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DNA Script Developing Enzymatic DNA Synthesis Technology

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NEW YORK (GenomeWeb) – French startup DNA Script has been developing a new technology for synthesizing DNA that relies on modified nucleotides and enzymes. The firm plans to start an early-access program for its platform, which promises to be complementary to traditional chemical synthesis and have a variety of applications, as early as the end of 2019.

Last month, the Paris-based company said that it had reached a technical milestone — being able to synthesize a 150-base oligonucleotide with up to 99.5 percent efficiency per base addition, essentially equivalent to traditional chemical synthesis.

The firm also announced the opening of a US subsidiary in Cambridge, Massachusetts this month, along with three new members to its executive team, and it plans to at least double its headcount over the next 18 to 24 months.

DNA Script joins a number of other companies that are developing enzyme-based alternatives to the well-established chemical DNA synthesis process, among them Molecular Assemblies in San Diego and University of California, Berkeley-spinout Ansa Biotechnologies, which each have developed their own variation of the approach and have secured related intellectual property.

"There is an urgent need for improved methods of oligonucleotide synthesis, and DNA Script's recent progress represents an important milestone for the field," said Dan Arlow, CEO of Ansa Biotechnologies.

DNA Script was founded in 2014 by three engineers — CEO Thomas Ybert, COO Sylvain Gariel, and CTO Xavier Godron — who wanted to apply their expertise in the life sciences. All three had met at French energy company Total, which at the time was interested in engineering microorganisms to turn them into oil producers. "We are really coming from the synthetic biology industry," said Gariel, adding that their vision for the company was to be able to "program a microorganism the way you program a computer."

What they saw as one of the limiting steps, he said, was the ability to design and synthesize DNA as easily and with as much automation as reading DNA with a next-generation sequencer. "The reading part of biology was becoming easier and easier, but on the other end, the writing part was still more difficult," Gariel said. "Our dream was to build a synthesis box that would do the exact inverse of an Illumina [sequencing] box, which is to make DNA."

To do that, they decided to move away from chemical synthesis that relies on nucleoside phosphoramidite building blocks — an approach that has been the standard for the past several decades — and focus on an enzymatic DNA synthesis method. After developing their concept on paper, "the first thing we did, we created a company, filed a few patents, and started working on the technology," Gariel recalled. However, after getting some initial results quickly, "we realized that we needed a lot of academic knowledge on the building blocks of the technology," specifically polymerase enzymes and modified nucleotides.

For that, they teamed up with researchers at the Pasteur Institute in Paris who had been studying polymerase enzymes for the last 40 years or so. "We're kind of a strange case in the sense that we're not a spinoff but kind of a spin-in," Gariel said, allowing the company to benefit from the expertise and resources of the academic institution.

After spending more than three years within the Pasteur Institute, DNA Script moved into its own space, about a 10 minute drive from the institute, and has since grown to about 35 employees.

The firm also has raised $27 million in funding so far. Initially supported by business angels, DNA Script in 2016 raised €2.5 million ($2.8 million) in seed funding from Sofinnova Partners, Kurma Partners, and Idinvest Partners. This was followed by a $13 million Series A round that was led by Illumina Ventures, with additional participation from Merck Ventures and existing investors. This spring, the company received $5.5 million in grants and refundable advances from the European Union's Horizon 2020 program and from Bpifrance, a French government bank, followed by a $2.7 million innovation grant from Bpifrance this summer.

Given its product development plans, the company might need to raise additional funding, but probably not before the end of 2019, Gariel said.

DNA Script's enzymatic synthesis workflow starts with a DNA primer with a free 3' end that is bound to a solid support in a reaction chamber. Next, engineered polymerase and 3'-protected nucleotides — also known as reversible terminators — are added and the enzyme incorporates a single nucleotide into the DNA strand. In the next step, the blocking group is removed from the 3' end of the DNA, followed by a wash step, and the cycle starts again.

Gariel said the company uses different types of polymerases, which tend to be phylogenetically related. They include terminal deoxynucleotidyl transferase (TdT), the same enzyme that is used by both Molecular Assemblies and the UC Berkeley researchers and its spinout. However, unlike the Berkeley group, DNA Script does not tether the enzyme covalently to the nucleotide, and the DNA does not carry a molecular "scar" after the protecting group is removed.

