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China's Singleron Biotechnologies Pushes Single-Cell RNA-seq Into Clinical, Translational Studies

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This story has been updated to include more information on Singleron Biotechnologies' plans to obtain regulatory approvals. 

NEW YORK – Singleron Biotechnologies, a single-cell sequencing technology company based in China, is expanding into Europe, with eyes on eventually reaching the US.

The company opened offices in Cologne, Germany, in January and began building a lab there in March to offer its single-cell analysis services to European customers.

Looking to differentiate itself from market leader 10x Genomics, Singleron has built a workflow tailored for clinical and translational researchers.

"Clinical applications require complete solutions, from sample preservation to reports," said Nan Fang, Singleron's CEO and cofounder. Among the features designed with clinical studies in mind are a proprietary sample preservation buffer that can keep cells viable for 72 hours and a mix of enzymatic and chemical reagents to help separate cells in tissues that have traditionally been resistant to single-cell analysis — even skin, fat, and bone. The company has also developed a bioinformatics suite to help with analysis.

At the core of the platform is the Matrix, a microwell-based cell isolation and library preparation instrument that takes up to two high-throughput chips that can process either 10,000 or 30,000 cells or nuclei per run. Singleron has also developed kits for specific single-cell assays, including gene expression analysis, immune cell profiling, and RNA expression dynamics, as well as a multiplexing kit that allows combination of up to 16 samples.

In China, the platform has already taken hold, especially in hospitals, which make up around 80 percent of the company's customers, with pharma and basic researchers making up the rest. So far, Singleron's platform has been used in more than 2,000 research projects to sequence 50 million cells from more than 300 different sample types, said Yuanyuan Chen, Singleron's director of business development. At Nantong University School of Medicine, for example, five of the 11 researchers doing single-cell experiments use Singleron, according to Hao Chen, a professor and vice director of the school's Institute of Reproductive Medicine, who uses the company's services to study the male reproductive system.

Now, Singleron can offer its technology to European researchers looking to bring single-cell analysis into molecular diagnostics, potentially at a discount to existing methods.

Though its European operations are just getting started, the company has been active in China since 2018. Fang, formerly associate director of global R&D at Qiagen and deputy general manager at Novogene, had known Rong Fan, a Yale University professor who has started several companies to commercialize molecular technologies developed in his lab, since a 2014 collaboration. "We had both been very enthusiastic about single-cell," Fang said. "In 2017, we saw rapid adoption of high-throughput, single-cell sequencing, and we both saw more clearly the commercial potential. We wanted to be part of this trend we saw coming." Both of them wanted to develop single-cell sequencing for clinical applications, she said, and had some idea on how to improve it for that purpose.

In its first year, Singleron focused on developing Fan's single-cell freeze-thaw lysis directed toward 3' mRNA sequencing technology, which was published in 2019, into a viable commercial product. The single-cell gene expression product uses unique molecular identifier (UMI)-barcoded beads to capture 3' mRNAs by their poly-A tails. By the beginning of 2019, the firm launched a manual library preparation kit for use with microwell chips and began offering it as a service. Over the course of that year, the researchers worked on automating their library preparation, which became the Matrix instrument, which they launched in the middle of the COVID-19 pandemic.

"We had the design freeze at the end of March 2020, but I was still in quarantine after coming back to China from the US," Fang said. "Engineers had to show the final product to me by video." The company also had to deal with suppliers who could not deliver components on time.

Now, the company has 340 employees, Fang said, and has raised nearly $100 million, including a $30 million Series A financing round in September 2020, led by Lilly Asia Ventures and joined by new investors CDH Investments, Arch Venture Partners, Tencent, and CDG Capital and existing investor Sherpa Healthcare Partners and China Growth Capital.

Fang said the firm plans to use the funds to build good manufacturing practice facilities for instruments and reagents and to get regulatory approval for its products. The firm has obtained Class I medical device registration in China and CE marking in Europe, but is pursuing Class II and III registrations and CE-IVD marking. "Of course, we also plan to develop more products, both instruments and reagents, and to explore overseas markets," she said, which "definitely" includes the US.

Already, the firm has signed a collaboration agreement with Agilent Technologies. Fang said her team would look at ways Agilent quality control and targeted sequencing products could be used in Singleron's workflow.

