Scientists at the Broad Institute of MIT and Harvard have taken a significant leap forward in gene-editing technology, making it capable of inserting or substituting entire genes in the human genome with remarkable efficiency. This advancement, led by Broad core institute member David Liu, could pave the way for groundbreaking therapies for genetic disorders such as cystic fibrosis.

The new method integrates prime editing, capable of making extensive edits up to 200 base pairs, with novel recombinase enzymes that insert large DNA sequences at specific genome sites. This combination, known as eePASSIGE, surpasses previous methods in efficiency and has been detailed recently in 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,” said Liu, the senior author of the study. “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.”

Targeted Gene Integration: A New Horizon

Graduate student Smriti Pandey and postdoctoral researcher Daniel Gao, both from Liu’s group, were the study’s co-first authors. The research was a collaboration with Mark Osborn’s team at the University of Minnesota and Elliot Chaikof’s team at the 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 of the body before administering them to patients to treat disease, among other applications,” Pandey noted.

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

Toward Clinical Applications

Liu’s lab had previously developed a method called twinPE in 2021, laying the groundwork for prime editing-assisted site-specific integrase gene editing (PASSIGE). The biotech company Prime Medicine, co-founded by Liu, soon began using PASSIGE to develop treatments for genetic diseases. Although PASSIGE was a significant step, its editing efficiency was not sufficient for treating most genetic diseases.

Liu's team identified the recombinase enzyme Bxb1 as the limiting factor and utilized phage-assisted continuous evolution (PACE) to develop more efficient variants. The evolved Bxb1 variant (eeBxb1) enhanced eePASSIGE’s efficiency, achieving an average integration of 30 percent of gene-sized cargo in mouse and human cells, significantly surpassing the original technique and other contemporary methods.

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

Liu’s team is now focused on combining eePASSIGE with delivery systems like engineered virus-like particles (eVLPs) to overcome traditional barriers in the therapeutic delivery of gene editors within the body. This innovative approach could potentially revolutionize the field of gene therapy, offering hope for effective treatments for a myriad of genetic diseases.