Two new studies offer more validation that prime editing and base editing have the potential to permanently fix a gene variant associated with the rare disease phenylketonuria.
In two separate papers published Nov. 2 in The American Journal of Human Genetics and Human Genetics and Genomic Advances, researchers at the University of Pennsylvania showed that prime and base editing could each repair the gene mutation that leads to PKU. This reversed the resulting pathology in both cases, correcting levels of a key protein enough to keep mice with PKU healthy. The data was also presented the same day at the American Society of Human Genetics' annual meeting.
“This research…demonstrates the feasibility of using gene editing to permanently correct the most common genetic variant associated with this condition,” Kiran Musunuru, M.D., Ph.D., a senior author on both studies, said in a press release. “While there are still challenges to overcome, these findings open the door to potential new treatments that could significantly improve the lives of PKU patients.”
While PKU is rare, it can be debilitating. The condition is caused by mutations in a gene called PAH, which encodes a liver enzyme by the same name to metabolize the amino acid phenylalanine. In PKU, mutations in PAH prevent it from encoding the enzyme. With nothing to break it down, phenylalanine builds up in the blood and brain, putting patients at risk of permanent brain damage if levels get too high.
PKU is most often diagnosed in infancy and requires lifelong dietary restrictions to keep phenylalanine levels in check. While the past decade has seen new strides in drug treatments—such as BioMarin’s drug Kuvan, which may eventually be joined by a competitor from PTC Therapeutics—pharmaceutical interventions don’t work for everyone. Even when they are effective, patients must still avoid foods that are high in protein.
As gene editing technology becomes more accessible, companies have looked for better solutions. BioMarin’s gene therapy candidate, BMN 307, is currently in a phase 1/2 clinical trial. Until recently, so was Homology Medicine’s HMI-103.
While the nuances of BioMarin’s and Homology’s approaches differ, they both involve integrating a new gene sequence in the PAH gene so it produces the enzyme efficiently again. That’s different from base and prime editing, which alters the mutation itself.
“To borrow an analogy from [CRISPR researcher] Fyodor Urnov, if you have a flat tire on your car, and you’re only on three tires, standard gene editing is like sticking an extra wheel on your car and hoping it works well,” Musunuru told Fierce Biotech Research in an interview. “Whereas base and prime editing is actually replacing the flat tire—you’re back to your normal four tires in the right places, and your car is going to work as well as it did before.”
Both of the Musunuru lab’s new studies focused on a severe form of PKU that stems from the missense mutation PAH c.1222C>T, where a cytosine nucleotide has been incorrectly replaced by a tyrosine nucleotide in the PAH gene. In the prime editing study, which appears in The American Journal of Human Genetics, the researchers started by verifying that patients with that mutation had poor control over phenylalanine levels, then showed it was possible to use prime editing to correct the mutation in human liver cells. They then proceeded to test the approach in humanized mouse models of PKU, delivering the therapy via an adeno-associated viral vector, or AAV.
Delivering the prime editors to the mice’s livers reduced phenylalanine to normal levels in all of them within around three weeks of treatment, with no adverse effects on their liver function. This was the case even in mice with only one copy of the alleles the researchers were editing, a good sign of the therapy’s potency.
In the second study, published in Human Genetics and Genomic Advances, the researchers tested base editors delivered in a newer lipid nanoparticle, or LNP, delivery system that’s similar to the one used in the mRNA vaccines for COVID-19. LNP delivery is an active area of research in the gene therapy space, as the approach offers several advantages over AAVs—including a lower risk of immune-related side effects, Musunuru explained.“We were doing [AAVs] in the 2010s, but it’s the 2020s,” he said. “We have better ways of doing this now.”
Following the same trajectory as in the prime editor study, the researchers first demonstrated that the base editors could correct the mutation in liver cells, then moved to mice. In this case, the treatment worked much more rapidly: Phenylalanine levels were reduced to normal within just 48 hours of administration in all of the treated mice. While the researchers didn’t monitor them long-term, previous studies using a similar approach have shown that the changes endure for at least six months, the researchers noted in their paper.
It wasn’t possible to compare the editing efficiency of the prime editors and the base editors head to head because they were delivered via different methods—and delivering prime editors in lipid nanoparticles isn’t ready for prime time, based on his lab’s work, Musunuru said. While prime editing is more versatile than base editing, it’s also more complex and takes longer to complete. Although both AAVs and the LNP delivery system use guide RNA, the one in the LNPs works in short bursts of activity then breaks down, so it might not be sticking around long enough for editing to take place.
Size might be another factor, Musunuru added. Prime editors are larger than base editors, so the guide RNA used with them is bigger as well—and creates a bigger challenge for packing it into LNPs, he said.
“I’m sure we’ll eventually get to the point that lipid nanoparticles work as reliably well with prime editors as they currently do with base editors, but it will require quite a bit of optimization,” he said.
Meanwhile, the base editors in LNPs are closer to being ready to study in humans, though there’s still more work to be done, Musunuru said. On top of that, his lab is working on figuring out how to make prime and base editing work on a variety of mutations. He’s especially interested in developing “plug-and-play” prime and base editors that can be useful even for very rare forms of PKU, as well as other diseases.
“We’d like to be able to treat patients who have their own unique mutations—really get to the point where we can craft a personalized editing therapy for any patient in need,” Musunuru said.