The modified nucleotides the company utilizes have a protecting group at their 3' end that prevents the polymerase from adding more than one base at a time. "We have a favorite chemistry" for these, Gariel said, but he declined to reveal specifics. The firm has exclusive licenses related to both its enzymes and nucleotides, he added.

Earlier this year, for example, DNA Script exclusively licensed technology around 3'-protected nucleotides from Dynamic Combinatorial Chemistry (DCC) and signed an agreement allowing it to commercialize additional technologies from DCC. Those modified nucleotides were originally developed by Steven Benner, a researcher at the Foundation for Applied Molecular Evolution, and developed by DCC's affiliate, Firebird Biomolecular Sciences. In addition, DNA Script co-patented enzymes it jointly developed with the Pasteur Institute. 

An important aspect of the technology was to make sure the 3'-protected nucleotides terminate the polymerase reaction completely. However, good nucleotide terminators tend to be poorly incorporated by natural polymerases, Gariel explained, so the company also needed to engineer the enzymes to work well with the 3'-protected nucleotides.

Last month at the SynBioBeta 2018 conference, DNA Script presented data showing that it could synthesize a 150-base oligonucleotide, with each addition reaching up to 99.5 percent efficiency, similar to chemical DNA synthesis. By next year, the company said, it expects to synthesize oligos of several hundred nucleotides, and eventually up to a kilobase.

Ansa's Arlow said there are two main challenges to DNA Script's approach. One is getting the TdT enzyme to incorporate modified nucleotides "in a reasonable amount of time" and the other is ensuring the stability of the blocking group during the extension step and the clean removal of the group in the deblocking step, also within a short amount of time. "For accurate synthesis of long DNA molecules, both of these steps must consistently have extremely high yields (>99.5 percent), and the deblocking step must not damage DNA," he said.

Ansa, for its approach, didn't have to modify the TdT enzyme, which is covalently bound to a nucleotide, and has shown that it can add a single nucleotide within 10 to 20 seconds. On the other hand, Ansa's approach requires a larger amount of enzyme, making it more expensive, but unless DNA needs to be synthesized in milligram quantities, "we do not believe that enzyme costs will be limiting," Arlow said.

Engineering the polymerase is a particularly difficult project, he said, and "it is an open question whether it will be practical to engineer the enzyme to have a similar speed on the [reversible terminator nucleotides] as wild-type TdT."

Gariel said that each nucleotide addition cycle takes DNA Script around five minutes at the moment, which the firm is working to reduce to three and eventually to two minutes.

The company currently uses different types of hardware in its laboratory, ranging from a standard oligo synthesizer to customized instruments, and manufactures oligos in 96-well plates. It then analyzes the quality of the products by gel electrophoresis, next-gen sequencing, and mass spectrometry. It currently synthesizes about 1,000 oligos a week, Gariel said.

The company has also started working on a commercial hardware platform and plans to ship a precommercial instrument to beta testers by the end of 2019 or early 2020. "We're very much focusing on molecular biology applications," Gariel said, adding that "there is already significant interest from people in the molecular biology, genomic, and synthetic biology field. Our goal is to be the first reaching a commercial stage with the instrument that we're working on."

Besides improving the performance of the chemistry, one challenge will be to make the technology robust enough so it can work in an industrial environment. "There is a lot of development work to be done to make sure that our technology is 99.9 percent reliable, because you want to be able to run this in an instrument that's going to be on the bench of your customers," he said.

The company plans to reveal details of its technology at some point after launching its platform but wants to focus first on publishing applications with academic partners. "It's our secret sauce and we want to make commercial use of this technology, so we have to protect … the advance we think we have over our competitors," Gariel said.

DNA Script plans to focus initially on applications in molecular biology and genomics but doesn't disclose specifics at the moment. After that, the plan is to move to applications related to protein engineering and synthetic biology "in the wider sense of the term," and, longer term, RNA drugs, whole-genome synthesis, and DNA data storage, Gariel said.

Also, while DNA Script is developing its own hardware platform for certain markets, it is considering partnering for specific applications with others, such as Twist Bioscience or Agilent Technologies, which already have such a platform. "High-throughput hardware is often very tricky, and this is something we're interested in from a business standpoint," Gariel said.

"We're really excited to be part of this new generation of DNA synthesis technologies," he said, which has advantages in many areas, such as making DNA libraries, synthesizing very long fragments, and offering fast turnaround times. "It will not necessarily replace phosphoramidite chemistry," he said, which still has advantages in areas such as modified DNA, "but I think it's very complementary."

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