NEW YORK – A team of researchers from the California Institute of Technology has developed a new nanoparticle transport system that can achieve programmable RNA export from living mammalian cells.
Described in a Cell paper published last month, the mechanism, termed controlled output and uptake of RNA for interrogation, expression, and regulation (COURIER), allows researchers to harvest target RNA without destroying cells, potentially opening the door for research and therapeutic applications such as studying temporal cell population dynamics and cell-to-cell RNA delivery.
"We care about how cells change over time," said Felix Horns, a postdoctoral researcher at CalTech and the first author of the study. "But our ability to study that has been held back by the fact that we need to kill the cell to get the RNA out."
According to Horns, the genesis of COURIER was inspired by some viruses' ability to package and secrete cargo RNA molecules using protective nanoparticles.
"One day, I was sitting in journal club with a colleague of mine, and he was describing the mechanisms that SARS-CoV-2 uses to package its genome into its own viral particles," he said. "I thought that sounds pretty useful, maybe we could use that to package other RNAs that we want into virus-like particles."
Horns and his team started out by trying to use the existing viral packaging mechanisms in nature for this study. However, they faced a challenge in that these natural exporter systems were not very specific for the target RNA molecules.
Therefore, a "major accomplishment" in developing COURIER was protein engineering, Horns said. To improve the RNA exporters' specificity, the team sought to replace the native RNA recognition system in the exporter nanoparticles with the MS2 bacteriophage coat protein (MCP), a classic RNA-binding protein. By fusing MCP with the capsid, the researchers designed a way to target RNA cargo and package them into virus-like particles (VLPs) that are secreted from the cells.
"It's really a challenge to find designs that are both efficient and specific for exporting RNA," Horns said. "We actually generated a panel of several dozens of these engineered proteins, where we fused the RNA binding protein at different places and replaced different domains, and we tested them all to see which ones are capable of exporting RNA."
To systemically quantify the RNA export efficiency and specificity, Horns and collaborators devised a benchmark framework using RT-qPCR and sequencing. While qPCR allowed the team to study the exporters' efficiency by measuring the number of RNA molecules secreted, Horns explained, sequencing helped characterize how specific the system was by profiling the entire RNA content. In the end, the results showed the team's "very best exporters" can achieve about 80 percent specificity in exporting the target RNA molecules, he added.
The CalTech researchers also investigated the potential side effects of the exporters on cells. Overall, Horns said, the team "[has] not seen that these exporters cause any perturbations to the cell physiology," and the results showed that expressing these systems does not impact cell morphology, growth rate, or the transcriptome profiles of the cells.
In addition to the human HEK293 cells, the researchers further illustrated that the RNA exporter systems worked in human lymphoblast cells, T cells, mouse fibroblasts, and hamster ovary cells, demonstrating "the portability of RNA exporters across mammalian species and cell types," they noted.
Horns said there are two major applications for COURIER. For one, he pointed out that the technology can be used as a cellular reporter system to help track live cell population dynamics, which may be useful in biological contexts such as developmental biology and cancer.
In their study, for instance, the researchers engineered cells and tagged them with a diverse library of RNA barcodes that were targeted by the RNA exporters. By harvesting and sequencing the secreted RNA barcodes, the researchers were able to achieve "non-destructive monitoring of mammalian cell population dynamics with clonal resolution and high accuracy, reproducibility, and sensitivity," they said.
Another key application for the exporter mechanism is cell-to-cell RNA delivery. "The idea is that these systems allow one cell to deliver RNA directly to another cell," Horns said. "That's exciting because we think this unlocks a new paradigm for cell therapy."
To achieve that, the team incorporated fusogens, proteins that enable vesicle-cell fusion, into the nanoparticles in this study and illustrated that the COURIER systems are capable of delivering RNA from cell to cell.
"I think this is a great approach," said Junyue Cao, head of the single-cell genomics and population dynamics laboratory at Rockefeller University. While COURIER systems are principally different from conventional RNA sequencing approaches, Cao said, the technology could offer potential advantages by delivering temporal information while keeping the cells alive.
In addition, although the RNA exporters were shown to have a clonal resolution in the current study, with future technical development, this approach also could potentially achieve analysis at the single-cell level, Cao said. More importantly, he added that this tool could be beneficial for in vivo studies.
"For in vitro systems, you can always use microscopes or other approaches to track cellular dynamics, but for in vivo systems, that is always challenging," he explained. "I think it will be really exciting if you apply that to study the in vivo cell population changes."
Moving forward, Horns said one future direction is to continue developing these exporter systems so they can be "optimized and refined."
While the current study did not detect any perturbations asserted by the exporters on the cells, he said, it is "possible that there are more subtle perturbations that we were not able to detect, so that is something we will continue to think about."
Moreover, given that this study only focused on RNA exporters from a limited panel of cultured cell lines, it still remains to be seen whether their performance may vary in other cell types and organisms.
Another future step, according to Horns, is to apply the RNA exporters to help answer biological questions as well as to explore their therapeutic utility given their cell-to-cell delivery capability. CalTech has already filed patent applications pertaining to this application.
"We think that the ability to measure what cells are doing over time will open up new ways to look at many different aspects of biology," Horns said. "Especially on the therapeutic side, we're really excited about building this into a next-generation type of cell therapy."