Dr Tim Brears, CEO, Evonetix and Dr Raquel Sanches-Kuiper, Director of Biology, Evonetix explore applications of synthetic DNA in drug discovery and development, the benefits and limitations of current methods, and the opportunities offered by emerging DNA synthesis technologies.
On 16 April 16, 1956, Arthur Kornberg and his team of biochemists first isolated the enzyme now known as DNA polymerase I1, giving scientists the power to make DNA in the lab for the first time. Since then, DNA technology has developed exponentially and with it the ability to understand life and health. Science tends to progress in line with the tools available, and while DNA sequencing and genome editing have developed exponentially over the past two decades2, advances in the technologies underpinning DNA synthesis has been slow3.
Inherent limitations in current methods have prevented accurate, fast, scalable DNA synthesis. The most commonly used methods require significant hardware and post-synthesis quality control, meaning that DNA synthesis is typically outsourced to centralised services, taking control away from researchers and preventing quick iteration of experiments. Although there have been significant gains in recent years, centralised DNA synthesis providers must still balance speed with accuracy and throughput.
However, recent advances offer the opportunity for the full potential of synthetic biology to be realised for drug discovery research. The coming generation of chip-based technologies aim to provide quick, accurate DNA synthesis in a desktop device, completing the final component of the DNA toolkit and giving researchers full control over their experiments.
Here we explore the various applications of synthetic DNA in drug discovery and development, the benefits and limitations of current methods, and the opportunities offered by emerging DNA synthesis technologies.
Historically, drug discovery has relied largely on mining chemical libraries of small drug-like molecules, hoping to find a compound providing the perfect fit for the target of interest. Over the years, there has been a shift from screening natural products as a source of bioactive compounds to combinatorial and medicinal chemistry approaches, where additions or subtractions to the core molecular scaffold aim to optimise binding or reduce off-target effects.
While this has worked in many cases, the struggling pipelines of pharmaceutical companies aiming to develop truly novel antibiotic therapies against drug-resistant pathogens provide a startling glimpse into just how challenging finding novel compounds and targets can be4.
High-throughput screening is yet to realise its initial promise. The success of this approach is dependent on the quality of the compound library being screened, and chemical proteomics data now show that even the most apparently specific compound can have multiple off-target effects 5,6.
This deeper biological understanding has led to a transition from the ‘one drug, one target, one disease’ approach pioneered by Paul Ehrlich a century ago7 to a more subtle understanding of small molecules as complex drugs that bind multiple targets, have myriad potential toxic side effects and that are challenging to find even using high-throughput screening technologies.
Initial forays into a synthetic biology approach to drug discovery include the breakthrough finding that microorganisms and plants use huge molecular units to produce secondary metabolites, and that these enzymatic units can be artificially modulated in synthetic cells to produce new molecules with therapeutic potential8,9. This led to the first real use of synthetic biology in drug discovery, creating new chemical scaffolds with orthomolecular properties that increase the chances of being effective and non-toxic10.
The potential scope of synthetic biology is not just to improve on old techniques but to think of drug discovery in a new light. The ability to engineer genes and molecules to order, opens up new avenues for nano-drugs, vaccines and CRISPR-based gene therapies. Modified CAR-T cells that have been genetically engineered to produce specific receptors are already proving clinically successful11,12,13. However, the rate limiting step currently preventing these approaches from reaching their full potential is still the need to quickly and accurately produce large amounts of synthetic DNA on demand.
A new era of DNA synthesis
The core technologies for synthesising DNA today rely on phosphoramidite synthesis, a chemical method first established in the 1980s. The process builds a DNA strand by adding one nucleotide at a time until the desired sequence is achieved. While relatively efficient, the reaction requires a steady supply of unstable chemicals that makes synthesis costly.
Phosphoramidite synthesis has an accuracy of approximately 99.6%, which drops as the length of the synthesised strand increases, limiting the output to relatively short DNA lengths of around 200-300 nucleotides. Building longer DNA strands hugely increases the time required to identify error-free DNA in post-synthesis quality control.
It is also challenging to create certain types of DNA using this process, such as repetitive sequences14. GC-rich sequences require additional processing due to their tendency to form complex secondary structures. This is far from a minor issue, given that GC-rich regions are found in more than 60% of gene promoters in higher eukaryotic organisms, the majority of tumour suppressor genes, housekeeping genes and around 40% of tissue-specific genes15. Enzymatic DNA synthesis methods have gained interest in recent years, with potential for generating longer sequences, although the efficiency and accuracy of these techniques remain open questions.
