SBIR-STTR Award

Single cell gene expression assays have become important tools to identify functionalsubtypes of cells, as well as changes within specific subtypes resulting from diseases or treatments. However,most current methods require dedicated hardware and kits, making them expensive, and most are 3' based andthus cannot measure splice variants, gene fusions, expressed single nucleotide variants, or RNAs that are notpolyadenylated. Furthermore, there is no method to-date that enables investigators to measure low or even manymoderately expressed genes from single cells, which prevents measurements of many key biomarkers andimportant genes in molecular pathways and functions. Current methods also only measure a limited number ofgenes/cell and do not provide quantitative measurements of the abundance of those genes, and thus cannot beused to measure changes in expression level along the order of magnitude possible using bulk preparations ofcells, nor can they be used to carry out dose response experiments at the single cell level. Our approach willenable investigators to carry out single cell assays without purchase of proprietary hardware, and provide a levelof performance comparable to the profiling of bulk cell samples. Based on the commercial targeted geneexpression TempO-Seq® bulk cell assay, we will implement a TempO-LINC single cell assay which, based onpreliminary data, will provide data measuring the same genes (low, moderate, high expressed) and similarnumber of genes/cell as can be measured from bulk samples, quantitatively, providing a similar dynamicexpression range and normal distribution of counts as measured from bulk samples. TempO-LINC can be usedto process a few 100 single cells up to 100,000+ cells at a time. TempO-LINC data will be benchmarked againstboth bulk cell data and 10x Genomics single cell data. Furthermore, we will demonstrate utility to measure splicevariants and to provide single cell dose response data. These data will be generated using the commercial S1500surrogate whole transcriptome assay adapted to the TempO-LINC process. In a follow-on Phase II program, thehuman and mouse whole transcriptome assays will be implemented, the TempO-LINC assay commercialized,and additional applications demonstrated using cell lines, purified cells, and cells dissociated from tissues. Theperformance and capabilities of the TempO-LINC single cell assay will drive expansion of applications pursuedwithin the single cell field beyond identification of subpopulations of cells.

Public Health Relevance Statement:
Project Narrative Singe cell gene expression assays have become important tools to identify functional subtypes of cells and changes in the numbers of cells within each subtype, or of subtypes, resulting from disease and treatments. However, current methods require dedicated hardware and kits, making them expensive, and do not permit low expressed genes that may be key biomarkers to be measured, typically only measuring a limited number of genes/cell, typically not providing quantitative measurements of abundance or changes in expression level, and are often 3' based and so cannot measure splice variants or RNA such as long non-coding RNA that are not polyadenylated. We will demonstrate the feasibility of a novel TempO-LINC assay using a barcoding approach that does not require proprietary hardware, and demonstrate that this assay provides single cell data that measures the low and moderately expressed genes identified from bulk samples, is highly correlated to bulk cell benchmark assay data with respect to the number of genes/cell, represents the dynamic expression range of those genes within each cell, and can provide single cell dose response data - enabling single cell studies not previously possible to understand (e.g.) molecular mechanistic differences at the single cell level of disease, and drug efficacy and safety.

