The success of the Pfizer/BioNTech and Moderna COVID-19 mRNA vaccines has galvanized research on mRNA therapeutics. Rather than use mRNA to make antigens, the objective of mRNA therapeutics is to make proteins on demand inside patients’ cells within a tissue of interest at a level and duration sufficient to achieve treatment benefit. Applications are many and varied: enzyme replacement therapy for rare diseases, hormone production, monoclonal antibodies or immunostimulatory proteins for cancer, cytokines or transcription factors to treat autoimmunity. Linear mRNA is the template of choice, but it has drawbacks. Synthetic mRNA must be heavily modified to resist nuclease degradation and to avoid innate immune stimulation. Such mRNAs are inefficient to manufacture, difficult to properly incorporate into lipid nanoparticle carriers, and expensive. And modified linear mRNA is still relatively short-lived, limiting the amount of therapeutic protein produced per molecule delivered.
Circular RNA (circRNA) has emerged as an intriguing alternative, with Orna Therapeutics the most visible company advancing the technology. The circular structure can naturally arise from the ‘backsplicing’ of the splice site motifs flanking the 5′ and 3′ ends of pre-mRNA transcript exons. At first glance, circRNA is an unlikely drug platform. Until 10 years ago, such RNAs had been observed rarely in nature, as idiosyncratic forms of some RNA viruses or byproducts of splicing errors in eukaryotes—basically, transcriptional ‘noise’. They were thought to be translated into proteins only rarely.
Attempts to make artificial circRNAs, mainly to study their in vivo properties, date back at least to the 1980s. Biologists have long observed that circRNAs are long-lived; because they lack 3′ and 5′ ends, where nucleases generally initiate RNA degradation, they’re more stable than linear mRNAs. But only short RNAs could be made artificially, “so the field kind of died in the nineties,” says Orna co-founder and director of molecular biology Alex Wesselhoeft.
Wesselhoeft, as a PhD student in the lab of MIT biomaterials researcher Daniel Anderson, took on the challenge. Chemically synthesizing an entire mRNA sequence in vitro and then using ligases to circularize it is inefficient. Wesselhoeft adapted an enzymatic approach that splits a cyanobacterial ribozyme sequence in two and pastes it backwards onto an mRNA sequence, with the 3′ end upstream of the gene to be expressed and the 5′ end downstream. The mRNA then autocatalytically splices itself into a circle during in vitro transcription, without the need for the usual cellular protein splicing machinery. Wesselhoeft added spacer and duplex sequences that, by dampening internal splicing interference and bringing the ends together, boosted circularization efficiency for large molecules, up to 5 kilobases in length. Orna has since pushed this to over 10 kb.
Wesselhoeft confirmed that these circRNAs persist in cells longer than modified mRNA typified by the Pfizer and Moderna vaccines. The circRNA also produced larger amounts of protein than modified linear mRNA, over a longer period, in part due to the discovery of new cap-independent RNA translation sequences (IRESs, or internal ribosome entry sites), RNA elements that recruit ribosomes to internal regions of RNA. An IRES is considered less efficient than the canonical translation that begins at the 5′ cap of linear mRNA, but as circRNA lacks a cap, Wesselhoeft inserted IRES sequences and discovered that some performed better than a cap.
“What we saw was actually if you pick the right IRES, you could get a lot of protein out,” he says. “That really got us thinking that maybe this was a new technology that could take the entire field of mRNA to the next step.”
On the basis of these findings, in 2019 Wesselhoeft, Anderson and economist Raffaella Squilloni, previously an entrepreneur in residence at Harvard, founded Orna, which was seeded by MPM Capital. A $80-million series A closed in February 2021. Orna had raised a total of $120 million by April 2022 and employs >70 people at its Cambridge, Massachusetts facility.
CircRNA “is theoretically a really intriguing idea,” says Yale University RNA biologist Carson Thoreen. “Definitely the evidence out there so far does indicate that these are really much more stable molecules, and I think there’s potential that they’re much less immunogenic than their linear counterparts. The challenge is really engineering ways to maximize the amount of translation and protein production that can occur.”
