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TUCSON, AZ, United States

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 599.27K | Year: 2010

DESCRIPTION (provided by applicant): Sequencing is very powerful for identifying differences in genomic DNA that may regulate cell function and diseases, pre-dispose persons to certain diseases, or warn of adverse drug metabolism. It provides a basis for identifying differences in gene expression, though such applications have been limited and are problematic because each expressed gene can vary from a single copy per cell to 10's of thousands of copies. Further, its application to formalin fixed paraffin-embedded tissue (FFPE) is quite challenging. Millions of such samples, along with the corresponding treatment modalities and known clinical outcomes, are archived at clinical centers and hospitals. Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease. In addition, the utility of sequencing for routine experiments where array-based measurements are used today is limited by cost, and might also be limited by accuracy, sensitivity, and reproducibility of the sequencing data. This Phase I proposal develops and validates a new approach for addressing these challenges, Nuclease Probe Mediated Sequencing, provides a variation of candidate capture methods used to focus sequencing onto genes of interest, and addresses issues with array-based methods. The method proposed uses a lysis-only assay and either a variation of HTG's quantitative Nuclease Protection Assay (qNPA ) or ligation/exonuclease generated Padlock probes to generate DNA probes for sequencing by synthesis, combined with gene tagging and experiment tagging methods to permit pooling to provide cost reduction and higher sample throughput. PUBLIC HEALTH RELEVANCE: This Phase I project will address challenges to using sequencing for measuring gene expression. The proposed method will enable researchers to use archived fixed tissue samples. It will also lower the cost per sample and increase the number of samples tested per sequencing run, while potentially increasing the accuracy, dynamic range and reproducibility of measurement, expanding the utility of sequencing to measure gene expression and providing a more cost effective and precise platform than currently provided by high density array-based measurement.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.37K | Year: 2010

This Phase I proposal will develop and validate assays to measure the metabolism of compounds and their affect on liver cells using a multiplexed gene expression assay platform, the quantitative Nuclease Protection Assay (qNAP¿) that can measure the expression of up to 100 mRNA and miRNA and is a simple, robust and highly precise assay that provides high sample throughput. Two cell systems will be evaluated, HepaRG cell line and primary hepatocytes. The genes associated with metabolism are known, and they will be incorporated into the assay. miRNA associated with metabolism are not known, and they will be identified using a whole transcriptome miRNA assay. The final screening assay will incorporate both mRNA and miRNA, and will be performed in a 384-well format. The data generated will be dose response data so that differential metabolism can be assessed based of what genes are regulated as well as the EC50 at which they are regulated. The significance is that this assay will enable the screening of compounds for metabolism on a cell system that mirrors in vivo liver function and assess all the pathways by which compounds are metabolized, including Phase I and Phase II metabolism, and transporters, so that not only will the primary compound effects be monitored, but also the effect of metabolites produced by the cell system will be assessed. This will be done ins a single assay, something that cannot be done with enzyme assays. Acquiring dose response data and EC50's at the level of gene expression is highly novel, and brings gene expression up to the level where metabolism enzyme assays are today.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 600.00K | Year: 2010

DESCRIPTION (provided by applicant): There is currently no good way to screen for early stages of head and neck cancers caused by high risk types of human papillomavirus. The objective of this proposal is to develop a screening assay for dental patients to detect HPV+ head and neck cancer ( HNC ) pre-symptomatically, based on the use of viral and host gene biomarkers. The overall hypothesis of this proposal is that as the development of HPV-associated HNCs proceeds, a number of viral and cellular events occur that can be exploited as biomarkers. This combination of biomarkers creates a molecular signature for cells transformed by HPV and consequently could be used to identify individuals likely to develop cancer. More broadly, the same is true of HNC not associated with HPV infection. Furthermore, we propose that, due to the anatomical location of these tumors, the probability that cells from these cancers can be found in saliva in sufficient numbers for the detection of these biomarkers is high, though another accessible fluid for screening would be blood. Evidence for this hypothesis has been published. We will begin by profiling the gene expression changes associated with HPV+ and HPV- HNC using a new assay on a high density array platform, the qNPA assay. In this assay, we will validate genes implicated by previous studies as putative biomarkers, and also test viral DNA and mRNA coding for genes associated with HPV, additional genes that cover cellular pathways and receptors, and miRNA. We will use tissue samples and matched blood and saliva samples to identify those candidate biomarkers that can be found (ideally) in saliva and that have the potential to distinguish between HPV-associated HNC, HNC not associated with HPV, and controls. The overall goal of this proposal is to develop a rapid, accurate and cost- effective diagnostic tool for the early identification of pre-cancerous and cancerous lesions in the head and neck area, using saliva as a sample source, with Phase I SBIR being the identification of signature genes using high-density array measurements. It is the objective of this program that the ultimate diagnostic test panel developed in a follow-on Phase II will be performed by reference laboratories on samples collected in dental offices as part of routine patient assessments to identify HPV infection and HNC. Phase I will identify putative DNA, mRNA and miRNA biomarkers for use in the diagnosis of oral HPV and the early diagnosis of HPV+ HNC. It is anticipated that, based on these results, a focused diagnostic array test (or tests) can be designed for the low-cost ArrayPlate microplate platform (Phase I, Aim 4) to be developed and validated in a Phase II grant application, which will also fund the testing of samples of an accessible fluid (expected to be saliva, but alternatively blood) from a large number of dental patients. It is anticipated that this overall program will result in validated diagnostic tests for: i) oral HPV; ii) HPV+ HNC; iii) HPV- HNC; and for iv) the pre- symptomatic identification of HNC. PUBLIC HEALTH RELEVANCE: At this time, there is no good way to screen for head and neck cancers ( HNC ) that are caused by Human Papillomaviruses (HPV). We propose that as the virus infects these cells and cancer begins to develop, changes in the mRNA and miRNA expressed in the affected cells occur that can be detected and used as biomarkers for the development of cancer, and further, that these can be detected in easily-accessible material, such as blood or saliva. This proposal is designed to develop a panel of biomarkers that could be detected in saliva, thus making it much easier to identify those individuals who are developing or are at risk for developing HNCs, especially those associated with HPV. The goal is to make it possible to quickly and accurately detect early stage HPV-associated HNC in individuals during routine dental visits.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 299.44K | Year: 2008

