Periodic Reporting for period 1 - LEVERAGE mRNA (Laboratory Evolution of Virus-likE pRotein cAGes for Eukaryotic mRNA delivery)
Periodo di rendicontazione: 2019-05-01 al 2021-04-30
Our genetic information is encoded in nucleic acid (NA) molecules. NAs are promising candidates for treating disease by stimulating, inhibiting, or replacing other NAs or proteins inside of eukaryotic cells—a process known as “gene therapy.” However, methods to deliver genes efficiently and safely into cells are still lacking. One way to transport NAs into cells is by packaging them into a hollow container made of proteins, a method that has been successfully hijacked by viruses for a long time. Because viruses are experts at selectively packaging and delivering their genomes across membranes, recreating these processes in the lab might provide inspiration on how to design better NA delivery vessels for biomedical applications. If successful, many genetically linked diseases that are caused by mutated or missing genes could become curable.
The protein cage I chose to study as a possible NA delivery vehicle originates from the cage-forming bacterial protein lumazine synthase found in Aquifex aeolicus (AaLS). AaLS consists of 60 identical subunits and assembles into symmetrical, porous shells. The cage functions as a cellular nanoreactor and catalyzes the penultimate step of riboflavin biosynthesis. AaLS has no affinity for NAs. Protein engineering and directed evolution were used to alter AaLS’s packaging preferences and make it package its own ribonucleic acid (RNA) genome. After four rounds of evolution, a new AaLS variant called NC-4 emerged. This variant exhibited a 240-subunit, T=4 structure consisting of interlaced trimeric units that bears a striking resemblance to some natural viral capsids.
With this exciting result in hand, my main objectives were to evaluate the RNA packaging properties of the four protein cage generations and utilize this knowledge to expand the cages’ ability to package other RNAs. Specifically, I asked (1) what RNAs and how much of them were packaged in the evolved NC-4 nucleocapsid, (2) how the RNAs were packaged into the protein cages in bacteria and in vitro, and (3) if NC-4 could encapsulate other non-native NAs.
The successful conversion of a bacterial enzyme into a nucleocapsid that efficiently packages and protects its own encoding mRNA opens up exciting new research avenues in the area of protein engineering and drug delivery. Directed evolution is a powerful tool not only for optimizing binding or enzyme activity, but also for recreating more complex biological processes in the lab. Although work is still ongoing, the major milestones of the outlined objectives have been fulfilled, some of which have already been published as further described below.
The protein cage I chose to study as a possible NA delivery vehicle originates from the cage-forming bacterial protein lumazine synthase found in Aquifex aeolicus (AaLS). AaLS consists of 60 identical subunits and assembles into symmetrical, porous shells. The cage functions as a cellular nanoreactor and catalyzes the penultimate step of riboflavin biosynthesis. AaLS has no affinity for NAs. Protein engineering and directed evolution were used to alter AaLS’s packaging preferences and make it package its own ribonucleic acid (RNA) genome. After four rounds of evolution, a new AaLS variant called NC-4 emerged. This variant exhibited a 240-subunit, T=4 structure consisting of interlaced trimeric units that bears a striking resemblance to some natural viral capsids.
With this exciting result in hand, my main objectives were to evaluate the RNA packaging properties of the four protein cage generations and utilize this knowledge to expand the cages’ ability to package other RNAs. Specifically, I asked (1) what RNAs and how much of them were packaged in the evolved NC-4 nucleocapsid, (2) how the RNAs were packaged into the protein cages in bacteria and in vitro, and (3) if NC-4 could encapsulate other non-native NAs.
The successful conversion of a bacterial enzyme into a nucleocapsid that efficiently packages and protects its own encoding mRNA opens up exciting new research avenues in the area of protein engineering and drug delivery. Directed evolution is a powerful tool not only for optimizing binding or enzyme activity, but also for recreating more complex biological processes in the lab. Although work is still ongoing, the major milestones of the outlined objectives have been fulfilled, some of which have already been published as further described below.
My results showed that over the four nucleocapsid generations (NC-1 to NC-4), the capsids packaged decreasing amounts of bacterial RNA and increasing amounts of their own genomes. The later capsid generations protected their packaged RNA from nucleases for longer time frames (at least three days) compared to less than one hour in the first and second generation. This observation was in line with structural information that showed that NC-1 and NC-2 exhibit large pores, while NC-3 and NC-4 exist as closed-shell structures with small pores and interwoven trimeric subunits that stabilize and protect the RNA cargo. Next, we asked why NC-4 is so much better at packaging its genome (two to three copies per capsid) than NC-3 (less than one copy per capsid) even though they share virtually identical protein structures. In collaboration with Profs. Reidun Twarock (University of York, UK) and Peter Stockley (University of Leeds, UK), we identified a cassette of RNA stem-loops that act as packaging signals to template capsid assembly around the NC-4 RNA genome. An equivalent packaging cassette was not identified in NC-3. Naturally occurring ssRNA viruses employ the same strategy to package and assemble around their genomes. This project was recently published in Science. Research is ongoing to deepen our understanding of the RNA packaging specificity and assembly behavior of these artificial virus-like cages.
This study represents a miniature laboratory simulation of virus evolution. A bacterial protein was evolved to encapsulate and protect its own genome. This process supports one hypothesis of viral evolution that states that early viruses arose in primitive cells and recruited host proteins to package and protect their genomes. The results generated in this project also have important implications for the development of virus-like particles for biomedical applications, such as vaccines and gene delivery vehicles.