Harvard and MIT Researchers Pursue Improved RNA and Cellular Therapies with eToehold Technology

By Paul Nicolaus

December 9, 2021 | A group of cell engineers and synthetic biologists at Harvard University and Massachusetts Institute of Technology (MIT) has developed a tech tool called “eToeholds” that could help improve the safety and efficacy of RNA and cell therapies, in addition to enabling new forms of detection. 

The eToeholds are RNA-based sense-and-respond devices that only allow the expression of a linked protein-coding sequence when a particular cell-specific or viral RNA is present. 

The researchers believe their line of work could help enhance therapeutic and diagnostic approaches in humans, plants, and other types of organisms. They also see potential as a basic research and synthetic biology tool.

In a recently published Nature Biotechnology study (DOI: 10.1038/s41587-021-01068-2), the researchers pointed out that their approach moves beyond existing RNA-based sensor systems by expanding the length and specificity of trigger sequences and enabling “dose responsiveness to triggers of varying quantity.”

“We’re at a very exciting time around harnessing RNA as a therapeutic molecule,” senior author James Collins, a core faculty member at Harvard’s Wyss Institute for Biologically Inspired Engineering and professor of medical engineering & science at MIT, told Bio-IT World.

A key feature of the work detailed in this paper is that it demonstrates the capability of introducing control to RNA therapeutics, Collins explained. More specifically, it involves introducing a level of control that will enable RNA therapeutics to be used in a cell or tissue-specific way via lipid nanoparticles or gene therapy. 

The next generation of life-saving drugs will be complicated, Evan Zhao, a research fellow at Harvard’s Wyss Institute and co-first author of the study, told Bio-IT World. There will be a need for scientists to design them in a way in which they are smart and capable of responding to their environments. 

In this paper, Zhao said, he and colleagues put forth technology behind the messenger RNA (mRNA) vaccines that have emerged in response to the coronavirus pandemic. “We give those technologies the capability to sense their surroundings and try to adapt their therapeutic capability after sensing their surroundings,” he added.

Targeted Approach Looks to Reduce Unwanted Side Effects

Scientists have considered RNA as a biomaterial that could be used to come up with new forms of biomarkers, therapies, and (as the pandemic has shown) vaccinations.

A synthetic RNA molecule delivered into a cell essentially tells it to produce a particular protein for diagnostic, therapeutic, or other purposes. To steer clear of side effects, though, researchers have traditionally faced the tricky task of allowing only the cells that cause a disease—or only the cells affected by it—to express that protein. 

In their study, the group noted that their capacity “to initiate translation of a desired protein in response to the presence of cell-type-specific or cell-state-specific RNA transcripts” has therapeutic potential considering many therapies are hindered by unwanted and potentially harmful side effects. 

“The ability of an eToehold module to translate a protein or protein-based precursor in response to an mRNA signature will help address this challenge by restricting activation of a desired therapy to specific target cells,” they added. 

A lot of therapy involving drug delivery runs the risk of off-target side effects, explained co-first author Angelo Mao, a technology development fellow at Harvard’s Wyss Institute. There is often a desire to deliver a drug to specific tissues or cells within the body without impacting other tissues or cells. But if the drug is active in different types of cells, it can cause ill health and sickness in patients. 

“One of the capabilities of the eToehold is that it prevents the creation of a therapeutic protein in non-target cells by reading the RNA that’s present inside cells,” he told Bio-IT World. 

An aspect that makes the technology especially attractive is that it is “so readily programmable,” said Mao, while highlighting its flexibility and versatility. “We can very easily design the eToehold to detect theoretically any RNA molecule that we’re interested in detecting, and also design it to produce any protein of interest that we want to produce,” he added. 

Wyss Founding Director Donald Ingber, a professor of vascular biology at Harvard Medical School and Boston Children’s Hospital and a Harvard professor of bioengineering, highlighted how this research could wind up positively impacting the lives of patients down the road.

The study shows how this research group is coming up with “innovative tools that can advance the development of more specific, safe, and effective RNA and cellular therapies,” he said in a press release announcing the research advance.

eToehold Potential Could Go Beyond Human Applications

The recently published study also highlighted the potential to extend the eToehold technology to organisms other than humans. 

The researchers took internal ribosome entry site (IRES) elements and engineered them into devices that can be programmed to sense trigger RNAs in yeast and plant cells, in addition to human cells. 

