pieces of cells. DNA Thousands of base pairs in length at specific locations in the genome. This system, called eePASSIGE, can perform gene-sized edits several times more efficiently than other similar methods, and is reported in Nature of biomedical engineering.

“To our knowledge, this is one of the first examples of programmable targeted gene integration in mammalian cells that meets key criteria of potential therapeutic significance,” said Liu, lead author of the study, the Richard Mirkin Professor and director of the UCLA Research Center. Mirkin Professor of Transformative Technologies in Healthcare at Broad, is a professor at Harvard University and an investigator at the Howard Hughes Medical Institute. “Given these efficiencies, we anticipate 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 into the clinical setting.”

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

“This system offers promising opportunities for cell therapies, as it can be used to precisely introduce genes into cells outside the body before giving them to patients to treat diseases, among other applications,” Pandey said.

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

Major improvements

Many scientists have used prime editing to efficiently stabilize changes in DNA up to tens of base pairs in length, which is sufficient to correct the vast majority of known disease-causing mutations. But inserting entire intact genes, often thousands of base pairs long, into their original location in the genome has been a long-standing goal in gene editing. Not only could this cure many patients regardless of what mutation they have in the disease-causing gene, but it would also preserve the surrounding DNA sequence, making it more likely that the newly found gene would be properly regulated, rather than overexpressed. Or too little, or at the wrong time.

In 2021, Liu’s lab reported a major step toward this goal and developed a key editing approach called TwinPE that installed recombinant “landing sites” in the genome, and then used natural recombinant enzymes such as Bxb1 to catalyze the insertion of new DNA into the primary cell. Edited target sites

Soon, the biotech company Prime Medicine, which Liu co-founded, began using the technology, which they called PASSIGE (primer-assisted integrative site-specific gene editing), to develop treatments for genetic diseases.

PASSIGE installs the modifications in only a fraction of cells, enough to treat some, but perhaps not most, genetic diseases that result from the loss of a functioning gene. So, in the new work reported today, Liu’s team set out to enhance PASSIGE’s editing efficiency. They found that the recombinant enzyme Bxb1 was the reason for the reduction in PASSIGE efficiency. They then used a tool previously developed by Liu’s group called a step (phage-assisted continuous evolution) to rapidly develop more efficient versions of Bxb1 in the laboratory.

A newly developed and engineered Bxb1 variant (eeBxb1) has improved the eePASSIGE method to incorporate an average of 30 percent more gene-sized cargo into mouse and human cells, four times more than the original technology and about 16 times more than another recently published method called splice.

“The eePASSIGE system provides a promising foundation for studies that integrate intact gene transcripts into our chosen loci 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 bringing the benefits of targeted gene integration to patients.”

With this goal in mind, Liu’s team is now working to combine eePASSIGE with delivery systems such as Virus-like particles (eVLPs) that may overcome Hurdles Which has traditionally limited the therapeutic delivery of gene editors into the body.

Reference: “Efficient site-specific integration of large genes into mammalian cells via ever-evolving recombination and primer editing processes” by Smriti Pandey, Chen D. Gao, Nicholas A. Krasnow, Amber McElroy, Y. Allen Tao, Jordyn E. Dube, Benjamin J. Steinbeck, Julia McCreary, Sarah E. Pierce, Jacob Tollar, Torsten B. Meissner, Elliot L. Chekhov, Mark J. Osborne, and David R. Leo, June 10, 2024, Nature of biomedical engineering.
doi: 10.1038/s41551-024-01227-1

This work was supported in part by National Institutes of HealthThe Bill & Melinda Gates Foundation and the Howard Hughes Medical Institute.