Base editing
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Scientists at the Broad Institute of MIT and Harvard have improved an existing gene editing technology they say is capable of inserting entire genes in the genomes of human cells, an advancement with broad therapeutic implications.

The advance, from researchers in the lab of David Liu, a core member of the Broad, could help researchers develop future single-gene therapeutics for complex genetic diseases such as cystic fibrosis and others which are caused by hundreds or thousands of mutations in a single gene. The new method can make a range of edits from 100 to 200 base pairs, which allows them to insert an entire healthy copy of the damaged gene at its native location in the genome—a vast improvement over current methods that target each mutation using gene-editing techniques that are only capable of making smaller edits.

“Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities,” the researchers reported in their new paper published today in Nature Biomedical Engineering.  “Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases.”

Graduate student Smriti Pandey and postdoctoral researcher Daniel Gao, both in Liu’s group, were co-first authors on the study. The team also collaborated with researchers at the University of Minnesota and the Beth Israel Deaconess Medical Center.

“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 senior author Liu, “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.”

Improving prime editing

Installing changes in DNA that are dozens of base pairs in length have been used by scientists and are sufficient to correct most known pathogenic mutations. While these can be effective, the goal of gene editing scientists has long been to develop a method of introducing an entire gene—thousands of base pairs in length—in their native location. This could allow for a single treatment for patients irrespective of the specific mutation they have in their disease-causing gene. Notably, this approach would also preserve the surrounding DNA sequences to ensure the new gene is properly regulated rather than expressed too much, too little, or at the wrong time.

Three years ago, Liu’s lab took a vital step in this direction with the development of the approach called twinPE that placed recombinase “landing sites” in the genome and then used recombinase enzymes such as Bxb1 as catalysts for inserting new DNA in prime editing target sites, which it now calls PASSIGE. Initially, this method was only capable of inserting edits in a smaller fraction of cells.

The ensuing work that led to the publication today, details the Liu lab’s efforts to boost the editing efficiency, in which the researchers discovered that Bxb1 limited the editing efficiency of PASSIGE. The team then turned to another tool PACE (phage-assisted continuous evolution), also developed in the Liu lab to evolve more efficient versions of Bxb1. The allowed them to create an engineered versions called eeBxb1 to improve the eePASSIGE method to integrate an average of 30% of gene-sized cargo in mouse and human cells, roughly 16 times more than another recently published prime editing approach.

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

The next steps in the development of this method with an eye toward human therapeutics is working to integrate eePASSIGE with various technologies that can overcome challenges of delivering gene therapies into the human body.

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