Singleron has licensed additional IP from Yale University but declined to disclose what those patents covered. It is pursuing clinical regulatory approvals in both China and Europe for Matrix.

Singleron joins a market dominated by 10x Genomics, a company with enviable brand recognition that has also become the measuring stick for single-cell data quality. Other companies offering single-cell RNA-seq solutions include Bio-Rad Laboratories, 1CellBio, and Parse Biosciences.

Performance-wise, the Singleron workflow is similar to published data from 10x Genomics, said Hao Chen, especially in terms of the number of genes per cell. While he hasn't used data from the 10x platform, he has used the Smart-seq 2 single-cell RNA-seq protocol. For the samples he sends, often from epididymis and testis, Singleron returns about 2,000 genes per cell at the median. "Epididymis samples are hard to collect, so maybe there is something that damaged the cell," he said. His experiments targeted between 5,000 and 10,000 cells per sample to keep sequencing costs reasonable.

For Singleron's gene expression assay, sensitivity, as measured in UMIs and genes per cell, is similar to 10x, Yuanyuan Chen said. "We're not claiming we're much better, but at least our performance is at the same level," she said.

The Matrix instrument has a cell capture rate between 30 and 60 percent, depending on chip types. The whole workflow takes about a day, "eight to nine hours for an experienced person," she said, but it can be split in the middle after generating cDNAs. Another differentiator is that the method works with multiple next-generation sequencing platforms, including Illumina's but also BGI's.

For now, in China, Singleron's method is cheaper than 10x's, at about RMB 20,000 ($3,086) per sample, Hao Chen said. "To my knowledge, 10x will go up to RMB 25,000 per sample."

Singleron's Yuanyuan Chen claimed that the firm is able to provide kits for about 30 percent less than 10x Genomics for its 10,000-cell chip. "If customers get our high-density chip, they're basically paying about the same cost [as 10x] but getting three times more cells," she said. (However, 10x recently released a multiplexing kit that enables gene expression profiling for up to 30,000 cells per lane on a reagent chip, up from 10,000 cells.)

While cost is always a concern, the ability to help with clinical studies is a major selling point. "That is where I see the advantage of this technology in comparison with 10x," said Margarete Odenthal, a professor at the Center for Molecular Medicine Cologne who is trying to bring single-cell sequencing into the molecular pathology lab. "Initially, we would like to establish this technology in cancer pathology, but later I can imagine that single-cell sequencing analysis will also be of great importance in chronic inflammatory diseases or other diseases," she said.

She has collaborated with the company and researchers in China on a study of non-small cell lung cancers using the Singleron platform and is working with the firm now to bring the technology into her lab.

In a paper published last month in Nature Communications, the researchers reported findings from 42 samples, showing that lung squamous carcinoma has a higher rate of inter- and intra-tumor heterogeneity than lung adenocarcinoma.

"RNA is very sensitive to degradation," and cells can go into apoptosis during processing, making them no longer representative of the sample, Odenthal said. The ability to preserve cells for up to three days can address these quality issues and allows sample collection away from the processing lab. It would also provide a chance to justify using single-cell analysis for diagnosis. "It's very expensive, sometimes it isn't worth doing it," she said. "With these three extra days, we can at least do some histological analysis."

She added that the method works well with several different tumor types. Lung adenocarcinomas were easily dissolved, she said, despite often coming from samples with lots of extracellular matrix and scarred tissue. Being in the same city as Singleron's European operations, Odenthal has been able to get people from the company to come to her lab and help out as they set up the workflow, and the preset bioinformatics analysis was appreciated as well.

Hao Chen said that he's able to get results from samples that might not have worked on other platforms. "In general, most companies require that the cell viability in a sample is above 90 percent," he said. "However, in real experiments, it was not easy to get such high viability for specific tissues, so our experiments will go on to the next step if the viability is greater than 85 percent. Singleron's tissue dissociation solution consistently gives us results that meet such requirements and generate good sequencing results." Turnaround time for each sample was between 20 and 30 days and his interactions with the company were always "professional and efficient," he said. "I can always get an update on project status."

With the company's technology now out in the world, Fang said she thinks clinical implementation of single-cell analysis "is not so far away." She pointed to the LifeTime initiative, a European-led precision medicine effort and its focus on single-cell technologies, as an example. "This will happen probably sooner than a lot of people believe at the moment," she said.

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