Viruses have long been scientists’ unwitting vessels for moving genome editing machinery into cells. Now, they face new and improved competition: engineered virus-like particles, or eVLPs.
In a study recently published in Cell, researchers presented a souped-up version of a VLP — a non-infectious skeleton of a virus lacking a genome — that can be packed full of the proteins needed for genome editing (1). With careful optimization, they made the VLP better at delivering its cargo to cells, not only in a test tube, but also in living organisms.
“Delivering those machines into the nucleus of the right types of cells in an animal requires a symphony of complicated events to all take place in a carefully orchestrated manner,” said David R. Liu, a gene therapy researcher at the Broad Institute of MIT and Harvard and senior author of the study. “The challenges… help explain why there are relatively few efficient in vivo VLP delivery reports.”
Editing a genome requires delivering a complex of RNA and protein to the cell, and these have typically been delivered in the form of DNA packaged in a viral vector. But when a virus injects its DNA into a cell, it can stick around for months, making it harder to control how much base editor the cell produces. Having too much base editor around increases the chance of harmful off-target edits.
VLPs were originally developed to overcome this limitation of using actual viruses. They still have the proteins that the virus uses to enter cells, but they can hold the shorter-lived protein complexes themselves instead of the genetic material that encodes them. “You have to basically build up the virus from scratch,” Liu said.
VLPs still weren’t perfect, though, especially when used for in vivo editing in a living organism; they successfully edited very few cells.
This made Liu and his team wonder what might be hamstringing the VLPs. They came up with a list of VLP characteristics to improve and then systematically tested what happened when they tweaked each part of the protein skeleton. Three changes in particular packed a punch: the ratio of components in the VLP, the way the base editor was loaded into the VLP, and the way it was released upon infecting the target cell.
The resulting eVLP carried an average of 16-fold more base editor than previous versions and had an optimized mechanism for cutting the base editor loose. Baisong Lu, a gene therapy researcher at Wake Forest School of Medicine who studies VLPs but wasn’t involved in this study, was especially excited to see Liu’s solution for inserting base editors into VLPs. Liu tagged the base editors with a nuclear export sequence, which made them easier for VLPs to scoop up as they formed in factory-like “producer cells.” Combined, these changes increased the editing efficiency of each particle eight-fold in test tube experiments.
When they tried to edit pathogenic genes in mouse liver and retina — two of the most advanced applications of in vivo CRISPR — they successfully edited enough cells to treat the animals’ illnesses. Importantly, they saw minimal measurable off-target editing.
Lu was happy to see that VLPs can be delivered systemically for in vivo gene therapy, but he still has questions about how easily large quantities of eVLPs can be produced and whether they will meet the demands of genome editing in humans.
“The big question is whether these particles can be used in clinical trials,” Lu said.
Liu’s team used VLPs based on one particular type of retrovirus in this study. They’re now testing hundreds of other viruses that might be better at targeting certain cell types as potential models for VLPs.
Banskota, S.*, Raguram, A.*, Suh, S.* et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250-265 (2022). *all authors contributed equally