Engineers develop highly precise gene editor for safer cystic fibrosis treatments

Engineers at the University of Pennsylvania and Rice University have refined a technology for editing individual genetic “base pairs” to a new level of precision, opening the door to safer, more reliable therapies for a wide range of genetic diseases, and to potential treatments for some cystic fibrosis patients that may yield better outcomes than existing therapies.

Unlike infectious diseases, many of which respond to the same treatments – like antibiotics that neutralize multiple types of bacteria – genetic diseases often require extremely specific therapies, which can vary from patient to patient, even for the same disease.

“More than a thousand different genetic mutations can cause cystic fibrosis,” says Xue “Sherry” Gao, Presidential Penn Compact Associate Professor in Chemical and Biomolecular Engineering (CBE) and in Bioengineering (BE) within Penn Engineering, and co-senior author of a new paper in Molecular Therapy describing the advance. “The fact that different mutations require distinct corrective tools highlights the importance of precision medicine.”

In other words, to treat conditions like cystic fibrosis, researchers need to develop a suite of tools, rather than a single therapy. But even when scientists know exactly which DNA letter they want to change, today’s gene-editing technologies can unintentionally alter nearby letters as well, introducing “bystander” mutations that raise safety concerns.

“It’s a bit like editing a document,” says Gao. “We can already identify and replace a particular letter in a specific word. How do we change only that one letter without accidentally altering the letters next to it?”

The challenge of swapping Cs for Ts

One common cause of genetic diseases, including cystic fibrosis, is the accidental substitution of one nucleotide base – a single “letter” in the genetic code – for another.

“In some cases, the letter should be a T,” says Tyler C. Daniel, a Penn Engineering doctoral candidate in CBE and co-first author of the new paper, referring to thymine, one of the four bases in human DNA, along with adenine (A), guanine (G) and cytosine (C). “Instead, it’s a C, which can impair or completely abolish the function of the gene, leading to disease.”

While it’s already possible to use editors to change the C to a T, including a base-pair editor the same researchers devised in 2020, and even to selectively modify just one of two adjacent Cs, problems arise when multiple pairs of cytosines appear close together, in “CC … CC” patterns, separated by just a few other base pairs.

The challenge is hardly theoretical: among the tens of thousands of known disease-causing C-to-T and T-to-C mutations this type of base-pair editor can address, three-quarters involve multiple cytosine pairs clustered together.

The issue is precision. How do you restrict the editor so it only modifies the targeted letter C you want and leaves its neighbors alone?”

Tyler C. Daniel, a Penn Engineering doctoral candidate in CBE

Engineering a more exact editor

In order to change letters in DNA, base-pair editors combine two essential functions: one component that locates a specific sequence in the genome and another that modifies DNA. Those two parts are physically connected by a segment of molecules known as the “linker.”

Just as the length of a dog’s leash determines how far it can stray from its owner, the linker’s properties dictate how freely the enzyme that carries out DNA editing moves at the target site.

By shortening and stiffening the linker, the team effectively limited the enzyme’s reach. “We essentially tightened the leash to ensure only our target was edited,” says Daniel.

The researchers also modified how strongly the base-pair editor interacts with DNA, weakening its tendency to act on neighboring letters.

In laboratory tests in human cells, the redesigned editor led to dramatic reductions in unintended bystander edits: The most accurate variant decreased bystander mutations by more than 80%, while retaining high levels of the original editor’s activity at the target site.

Toward a permanent cystic fibrosis treatment

Cystic fibrosis is caused by mutations in a gene that controls how cells lining the lungs move salt and water in and out. When that process breaks down, thick mucus builds up in the lungs, making it hard to breathe and leaving patients vulnerable to repeated infections.

While drugs developed in recent years, such as Trikafta, have transformed life for many people with cystic fibrosis, the medicines must be taken daily and can carry staggering annual costs. Because many of the mutations that cause cystic fibrosis involve changes to just a single DNA letter, base-pair editors could, in principle, help treat the disease, even when Trikafta is ineffective – but only if they can avoid causing harmful off-target mutations.

“We were able to introduce specific cystic-fibrosis mutations into human epithelial cells relevant to the disease, generating cell models that will improve our understanding,” says Gang Bao, Foyt Family Professor of Bioengineering at Rice University and a co-senior author of the study. “We were also able to reverse those mutations and show improved cellular functions using the same editor, demonstrating the level of pinpoint gene-editing control this technology now offers and the potential of base-pair editors to treat the disease.”

The work remains at an early, preclinical stage. Still, at several cystic fibrosis-related genetic sites affecting a subset of cystic fibrosis patients, the refined editor reduced unintended bystander edits from as high as 50–60% to less than 1%, while largely preserving the desired DNA change.

By directly repairing the underlying genetic error, the method points toward the possibility of a long-lasting, potentially permanent treatment. “The more precise we can make these tools,” Bao adds, “the greater their potential to change how we treat genetic disease with a high level of efficacy and safety.”

A broader toolkit for genetic disease

Beyond cystic fibrosis, the refined base editor could help researchers tackle a wide range of genetic diseases caused by single-letter DNA changes. Because the tool allows scientists to introduce – and correct – specific mutations with much greater precision, it offers a powerful way to study how individual genetic variants affect disease and drug response.

That capability is especially valuable for rare mutations, which may affect only a small number of patients and are difficult to study through large clinical trials. By creating accurate cellular models of those mutations in the lab, researchers can test existing drugs, explore new therapeutic strategies and begin to identify which treatments are most likely to work for specific patients.

“The ability to precisely model disease-causing mutations gives us a much clearer window into how those mutations behave, including how they might respond to different therapies,” says Gao. “That kind of insight is essential for moving toward more personalized approaches to treating genetic disease.”