A team of synthetic biologists and cell engineers led by researchers at the Wyss Institute for Biologically Inspired Engineering and Massachusetts Institute of Technology (MIT), has designed a technology that makes it possible to selectively turn on gene therapies in target cells, including human cells. The approaches harnesses small, versatile devices known as eToeholds, which are built into RNA, and enable expression of a linked protein-coding sequence, only when a cell-specific or particular viral RNA is present.

This highly targeted approach, which is based on a genetic element used by viruses to control gene translation in host cells, could feasibly help to avoid some of the side effects of some treatments that affect the entire body. The researchers suggest that the eToehold platform could unlock multiple opportunities for developing more targeted types of RNA therapy, for designing in vitro cell and tissue engineering approaches, and platforms for sensing diverse biological threats in humans and other higher organisms. In cancer therapy, for example, it may be possible to harness the eToehold approach to design a system that identifies cancer cells and then produces a toxic protein, but only inside those cancer cells.

“This brings new control circuitry to the emerging field of RNA therapeutics, opening up the next generation of RNA therapeutics that could be designed to only turn on in a cell-specific or tissue-specific way,” said James Collins, PhD, the Termeer Professor of Medical Engineering and Science in MIT’s Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering. “eToeholds could enable more specific and safer RNA therapeutic and diagnostic approaches not only in humans but also plants and other higher organisms, and be used as tools in basic research and synthetic biology.”

Collins is senior author of the team’s study, which is published in Nature Biotechnology, and titled, “RNA-responsive elements for eukaryotic translational control.”

In cells, the basic function of RNA is to translate the information encoded in genes, into proteins. However, the structural complexity and relative stability of RNA has also led scientists to consider this biomaterial as the basis for new types of therapy, synthetic biomarkers, and vaccines, as already evidenced with development of the first mRNA vaccines against SARS-CoV-2.

Delivering a synthetic RNA molecule into a cell essentially instructs the cell to produce a desired protein, which can then carry out therapeutic, diagnostic, and other functions. A key challenge for researchers has been how to ensure that only those cells causing or affected by a specific disease will express the protein. This capability could then help to reduce unwanted side effects that can be associated with production of the protein in un-affected cells and tissues.

In 2014, Collins’ team, together with that of Wyss Core Faculty member Peng Yin, PhD, successfully developed toehold switches for bacteria, which are expressed in an off-state and respond to specific trigger RNAs by turning on the synthesis of a desired protein by the bacterial protein synthesizing machinery. The system works by introducing the toehold RNA molecule, which binds to the ribosome-binding site of an mRNA molecule that codes for the specific protein of interest. (The ribosome is where proteins are assembled based on mRNA instructions.) This binding then prevents the mRNA from being translated into protein, because it can’t attach to a ribosome.

The RNA toehold also contains a sequence that can bind to a different mRNA sequence that serves as a trigger. If this target mRNA sequence is detected, the toehold releases its grip, and the mRNA that had been blocked is translated into protein. This mRNA can encode any gene, such as a fluorescent reporter molecule. That fluorescent signal gives researchers a way to visualize whether the target mRNA sequence was detected.

However, the bacterial toehold design cannot be used in more complex cells, including human cells, with their more complicated architecture and protein synthesizing apparatus. For their new study, the researchers set out to try to create a similar system that could be used in eukaryotic cells, including human cells. Because gene translation is more complex in eukaryotic cells, the genetic components that they used in bacteria couldn’t be imported into human cells. Instead, the researchers took advantage of a system that viruses use to hijack eukaryotic cells to translate their own viral genes. This system consists of RNA molecules called internal ribosome entry sites (IRES), which can recruit ribosomes and initiate translation of RNA into proteins.

“These are complicated folds of RNA that viruses have developed to hijack ribosomes because viruses need to find some way to express protein,” explained co-first author Evan Zhao, PhD, who is a Postdoctoral Fellow on Collins’ team, and who teamed up with co-first author and Wyss Technology Development Fellow Angelo Mao, PhD, to combine their respective areas of expertise in synthetic biology and cell engineering.

IRESs are effectively sequences found in viral RNA that allow the host cell’s protein-synthesizing ribosomes access to a segment of the viral genome next to a sequence encoding a viral protein. Once latched on to the RNA, ribosomes start scanning the protein encoding sequence, while simultaneously synthesizing the protein by sequentially adding corresponding amino acids to its growing end.

