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 Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease.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: 149.97K | Year: 2010
A molecular test using FFPE tissue from patients to distinguish basal-like breast cancer from other subtypes will be developed and confirmed. The test will be based on a commercial platform, the lysis-on ly quantitative Nuclease Protection Assay (qNPA 1M). Current gene expression signatures described for breast cancer, in particular those for distinguishing basal-like breast cancer from other subtypes, will be incorporated into a profiling qNPA assay along with expressed SNP's and gene fusions associated with breast cancer and miRNA discovered by profiling on a whole transcriptome qNPA microarray. The comparative standard will be a 50 gene signature extensively investigated in the literature, which is the basis for the definition of basallike breast cancer. This signature will be among those incorporated into the profi ling assay. Samples used will include two archived sets for which the ERJPR and HER2 status as well as outcome are defined, one set predominately Caucasian, the other predominately African American. The program will provide a ~qNPA Basal Breast Cancer Classifier", the qNPA BBCC. The significance of the qNPA BBCC test will be low cost, simplicity, specificity, accuracy, reproducibility, sensitivity, robustness, and use of clinical samples as they are collected today from biopsy and surgery - FFPE and HandE stained FFPE. The platronm will be able to run diagnostic tests for other forms of cancer. The qNPA BBCC will identify the basal-like and other subtypes, providing patients information as to the prognosis of their disease and oncologists information for selecting the ~ best therapy" for individual patients. The test will be used to select patients for clinical trials, aid ing in the development of new therapies and potentially serving as a companion diagnostic
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: 309.02K | Year: 2011
DESCRIPTION (provided by applicant): miRNA biomarkers related to Alzheimer's disease (AD) that may be useful as a diagnostic tool or in elucidating the mechanisms of disease and discovery of novel therapeutics will be identified using a novel whole transcriptome qBead X-MAP-based assay (not yet launched commercially). Biomarkers will be identified from brain formalin fixed (FFPE) tissue and matched serum samples of AD patients, patients diagnosed Parkinson's disease and dementia (PD), patients with cerebrovascular disease neuropathologically diagnosed as vascular dementia (VaD), and normal controls (NC). The results will be validated using matched frozen tissue and both the qBead assay and PCR. The qBead X-MAP assay utilizes the quantitative Nuclease Protection Assay (qNPA ) and measures both miRNA and mRNA. It is very precise and sensitive. This program will exploit the qBead assay to validate the utility of FFPE and serum for identification of AD biomarkers, to confirm recent reports of altered miRNA levels in frozen brain of AD patients, and to provide an early example of an application of the qBead whole transcriptome miRNA assay. In Phase II mRNA biomarkers will be included and the studies expanded to a larger training set of AD, PD, VaD and NC FFPE, serum, buffy coat, and plasma samples to expand the biomarker set and then test an independent set of samples to confirm/validate the biomarkers. PUBLIC HEALTH RELEVANCE: miRNA biomarkers of Alzheimer's disease (AD) useful for diagnosis, for identifying mechanisms of disease, and as targets for drug discovery will be identified from brain FFPE and serum using a novel whole transcriptome qBead X-MAP-based assay that has not yet been launched commercially. The qBead X-MAP assay utilizes the quantitativeNuclease Protection Assay (qNPA ) and measures both miRNA and mRNA. It is very precise and sensitive and will enable the identification of low abundant miRNA and those that change by as little as 1.2-fold. Such small changes can be particularly relevant intissues where a 1.2-fold change in a target population of cells making up only 40% of the sample may reflect a 50% change in those cells.
High Throughput Genomics, Inc. | Date: 2011-03-02
The present invention relates to methods useful for concurrently performing assays for detecting nucleic acids and employing nuclease protection fragments. The assays may be using repeated arrays of probes. A surface, which comprises a plurality of test regions, at least two of which, and in a preferred embodiment, at least twenty of which, are substantially identical, wherein each of the test regions comprises an array of generic anchor molecules may be used. The anchors are associated with bifunctional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion which is a probe specific for a target of interest. The resulting array of probes is used to analyze the presence or test the activity of one or more target molecules which specifically interact with the probes. A sample to be tested is subjected to a nuclease protection procedure before it is contacted with a combination of the invention.
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.
High Throughput Genomics, Inc. | Date: 2011-03-16
A method and apparatus for analyzing molecular structures within a sample substance using an array having a plurality of test sites upon which the sample substance is applied. The invention is also directed to a method and apparatus for constructing molecular arrays having a plurality of test sites. The invention allows for definitive high throughput analysis of multiple analytes in complex mixtures of sample substances. A combinatorial analysis process is described that results in the creation of an array of integrated chemical devices. These devices operate in parallel, each unit providing specific sets of data that, when taken as a whole, give a complete answer for a defined experiment. This approach is uniquely capable of rapidly providing a high density of information from limited amounts of sample in a cost-effective manner.
High Throughput Genomics, Inc. | Date: 2011-02-11
Scientific equipment for use in genetic and genomic analysis, namely, nucleic acid arrays, scanners, autoloaders, and software for analyzing data. Systems, arrays and reagents for medical research and clinical diagnostic use.