A team of researchers has adapted engineered virus-like particles (eVLPs)—that they had previously designed to carry base editors—to deliver prime editors to cells in mice at a high enough efficiency to rescue a genetic disorder. The eVLPs and parts of the prime editing protein and RNA machinery were both re-engineered to boost editing efficiency up to 170 times in human cells compared to the previous eVLPs that deliver base editors. The system was used to correct disease-causing mutations in the eyes of two mouse models of genetic blindness, partially restoring their vision. Prime editors were also delivered to the mouse brain without any detection of off-target editing.
“This study represents the first time to our knowledge that delivery of protein-RNA complexes has been used to achieve therapeutic prime editing in an animal,” said David Liu, PhD, professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute and a Howard Hughes Medical Institute investigator.
This work is published in Nature Biotechnology, in the paper, “Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.”
Prime editing, described in 2019 by Liu’s group, allows for longer and more diverse types of DNA changes than other types of editing. The prime editing system has three components: a Cas9 protein that can nick DNA; an engineered prime editing guide RNA (pegRNA) that specifies the location of the edit and also contains the new edited sequence to install at that location; and a reverse transcriptase that uses the pegRNA as a template to make specific changes to the DNA.
Researchers have used a variety of methods to deliver these molecular machines to cells, including lipid nanoparticles and viruses. Virus-like particles (VLPs), composed of a shell of viral proteins that carry cargo but lack any viral genetic material, have also been of particular interest. But VLPs have yielded modest delivery outcomes in animals, and have to be specifically engineered for each different type of cargo to efficiently deliver to cells.
“The prime editor cargo must be efficiently packaged into eVLPs when the particles form but must also be efficiently released from the particles after target cell entry,” said Aditya Raguram, PhD, a former Liu lab graduate student. “All of these steps have to be carefully orchestrated in order to achieve efficient eVLP-mediated prime editing.”
“When we combined everything together, we saw improvements of roughly 100-fold compared to the eVLPs that we started with,” said Liu. “That kind of improvement in efficiency should be enough to give us therapeutically relevant levels of prime editing, but we didn’t know for sure until we tested it in animals.”
The researchers first tested the system in mice to correct two different genetic mutations in the eyes. One mutation, in the gene Mfrp, causes a disease called retinitis pigmentosa that leads to progressive retinal degeneration. The other, in the gene Rpe65, is associated with blindness seen in the condition known as Leber congenital amaurosis (LCA) in humans.
In both instances, the eVLPs corrected the mutation in up to 20% of the animals’ retina cells, partially restoring their vision. The research group also showed that the eVLPs loaded with prime editing machinery could effectively edit genes in the brains of living mice. Nearly half of all cells in the cortex of the brain that received the editing machinery showed a gene edit.
“The gene editing field largely agrees that, moving into the future, gene editing machinery should ultimately be delivered as proteins to minimize potential side effects and we’ve now shown an effective way to do that,” said Liu. “We plan to continue to actively work on improving eVLPs and adapting the technology to target other tissue types within the body.”
