July/August 2022 • PharmaTimes Magazine • 28-29
// THERAPIES //
Synth revival
In the new world enzymatic DNA synthesis will accelerate drug discovery and development
While it may not be as visible as high-content screening or lead optimisation, DNA synthesis is a critical tool in the drug discovery process. The recent rise of mRNA-based vaccines and therapies has made gene synthesis even more important in discovery and development pipelines.
Decades ago, scientists found the process of synthesising DNA too error-prone, labour-intensive and hazardous to perform in-house. Since then, pharmaceutical and biotech researchers have ordered their oligos or genes from DNA synthesis service companies.
Unfortunately, outsourcing gene synthesis has drawbacks – especially for pharma and biotech scientists, who are constantly looking for ways to accelerate their efforts. Vendors operate with turnaround times that can span weeks or even months and higher-quality products tend to be very expensive.
But now, new technology advances have finally made it possible to put DNA synthesis capabilities where they belong – back in the lab. They are based on a new approach to building DNA that uses enzymatic processes rather than chemicals to construct oligos, genes or even entire genomes. With enzymatic DNA synthesis (EDS), scientists in pharmaceutical and biotech companies can safely bring gene synthesis into their workflows, giving them better control over timelines, costs and intellectual property.
The right notes
There are a number of areas throughout the drug discovery and development pipeline where it’s useful to synthesise DNA fragments, clones or even entire libraries of variants.
With the development of mRNA-based vaccines and therapies, for example, synthesising DNA constructs based on a target sequence can significantly shorten the timeline. In some cases, scientists have gone from identifying the sequence to having vaccine candidates ready for testing in just a few weeks.
In addition, scientists may use synthetic clones or variant libraries for discovery biology studies, engineering of proteins or antibodies and epitope mapping, among other key applications. A reliable supply of synthetic DNA can also support key activities in identifying, validating and optimising targets.
Synthesised DNA has been used to design and assemble nanobody sequences for potential use in vaccines, identify important T cells that can express specific receptors for the development of adoptive cell therapies and model disease with a CRISPR-enabled knockout system for specific tissues. An impressive roll call by anyone’s estimations.
In-house music
As synthetic DNA is increasingly used in drug discovery, the industry’s reliance on outsourcing is more and more problematic. Challenges associated with accuracy, cost, turnaround time, and waste have made it more important than ever to find a feasible, in-house approach to DNA synthesis.
Accuracy may be the most obvious of these limitations. DNA constructs are built in cycles, with each reaction based on what was built in the previous reaction. Because of that, even seemingly minuscule coupling inefficiencies get compounded with each additional cycle, eventually snowballing into relatively high error rates. Industry-leading accuracy rates might fall to just 60% after the repeated cycles required to build a simple 100-mer.
Subpar accuracy translates to another problem – high costs. In order to overcome low fidelity rates, scientists clone DNA fragments into vectors, assemble them and then sequence the resulting genes to find out whether the product actually matches the desired sequence. These extra steps add significantly to the cost of using synthetic DNA.
They also lengthen the time before DNA can be incorporated into the drug discovery and development pipelines. When added to the weeks or months it may take a vendor to build and ship the constructs, the overall turnaround time is unacceptable for an industry under pressure to accelerate the release of new therapies and vaccines.
Unfortunately, even if the cloning, assembly and sequencing steps could be eliminated, the chemical DNA synthesis techniques used by vendors are quite expensive. While the typical cost per base of about ten cents may seem trivial, it adds up quickly: ordering enough oligos to create an entire gene could cost about $300, while buying enough DNA to build a small genome could run to $200,000 or higher.
Last but certainly not least, conventional approaches to DNA synthesis rely on toxic chemicals that result in hazardous waste by-products. The purchase of a 96-well plate of oligos – enough to build a gene or two – generates huge amount of toxic waste.
It’s one of the reasons so few scientists choose to build their own DNA; properly disposing of this hazardous waste is difficult and expensive. But allowing service providers to synthesise more and more DNA using these environmentally destructive methods is not a sustainable solution either.
Much about chemistry
The challenges of conventional gene synthesis can be addressed by shifting to an enzymatic approach, which would also make it feasible to implement DNA and RNA synthesis capabilities in-house for on-demand use.
EDS-based technologies leapfrog their phosphoramidite chemistry-based predecessors, using natural or engineered versions of enzymes to create strings of nucleic acids in the desired sequence. Most EDS approaches rely on terminal deoxynucleotidyl transferase (TdT), a naturally occurring enzyme, in a process that involves no toxic chemicals.
But TdT-based techniques are hindered by the enzyme’s inefficiency and permissiveness in adding bases that may not be the right ones. Costs remain high because scientists must use significantly more reagents to overcome the efficiency bottleneck and implement time-consuming quality control steps to ensure accuracy.
Currently, synthetic DNA from TdT-based approaches costs an order of magnitude more per base than conventionally made constructs. Still, EDS technology based on TdT has much room for improvement. Continued efforts to optimise the enzyme or replace it will likely reduce costs and boost accuracy.
In our DNA
In the shorter term, a hybrid approach might offer more benefit. It pairs one-time use of a conventional gene synthesis vendor to build a universal library of small building blocks that can be contained within just a few high-density microtiter plates. That library serves as the foundation for the EDS-based part of the process, in which the building blocks are enzymatically stitched together into longer and longer oligos using an automated benchtop instrument.
This approach takes fewer reactions to build a construct, making it faster, more cost-effective and less susceptible to errors. And because unused building blocks from the universal library can be accessed over and over for millions of DNA synthesis cycles, the process minimises the production of hazardous waste – and ensures that the in-house, EDS-based workflow generates only harmless aqueous waste.
EDS technologies – whether they’re based on this oligo ligation approach or future iterations of TdT-based techniques – will be essential for restoring gene synthesis capabilities.
In the final analysis, producing transformative DNA or RNA for pharma labs in hours rather than months will help scientists get new therapies and ignite the market much more rapidly.
Dan Gibson is Chief Technology Officer at Codex DNA
PharmaTimes
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