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Ansa Biotechnologies Accelerating Enzymatic DNA Synthesis Technology as a Commercial Service


BALTIMORE – Fresh off an oversubscribed $68 million Series A financing, Ansa Biotechnologies is poised to commercialize its enzymatic DNA synthesis technology into a service in the increasingly crowded synthetic DNA race.

The Emeryville, California-based synthetic biology company is developing a polymerase-nucleotide conjugation approach for de novo DNA synthesis. Like other polymerase-based methods, the approach relies on an engineered terminal deoxynucleotidyl transferase, or TdT, to fuse nucleotides together without a template one at a time. But instead of attaching a reversible terminator to the 3' end of the nucleotide to curb each cycle, Ansa tethers the nucleotide to the TdT enzyme via a molecular linker to prevent further additions.

The company claims this method provides "significant advantages" in terms of cost, speed, and flexibility over other enzymatic DNA synthesis practices — but it is not ready to disclose specific performance metrics publicly at this point.

A University of California, Berkeley spinout, Ansa was created by cofounders Daniel Lin-Arlow and Sebastian Palluk based on their graduate work on enzymatic DNA synthesis. The duo started to work together at UC Berkeley in 2015 and described their invented method in a 2018 Nature Biotechnology paper.

According to Lin-Arlow, the tethering approach that Ansa uses comes with several advantages. For one, he said it is "really hard" to get TdT to work with 3' blocked nucleotides. By eliminating the need for reverse terminators, the enzyme can incorporate the nucleotide into the growing oligo more efficiently. In addition, he argued there are not a lot of three prime blocking groups that are readily compatible with TdT. Meanwhile, he said the "huge literature" on existing linkers affords researchers "an enormous chemical space" to search for a best-performing linker within the capacious enzyme cavity where the linker is anchored.

That said, Lin-Arlow noted that Ansa has already tested hundreds of linkers to narrow down the optimal candidates. The company also "genetically introduced" an attachment site on the TdT enzyme and developed conjugation chemistry that is "extremely specific" to that residue to ensure only one nucleotide can be attached to each TdT, he said.

While Lin-Arlow declined to detail the specific performance of Ansa's technology at this point, he purports the company's approach has the highest cycle efficiencies of any methods that he is aware of. He also claimed the method is "unambiguously superior" in terms of length and accuracy compared to the ​​traditional phosphoramidite-based chemical DNA synthesis.

Perhaps, one potential pitfall for linker-based technology is that it tends to leave residual atoms on the nucleotide, or a scar, after cleavage, possibly obstructing the downstream workflows. To that, Lin-Arlow said the company has developed conjugate sets that both leave scars and do not, though of varying efficiencies.

Lin-Arlow acknowledged that scarred DNA may impact certain applications that require raw synthesized oligos, such as primers. But he said primer production is not really a focus for Ansa. He further argued that for gene synthesis, which is a major area of interest for Ansa, the scars will not be a concern since the initial oligos will be PCR amplified post-synthesis, producing natural, scarless DNA for the downstream.

But, according to Emily Leproust, CEO and cofounder of synthetic DNA maker Twist Bioscience, post-synthesis PCR using scarred DNA will likely have a higher error rate compared with using native DNA. "The scars on each base will interfere with the intramolecular bonds as the bases pair during the PCR steps," said Leproust in an email. "This will likely mean a higher risk of mutation, insertion, or deletion."

Based in South San Francisco, California, Twist also announced earlier this year its own linker-based enzymatic DNA synthesis method. However, Twist claimed its method is scarless, fabricating DNA identical to its natural counterpart. "For Twist, we opted to skip that risk entirely by creating scarless DNA," Leproust said.

Moreover, she said post-synthesis PCR necessitate primers, which may further complicate downstream applications as they must be removed before the next steps.

Similar to Ansa, Twist also declined to disclose the overall error rate or the length of oligo achieved by its enzymatic synthesis method. But Leproust said she expects the technology, when fully optimized, to match or exceed the error rate of Twist's current phosphoramidite DNA synthesis, which is 1 in 2,000 for oligos up to 300 bases and 1 in 7,500 for non-clonal fragments.

