Engineering selectivity into human RNA therapies with eToeholds

creating eToeholds for safer RNA therapies

Date: 3rd November 2021

Delivering RNA into cells has attracted much attention over recent years for therapeutic and diagnostic applications. Their structural complexity and relative stable properties lend themselves well to becoming highly valuable biomaterials.  However, a key challenge has been specificity, where only the cells causing or affected by a specific disease can express the therapeutic protein via the RNA therapy. This ability could significantly streamline production of the protein in the body and avoid unwanted side effects.  Now, researchers have developed eToeholds – small versatile devices engineered into RNA that enabled expression of a linked protein-encoding sequence only when a cell-specific or viral RNA was present.  This new technology opens up opportunities for targeted types of RNA therapy, in vitro cell and tissue engineering approaches, and the sensing of diverse biological threats.

Synthetic biology techniques for sensing and responding to specific intracellular RNAs are desirable for therapeutic and diagnostic applications, as they provide a means to discriminate and target specific cells, tissues and organisms and can serve as building blocks for sophisticated genetic circuits. One such technology recently developed was toehold switches (synthetic RNAs that mimic messenger RNAs) for bacteria.

They contain a recognition sequence (toehold) for a specific stimulus in form of a specific “input” RNA, and a recognition sequence that the ribosome needs to bind to initiate the translation of a fused protein-coding sequence. In the absence of the “input” RNA, the toehold switch is kept in its OFF state by forming a hairpin structure that uses part of the input recognition sequence and the ribosome recognition sequence, thereby keeping it inaccessible. The toehold switch is turned on when a stimulating input RNA binds to the toehold and induces the hairpin structure to open up, giving the ribosome access to its recognition sequence which then starts the synthesis of the encoded protein downstream.  However, these bacterial toeholds were designed for use in relatively simple cells, and applications have not yet transferred in more complex cells such as human cells.

Now, researchers at the Wyss Institute for Biologically Inspired Engineering and Massachusetts Institute of Technology (MIT), US, led by James Collins, have taken IRES (internal ribosome entry sites) elements, a type of control element common in certain viruses which can harness the eukaryotic protein translating machinery, and engineered them from the ground up into versatile devices that can be programmed to sense cell or pathogen-specific trigger RNAs in human, yeast, and plant cells.  Opening avenues for more specific and safer RNA therapeutic and diagnostic approaches not only in humans but also plants and other higher organisms, and that can be used as tools in basic research and synthetic biology.

To start the team forward-engineered IRES sequences by introducing complementary sequences that bonnd to each other to form inhibitory base-paired structures, preventing the ribosome from binding the IRES.  These inhibitory hairpin loop-encoding sequence elements overlapped with specific regions that were complementary to know trigger RNAs (trRNAs), which when present bound to the eToeholds, broke open the hairpin loop, and thus enabled activation of the ribosome and protein production.

Initial validation experiments showed that eToeholds were functional in human and yeast cells, as well as cell-free protein-synthesising assays. Using fluorescent reporter genes linked to the eToeholds they demonstrated up to 16-fold induction of the reporter in the presence of the trRNAs.

The team engineered and tested a variety of eToeholds that could serve as live cell biosensors for viral infection such as those that specifically detected Zika virus infection or the presence of SARS-CoV-2 viral RNA in human cells, and produced either nanoluciferase or an Azurite fluorescent protein as a readout.  They also designed eToeholds to detect hepatitis C virus, poliovirus and enterovirus, suggesting the system could respond to infection status.

They also explored the potential of eToeholds for sensing endogenous transcripts, designing them to be triggered by cell-specific RNAs for example tyrosinase (Tyr) mRNA, which is abundant in melanin-forming cells.  These Tyr-sensing eToeholds showed a 3.6 fold increase in reporter signal when transfected into murine melanoma cells compared to control cells demonstrating their ability to recognise specific cell types.  Further experiments also showed that cell state could also be detected.

Conclusions and future applications

The team, here have developed eToeholds, an RNA-based eukaryotic sense-and-respond module, based on modified IRES elements that permit the translation of a desired protein in the presence of specific trRNA. Validated in several systems including fungal, plants and mammals, it suggests these eToeholds will have a broad range of applications in biotechnology.

The eToeholds could detect various intracellular RNAs that were either introduced by transfection or infection, such as viruses, and endogenous transcripts, such as those indicative of cell state or cell type.

The system shows great flexibility such that a variety of desired proteins could be produced in the presence of specific cell-types or cell-state RNA transcripts, meaning it has considerable therapeutic values.  Moreover, as the eToeholds and associated chosen proteins can be encoded in more stable DNA molecules for introduction into the cell, it expands the possibilities of eToehold delivery yet further to target cells, with the aim of significantly reducing off-target effects.

The researchers envisage this innovative system can advance the development of more specific, safe, and effective RNA and cellular therapies, and so positively impact on the lives of many patients.

With this in mind we are currently seeing a range of RNA therapies designed to treat disease. A new mRNA ‘universal’ therapeutic strategy, using lipid nanoparticles, has been shown to ameliorate cystic fibrosis and can be administered intranasally.  Two other RNA therapies have recently entered clinical trials, an antisense RNA oligonucleotide (AON) called sepofarsen has been used to reverse childhood blindness whilst a mRNA cytokine therapy which eradicates tumours and promotes antitumour immunity is also being explored. Efforts to deliver safer therapies as seen here with eToeholds, with less off target or systemic toxicities will be a welcome addition to the research tool box, and promises to open new avenues for the treatment of disease, biosensing and diagnostics.


For more information please see the press release at the Wyss Institute


Zhao, E.M., Mao, A.S., de Puig, H., Zhang, K., Tippens, N.D., Tan, X., Ran, F.A., Han, I., Nguyen, P.Q., Chory, E.J., et al. (2021). RNA-responsive elements for eukaryotic translational control. Nature Biotechnology.