Engineered Enzyme Boosts Gene Editing Efficiency for Potential Cystic Fibrosis Treatment

Researchers at the Broad Institute of MIT and Harvard have enhanced a gene-editing technology, enabling it to efficiently insert or substitute entire genes in the genome of human cells. This advancement holds promising potential for therapeutic applications.

The discovery, made in the lab of David Liu, a member of the Broad Core Institute, may one day aid in the development of a single gene therapy for illnesses like cystic fibrosis, which are brought on by any one of 100 or 1000 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 newly 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 approach, named eePASSIGE, is described in the journal Nature Biomedical Engineering and has the ability to make gene-sized modifications multiple times more efficiently than other comparable techniques.

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. 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 R. Liu, Study Senior Author and Professor, Howard Hughes Medical Institute, Harvard University

Liu is also a Richard Merkin Professor and the Director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad.

The study's Co-first Authors were graduate student Smriti Pandey and postdoctoral researcher Daniel Gao. Mark Osborn's group from the University of Minnesota and Elliot Chaikof's group from the Beth Israel Deaconess Medical Center 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, Study Co-First Author, Merkin Institute of Transformative Technologies in Healthcare, Broad Institute

Gao said, “It is 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.”

Prime Improvements

Prime editing has been widely utilized by scientists to effectively insert DNA modifications up to 100 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 potentially treat a large number of patients and 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 in achieving this goal was reported in 2021 by Liu's lab, which created a prime editing technique known as twinPE. This technique created recombinase “landing sites” in the genome and then employed natural recombinase enzymes, like Bxb1, to catalyze 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 treat some genetic illnesses caused by the loss of a functional gene, but probably not the majority. Thus, the goal of Liu's team's recently published work 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 30% of gene-sized cargo in human and mouse cells, approximately 16 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 R. Liu, Study Senior Author and Professor, Howard Hughes Medical Institute, Harvard University

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