Rather than iteratively improving current technologies, the next generation of DNA synthesisers is creating a paradigm shift in how and where DNA can be synthesised. New methods create DNA strands using multiple parallel micro-reactions on silicon chips, compatible with both chemical and enzymatic synthesis techniques. The coming generation of desktop DNA synthesisers are based on precision thermal control chips with thousands of independently temperature-controlled ‘pixels’ or ‘virtual wells’, each one of which acts as a DNA synthesis site within a continuously flowing liquid.
Base by base, DNA strands are built and selectively released from the surface for on-chip assembly. Error correction is inherently built into the process through thermal purification: any mismatches between partially assembled DNA strands are detected and removed at a specific temperature, weeding out strands with mistakes without the need for time-consuming and costly complex post-synthesis clean-up processes, such as cloning, screening and sequencing. The end result is a high yield of high-fidelity DNA generated through an automated assembly process.
As well as scaling up production of accurate, long strands of DNA through parallelisation, this silicon chip approach also offers the opportunity to shrink down the synthesis devices themselves. The coming generation of desktop synthesisers will open up access to synthetic DNA, making biological engineering as accessible and widespread as next-generation sequencing.
For drug developers, there are applications from high-throughput screens to the rapid evolution and prototyping of new genes, pathways and molecules, such as enzymes or antibody variable domains. Experimental nucleic acid-based therapies could be rapidly synthesised including CAR-T, CRISPR guides, vaccines and eventually RNA.
DNA synthesis could also yield results for precision and stratified medicine approaches, with rapid design and synthesis of personalised custom DNA probes to identify specific genetic features via liquid biopsy, followed by interrogation with next generation sequencing for diagnosis or monitoring of response.
A powerful tool for drug discovery research
The coming wave of rapid, scalable, accessible DNA synthesis offers exciting opportunities for researchers working in drug discovery and development.
Library generation and screening
Firstly, DNA synthesis could aid the traditional drug discovery process by generating libraries of genetically encoded molecules such as peptides. Although it is possible to chemically synthesise some (but not all) of these kinds of compounds, genetic encoding hugely simplifies this process much easier. It also makes hit identification and deconvolution more straightforward, particularly in combination with DNA barcoding. One successful example comes in the form of DNA-encoded peptide or chemical libraries, which have significant potential to solve long-standing issues in drug development for challenging targets such as protein-protein interactions 16,17.
This concept of DNA-encoded library screening is enhanced further through the use of randomly generated or rationally designed synthetic oligonucleotide libraries. These allow unique DNA ‘barcodes’ to be generated for high-throughput screening applications, such as sequencing, genetically encoded compound screens and much more. As well as being used to identify compounds, the DNA barcode can contain additional information, such as the template used to make that particular molecule 18.
From the ability to create synthetic gene circuits to evolving and screening enzymes for therapeutic use, access to rapid, accurate DNA synthesis allows faster and more efficient profiling of many more targets and leads than would not be possible otherwise. For example, synthetic gene circuits in bacteria can be constructed to probe the complex interacting pathways, genetic variants and stochastic molecular fluctuations that lead to antimicrobial resistance19.Such circuits are challenging to construct quickly, accurately and cost effectively using conventional methods, but access to rapid, reliable DNA synthesis could allow creation of multiple versions of genetic components with greater diversity and quick optimisation.
Moving along the drug discovery journey, once a hit compound has been identified it is still necessary to make sufficient amounts for further study and ultimately for commercial manufacture. This can be challenging, particularly for naturally occurring compounds and complex biomolecules that are difficult to recreate through chemistry alone9.
One example is terpenoids – a large and diverse group or organic plant-derived molecules, many of which have interesting biological properties. The antimalaria drug artemisinin is a terpenoid that is costly and difficult to isolate from the natural source and hard to make through chemical synthesis. Instead of isolating from nature, scientists have genetically modified the yeast Saccharomyces cerevisiae to produce artemisinic acid, a precursor of artemisinin, making this life-saving drug much quicker and cheaper to manufacture 20,21.
Another example is paclitaxel – one of the most successful anti-cancer drugs to date, which is manufactured from raw material harvested from mature yew trees. Adequate supply of paclitaxel is a serious issue, and many species of yew are now endangered as a result 22,23.
While recombinant DNA is an established tool for creating biologically active compounds in new hosts, this could be greatly enhanced by the use of synthetic DNA technology to generate large numbers of enzyme variants for high-throughput screening or evolution studies. Chassis organisms require the introduction and testing of multiple genetic components, which would be significantly easier with rapid, accurate DNA synthesis 34. Once optimised, therapies could be produced quickly, accurately and at scale. This would not only allow the development and manufacture of novel therapeutics but could also identify more cost-effective, efficient and sustainable ways to make existing drugs 24.
Genome engineering also holds great promise in the drug discovery research landscape. The first synthetic gene was created 40 years ago, and the capacity to construct DNA sequences has doubled every three years since then. Plasmids were constructed in the 1990s followed by viruses in the early 2000s, then gene clusters and bacterial chromosomes. Research teams have constructed whole genomes of Escherichia coli and Salmonella typhimurium, each around 4 Mb.
Now the Genome Project-Write (GP-Write), formed in 2016 with groups from industry and academia, aims to engineer chromosomes and even whole genomes of higher eukaryotes, with a specific goal of developing a virus-resistant human cell line. Being able to engineer pathogens or cells with partly or whole synthetic genetic components, offers new opportunities for studying the underlying mechanisms of disease and revealing new drug targets 25.
CRISPR/Cas9 genome editing is a fast-growing area of research, with the market for this therapeutic gene editing technologies predicted to reach $4 billion by 2024. While CRISPR technologies are already revolutionising how scientists can manipulate the genetic code of an organism to better understand function, adding the power of DNA synthesis to genome engineering opens up even greater possibilities.
CRISPR and other gene editing tools require the production of guide RNAs, which are created from DNA templates. As a research tool, rapid on-demand synthesis of many thousands of different templates across the whole genome enables efficient unbiased target identification and validation through functional genomics approaches, giving new insights into how to target molecular processes to prevent or cure disease 26.
One area of interest is synthetic lethality, building on the success of PARP inhibitors in BRCA-deficient cancers27. DNA synthesis at greater scale with sequence freedom and accuracy would enable unbiased systematic functional analysis of the combined effects of thousands of variants across a genome and revealing novel pathways for therapeutic intervention.
Opportunities for novel therapeutics
Along with the tools to do better research in the pursuit of novel drugs, synthetic DNA technology also has a more direct role to play in the therapeutic arena.
Gene and cell therapies
The most obvious application here is DNA itself, in the form of gene therapy, which is being investigated for a wide range of diseases from metabolic disorders to skin conditions. Aided by developments in delivery technology, such as lipid and polymer nanoparticles, and gene editing tools like CRISPR, the ability to reliably synthesise DNA to order opens the door to the rapid production of custom genetic therapeutics.
Oligonucleotide DNA aptamers, from 20 to 60 nucleotides in length, have attracted a lot of attention for their ability to specifically bind to targets from inorganic molecules to large protein complexes or even whole cells, with significant diagnostic and therapeutic potential. While the basic capacity to synthesise aptamers has been available for around two decades, the ability to do so quickly on demand offers significant potential in the drug discovery arena28,29.
Cell therapies are a promising modality for currently incurable or hard to treat diseases and personalised therapies, with autologous CAR T cell therapies already proving their value for treating certain types of cancer in the clinic 30. CAR T cells are created by delivering DNA via plasmids or viral vectors into the patient’s own immune cells in vitro – an expensive and time-consuming technique that puts the technology out of reach of many.
A means of quickly producing CAR T cells would be of huge benefit and allow faster research into effective means of using these cells. Promising recent results suggest that synthetic DNA nanoparticles can achieve T cell reprogramming in vivo, opening up the exciting possibility of ‘on demand’ cell therapies for cancer patients at lower cost and faster speed than current approaches 31.
There are also exciting applications for ‘bugs as drugs’ 32 – genetically modified microbes designed to detect infections or disease and produce biologically active molecules. Not only does the gut microbiome play a role in digestive health, metabolism and infection, but it is also increasingly linked to many other aspects of wellbeing, including immunity and mental health.
Microbial communities elsewhere in the body, including the mouth, skin and vagina, also have close connections to health and disease. Using synthetic DNA technology to rationally design and synthesise novel bacterial genes and genetic circuits is a powerful tool for exploring this novel therapeutic space.
Antibodies, enzymes and molecular therapeutics
Not only are antibodies versatile tools in detection assays and diagnostic tests, they can also be used as therapies for cancer, autoimmune conditions and other diseases33. Monoclonal antibody drugs represent a global market of greater than $100 billion and over 80% of biopharmaceuticals. Discovery of novel antibodies often involves high-throughput phage display library screening to find those with the required characteristics34.These libraries currently suffer from issues with accuracy and sequence bias, where certain antibodies cannot be created because of technical limitations, and better options are needed to generate diverse antibody variants.
This process could not just be improved but totally revolutionised through the use of high-throughput DNA synthesis at scale to create diverse, rationally designed, codon optimised antibody libraries encoded directly from DNA. The use of synthetic libraries gives researchers greater control over the exact sequences used in construction of the antibodies, creating greater potential to target otherwise difficult targets. In this way, researchers can quickly generate a library with greater diversity in an array format, allowing efficient ‘design, test, build’ cycles.
Antibodies are an attractive option for so-called ‘undruggable’ targets, which lack a binding pocket typically required for small-molecule drug binding, as well as protein-protein interactions and immunological applications. G-protein coupled receptors (GPCRs) are a notable example, where more than 50% of those discovered are currently untargeted with small molecules but may be amenable to antibody blockade. Synthetic biology would allow researchers to precisely control the creation of domain libraries with high diversity, directed at previously untargeted epitopes, significantly reducing the time between lead identification and antibody validation.
Synthetic DNA technology also expands the possibilities for the development of improved or entirely novel molecular therapies such as enzymes and RNA catalysts. DNA synthesis can also be combined with other advancing technologies, such as the increased capacity to predict protein folding from amino acid sequences using artificial intelligence (AI) software like DeepMind35 to inform the design and creation of novel biomolecules with desirable properties.
Vaccine development is another area where synthetic biology could bring significant benefits. The power of synthetic DNA technology, combined with a number of recent advances in delivery and immunogenicity, has renewed interest in DNA vaccines not only for protection against infection but for therapeutic cancer vaccines and the in situ production of biological drugs36. Synthetic DNA is also required in the development stage of many types of vaccine, including DNA, mRNA and viral vector vaccines, and has a vital part to play in the fight against existing and emerging infectious diseases. Rapid DNA synthesis would make it possible to go from the genetic sequence data of a novel pathogen to having a range of potential vaccine candidates for testing in a matter of days in an outbreak situation, such as the recent COVID-19 pandemic37.
Finally, synthetic DNA has a number of applications in the sphere of diagnostics. PCR-based tests for pathogens rely on oligonucleotide DNA primers. The experience of the COVID-19 pandemic has shown that being able to rapidly synthesise DNA to order is crucial for the fast development and roll-out of testing in outbreak situations. Having access to widespread, affordable, accurate DNA synthesis technology can therefore play a vital role in infection surveillance and public health.
Targeted sequencing for mutation or allele detection through next-generation sequencing is another application of synthetic DNA in diagnostics and patient stratification in oncology and other diseases. However, the challenge of capturing faint target signals from the abundant noise created by the rest of the genome is an ongoing challenge in molecular diagnostics. Being able to rapidly generate large numbers of accurate custom synthetic oligonucleotides for multiplexed PCR, targeted sequencing or allele detection would be a significant advantage in this space38.
The next generation of accessible, accurate desktop DNA synthesisers do not just offer a step forwards but rather a paradigm shift in the life sciences. If the technology holds true to its promise, it could democratise and transform life sciences research in the 21st century in the same way as the laboratory PCR machine did in the 1990s.
Accessible rapid synthetic DNA technology opens the horizon to unprecedented possibilities, from the creation of novel therapeutics to doing research at greater scale and with increased efficiency and predictability. Over the past two decades we have seen huge progress in DNA sequencing, multi-omics technologies and gene editing technologies, all of which have had a huge impact on drug development and human health. The coming era of on-demand accurate DNA synthesis is the final key to unlocking the potential of drug discovery research at scale and realising the fruits of this progress for global health.
Volume 22, Issue 2 – Spring 2021
About the authors
Dr Tim Brears, is CEO, EvonetixPrior to his appointment at Evonetix in 2017, Brears served as Chief Executive of bioscience companies including Xention, and Gendaq. He spent ten years in the US, initially as a long-term fellow of the European Molecular Biology Organisation at Rockefeller University, New York, and subsequently as Director of Licensing at Ciba-Geigy (later Novartis) Agribusiness in Research Triangle Park, North Carolina.
Dr Raquel Sanches-Kuiper is Director of Biology, Evonetix. Sanches-Kuiper is an experienced R&D leader with a track-record of taking new ideas from concept phase to commercialised products in the Next Generation Sequencing (NGS) space. Before joining Evonetix, Sanches-Kuiper was at Solexa (then Illumina) from 2002 as a Protein Engineer and major contributor to its technology.
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