DESCRIPTION (provided by applicant): The objective of this Phase I 2nd resubmitted proposal is to demonstrate the feasibility of establishing a screening assay and platform (SP-qNPA Microfluidic Screening Platform) that monitors host cell response to detec t exposure to, and provide diagnosis of wellness and potentially specific infectious agents (NIAID category A, B and C pathogens), determine the stage of infection, and either indicate patients to be isolated and followed up with more testing, or even perm it selection of the best treatment and monitoring of treatment course. The screening platform will perform a novel array-based multiplexed assay of gene expression (the quantitative Nuclease Protection Assay, qNPA ). The assay will be performed on a microf luidic platform. For Phase I that microfluidic platform is an adaptation of a current operational system that currently performs PCR and ELISA assays, developed by our collaborator, the Applied Nanobioscience Center (ANBC) of the BioDesign Institute, Arizo na State University. A programmable open format universal array that can be changed to measure any set of molecules by the simple addition of an appropriate reagent will be used to perform the screening SP-qNPA assay. The platform will provide a fast tur n around time (TAT) and enable the use of standard clinical samples (anti-coagulated blood, fixed tissue), not just samples specially prepared for the measurement of RNA. The assays of gene expression and protein only require lysis of the sample. Thus, thi s approach eliminates the need to extract RNA or carry out any biosynthetic step, such as reverse transcription or gene amplification, in order to measure RNA, greatly simplifying the assay. The sensitivity will be lt3,000 target RNA molecules and lt1ng/ml protein. The assays will be developed and integrated into the microfluidic platform as a b-test breadboard to permit performance to be determined as go/no-go criteria to proceed with a Phase II proposal. Thus, Phase I is designed to validate performance a nd address the major risk factors involved in the implementation of the qNPA to measure gene expression. Adaptation of the microfluidic compatable qNPA will be developed first in the same microplate format as the current commercial ArrayPlate qNPA is perfo rmed, benchmarked against the current assay, and then transferred into the microfluidic hardware for optimization, determination of go/no-go performance criteria, and further benchmarking against the current ArrayPlate qNPA. Ultimately, it is anticipated t hat the screening platform hardware costs will be lt 2,000, the cost/test will be lt 5 each, and the platform will be compact, easily transported, battery powered, robust, and capable of carrying out new response just-in-time tests simply by formatting dif ferent reagents. This screening platform will permit novel diagnostics to be developed that are useful in screening blood donations, and possibly for identifying and staging infectious diseases and all types of progressive disorders and to provide early di agnosis of the onset of adverse events such as transplant rejection or monitoring the onset of flares in lupus, as well as for identifying exposure to chemical agents and radiation using standard clinical samples.

High Throughput Genomics, Inc. | Date: 2010-11-03

The present invention provides a new approach, quantitative Nuclease Protection Sequencing (qNPS), for addressing several challenges that face sequencing and which provides improvements for research and diagnostic applications. The method uses a lysis-only nuclease protection assay to generate nucleic acid, e.g., DNA probes for sequencing, which can be coupled to gene-specific tags to permit the identification of the gene without necessitating the sequencing of the nuclease protection probe itself and/or can be coupled to experiment-specific tags whereby samples from different patients can be combined into a single run. The disclosed qNPS makes sequencing fixed or insoluble samples possible and affordable as a research and discovery tool and as a diagnostic test.

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