Agreeing with Leproust, Thomas Ybert, CEO and cofounder of DNA Script, also thinks the post-PCR synthesis step can be cumbersome. "Of course, you can do post-[synthesis] processing, but it's going to increase complexity, increase price, and it's not going to be amenable to any type of business model or any type of user," he said.

Having already commercialized its enzymatic DNA synthesis technology into its Syntax benchtop DNA synthesizer, DNA Script's approach, unlike Ansa's and Twist's, involves a 3' reverse terminator to switch on and off nucleotide incorporations during synthesis.

Ybert acknowledged that it may be the case that there are fewer reverse terminators compatible with TdT. However, he said that is not really a concern for DNA Script since the company has already locked in a proprietary reverse terminator group, ONH2, that is exclusive to DNA Script.

While Lin-Arlow emphasized that stability and deprotection efficiency are the major chokeholds for a reverse terminator to achieve high-fidelity DNA synthesis, Ybert noted both are not issues for DNA Script's blocker.

According to Ybert, with DNA Script's deprotection step, nucleotides that remain protected are below the level of detection. As for stability, he said DNA Script has conducted "extensive stability studies," including expedited studies where nucleotides are exposed to harsh temperature or chemical conditions as well as real-time studies to evaluate the nucleotides' optimal storage or handling condition.

Meanwhile, Ybert said the linker-based approach does not inherently change the equation and faces the same stipulations, namely, the linker also has to be solidly stable and cleaved efficiently to prevent synthesis mishaps.

Lin-Arlow said the company observed the best cleaving results when the linker was digested enzymatically. But he did not disclose the detailed linker enzymology, other than saying it is a type of hydrolase that works under neutral pH.

He also said Ansa's enzymatic cleavage approach, which is congruous with the company's goal to achieve fully enzymatic DNA synthesis, protects DNA from being damaged by the inclement chemical conditions often deployed to deprotect the reverse terminators.

But Ybert tends to disagree. Any reaction, if not done right, has the potential to damage the DNA, he said, while he argued that the fact that some linker-based methods are leaving scars is already damaging the DNA.

Unlike DNA Script, which primarily focuses on the instrument market, both Twist and Ansa said they plan to pursue a service model for their enzymatically manufactured DNA in a centralized facility. "We're not going to sell the [DNA] printer," said Lin-Arlow. "It's a lot more complicated and expensive to build an instrument that works in someone else's hands."

He added that a centralized production system with standardized quality control can ensure high-quality DNA being shipped to the customers. Most importantly, he said the model allows the company to screen for potential biosecurity hazards, avoiding the technology ending up in the wrong hands.

Ybert said DNA Script, though pursuing a decentralized model, is also taking the biosecurity issues "very, very seriously." He said the company has hired a dedicated biosecurity team and is working with regulatory agencies both in Europe and the US.

According to Ybert, DNA Script's two-pronged strategy for biosecurity involves screening for customers first and then screening for their sequences. "Everyone who is using Syntax is sending information to our cloud platform," he said, adding that the company is keeping tabs on the DNA sequences rendered to the platform for synthesis.

In addition, Ybert said the Syntax platform is a closed system, meaning the company will only sell the proprietary reagents to registered accounts. Without the reagents, people will end up with "a very expensive piece of metal" that they cannot use, he said.

With the money injected from its Series A funding round, Lin-Arlow said Ansa is geared up to build high-throughput synthesizers for internal use so the company can generate enough DNA to launch its commercial service.

The company, which currently has more than 30 employees, also plans to expand its R&D and manufacturing facilities and grow its workforce across biochemistry, chemistry, engineering, bioinformatics, and operations.

While Ansa currently places an emphasis on gene synthesis and considers industry researchers its first target customers, Lin-Arlow said the company is also increasingly drawn to pharmaceutical applications, especially producing longer and more complex DNA constructs for vaccine development, gene therapy, cell therapy, and biologic drugs discoveries.

Although the company has yet to ship any enzymatically synthesized DNA to customers, Lin-Arlow said Ansa is in discussions with several companies about pilot projects.

He also declined to provide a clear commercialization timeline at this point. "What I can say is that this [Series A] money will get us to launch," he said. "But I'm not ready to give you a launch date yet."