Prime Editing Repairs Rare Brain Mutation in Mice

Scientists at The Broad Institute of MIT and Harvard and The Jackson Laboratory have used a precise form of gene editing to target the underlying mutations behind the rare neurological disorder, alternating hemiplegia of childhood (AHC).

In mice, prime editing strategies efficiently corrected mutations in the Atp1a3 gene, which encodes a subunit of the neuronal sodium pump. This plasma membrane protein complex is responsible for the transport of sodium and potassium ions across the plasma membrane in neurons and is therefore essential for healthy nerve cell function.

Since Atp1a3 mutations are found in 70% of AHC cases, this proof-of-concept study – published in Cell – opens a promising path for developing new therapies, the authors said. It also marks the first successful use of prime editing to treat a neurological disease in animals.

Expanding the genome editing toolbox with prime editing

Following the development of CRISPR-Cas as a genome-editing tool, researchers have been working to expand the toolkit with more precise and diverse editing methods. David R. Liu, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, is a renowned contributor to this field.

Liu and colleagues created prime editing – the “newest kid on the block” in gene editing – back in 2019. Also referred to as “search-and-replace editing,” prime editing overcomes some of the limitations associated with base editing, a technique that was also developed by Liu’s lab in 2016.

“Prime editing is often referred to as a ‘search-and-replace’ method because prime editors are directed to engineer a mutation of interest at a specific site in the genome by virtue of the instructions encoded in a prime editing guide RNA (pegRNA),”  Dr. Francisco Sánchez-Rivera, assistant professor of biology at the Massachusetts Institute of Technology (MIT), told Technology Networks. Sánchez-Rivera’s research uses prime editing to screen the effects of cancer mutations.

“These pegRNA molecules contain both a protospacer (the ‘search’ sequence) and a 3′ extension sequence (the ‘replace’ sequence that dictates the mutation to be installed at the site). Because the mutation is encoded in a pegRNA, prime editing can be used to engineer any type of single nucleotide variation and small indel,” he continued.

Prime editing could represent a “one and done” treatment for rare genetic disorders in the future. While the first human trial testing its use as an ex vivo therapeutic for chronic granulomatous disease is underway, Liu and colleagues sought to establish whether in vivo prime editing could be safe and efficacious as an in vivo treatment for central nervous system (CNS) disorders.

AHC is a rare disorder first reported in 1971. It presents as repeated episodes of weakness or paralysis that can affect one or both sides of the body and seizures. Infants born with AHC also experience developmental delays and intellectual disability, for which there is no cure.

While over 50 pathogenic variations of the gene Atp1a3 are linked with AHC, there are three mutations known to be associated with over 65% of cases. Typically, gene editing therapies target one mutation at a time, such as the recent treatment of baby KJ Muldoon using a base editing approach. Liu and colleagues developed a framework to test prime editing strategies targeting five ATP1A3 mutations – including the most common – all at once using an adeno-associated virus delivery system.

“We developed a robust framework to correct multiple mutations in parallel,” said Dr. Alexander Sousa, a postdoctoral fellow in the Liu lab and co-first author. “This effort was really about creating a blueprint that could be rapidly applied to other rare diseases too.”

Prime editing reverses disease traits in mouse models

After demonstrating that their approach repaired ~90% of mutations in cells cultured from AHC patients without adversely affecting other areas of DNA, Liu and team moved on to test injecting the therapy directly into the brains of mice models. In collaboration with the Jackson Laboratory, they tested the safety and efficacy of prime editing in two models carrying similar Atp1a3 mutations observed in humans with AHC.

“Dual AAV-mediated delivery to the mouse CNS as a P0 intracerebroventricular (ICV) injection at a dose below that of an FDA-approved AAV9 drug efficiently corrects the Atp1a3 D801N and E815K mutations, rescues hippocampal Atp1a3 ATPase activity, ameliorates multiple behavioral defects and greatly extends lifespan in Atp1a3 D801N mice,” the researchers said.

Previously, researchers were unsure whether correcting Atp1a3 after birth would be effective, given the protein’s important role in brain development.

“The results really exceeded our expectations,” said Dr. Holt Sakai, postdoctoral researcher in Liu’s research group and co-first author. “It was incredibly exciting to see that data.”

“This is a powerful proof of concept,” he added. “It shows that we can use prime editing to treat genetic brain diseases, and it lays the groundwork for translating this approach to the clinic.”

Though this research provides a template for in vivo prime editing to target rare CNS disorders, additional studies are required to optimize the method. In this study, the therapy was delivered directly to the brains of mice shortly after birth. Liu and his team plan to explore a less invasive route of administration and evaluate the potential effectiveness of treatment at later stages of life.

Reference: Sousa A, Terrey M, Sakai H, et al. In vivo prime editing rescues alternating hemiplegia of childhood in mice. Cell. 2025. doi: 10.1016/j.cell.2025.06.038