Orna’s lead program is ‘in situ CAR’ (isCAR) therapy for cancer. CARs, or chimeric antigen receptors, express an antibody-like fusion protein linked to a T cell receptor intracellular domain, using antigen binding to activate T cells against the target. Five autologous engineered CAR-T cell therapies are FDA approved for blood cancers, all involving ex vivo cellular engineering via lentiviral transduction before reinfusion. Pre-treatment chemotherapy-induced lymphodepletion creates a niche for CAR T cell expansion. Besides the enormous expense, “it’s hard to control how much expansion of those cells has taken place inside the patient,” says Orna CEO Thomas Barnes. Runaway expansion leads to cytokine release syndrome, a severe side effect.
Orna uses immunotropic (immune-cell specific) lipid nanoparticles, in-licensed from an unnamed academic investigator, to deliver the CAR-encoding circRNA in vivo. With this system, “biomanufacturing takes place inside the patient,” says Barnes. That makes immune cell expansion more predictable. Unlike current CAR-T therapies, “it’s not a living drug,” says Barnes. “It’s going to behave like a classic drug … with a half-life; it’s going to have peak expression, and it’s going to taper off. And, lastly, no lymphodepletion.” The result, in theory, is a cheaper and safer treatment.
Orna has now shown that its circRNA can drive protein expression to levels that have therapeutic value in an animal model of human disease. At the May meeting of the American Society of Gene & Cell Therapy, Barnes reported that five doses of Orna’s anti-CD19 isCAR fully eradicated tumors in a mouse xenograft model of acute lymphoblastic leukemia.
Barnes suggests that its immunotropic lipid nanoparticles can also deliver circRNAs for autoimmunity. “And we have a second delivery solution that we’ve in-licensed that is primarily hepatropic, but also myotropic to a lesser degree,” he says. At the ASGCT meeting, Barnes reported that its lipid nanoparticle, delivered intravenously in a mouse model of Duchenne muscular dystrophy, could induce limited expression in muscle of a short version of the dystrophin protein.
CircRNA has two main theoretical concerns. One is that Orna’s circularization method unavoidably leaves behind small fragments of cyanobacterial ribozyme mRNA at the ligation junction. “It’s possible that some of those remnants may, at least in some contexts, be immunogenic,” says Thoreen. “That would be bad from any kind of therapeutic standpoint because they’ll limit any kind of protein that could be produced from those RNAs.” Such an outcome is very unlikely, counters Wesselhoeft. The sequence remnants “are relatively short and noncoding,” he says. “You’re not going to be creating a new synthetic peptide in the cell, through translation.”
The other theoretical concern is an immune response against the circular RNA itself, which would also limit translation. Wesselhoeft and Anderson have reported that unmodified circRNA is less immunogenic than unmodified, capped linear mRNA in cells by monitoring cytokine and chemokine release into the culture medium and circRNA protein expression stability and in vivo translation. However, the process of making circRNA generates noncircular byproducts — linear mRNA, double-stranded RNA — that do trigger an immune response. But Orna maintains that circRNA, once rigorously purified, does not. “The circles can be purified without this other stuff present,” says Barnes. And “the stimulation is not there.”
But whether purification is enough somewhat controversial. “Other people have disputed that,” says Thoreen. “I think part of the uncertainty really derives from uncertainty over what it is in the first place that’s triggering that response. So it could be that there are some sequences of RNA, it doesn’t really matter whether they’re in a circular context or a linear context, they’re still going to be immunogenic.”
If the circRNA story plays out as Orna expects, it could become a highly competitive platform for mRNA therapeutics. “Circles are the way to go,” says Barnes. “In every way they’re advantageous: they’re cheaper, faster, you can make them larger, you can express higher, they formulate better. It’s the gift that keeps on giving.”