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Mission Bio Harnesses Droplet Microfluidic Tech for Single-Cell DNA Analysis; Focuses on AML


SAN FRANCISCO (GenomeWeb) – Mission Bio has entered the single-cell analysis field, hoping to set itself apart from the competition with a platform that focuses on analyzing DNA rather than RNA from single cells and by developing targeted panels.

The South San Francisco, California-based startup earlier this week announced a $10 million Series A financing round, and plans to broadly launch its instrument, dubbed Tapestri, along with an acute myeloid leukemia panel in December.

Mission Bio's technology is based on proprietary droplet microfluidic technology for single-cell sequencing, some of which it licensed from the University of California, San Francisco. Adam Abate, a cofounder of Mission Bio and principal investigator in the department of bioengineering and therapeutic sciences at UCSF, led a study published in Nature Biotechnology earlier this year describing the technology.

Although that study described its use for sequencing environmental samples, the Mission Bio team has modified it to work on human cells for its first application and has developed a targeted acute myeloid leukemia panel.

Charlie Silver, CEO of Mission Bio, said that the firm plans to follow up on the initial instrument and AML panel launch with consumables that enable customers to design their own targeted panels.

The Tapestri instrument has a list price of $79,500 and a throughput of up to 10,000 cells, while the AML panel will cost around $795. The panel consists of 40 amplicons that target AML-related gene regions and controls to measure allele dropout.

In one run on the Tapestri instrument, up to 10,000 cells can be processed and prepared for targeted sequencing. Depending on the cell throughput, running the AML assay on a MiSeq instrument will yield approximately 40x to 80x sequence coverage per amplicon.

Mission Bio also provides bioinformatics software to interpret the final sequence results. The consumable is a microfluidic cartridge. Cells are captured into microdroplets where they are lysed and digested. Then, the AML amplicon library is created with cell-specific tags and Illumina sequencing adaptors are added.

As described by the UCSF team in their Nature Biotechnology study, the researchers made use of hydrogel microspheres, or microgels, that trap DNA molecules but are permeable to enzymes and small molecules. Cells are captured in the microgel by merging a cell suspension stream with an agarose stream to form a droplet. Because the droplet is permeable to enzymes, the DNA is purified and fragmented within the droplet. Next, a microfluidic device is used to merge the microgels with a droplet containing PCR reagents and a droplet containing a barcode.

Silver said that one key piece that Mission Bio developed was a protease workflow, which digests the nucleosomes, enabling better access to the DNA.

What sets Mission Bio's technology apart from other single-cell technologies such as 10x Genomics', Fluidigm's, or the Drop-seq methodology is that it is focused on DNA analysis rather than RNA, Silver said. "It's the first platform that does high-throughput single-cell analysis of DNA," he said.

He said that the firm decided to first focus on AML because its goal is to make a dent in precision medicine. And although much is already known about the genes involved in AML, there is evidence that a better understanding of the clonal evolution and heterogeneity within AML could help design better treatments or make better predictions about a patient's prognosis.

"There's evidence that clonal evolution in response to treatment has important implications for the outcome of disease and potential treatment options," Silver said.

Already Mission Bio has been working with early access users at Stanford University and MD Anderson Cancer Center.

Koichi Takahashi, an assistant professor at MD Anderson, said that his lab has tested the technology on multiple samples from three AML patients and plans to expand on that initial proof-of-principle study. For that initial work, Mission Bio processed and ran the samples, getting about 5,000 single cells per run. Going forward, he said that his lab plans to install the Tapestri instrument.

Takahashi said that his lab had first done bulk sequencing under a research protocol on bone marrow collected from AML patients at diagnosis, remission, and relapse. For one patient, for which he has received the Mission Bio data, the mutations that were found in bulk sequencing were also found in the single-cell data.

That patient had three notable known AML mutations — to TP53, DNMT3A, and a tandem duplication in FLT3. Takahashi said his team wanted to see whether single-cell sequencing could shed any light on which clones were responsible for the patient's relapse.

"Relapse is the biggest problem we face" in fighting AML, Takahashi said. Approximately half of all AML patients relapse "and we're still trying to figure out why."  The hypothesis is that treatment eliminates some, but not all of the clones, leaving resistant clones that eventually cause relapse, he said. By using single-cell sequencing, he said he hopes to be able to more precisely identify which clones are responsible for relapse.

In addition, Takahashi said, by looking at single cells it is possible to distinguish which mutations are heterozygous and which are homozygous, which can help identify which mutations are the founder mutations.

In this case, single-cell sequencing revealed that although the clonal architecture between the pretreatment and relapse sample was very similar by both bulk and single-cell sequencing, single-cell sequencing revealed more details about the variant allele frequency and the number of mutated cells. For instance, in the pretreatment samples, although TP53 mutations affected the most number of cells, there was a higher frequency of DNMT3A mutations because those mutations were predominantly homozygous. "This opens up new questions about clonal heterogeneity. Should we define founder mutation by the number of affected cells or the number of affected alleles? There's not a clear answer to this," Takahashi said.

Takahashi stressed that because these were results from just one case it was too early to make any conclusions, but he said that it would help lay the groundwork for future studies. He plans to expand the study to additional patients and to investigate whether there are differences in chemosensitivity between cells with homozygous versus heterozygous mutations. In addition, he said, he is also interested in using the Tapestri instrument to see whether it can help identify minimal residual disease.

He said that Mission Bio's technology fit his needs because it looks at DNA instead of RNA and also because it is targeted. Since his lab has already done bulk sequencing on its samples, he said he isn't looking to discover new mutations but to track the mutations already identified.

Another early access user, Liwen Xu, senior research scientist at Stanford University, said that in a pilot study involving samples from two AML patients at three different time points, the technology worked well, delivering consistent results. Similar to Takahashi, she said the single-cell technology enabled them to identify clonal expansion.

In her study, she examined blood samples from patients before they received a bone marrow transplant, after, and then again when they relapsed. Xu ran the samples on the Tapestri instrument and sequenced them on the Illumina MiSeq. The single-cell data allowed the researchers to clearly see that in one patient only the clones with the TP53 mutation expanded. The TP53 mutation wasn't wiped out by chemotherapy or the bone marrow transplant, Xu said, and continued to expand. Meanwhile, all the other clones either stayed the same or decreased.

In the second patient, sequencing identified an SF3B1 mutation, which has previously been associated with AML. Before the bone marrow transplant it was found at a low frequency. The mutation persisted after the bone marrow transplant and the researchers also identified some cells that had both SF3B1 and TP53 mutations. "We're not sure whether the TP53 mutation was present in the original patient cells or whether it was a newly acquired mutation," Xu said. But again, only the cells with the TP53 mutation continued to expand.

Xu said that single-cell sequencing also enabled them to more closely examine the percentage of patient-derived cells and donor-derived cells and how those proportions changed following treatment. In this study, both patients had partial donor chimerism, she said. And following the transplant, the percentage of patient-derived cells expanded.

Xu said she is now discussing with other groups at Stanford how they want to use the Mission Bio technology in their studies and the types of information they want. She said that being able to profile single cells would help better understand clonal evolution, which could lead to identifying new targets or better understanding how and why patients develop resistance.