NEW YORK (GenomeWeb News) — Mission Bio, a startup founded last year to commercialize PCR-activated cell sorting (PACS) technology developed at the University of California, San Francisco and the California Institute for Quantitative Biosciences (QB3), has been awarded a pair of National Institutes of Health grants to do just that, PCR Insider has learned.
In addition, researchers from the company and UCSF recently published the first description of the PACS technology in a peer-reviewed scientific journal, demonstrating the ability to sort single cell lysates in individual droplets based on mRNA expression, enrich cancer cells out of mixed populations based on expression of known cancer-associated genes, and confirm those findings with downstream RT-qPCR and next-generation sequencing analysis.
In the PACS method, developed in the laboratory of UCSF and QB3 researcher and Mission Bio co-founder Adam Abate, up to 1 million individual cell lysates are isolated in discrete aqueous picoliter- or sub-picoliter-volume droplets suspended in oil, essentially creating individual test tubes for cells.
The individual cell lysates are then subjected to multiplexed TaqMan PCR assays so as to interrogate them for the expression of specific combinations of transcripts, mutations, or non-coding RNAs. In addition, the cell lysates can then be sorted into different containers based on unique transcriptional signatures signified by fluorescence markers and subsequently recovered for additional downstream analysis.
The method, which Abate described in a presentation earlier this year at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference, lies somewhere at the intersection of fluorescence-activated cell sorting and droplet-based digital PCR, and has a number of potential molecular biology applications.
Mission Bio was founded last year to identify and cultivate those applications, and the newly awarded grants, both administered by the National Human Genome Research Institute, will go a long way toward supporting that goal, Abate and Mission Bio CSO Dennis Eastburn, told PCR Insider this week.
Mission Bio declined to disclose the amount of funding it is receiving through the grants, which are not yet published in the NIH grant database. However, Eastburn, who is principal investigator on both awards, noted that one is a Small Business Innovation Research grant that will support the general development and commercialization of the PACS method. The second award is a Small Business Technology Transfer grant designed to support work at both Mission Bio and Abate's UCSF lab to develop and commercialize a product for targeted genome enrichment based on the PACS method. "That's really a separate product," Eastburn said. "We're doing preliminary development on it. What we're really focused on [at Mission Bio] is PACS."
The new paper describing the technology and demonstrating various applications was published last month in Nucleic Acids Research. Using PACS, Abate, Eastburn, and UCSF-QB3 researcher Adam Sciambi were able to identify and sort DU145 prostate cancer cells from a heterogeneous population by performing more than 132,000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA.
Vimentin, the researchers explained in the paper, is an intermediate filament protein known to participate in epithelial-to-mesenchymal transitions and can serve as a biomarker for some cancer cell types. Based on prior bulk qRT-PCR analysis, vimentin is expressed at an approximately 380-fold higher level in DU145 cells than in Raji cells, which the UCSF-QB3 researchers used as a control and abundant "background" cell type for their DU145 enrichment experiments.
Furthermore, the researchers were able to isolate DNA from pre- and post-sorted droplet emulsions to validate their enrichment experiment findings using both qRT-PCR and sequencing. The ability to perform these analyses following the cell sorting portion of experiments is a major advantage of PACS, Abate noted.
Reflecting on his presentation earlier this year at Tri-Con, Abate said that at the time his lab hadn't yet demonstrated enrichment with sequencing of the sorted cells. "That's something we showed in this paper," he said. At the time of his Tri-Con talk, PACS "was all based on gene expression, so we were sorting cells based on mRNA expression. Since then … we are confirming correct enrichment by doing qRT-PCR and sequencing SNPs in the genomes of these cells."
Another development demonstrated in the Nucleic Acids Research paper is the ability to use PACS to sort single cells based on a single copy of a gene in their genomes.
"To probe the sensitivity of our method and see if we could also detect genomic DNA sequences … we used a male and female cell population that were mixed together, and we used a TaqMan assay targeting a portion of the Y chromosome," Eastburn told PCR Insider. "We were specifically able to detect essentially single-molecule genomic DNA from the male cells … with TaqMan reactions and based on that TaqMan-generated fluorescence."
Eastburn said that this portion of their paper "shows the potential for the detection of SNPs in the genome and sorting based on that, and demonstrates the overall sensitivity of the method to detect very low-copy targets with PCR."
Mission Bio is now attempting to partner with academic and commercial collaborators to apply PACS to address "various questions across life science research," Eastburn said. The company is not yet inclined to identify specific promising applications for PACS, but Abate noted in his Tri-Con presentation earlier this year that some of the early applications for PACS being explored in his lab and at Mission Bio included examining how stem cell markers such as LGR5 and Myc affect cancer development; developing a PACS assay for bacteria that eat perchlorate for environmental markets; and investigating latent HIV infection in blood cells.
Eastburn noted this week that the technique is "analogous to FACS, so it's a general new sorting capability that you could imagine being applied to many different areas of biology."