Despite the recent progress in targeted cancer therapies, the frequent development of drug resistance has proved to be a major hindrance. Rare mutations acquired by a small population of cells could benefit tumorigenesis and be cancer’s ticket to back door entry. However, identifying them is a challenge owing to the high error rates of current sequencing tools. A powerful sequencing method dubbed ‘Duplex Sequencing’ could significantly minimize this problem. By sequencing both strands of individual DNA molecules, Duplex Sequencing can identify ultra-low frequency mutations with a 10,000-fold higher resolution. We interviewed Dr. Jesse Salk, CEO of TwinStrand, to learn more about the technique and its potential in advancing cancer research.
From Concept to Reality
The foundation of TwinStrand Biosciences was laid almost a decade back when a medical student and postdoc from the lab of Dr. Lawrence Loeb were busy discussing science on their way to an ice climbing trip. Dr. Jesse Salk and Dr. Michael Schmitt were pondering ways to detect ultra-low frequency mutations that arise from mutagenic chemicals or cancer. At that time, next-generation sequencing (NGS) was already widely used, however, it had an error rate of ~1%, which can equate to hundreds of millions of sequencing lapses per run. While such an error rate is tolerated in some applications, it could be detrimental when searching for single mutant molecules mixed with millions of normal ones, such as in the case of residual cancers.
Eventually, they hatched the idea of sequencing both strands of each DNA double helix to make more accurate sequence calls. The principle was that if they detect a mutation on one strand but not the other, it could be recognized as a sequencing error. “The concept was very simple, but it took a great deal of time to think about how you would practically do it,” said Dr. Salk. When they started experimenting with this concept, they had to write several custom programs and analyze extremely large data sets. A lot of hard work eventually translated into a technology that demonstrated an error rate of less than one mistake per million nucleotides sequenced, in contrast to standard methods that yielded one mistake every few hundred nucleotides. Theoretically, now they could sequence the entire genome without a single error. They had hit the jackpot.
However, the technology was not market-ready and needed further polishing. The idea behind launching TwinStrand was to take the academically prototyped Duplex Sequencing technology and make it more robust, efficient, and broadly usable for a wide range of applications.
Slow Adoption of the Technology
Dr. Salk recalls that the initial adaptation of the technology was difficult as it had just come out of the lab and the biochemistry was rough around the edges. Moreover, the software they had built for data analysis was not user friendly. Since the launch of TwinStrand, they refined the technology and brought professional cloud-based software to the market that anyone could use. In his words, “We have built software that is not only accessible to sophisticated users who can work with command-line data formats but also to someone who is just familiar with working with Excel spreadsheets.” This caught people’s eyes and increased the visibility of the product.
Another thing that helped with adoption was the cost of sequencing. Back in 2007, the cost of sequencing 1 million bases of DNA sequence was approximately $1000. As NGS technologies progressed, the cost came down to ten cents. In Dr. Salk’s opinion, when performing “incredibly deep sequencing to detect very rare variants, a fair amount of sequencing needs to be done. The order of magnitude decrease in the raw cost of NGS since the Duplex Sequencing’s invention made a big difference in its feasibility.”
In addition to that, the adoption was facilitated by an improved understanding of genome biology. Dr. Salk points to those times when it was believed that “every cell in our body has the same genetic sequence.” But now we know that is not the case. “There is an ongoing evolution throughout our bodies and core processes that we can only see now.” The DuplexSeq technology has been facilitating the identification of these small, albeit important changes. The technology has found use in the early detection of cancer, improved understanding of drug resistance in cancer, and assisting in forensic applications, among many other fundamental discoveries.
Overall, the combination of optimization, lower cost of sequencing, and a better understanding of the importance of somatic genetic heterogeneity in biology have impacted the acceptance of the technology, and Dr. Salk hopes that it continues in the future with more and more people using it.
Turn of the Tide
Despite the slow adoption, the company has seen tremendous growth in the last four years. “We have gone from a one-person research and development organization to a commercial company with 40 people,” he said. TwinStrand recently received its Series A financing led by the Madrona Venture Group early this year. The funding successfully boosted commercial and operational teams to turn foundation science into a meaningful product.