Project Terms:
Archives ; Bar Codes ; barcode ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Complementary DNA ; cDNA ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; Cells ; Cell Body ; Disease ; Disorder ; Gene Expression ; Gene Fusion ; Genes ; Goals ; Human ; Modern Man ; Ligase ; Ligase Gene ; Synthetases ; Ligation ; Closure by Ligation ; Methods ; Mus ; Mice ; Mice Mammals ; Murine ; Noise ; Normal Statistical Distribution ; Gaussian Distribution ; Normal Distribution ; Nucleotides ; Oligonucleotides ; Oligo ; oligos ; Research Personnel ; Investigators ; Researchers ; RNA ; Non-Polyadenylated RNA ; RNA Gene Products ; Ribonucleic Acid ; RNA Splicing ; Splicing ; Signal Transduction ; Cell Communication and Signaling ; Cell Signaling ; Intracellular Communication and Signaling ; Signal Transduction Systems ; Signaling ; biological signal transduction ; Species Specificity ; Specificity ; Sulfhydryl Compounds ; Mercaptans ; Mercapto Compounds ; Thiols ; sulfhydryl group ; Suspensions ; Suspension substance ; Time ; Tissues ; Body Tissues ; Measures ; In Situ Hybridization ; in situ Hybridization Genetics ; in situ Hybridization Staining Method ; base ; crosslink ; cross-link ; detector ; improved ; Phase ; Variant ; Variation ; data quality ; Measurement ; tool ; programs ; Protocol ; Protocols documentation ; In Situ ; Best Practice Analysis ; Benchmarking ; adduct ; Performance ; drug efficacy ; PBMC ; Peripheral Blood Mononuclear Cell ; novel ; sorting ; Sorting - Cell Movement ; Single Base Polymorphism ; single nucleotide variant ; Single Nucleotide Polymorphism ; Gene Expression Monitoring ; Gene Expression Pattern Analysis ; Transcript Expression Analyses ; Transcript Expression Analysis ; gene expression analysis ; gene expression assay ; transcriptional profiling ; Gene Expression Profiling ; Sampling ; response ; drug development ; Functional RNA ; Non-Coding ; Non-Coding RNA ; Non-translated RNA ; Noncoding RNA ; Nontranslated RNA ; noncoding ; Untranslated RNA ; Genomics ; cell preparation ; RNA Polyadenylation ; Polyadenylation ; preventing ; prevent ; CD62L ; HLHRC ; LAM-1 gene ; LECAM1 ; LNHR ; LSEL ; LYAM-1 ; LYAM1 ; Selectin L Gene ; TQ-1 ; SELL gene ; Address ; Dose ; Crosslinker ; Data ; Mouse Cell Line ; Sum ; Cellular Assay ; cell assay ; Transcript ; Molecular ; Process ; Pathway interactions ; pathway ; cost ; Coupling ; innovation ; innovate ; innovative ; combinatorial ; translational medicine ; Biological Markers ; bio-markers ; biologic marker ; biomarker ; transcriptome sequencing ; RNA Seq ; RNA sequencing ; RNAseq ; single cell sequencing ; differential expression ; differentially expressed ; transcriptional differences ; transcriptome ; global gene expression ; global transcription profile ; experimental study ; experiment ; experimental research ; safety assessment ; medication safety ; drug safety ; pharmaceutical safety ;

Single cell gene expression assays have become critical tools for identifying functional subtypes of cells,as well as changes within specific subtypes resulting from development, disease or therapy treatment. However,most current methods are expensive, low in sample throughput and rely on oligo-dT primed reverse transcription.Furthermore, there is no method to-date that enables investigators to consistently measure low expressed genesfrom single cells, which prevents measurements of many key biomarkers and important genes and molecularpathways. Another problem with existing single-cell sequencing methods is that many of the cell-associatedreads either fail to map to transcripts or map to uninformative ribosomal and mitochondrial genes. Theaforementioned limitations have all contributed to most single-cell sequencing studies being restricted torelatively small numbers of samples and have prevented their wider scale adoption in biopharma, clinical,translational and applied markets. In Phase 1 SBIR development, we implemented a novel combinatorial split-pool-based workflow,TempO-LINC, that adds cell-identifying molecular barcodes onto high-sensitivity gene expression probes. Allprobes within the same cell receive an identical barcode, enabling the reconstruction of single-cell geneexpression (and protein) profiles across tens of thousands of cells. TempO-LINC is an instrument free approachthat multiplexes 96 1,000 cell samples per run, has a simple workflow that is amenable to automation and hasthe highest gene detection rate of any commercial single-cell platform. Critically, TempO-LINC reduces both theper sample assay cost as well as associated sequencing costs; furthermore, although we show the assaysimultaneously profiles the whole transcriptome (23,000 transcripts), it can also be targeted to measure onlygenes/pathways of interest - dramatically reducing sequencing costs yet further. All of these features positionTempO-LINC as a potential disruptive technology that researchers, clinicians and drug developers can use fornew large-scale applications/studies using single-cell sequencing across thousands of samples while obtainingimproved data quality. Our objective in this Phase 2 proposal is to take the basic TempO-LINC platform that we are currentlyrunning and ready it for commercialization by optimizing the sensitivity, workflow, multi-omic capability andsoftware. To demonstrate the unique capabilities of TempO-LINC, we will benchmark it against 10X Chromiumdata using PBMCs and demonstrate a high-impact application of our technology with our collaborators/partners.Our approach relies on existing commercial TempO-Seq assays/technology, Phase 1 data that far exceeded allmilestones, and a solid foundation of intellectual property. BioSpyder's TempO-LINC constitutes a powerfulsingle-cell platform with high potential for technical success, which will fill a critical need in the marketplace.

Public Health Relevance Statement:
Project Narrative The analysis of rare and biologically important cell subtypes presents a common challenge in development and disease, where phenotypically variant cell populations can significantly affect the response to therapy and impact patient outcomes. High-throughput single-cell sequencing has proven to be the most successful approach for identifying unique cell subtypes and resolving complex cellular heterogeneity The vision of BioSpyder is to commercially launch a low cost, high-throughput and targeted single-cell multi-omic platform, TempO-LINC, that provides numerous advantages over existing commercial platforms and will enable new studies relying on single-cell sequencing in basic, biopharma, clinical, translational and applied markets.

Project Terms: