Gene editing tech that inserts entire genes in cells may aid CF treatment

Inserting healthy CFTR gene into genome could treat nearly all CF cases

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by Steve Bryson, PhD |

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Illustration of a DNA strand.

Scientists at the Broad Institute of MIT and Harvard have created a gene editing technology that may be able to insert or substitute entire genes in human cells, which could pave the way for new gene therapies for diseases such as cystic fibrosis (CF).

The approach could efficiently insert a healthy copy of CFTR, the gene mutated in CF, in its native location in the genome, potentially treating nearly all CF patients, regardless of their mutation type.

“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 David Liu, PhD, the study’s senior author and director of the Merkin Institute of Transformative Technologies in Healthcare at Broad, in a university news release. “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.”

Liu is also a professor at Harvard University and a Howard Hughes Medical Institute investigator. The study, “Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing,” was published in Nature Biomedical Engineering.

More than 2,500 different mutations in the CFTR gene have been reported so far, all of which disrupt the production or function of the CFTR protein. The defects lead to the buildup of thick mucus in various organs, such as the lungs and digestive system.

Replacing the defect gene with a full-length healthy copy in its natural location within the genome could act as a single therapeutic strategy for patients with different CFTR mutations. The approach also has the potential to avoid excessive gene activity associated with gene therapies delivered using viral vectors, which can induce harmful side effects.

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Gene Editing Corrects CF Mutation in Multiple Organs All at Once

A new gene editing technique

Prime editing is a type of gene editing technique that lets precise changes be made in DNA and has been used to efficiently correct many known disease-causing mutations. Current prime editing technology can only replace up to about 100 or 200 nucleotides, the building blocks of DNA.

Introducing an entire healthy gene, often thousands of nucleotides long, in its natural location within the genome has been a long-standing goal in gene-editing therapeutics. Along with treating nearly all patients regardless of their mutation, it can preserve the surrounding DNA sequences and increase the likelihood that the new gene’s activity is properly controlled.

Liu’s lab recently developed a prime editing approach that uses naturally occurring recombinase enzymes, proteins that can cut and paste DNA at specific locations in the genome.

Dubbed PASSIGE, for Prime Assisted Site Specific Integrase Gene Editing, the technology supported the launch of the biotech company Prime Medicine, cofounded by Liu, to develop treatments for genetic diseases. Earlier this year, the Cystic Fibrosis Foundation announced new funding of up to $15 million for the company to further develop its technology.

Still, PASSIGE edits only between 10 to 20% of cells, which may not be enough to treat all genetic diseases.

Here, Liu’s team set out to boost PASSIGE’s editing efficiency, which was limited by the recombinase enzyme called Bxb1. Applying a tool developed by Liu’s group called phage-assisted continuous evolution (PACE), Bxb1 was selectively evolved to improve PASSIGE efficiency.

Among dozens of Bxb1 variants with improved activity, one evolved variant, called eeBxb1, improved gene integration efficiencies.

Applying eeBxb1 to PASSIGE, now referred to as eePASSIGE, improved the average gene integration efficiencies at therapeutically relevant sites in human cell lines by 23%, which was 4.2 times higher than normal Bxb1. Moreover, eePASSIGE also outperformed another gene editing technique called PASTE by 16 times.

Using human fibroblasts, the most common type of cell in connective tissue, eePASSIGE outperformed PASSIGE by 14 times on average at two therapeutically relevant sites in the genome, with integration efficiencies up to 30%.

Regarding CF, eePASSIGE improved the insertion of the CFTR gene compared with PASSIGE alone, also significantly outperforming PASTE.

“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 toward realizing the benefits of targeted gene integration for patients.”

“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.” co-first author Daniel Gao, PhD, said.