However, the key factor that has led to their success is the innovation that has been the guiding principle of the company. “Our philosophy is that science comes first. So, although we are moving products through the pipeline, we are also innovating and looking at the next generation of tools. And our partnerships with biotechnology and pharmaceutical companies, regulatory agencies, academics, and others are helping to further drive this innovation.”
Initially, TwinStrand started with a focus on one product but has grown to add many more to its portfolio. Dr. Salk attributes that to open collaborations and science-driven innovations. “Our company has become known for being science-driven and high quality. We are always keeping the quality up and always keeping our eye on what’s the next thing we can do to make our tool even better or make something new or different for scientists and medical researchers,” he said proudly. TwinStrand’s scientists have authored more than 15 peer-reviewed Duplex Sequencing-related articles and have developed a portfolio of over 45 patents and patent applications.
Duplex Sequencing has found its way into several new and existing biological problems. Dr. Salk believes that “We are really pushing the envelope in terms of how we are using the technology and how this could impact not just cancer diagnostics but also global cancer burden via individual risk stratification and public health measures globally.” One of the applications the company is currently focusing on is identifying residual disease. Cancer treatments are not perfect and may leave some residual cells behind after treatment. Due to their small numbers, detection of these cells requires highly sensitive laboratory methods that can uncover one cancer cell among one million normal cells. This is extremely important as it may inform treatment decisions that prevent future cancer relapse. Duplex Sequencing is uniquely capable of detection at this level, as it has a 100-fold higher resolution than even the best other error-corrected NGS methods.
Another application where the technology is used is in testing chemical-induced mutagenesis in humans. “Every drug or chemical that goes into humans has to be tested for its carcinogenic potential. The standard way of doing this is through animal studies that take years to detect the formation of tumors. With Duplex Sequencing, we are able to directly identify induced mutations at levels of one-in-10 million within days of exposure.”
Taking it a step further, Dr. Salk plans to apply Duplex Sequencing on human samples to spot unrecognized mutagenic exposure in people and hopes to use this to help affected individuals get the care and to aid the public health authorities in finding and eliminating carcinogens in the environment. However, he acknowledges that obtaining tissue samples is impractical, and turning to liquid biopsies for exposure detection will be the future. He believes that Duplex Sequencing might well become the gold standard for both mutagenic exposure detection and cancer detection using liquid biopsy.
Identifying unmet needs in genomics, the company is also working to help advance new drug technologies like therapeutic stem cells, immunotherapies, and CRISPR-based gene editing with partner organizations.
Future of TwinStrand
Besides TwinStrand, several other big players, including Roche, Illumina, Promega, and ThermoFisher, are developing error-corrected NGS technologies and creating competition. However, Dr. Salk believes that his group has a clear advantage over others with their understanding of technology and the extent of patent protection. “We deeply understand the science and applications of technology. When we embark on partnerships with pharmaceutical companies, clinical trial networks, regulators, and academics, we bring both our technical and scientific savvy, as well as a strong clinical understanding to help solve their unmet needs.”
He explained that TwinStrand’s strength lies in the quality of their science and firm focus on the applications of the technology. Over time they have built a solid reputation that has received recognition from important stakeholders. “We have built ourselves on the credibility of science. I think that this is part of what makes us unique and distinguishes us from other very good companies out there,” he added.
In conclusion, Dr. Salk shared how the COVID-19 pandemic has impacted the company due to the strict lockdown measures in place. However, he prefers to treat it as an opportunity to help understand the virus, build new safe treatments, and find other ways to assist with the crisis. That is a quality he shares with his grandfather Dr. Jonas Salk, discoverer of the first successful polio vaccine. With such an inspirational figure within the family, Dr. Salk’s TwinStrand has a rich legacy to emulate.
Writer: Ruchi Jhonsa, Ph.D.
Interviewer & Editor: Rajaneesh K. Gopinath, Ph.D.
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