While Kiran Musunuru, MD, PhD, is already in the process of using prime editing—a search and replace genome editing technology that doesn’t create double-strand breaks or use donor DNA—in his academic laboratory to treat genetic disorders (and is having success in some cases in tackling mutations for rare metabolic disorders that aren’t amenable to other editing tools like base editing) he says it’s all for naught if the editing isn’t efficient in vivo.

“It’s no good if you can solve something by prime editing but only get 5% editing efficiency in vivo because that’s not going to help you with most diseases,” Musunuru, professor of medicine at the University of Pennsylvania, director of the genetic and epigenetic origins of disease program at the Cardiovascular Institute, and the scientific director of the Center for Inherited Cardiovascular Disease Perelman School of Medicine, told GEN. “So, it’s very important that to get the efficiency up into the double digits, certainly, and really high double digits. While there have been several studies that have shown prime editing in living animals, the efficiencies have been very poor (in the single digit percentages).”

Now, a new Nature Biotechnology article from the lab of prime editor pioneer David Liu reveals a high-efficiency, therapeutically-relevant prime editing in vivo in mice with a dual-AAV system, demonstrating prime editing in the mouse brain (up to 42% efficiency in the cortex) and liver (up to 46%) by installing putative protective mutations in vivo for Alzheimer’s disease in astrocytes and for coronary artery disease in hepatocytes, respectively.

The perfect pairing for gene editing

As of now, there are only two clinically viable approaches to putting a gene editor into the body of a living human patient: AAV and lipid nanoparticles (LNPs). In recent months, some companies have talked about unpublished work using LNPs for in vivo gene editing, like Tessera Therapeutics’ announcement at JPM 2023 of data showing “clinically relevant levels of in vivo rewriting in the genome of liver cells of non-human primates after a single administration.”

Senior author and the man behind prime editing, David Liu, told GEN that some pharmaceutical companies are moving away from AAV for gene therapy, in part because of the challenges associated with re-dosing AAV. AAV provokes in some patients antibodies that can impede the efficacy of subsequent doses. This problem may not be an issue for gene editing therapeutics because the expectation is patients will only need a single dose.

“AAV is one of the only clinically validated ways to deliver macromolecules into a variety of tissues such as the heart and brain,” said Liu, who is Richard Merkin professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute of MIT and Harvard, the Thomas Dudley Cabot professor of the natural sciences at Harvard University, and a Howard Hughes Medical Institute (HHMI) investigator. “There are many delivery technologies being pursued, including LNPs and engineered virus-like particle (eVLP) systems. AAVs offer a very attractive intersection of properties, including clinical validation, delivery to a variety of different tissue types of therapeutic interest, and a relatively well-understood safety profile, even though at high doses they have provoked toxicity in patients.”

Since prime editors are quite big, encoding prime editors requires more than one AAV vector. The DNA encoding a prime editor has to be split into two different AAV vectors that each express a different piece. The problem is that those two pieces have to be designed so that the translated protein components will come together spontaneously and then assemble into the full prime editing protein. And then the prime editor guide RNA (pegRNA) and often a nicking guide RNA have to also be put into the AAV, along with the components to express all of the RNAs. The two co-first authors, Jessie R. Davis and Samagya Banskota, figured out how to squeeze DNA encoding the prime editor, pegRNA, and nicking guide RNA into two AAV vectors that, when injected into mice, edited cells in therapeutically relevant organs at pretty high levels without off-target effects.

Liu thinks that ultimately the best way to demonstrate broad applicability for a technology, whether it’s a delivery technology or an editing technology, is to show that it can be used to install or correct mutations that weren’t chosen because they’re easily or commonly edited but because of their importance to biology and medicine. And that is exactly what makes prime editing so useful—the fact that it is essentially sequence-agnostic and can make any nucleotide change.

Another aspect of the study was to understand the effect age has on editing efficiencies because, in prior studies and other contexts, it can actually make a big difference as many editing technologies work better in newborn and young mice. According to Musunuru, who is a co-author of the article, part of that has to do with delivery, and part of it has to do with the proliferation and state of the targeted cells.

“It was nice to see in this study the ability to give the same treatment to older animals and still be able to get the editing done because that means this isn’t restricted to very young patients or newborns,” said Musunuru, who is also a co-founder of biotechnology company Verve Therapeutics, which announced last year the first dosing of a patient with Verve-101, an investigational in vivo base editing medicine for the treatment of heterozygous familial hypercholesterolemia (and has since been placed on hold by the FDA).

This pairing is something that will probably be quite relevant and useful in adult patients who have had a longstanding genetic disease or are starting to experience clinical symptoms from a long-brewing genetic disease that they may not even realize they had for all these years and it’s only showing up later in life, which is the case for many neurodegenerative diseases like Alzheimer’s. “It’s remarkable that you can do something as precise and versatile as prime editing and actually deliver it into living animals using a therapeutically relevant vector,” said Musunuru. “This is something you could imagine using in a human being in the fullness of time, and actually get such high-level editing.”

Musunuru believes that this opens the door to being able to create a wide variety of prime editing therapies, which will allow a much larger number of diseases and groups of patients to be addressed than has been before with editing technologies. “The next few years are going to be incredibly exciting because the ability to really be able to take prime editing into small animals, and then soon enough into large animals, and eventually give it a couple of years into human beings, we’re going to be able to do so many amazing things,” said Musunuru. “It’s going to be transformative for the practice of medicine.”

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