3d illustration of a part of an RNA chain to illustrate new RNA therapeutics
Credit: Christoph Burgstedt /Getty Images

RNA therapeutics that will switch on in response to different physiological cues can be produced by adding a sensor that can respond to the cue as needed, according to research from the Wyss Institute at Harvard University and MIT.

Using a “sense-and-respond circuit” they call detection and amplification of RNA triggers via adenosine deaminases acting on RNA (ADARs), or DART VADAR, the research team were able to design a prototype therapy that could trigger the translation of a therapeutic protein in response to the presence of a specific molecular marker of disease or cell type.

“I am particularly excited by the fact that our DART VADAR system is a clinically relevant, compact RNA-based circuit that enables one to direct therapies in a highly programmable manner to specific cell types and cells in certain states, thereby minimizing off-target effects,” said lead investigator Jim Collins, a professor at MIT and faculty member at the Wyss Institute.

Several RNA therapies are already on the market, notably small interfering RNAs developed by pioneer biotech Alnylam, and the two mRNA COVID-19 vaccines developed by BioNTech/Pfizer and Moderna during the pandemic have been very successful. However, truly targeted RNA therapies that will switch on in response to a stimulus are still in the future.

Collins and his team have been investigating ways to better control how RNA therapies produce the therapeutic proteins they are designed to make for a while. For example, in 2021 they published work describing ‘eToeholds’, small RNA-based markers that make use of internal ribosome entry sites to allow expression of a linked protein-encoding gene sequence only when a specific cell or viral sequence is present.

But the eToehold process was complex and time consuming and the current study, published in Nature Communications, came about because the team wanted to work out a way of keeping the sensor the same and just changing the target of interest.

“Our technology grew from the idea that we could decouple the elements of responsive RNA sensors—sensing, actuation, etc.—so it’s much easier to design circuits for new targets. Ideally, we wanted to be able to change the payload without modifying the sensor element every time,” said co-first author Raphaël Gayet, a research scientist at the Wyss Institute.

ADARs are enzymes that are able to bind to double stranded RNA and make a base edit converting adenosine molecule into inosine, which destabilizes the RNA structure and is linked to the cellular response to viruses.

Collins and team used the RNA editing ability of ADARs to create the new sensor and programmable therapy. Each single-stranded “circuit” contains the RNA sequence needed, a “stop” codon sequence of uracil-adenosine-guanine (UAG) in the middle of the RNA strand and a sequence coding for a fluorescent green protein. They also added the ADAR sequence to a later model to allow it to work in more tissues, as ADARs are not naturally found at high levels in all cells.

If the therapy binds a complementary target RNA strand it becomes a double-stranded. The A of the UAG sequence will ‘mismatch’ with a cytosine in the target strand, rather than uracil. This mismatch essentially frees up the adenine to be found and converted to inosine by ADARs. The resulting sequence is no longer a “stop” codon and translation can occur.

“What’s really exciting about this sensor is that the green protein signal sequence can be easily replaced with the sequence for any therapeutic gene that you want to express in response to the presence of a trigger RNA in the cell. So not only does this sensor detect targets, it can automatically respond to them without requiring user input, automating the delivery of a therapeutic payload at the cellular level,” said co-first author Shiva Razavi, a Postdoctoral Fellow at MIT.

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