In The Midst Of A Pandemic, Duplex Sequencing Comes Of Age

By Deborah Borfitz

July 15, 2020 | A technique for improving the accuracy of next-generation sequencing (NGS) by more than 10,000-fold could eliminate a good deal of the guesswork in advancing drugs to market, from better understanding the evolution of viruses and the toxicology of drugs to detecting resistance mutations in patients before they show clinical signs of disease relapse. Duplex Sequencing, which independently tags and sequences each of the two strands of a DNA molecule, made its debut nearly a decade ago but only in recent years found a home in real-world applications, says Jesse Salk, M.D., Ph.D., an oncologist as well as co-founder, CEO, and CSO of TwinStrand Biosciences.

As detailed in a 2012 paper (DOI: 10.1073/pnas.1208715109), Duplex Sequencing can detect ultra-rare mutations with an error rate of less than one per billion nucleotides sequenced and identify sites of DNA damage even when mutations are present in just one of the two DNA strands. Salk developed the technology with colleagues at the University of Washington where he earned his graduate and medical degrees.

Currently, the focus is on extracting useful, actionable information from low-frequency mutations in small- to medium-sized portions of the genome, such as those responsible for cancer, says Salk. Whole genome Duplex Sequencing projects can get prohibitively expensive because of the cost associated with making redundant copies of molecules. Inherited mutations wouldn’t require this much horsepower to detect.

The price of sequencing may not be a barrier for long. Since Duplex Sequencing was invented, the cost of NGS has already dropped by an order of magnitude, he says.

Eight years ago, when Salk was still in clinical training, Duplex Sequencing was an impractical option for anyone beside expert users, he says. All that changed over the last few years when his attention shifted to transforming the core technology into robust, highly efficient, and regulatory-grade assays—products that are easy to use and come with cloud-based software that required no programming know-how and can handle billions of nucleotides worth of data.

Research Applications

In February, TwinStrand Biosciences formally released its Duplex Sequencing platform for two research applications. One is specific to the detection of minimal residual disease in acute myeloid leukemia patients, and the other is a multi-species genetic toxicology assay for rapid identification of carcinogenic chemicals in preclinical testing as well as detection of patients who have been environmentally exposed.

The timing of the launch was somewhat fortuitous, as COVID-19 presented the company with some important opportunities related to the development of therapies that reduce the chance of evolving drug resistance and the safety screening of candidate compounds for genetic toxicity, says Salk. The potential for unintended consequences has historically been carried out in long-term studies on rodents, but with the novel coronavirus (like many cancers) “we don’t have the luxury of two years to wait on results.”

The genotoxicity application of Duplex Sequencing is also being applied to pre-clinical drug studies to rapidly identify safety signals and do so “very early on,” Salk continues. Patients who are critically ill need immediate treatment options but determining safety is critical from a broader public health perspective if broader use were to be considered.

In addition to pharma companies, TwinStrand Biosciences is actively engaged in toxicology research with academic groups (e.g., Duke University, University of Michigan, and Massachusetts Institute of Technology) and the research arms of the U.S. Food and Drug Administration and Health Canada, among others.

Other applications in the development queue include one identifying very early signals of ovarian cancer when the disease is at a curable stage, says Salk, adding that studies are already underway with trialists at Massachusetts General Hospital, the National Cancer Institute, and the Medical University of Vienna. The company is also working with biotechnology companies and therapeutic cell manufacturers in the development of cutting-edge CRISPR and CAR-T cell therapies “where there’s potential for mutations to occur where you don’t necessarily want them.”

The platform will continue to be adapted for compatibility with emerging next generation sequencing technologies, including nanopore long-read sequencing technologies, to allow broader access for customers, Salk says.

Monitoring Mutations

TwinStrand Biosciences most recently published an article describing how Duplex Sequencing was used to detect low-level, emerging resistance mutations up to five months prior to relapse among patients being treated with a targeted therapy for Philadelphia chromosome-positive acute lymphoblastic leukemia. The paper, which was published in the Blood Cancer Journal (DOI: 10.1038/s41408-020-0329-y), was a collaborative project with the MD Anderson Cancer Center, ARIAD Pharmaceuticals (now Takeda), Pennsylvania State University, and Fred Hutchinson Cancer Research Center.

Research results point to the relevancy of the technology for other molecularly targeted drugs, which are now being FDA-approved at the rate of close to one per month, says Salk. Over the long term, cancers that cannot be cured will be managed much like a chronic disease. As envisioned by Salk, this will include following the dynamics of “one-in-a-million” mutations in real time so disease relapse can be predicted and prevented with new drugs and therapeutic cocktails.

That way, he says, patients will get more effective therapies, they will be spared the toxicity of those that are unlikely to work, clinical trials will generate a lot more data to guide or improve risk stratification and costs for the broader healthcare system can be reduced.

Salk says an ongoing part of the strategy of TwinStrand Biosciences is to make Duplex Sequencing available to smaller groups of basic scientists and academicians who are critical to driving innovation. Moving forward, he’d also like to broaden the spectrum of users to include those in the fields of infectious disease, prenatal diagnostics, inherited genetic diseases and forensics who could be co-developers of a whole new set of use cases for the technology.

Other than a few of the older oncology patients he continues to treat at a local VA hospital, surprisingly few people bring up the fact that he is the grandson of famous physician and medical researcher Jonas Salk who developed one of the first successful polio vaccines. “I think of myself as a scientist first and foremost,” says the younger Salk, echoing what his grandfather could well have said. “What drives me most is coming up with new tools that help the broader scientific community. That’s what counts the most.”