We could turn circular RNA, a naturally stable but noncoding form of RNA, into a programmable "engine" that persistently expresses therapeutic proteins in the patient's own body?
We were able to engineer translatability into this closed-loop, circular RNA form, creating synthetic Endless RNATM (eRNA). eRNA can be programmed for long-lasting expression of diverse therapeutic proteins and has the potential to become a powerful new therapeutic platform capable of generating a diverse array of novel medicines for the world.
Existing protein therapeutic modalities have drawbacks that limit the diseases they can treat or the patients who can access them. Many of these drawbacks arise because protein therapeutics have to be made outside the body, then introduced into the patient – often repeatedly. These limitations make for complex and costly manufacturing, delivery, dosing, and patient access.
Many of the limitations of existing treatment modalities could be overcome if the body could be co-opted to produce its own therapeutic proteins. The application of secreted peptides and larger proteins, therapeutic antibodies in their many forms, vaccines, and intracellular enzymes could be significantly improved if they were made endogenously by a system that resulted in persistent expression and enabled repeated dosing.
Unlocking the full potential of RNA to create novel and superior programmable medicines is what drove Avak Kahvejian, Ph.D., General Partner at Flagship Pioneering, Noubar Afeyan, Ph.D., Founder and CEO of Flagship Pioneering, and the team at Flagship Labs. They set out to build upon their expertise in RNA science and further explore its potential to create the next generation of therapeutics.
Their journey began with a deep understanding of RNA and its many forms. They quickly homed in on an intriguing, naturally occurring circular form of RNA that, while very stable, is unable to engage the ribosome and start protein translation. The result was a great leap forward in human therapeutic development: eRNA, a versatile RNA platform that instructs cells to express a desired therapeutic protein persistently, allows for repeat redosing and multiple routes of administration, and does not trigger an unwanted immune response – attributes that have not been available with other therapeutic modalities.
"Biology is constantly evolving, along with the tools for probing it," said Kahvejian, the founding CEO of Laronde from 2017 to late 2020. "One of the greatest things about being at Flagship and having the ability to invent and create is having the freedom to challenge assumptions, pursue completely new and untested ideas, and work on unprecedented approaches. That's what gets us excited; we're not beholden to old dogmas or the status quo."
At Laronde, Kahvejian, Afeyan, and the Flagship Labs team started by looking at the world of RNA, which now encompasses much more than mRNA; all sorts of new RNA types have been found in recent years. The team was particularly intrigued by circular RNAs because of their unusual format: in circular RNAs, the free 5’ and 3’ ends found in linear RNA forms are joined together to form a closed loop. "When I first saw the circular format in the cell, I got very excited,” said Kahvejian. “These circular RNAs are very stable, which is a highly desired property."
Circular RNAs are naturally overrepresented in red blood cells and platelets, suggesting that they are long-lasting. This is in contrast to linear RNAs, which are intrinsically unstable and short-lived. Was the absence of free ends in circular RNA making them impervious to exonuclease digestion and therefore responsible for this amazing stability? However, circular RNAs are noncoding, meaning that they cannot translate their sequences into proteins.
The team began wondering what would happen if these closed-loop RNAs could be modified to engage the ribosome and were engineered to carry protein sequences that result in protein expression: Would a stable "coded message" emerge? Would the ribosome keep going around and around, reading the RNA in a "rolling circle" of translation?
"We believed if the RNA molecule were stable, and if we could get prolonged protein translation, then we could make a therapeutic platform – potentially a quite powerful therapeutic platform with a wide range of potential products," said Afeyan.
To probe these possibilities, the Flagship team had to apply inventive steps to engineer in translatability that, when combined with the stability of the closed loop, would enable a powerful and long-lasting translation engine.
The team's first challenge was getting the ribosome to engage with circular RNA, which lacks the free 5' end and cap structure the ribosome ordinarily needs to start reading RNA. The solution was inspired by canonical and non-canonical translation mechanisms, including those used by viruses, whose genetic sequences contain Internal Ribosomal Entry Sites (IRES) are able to engage the ribosome. This constituted a key inventive step: the Flagship team incorporated an IRES into circular RNAs and then worked to optimize its features – for example, how close to the coding info to place the IRES and how large the rest of the construct could be.
The actual "aha!" moment came in mouse experiments. The team performed head-to-head comparisons of an eRNA encoding the secreted form of the light-emitting firefly enzyme G-Luc, and an optimized mRNA encoding the same protein. Both RNAs expressed the protein in the animals' bloodstreams. However, the enzyme produced by mRNA began to disappear after 3-4 days, while the eRNA continued to produce the enzyme for several weeks.
According to Sophie de Boer, a member of the founding team of Laronde and Senior Associate, Flagship Pioneering, "To the best of our knowledge, this is a completely novel observation. The firefly enzyme has a half-life of twenty minutes, but eRNA molecules can have half-lives of days or even weeks; so the protein we measured several weeks out had just been made a few minutes before. This was a seminal moment: we had established completely different pharmacology that never existed for any therapeutic product before."
Between conception and launch, a Flagship team worked for four years, entirely in-house, to create and develop the eRNA platform. As a result, the platform's broad intellectual property (IP), which includes methods of translating closed-loop RNA utilizing IRES, is wholly owned by Flagship Labs and Laronde.
The real key to eRNA's power is the combination of programmability and persistence, which has never really been possible in the therapeutic world. Each closed loop of eRNA contains a cassette that codes for the desired therapeutic protein; the cassette can be digitally designed on a computer then inserted into the eRNA. Laronde can generate eRNA that produces a diverse array of protein modalities – secreted peptides and proteins, enzymes, antigens, channels, receptors, and antibodies – inside or outside the cell, simply by switching the protein-coding cassette on the eRNA. The product is essentially a "script" that runs in a cell and directs it to produce the desired protein.
The result is programmable, persistent protein expression within the patient's body. Unlike other RNA forms that only last in the body for a short amount of time, eRNA is long-lasting, continually working in the body far longer than other RNA modalities, injected peptides, proteins, and antibodies. Additionally, eRNA is not immunogenic and can be repeatedly redosed, opening up the potential to treat chronic or lifelong genetic diseases. eRNA is also amenable to multiple routes of administration. Together, these features could translate to patient benefits, such as less frequent dosing, easier administration, higher efficacy per dose, and greater accessibility for patients.
"You don't have to strive for integration as a way to achieve permanence, like gene therapy, to get a therapeutic benefit," said Diego Miralles, CEO-Partner at Flagship Pioneering and CEO of Laronde since December 2020, "eRNA’s long-lasting protein expression constitutes an engine that will run continuously in the cell, allowing the provision of lifelong protein replacement through infrequent, repeated dosing."
"eRNA gives Laronde the power to create medicines, intentionally and predictably," Kahvejian said. "It is nothing short of a tectonic shift in drug development and the treatment of diseases."
Laronde has already optimized its methods for building and manufacturing eRNA at scale and will initially pursue targets with well-established biology, where there are multiple opportunities to develop best-in-class medicines. The platform's power and scale will also allow Laronde to process multiple programs and products in parallel as it rapidly builds out its infrastructure and diverse pipeline.
Miralles continued, "When I first heard about eRNA and Laronde, I thought: If this is true, it’s the Holy Grail. This can really change the world. Then I saw just how robust and reproducible the data were, and I knew I had to join to help build a great company that could have an enormous, positive impact on millions of patients around the world."