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Cellular Research Set to Launch First Gene Expression Platform Using 'Molecular Indexing' Technology

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Cellular Research, a three-year-old startup founded by Bay Area molecular biology entrepreneurs including Stephen Fodor, Glenn Fu, and Stephen Quake, said this week that it will commercially launch its first gene expression platform at the Advances in Genome Biology and Technology meeting next week in Marco Island, Fla.

The new product, called the Pixel System, uses the company's so-called molecular indexing technology to provide absolute digital mRNA quantitation from single cells, and is expected to enable single-cell gene expression studies with sensitivity akin to digital PCR but without the inherently destructive nature of that assay.

In addition, Cellular Research plans to introduce sequencing-specific follow-on products later this year using the same core technology to enable researchers to quantitate libraries for sequencing and to measure the efficiency of sequencing results, among other applications.

Cellular Research was founded in 2011 with the goal of commercializing the molecular indexing technology, which was originally developed at Affymetrix but subsequently assigned to Cellular Research, Martin Pieprzyk, director of marketing at the company, told PCR Insider in a recent interview. Affy disclosed an IP licensing deal in February 2012, noting that it had assigned Cellular Research one patent application and related know-how, although at the time it did not specify the nature of the technology. Under that agreement Cellular Research was to pay Affy single-digit royalties on the sale of products covered by the technology as well as an annual minimum fee of $100,000 starting in December 2015.

Fodor, who retired as CEO of Affymetrix in 2009, established the company along with Fu, who formerly served as Affy's director of genotyping research. Fodor is now CEO of Cellular Research, and Fu serves as a senior scientist for the company, and the two also brought into the fold Stanford's Quake, whose long history of entrepreneurship in the field of microfluidics and genetic analysis has included co-founding Helicos Biosciences and Fluidigm.

Pieprzyk, himself a Fluidigm alum, explained that molecular indexing, the foundation upon which all of Cellular Research's products will be based, is similar to DNA barcoding techniques such as amplicon tagging for sample indexing in sequencing studies.

"Basically you take a barcode and assign it to a sample," Pieprzyk said. "After sequencing you basically use the barcode to deconvolute which sample the sequencing read came from. Our technology is similar in that we do use barcodes, but we effectively take the entire process one step back, so what we actually encode … are individual mRNA molecules at the time of reverse transcription."

Pieprzyk further explained the technology in the hypothetical context of a single cell containing 10 different transcripts. During reverse transcription, each of the 10 transcripts gets assigned a different barcode such that 10 different cDNA molecules, each containing a different barcode, are produced.

"You now have these 10 molecules that are unique in that they are represented by the different barcodes," Pieprzyk said. "Let's say we decided to run a standard PCR reaction [to] amplify the cDNA and end up with an average of 1 million copies of each of those 10 starting molecules. With qPCR, digital PCR, or sequencing, all we're effectively doing is counting the number of … final copies. That information doesn't actually tell us how many molecules we started with. It just tells us that after 20 rounds of qPCR, we ended up with [x number of] molecules.

On the other hand, since Cellular Research's technology associates a unique barcode with each molecule before it is amplified, all of the copies of a specific cDNA transcript contain the same barcode that a specific single molecule started with.

"That allows you to effectively eliminate any sort of PCR or library prep bias, because it doesn't matter if two different transcripts amplify at different efficiencies, or you have different library prep efficiency differences, because we're not counting the final number of copies — we're just counting the final number of barcodes," Pieprzyk said.

The product that Cellular Research is launching next week is a digital reader about the size of a small PC that leverages the technology to perform highly quantitative and precise gene expression analysis in a single-tube format "at a price point available to all researchers," although the company did not detail the platform's price.

"You can start off with a single cell, we encode all the mRNA molecules inside the cell with different barcodes; we amplify them; and then the instrument counts how many barcodes are associated with each specific mRNA target," Pieprzyk said.

