Scientists use directed evolution to develop better viruslike capsules

Viruses are pretty simple: They’re small DNA or RNA genomes enclosed in protein containers called capsids. Scientists have designed and engineered proteins that self-assemble into viruslike containers that hold cargoes such as drugs, vaccines, and biomolecules.

David Baker of the University of Washington and coworkers have now devised the first so-called nucleocapsids, artificial capsids that enclose their own RNA genomes. The development opens the door for researchers to use directed evolution, a repetitive protein mutation and screening technique, to optimize the properties of these protein containers for drug delivery applications (Nature 2017, DOI: 10.1038/nature25157).

In particular, the team optimized nucleocapsids to remain stable for long times and to protect their RNA cargoes from degradation while floating around in the bloodstreams of mice. The artificial nucleocapsids could be useful for delivering small molecules, biomolecules, or materials for therapeutic or nanomaterials applications. They could even provide housing for future synthetic lifeforms, the researchers say.

“This powerful new technology opens a new paradigm for the development of synthetic viral capsids,” says oncologist David T. Curiel of Washington University School of Medicine in St. Louis, whose group develops delivery vectors for gene therapy.

Baker and coworkers produce the capsids by engineering bacteria to carry DNA plasmids encoding two computationally designed proteins. The proteins form trimers and pentamers that self-assemble with each other and mRNA to form icosahedral capsids. The capsids incorporate the mRNA coding for their own proteins because it is the predominant form of mRNA in the cells. Positive charges line the inside of the capsids, enhancing their ability to hold onto and protect the mRNA, which is negatively charged.

In the directed evolution process, the researchers make targeted mutations in the two DNA genes, allow bacteria to produce capsids based on those genes, inject the capsids into mice, and then test desired properties of the capsids in the animals. Because each capsid carries the mRNA encoding its own proteins, the researchers can then reverse transcribe mRNA from the best-performing capsids into the two DNA genes, which then can be used in additional rounds of directed evolution. The researchers repeat the process until capsid properties improve to an optimal level.

The researchers chose to optimize three properties, which they improved significantly relative to those of the proteins they started with: The amount of RNA packaged per capsid increased about 133-fold, the fractional amount of encapsulated mRNA persisting six hours in mouse blood went from 4% to 71%, and capsid circulation time in the bloodstream improved from 5 minutes to 4.5 hours.

“It’s a very important breakthrough in synthetic biology and biotechnology,” says genomic nanomaterials designer Armando Hernandez-Garcia of the National Autonomous University of Mexico. The ability of the new capsids to recognize and incorporate their own genomes is a key factor that allows them to evolve into structures that could be useful for drug delivery or nanotechnology, he says.