Eukaryotic Toeholds (eToeholds) are engineered RNA-based control elements that, as in this example, can be specifically activated by viral “trigger RNAs” to enable synthesis of a reporter protein and thus signal the presence of the virus. I the future, eToeholds could be used to design safer and more specific RNA therapeutics, RNA diagnostics, and strategies to enrich therapeutic cell types in in vitro differentiation approaches. [Wyss Institute at Harvard University]
The researchers started with naturally occurring IRES from different types of viruses and engineered them to include a sequence that binds to a trigger mRNA. When the engineered IRES is inserted into a human cell in front of an output transgene, it blocks translation of that gene unless the trigger mRNA is detected inside the cell. The trigger causes the IRES to recover and allows the gene to be translated into protein. “we developed RNA-based eukaryotic modules, called eToeholds, that enable the regulated translation of in cis reporter genes by the presence of specific trRNAs. These eToeholds incorporate modified IRESs that are designed to be inactive until sense–antisense interactions with specific trRNAs cause activation,” the team explained.


“In this study, we took IRES [internal ribosome entry sites] elements, a type of control element common in certain viruses, which harness the eukaryotic protein translating machinery, and engineered them from the ground up into versatile devices that can be programed to sense cell or pathogen-specific trigger RNAs in human, yeast, and plant cells,” said Collins.

“We forward-engineered IRES sequences by introducing complementary sequences that bind to each other to form inhibitory base-paired structures, which prevent the ribosome from binding the IRES,” added Zhao. “The hairpin loop-encoding sequence element in eToeholds is designed such that it overlaps with specific sensor sequences that are complementary to known trigger RNAs. When the trigger RNA is present and binds to its complement in eToeholds, the hairpin loop breaks open and the ribosome can switch on to do its job and produce the protein.”

In a process of quick iteration, the scientists were able to design and optimize eToeholds that were functional in human and yeast cells, as well as cell-free protein-synthesizing assays. They used the technology to develop toeholds that could detect a variety of different triggers inside human and yeast cells. First, they showed that they could detect mRNA encoding viral genes from Zika virus and from SARS-CoV-2. One possible application for this could be designing T cells that detect and respond to viral mRNA during infection, the researchers further suggested. “We further show that stable cell lines expressing eToeholds can be used to sense natural viral infection (by Zika virus) and viral transcripts (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constructs). We also demonstrate the capability of eToeholds to discriminate different cell states and cell types by selectively activating protein translation based on endogenous RNA levels.”

They also designed toehold molecules that can detect mRNA for proteins that are naturally produced in human cells, and which could help to reveal cell states such as stress. As an example, they showed they could detect expression of heat shock proteins, which cells make when they are exposed to high temperatures. “We found that eToeholds could detect various intracellular RNAs, including those introduced by transfection or infection, and endogenous transcripts, such as those indicative of cell state or cell type,” the investigators stated. “The ability to initiate translation of a desired protein in response to the presence of cell-type-specific or cell-state-specific RNA transcripts, as demonstrated here, has considerable therapeutic potential.”

The researchers in addition demonstrated that they could identify cancer cells by engineering toeholds that detect mRNA for tyrosinase, an enzyme that produces excessive melanin in melanoma cells. This kind of targeting could enable researchers to develop therapies that trigger production of a protein that initiates cell death when cancerous proteins are detected in a cell.

“The idea is that you would be able to target any unique RNA signature and deliver a therapeutic,” Mao said. “This could be a way of limiting expression of the biomolecule to your target cells or tissue. We engineered eToeholds that specifically detected Zika virus infection and the presence of SARS-CoV-2 viral RNA in human cells, and other eToeholds triggered by cell-specific RNAs like, for example, an RNA that is only expressed in skin melanocytes. Importantly, eToeholds and the sequences encoding desired proteins linked to them can be encoded in more stable DNA molecules, which when introduced into cells are converted into RNA molecules that are tailored to the type of protein expression we intended. This expands the possibilities of eToehold delivery to target cells.”

The researchers believe that the eToehold platform could help target RNA therapies and some gene therapies to specific cell types, which is important as many such therapies are hampered by excessive off-target toxicities. “… the clinical utility of many nucleic acid-targeting therapies is hampered by excessive off-target toxicity,” they noted. “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.”

In addition, the platform could facilitate ex vivo differentiation approaches that guide stem cells along developmental pathways to generate specific cell types for cell therapies and other applications. The conversion of stem cells and intermediate cells along many differentiating cell lineages often is not very effective, and eToeholds could help with enriching desired cell types.

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