Further detailing the approach, Pieprzyk said that in a typical experiment, a cell is basically "dropped into something like a [reverse transcriptase] mix" whereupon the barcodes "get associated with every single mRNA molecular in the cell. During the reverse transcription process, the barcodes are poly-A tagged, and they effectively incorporate into the final cDNA product."

The technology that molecular indexing is most likely to draw comparisons to is digital PCR because of its ability to provide absolute molecule counts. However, Pieprzyk noted that a big difference between the technologies is that digital PCR is a destructive assay while molecular indexing is not.

"If we start with a single cell, and I want to measure GAPDH [using digital PCR], once I complete the measurements there is no sample left," he said. "I divide up the cell, count how many transcripts of GAPDH are there, and that's it. I can't go back and ask another question. The sample is destroyed."

With molecular indexing, the encoding reaction is an archived step, which means that after it takes place there is sufficient material leftover to keep sampling from the same pool. "We have almost unlimited ability to go back and continue sampling the same single cell," Pieprzyk said. "You can continue to run experiments in parallel, or come back and run experiments at a later time point."

This also means that once a molecular indexing reaction is complete users can run the sample through a subsequent qPCR assay or sequencing to compare results across platforms. Recovering sample for this purpose has been a major bugaboo of digital PCR, especially droplet-based methods.

Another key differentiator, he noted, is that the best digital PCR techniques currently available sample "at best 50 to 60 percent of the actual volume" due to microfluidics or droplet-based partitioning. With molecular indexing, "because you do this encoding reaction on the whole cell or sample … there is no sample loss at all."

From a market competitiveness standpoint, Cellular Research looks to be an early challenger to Fluidigm in the single-cell genetic analysis market — a market that Fluidigm has essentially created over the past few years with its digital PCR and single-cell sample prep products.

"The single strongest application that we're getting the most customer feedback on is single-cell sequencing or single-cell quantitation," Pieprzyk said. "Even though there is tons of interest around single-cell, and Fluidigm is pushing very hard on that, the reality is that up to now most people have only really looked at high-abundance genes. Things that are probably below 100 transcripts per cell have not really been examined." Cellular Research is betting that its technology will allow researchers to obtain absolute counts of minute variations in gene and transcript levels.

Although Cellular Research will initially focus on gene expression applications with its Pixel instrument, the company will also soon be launching a product designed to quantitate libraries for sequencing studies. In fact, at AGBT a company collaborator will be presenting a comparison of this application enabled by molecular indexing with commercial solutions from Fluidigm, Life Technologies, and Illumina, Pieprzyk said.

Researchers from the company, including Fu and Fodor, along with Stanford scientists have also recently published on this application. In a paper appearing this week in PNAS, the group described using the molecular indexing technology to improve quantitative aspects of RNA sequencing, such as correcting for amplification bias, distinguishing clonal replicates, and obtaining absolute measurements of gene expression. Further, they combined capture enrichment with molecular barcoding to sequence targeted genes and demonstrate low library preparation efficiencies that lead to a stochastic loss of low-abundance transcripts that can't be overcome by increasing sequencing depth, according to the paper's abstract. A detailed article about their work was published this week in In Sequence.

And Cellular Research has other irons in the fire, namely the development of a non-invasive test for Down syndrome using the molecular indexing technology. In January 2013 the company received a $360,000 grant from the National Institute of Child Health and Human Development for this application, and it received follow-on funding of $95,000 last month.

The researchers, led by Fu and in collaboration with Stanford's Ronald Davis, claim in the grant's abstract that their approach will count single molecules to detect fetal aneuploidy from cell-free DNA in maternal plasma, and will be "faster and less expensive than massively parallel DNA sequencing methods … yet exceed the statistical power required for the accurate diagnosis of fetal chromosomal anomalies such as trisomy 21."

Pieprzyk said that Cellular Research is completely privately funded and is not currently pursuing an investment series.

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