BALTIMORE – Flush with $12 million in recent seed funding, Harvard University biotech spinout EnPlusOne Biosciences is poised to usher enzymatic RNA synthesis into the commercial realm.
The Watertown, Massachusetts-based startup — with the backing of its cofounder, Harvard Medical School geneticist George Church — believes its enzymatic RNA synthesis platform technology, ezRNA, can "sustainably deliver therapeutic RNA at commercial scale." But it remains to be seen whether or when the nascent company will be able to start offering RNA products.
According to EnPlusOne Cofounder and CEO Daniel Wiegand, the company's enzymatic RNA synthesis technology was conceived during his time as a staff researcher in Church's lab at Harvard's Wyss Institute for Biologically Inspired Engineering.
"We realized that … RNA, in general, [is] very difficult to manufacture and access, so it really limited a lot of researchers out there," said Wiegand. "We wanted to build a platform to bring RNA to the world."
To develop the technology, about six years ago, Wiegand teamed up with Cofounder Jonathan Rittichier, now the CSO of the company, who was also a researcher in the Church group at the time. Later, Dan Ahlstedt, a fourth cofounder, joined the team from Harvard Business School about two years ago, and now serves as the firm's chief operating officer.
So far, most commercial RNA synthesis uses phosphoramidite chemistry. While the approach is "tried and true," Wiegand said, it does come with major limitations. One primary hurdle, he noted, is the ability to produce RNA at a large scale cost-effectively.
Because chemical RNA synthesis involves harsh reaction conditions, Wiegand said, a significant amount of time, money, and human resources are required downstream to purify the end products.
In addition, the highly protected building blocks in chemical synthesis can constrain the ability to produce modified RNA molecules for therapeutics. "The amount of actual therapeutic compound that you're trying to make is dwarfed by the amount of actual chemical structure you have to put on there to protect it," Rittichier said.
"What EnPlusOne is bringing is essentially a protecting-group-free kind of chemistry," he added. "Enzymatics can do cleaner reactions, and that's what EnPlusOne has."
The company has so far mostly kept the technical details from the public, but it has disclosed that the ezRNA technology is template-independent and can be carried out in an aqueous environment, which Wiegand said contains "cofactors, buffers — anything you need to get an enzymatic reaction to go."
According to Rittichier, EnPlusOne's enzymatic synthesis process involves two main steps for each cycle, during which enzymes incorporate NTPs into the growing oligonucleotides and a blocking group that prevents further extension is removed.
The cofounders did not reveal the name of the enzyme that incorporates the building blocks, other than saying it belongs to the family of terminal transferases. "We spent a long time at the Wyss Institute engineering this enzyme to be able to accept the special building blocks that we use to write the RNA," Wiegand said. "These enzymes are very picky; they don't like all the different pieces."
Previously, terminal deoxynucleotidyl transferase, or TdT, a vertebrate polymerase, has been commonly deployed for de novo enzymatic DNA synthesis. During that process, which also requires two main steps, TdT incorporates a nucleotide into the growing oligonucleotide, and the removal of a 3' reversible terminator on the last nucleotide enables the synthesis cycle to repeat. Wiegand said the company's enzyme is "kind of similar, but it is its own special thing that we engineered to do this work."
In terms of ezRNA's performance, each incorporation and deblocking step has 99.9 percent accuracy, which Wiegand said is comparable to that of the phosphoramidite chemistry. "We see that across the board for pretty much all the different bases and modifications that we are currently using to synthesize RNA," he added.
As for turnaround time, depending on what nucleotide is being added to the RNA molecule, a full reaction cycle could take half an hour to an hour, Ahlstedt said, although the team is trying to expedite the synthesis time. So far, on an automated system, the company has achieved up to 10 full cycles, according to Wiegand.
The platform can also accommodate a wide range of modifications when writing RNA molecules, which traditional chemical synthesis cannot easily achieve. "There are various modifications to the nuclear base that can't handle the deprotection conditions for phosphoramidite," Rittichier noted. "Since we are mild and in water, we can put these types of natural modifications on."
Wiegand said the ezRNA platform has so far achieved pretty much all modifications — such as 2′-fluoro or 2'-O-methyl — currently used in small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). "What's really interesting is that … we see that it can pretty much incorporate anything we throw at it," he added. "This is what we really think is the big promise of this technology."
Commenting on the method's cost, Ahlstedt said it is "very variable" and hard to nail down. For a plain, unmodified, and short oligonucleotide, "I think IDT is going to be beating us for a long time," he said, adding that when it comes to producing heavily modified RNAs, he believes EnPlusOne's approach would be "much cheaper." Meanwhile, for a moderately modified molecule, he thinks the costs are on the same order of magnitude.
"The platform is really intended to deliver novel modifications, difficult modifications, and RNA at scale for therapeutics," Ahlstedt said. "It tends to be the case that the cost of goods is not usually a consideration that these companies are worried about right now, it's usually the [ability to access] them."
He noted that EnPlusOne's current business model is to work with industry partners to manufacture RNA therapeutics with desired modifications that are not feasible or accessible at scale through chemical synthesis. To that end, Ahlstedt said the team has had "very good conversations" with players in the siRNA and the mRNA space, but he declined to name any existing or potential customers.
Regarding intellectual property, Wiegand said the company's enzymatic RNA synthesis technology is covered by one main pending patent, which is at "a very advanced stage" of the application process. According to the US Patent and Trademark Office database, a patent application titled Enzymatic RNA Synthesis (US20220145295A1) that lists Church, Rittichier, and Wiegand as co-inventors was filed in October 2019.
With $12 million in seed funding added to its balance sheet, which Ahlstedt said provides two years of runway, the company's goal moving forward is to continue developing the RNA enzymatic synthesis platform to be robust, reliable, and reproducible, the cofounders said. As part of that, the company is also hoping to make RNA of 100 nucleotides or greater in the next two years, but it did not offer a concrete timeline on when it will start offering products.
The firm, which currently has five employees, is also hoping to double its workforce by the end of next year, Ahlstedt said. In the long run, it also plans to become a large-scale Good Manufacturing Practice (GMP) manufacturer of RNA molecules, although the company did not disclose a concrete commercialization timeline.
Meanwhile, after years of development, the race to commercialize and supply enzymatically synthesized oligos still continues in the neighboring DNA field. A plethora of enzymatic DNA synthesis companies — including French startup DNA Script; University of California, Berkeley spinout Ansa Biotechnologies; San Diego-based Molecular Assemblies; and Twist Bioscience of South San Francisco, California — all have been using some version of engineered TdT to produce DNA de novo. So far, DNA Script is purportedly the only company that has reached the commercialization stage, with its Syntax benchtop DNA synthesizer launched under an early-access program earlier this year.
When it comes to enzymatic RNA synthesis, EnPlusOne's competition is still scant for now, although DNA Script has professed an interest in entering that space, as well. "Currently, we really don't have any competitors in the enzymatic RNA space," Wiegand said. "We are kind of in a unique space right now."