An improved prime editing system can do gene-sized edits in human cells at therapeutic levels

The discovery, made in the lab of David Liu, a member of the Broad Core Institute, may one day aid in developing a single gene treatment for illnesses like cystic fibrosis.

Now that a gene-editing technique has been refined, researchers at the Broad Institute of MIT and Harvard can insert or replace whole genes in human cells’ genomes with such efficiency that it may find utility in therapeutic settings.

The discovery, made in the lab of David Liu, a member of the Broad Core Institute, may one day aid in developing a single gene treatment for illnesses like cystic fibrosis, which are brought on by any one of hundreds or thousands of distinct gene abnormalities. Instead of developing a new gene treatment for every mutation using minor tweaks made by previous gene-editing techniques, they would use this new method to insert a healthy copy of the gene at its original place in the genome.

The novel technique combines recently created recombinase enzymes, which easily insert vast sections of DNA thousands of base pairs in length at particular spots in the genome, with prime editing, which can directly make a wide variety of modifications up to roughly 100 or 200 base pairs. This system is known as eePASSIGE.

The learnings were published in the journal Nature Biomedical Engineering.

To our knowledge this is one of the first examples of programmable targeted gene integration in mammalian cells that satisfies the main criteria for potential therapeutic relevance,

Who is senior author of the study, the Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad, a professor at Harvard University and a Howard Hughes Medical Institute investigator. “At these efficiencies, we expect that many if not most loss-of-function genetic diseases could be ameliorated or rescued, if the efficiency we observe in cultured human cells can be translated into a clinical setting.

David Liu

The study’s co-first authors were postdoctoral researcher Daniel Gao and graduate student Smriti Pandey from Liu’s group. Mark Osborn’s group from the University of Minnesota and Elliot Chaikof’s group from the Beth Israel Deaconess Medical Centre also collaborated on the project.

This system offers promising opportunities for cell therapies where it can be used to precisely insert genes into cells outside of the body before administering them to patients to treat disease, among other applications.

Smriti Pandey

It’s exciting to see the high efficiency and versatility of eePASSIGE, which could enable a new category of genomic medicines,

We also hope that it will be a tool that scientists from across the research community can use to study basic biological questions.

Daniel Gao

Prime editing has been widely utilised by scientists to effectively insert DNA modifications up to hundreds of base pairs long, which is enough to fix the great majority of known harmful mutations. However, a long-standing objective of the gene-editing community has been to introduce whole healthy genes—often thousands of base pairs long into the genome at their original place. Regardless of the mutation causing the disease, this could not only potentially treat many patients, but it would also preserve the surrounding DNA sequences, increasing the likelihood that the newly installed gene is properly regulated and not expressed excessively, insufficiently, or inappropriately.

A significant step towards achieving this aim was reported in 2021 by Liu’s group, which created a prime editing technique known as twinPE. This technique created recombinase “landing sites” in the genome and then employed natural recombinase enzymes, including Bxb1, to catalyse the insertion of fresh DNA into the prime edited target sites.

Liu co-founded the biotech business Prime Medicine, which quickly started developing cures for genetic illnesses using this approach, which they dubbed PASSIGE (prime-editing-assisted site-specific integrase gene editing).

A small percentage of cells are altered by PASSIGE, which is sufficient to cure some genetic illnesses caused by the absence of a functional gene, but probably not the majority. Thus, the goal of Liu’s team’s recently published study was to increase PASSIGE’s editing effectiveness. They discovered that the recombinase enzyme Bxb1 was the cause of PASSIGE’s efficiency limitation. Then, they quickly evolved more effective forms of Bxb1 in the lab using a technique called PACE (phage-assisted continuous evolution), which had previously been created by Liu’s group.

The resultant newly evolved and designed Bxb1 variation (eeBxb1) enhanced the eePASSIGE method to integrate an average of thirty per cent of gene-sized cargo in human and mouse cells, almost sixteen times more than that of another recently published approach called PASTE, and four times more than the original methodology.

The eePASSIGE system provides a promising foundation for studies integrating healthy gene copies at sites of our choosing in cell and animal models of genetic diseases to treat loss-of-function disorders,

We hope this system will prove to be an important step towards realizing the benefits of targeted gene integration for patients.

David Liu

To do this, Liu’s team is now integrating eePASSIGE with delivery methods like engineered virus-like particles (eVLPs) to potentially get around obstacles that have historically prevented the therapeutic distribution of gene editors within the body.


Source: BROAD Institute News

Journal Reference: Pandey, Smriti, et al. “Efficient Site-specific Integration of Large Genes in Mammalian Cells via Continuously Evolved Recombinases and Prime Editing.” Nature Biomedical Engineering, 2024, pp. 1-18, https://doi.org/10.1038/s41551-024-01227-1.


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