“The proven functionality of eToeholds in multiple domains of eukaryotic life, including fungal, plant, and mammalian systems, suggests its potential for broad utility in biotechnology,” the researchers pointed out.

In a plant, for example, the idea is that you could potentially program it to report on a pathogen infection, Collins said, or to both report and respond to it. 

He explained that the first scenario would involve programming the plant to produce a detectable molecule that would indicate the plant had been infected. The latter scenario, on the other hand, would mean making a molecule that could be detected and producing molecules that could kill the infection or stop its growth.

Earlier Toehold Work Led Up to Latest Design 

Years ago, Collins and colleagues came up with toeholds of a different sort. Detailed in a 2014 paper (DOI: 10.1016/j.cell.2014.10.002), these bacterial toeholds—a different design with different applications—were meant to function in bacterial cells and were extended to cell-free systems. 

This earlier work garnered interest from academic groups and investors who were intrigued about applications in the diagnostic space and potentially the therapeutic space as well, Collins said. The interest in therapeutic applications was almost exclusively geared toward developing toeholds that could function in human cells.

Because the bacterial toehold design could not be tweaked or modified for use with more complex cells, however, it helped motivate and lead to eToeholds—an entirely new design. 

In spirit, he said, the earlier toehold technology and the newer eToehold iteration are similar in that the researchers are pursuing synthetic biology designs for translational control. But a notable distinction is that eToeholds are geared toward eukaryotic cells. 

The new design makes it possible to produce RNA elements that could function in human cells and turn on in the presence of RNA sequences of interest. 

eToeholds Capable of Detecting Zika, SARS-CoV-2, and More

According to Zhao, he and colleagues forward-engineered IRES sequences by introducing complementary sequences that bind to one another and prevent the ribosome from binding the IRES. The hairpin loop-encoding sequence built into eToeholds is meant to overlap with specific sensor sequences that are complementary to trigger RNAs.

When present, the trigger RNA binds to its complement, the loop breaks open, and the ribosome can then “switch on” to produce the desired protein. 

In their study, the group reached up to 16-fold induction of reporter genes linked to eToeholds in the presence of their trigger RNAs compared to control RNAs. 

In other words, they “can get a very large output in response to the detection or the trigger event to flip on the switch,” Collins said. In response to an RNA produced by a specific cell type or associated with a pathogen infecting a particular cell type, they can get a substantial expression on the output of their RNA control element. 

This is useful, he continued, because it means they can produce plenty of a reporter signal that could be read out in a diagnostic application or a lot of protein for use in a therapeutic application. 

The eToeholds detected the presence of SARS-CoV-2 viral RNA in human cells as well as Zika virus infection. And other eToeholds are triggered by cell-specific RNAs, such as an RNA expressed only within skin cells that produce melanin. 

“Importantly, eToeholds and the sequences encoding desired proteins linked to them can be encoded in more stable DNA molecules,” Mao explained. When introduced into cells, they are converted into RNA molecules geared toward the intended protein expression. This broadens the possibilities of delivering eToeholds to target cells, he added.

Envisioning the Next Generation of eToeholds 

While the researchers foresee plenty of potential, they also acknowledged that their work isn’t finished just yet. Currently, their technology does have limitations, and they intend to pursue improvements moving forward. 

For instance, they are now looking to make it easier to program their eToeholds, and there are two primary components involved with this endeavor. The first involves developing “as small of a module as possible that behaves as robustly as possible,” said Zhao. 

As the group continues to build their system, they are attempting to turn this initial iteration into a tool that a therapeutic designer could take and turn into something that significantly improves their therapy. 

The other major component is geared toward moving beyond the current ability to sense just one transcript. “There’s only really one knob that we can turn for each of these designs,” he explained.

Zhao used the analogy of a vehicle to help illustrate. It’s kind of like the ability to turn a car left or right. Although that ability is beneficial, there’s still a desire to do more, such as accelerate or decelerate, when designing therapies. So they now have their sights set on adding other types of knobs. 

“Say you have a therapy that you want to turn on only in the presence of a small molecule or something that activates therapy,” Zhao said. “We are designing those things into the next generation of eToeholds.”

The group plans to continue to build upon this line of work by seeing how they might alter and expand the control in terms of handling multiple RNA inputs and different ligands that can trigger RNA switches, Collins added. And moving forward, they are looking to extend their technology toward clinical implementation.

Paul Nicolaus is a freelance writer specializing in science, nature, and health. Learn more at www.nicolauswriting.com.