Whole Genome qNPA Gene Expression Platform

DESCRIPTION (provided by applicant): The identification of gene targets for drug discovery, including AIDS research and mental health, relies heavily on the use of high density array whole genome platforms. It is also true for the discovery of biomarkers for diagnostic assays. The quantitative reliability and reproducibility of data from such platforms is low, requiring both extensive validation and limiting identification of genes to those that exhibit large differences in expression level between control and treated. It is important to identify novel biomarkers of the host and target cell response that can be used as a means to select the best therapy, monitor the effectiveness of therapy, to address key questions such as whether there are compensatory host cell mechanisms in certain individuals or cell types that help attenuated viruses survive, to develop new drugs, and even to predict safety. The objective of this Phase I proposal is to demonstrate the feasibility of establishing a high density array whole genome assay with vastly improved quantitative performance, the high density array whole genome quantitative Nuclease Protection Assay (qNPA(TM)), that has the performance of the current ArrayPlate qNPA. The whole genome qNPA will be run on an existing custom high density array platform (e.g. Agilent or Affymetrix). In this Phase I the development and validation of the whole genome qNPA will demonstrate that it provides an assay that can measure gene expression from lysed samples without extraction or gene amplification, including clinical samples that other whole genome assays cannot use with good result (e.g. archived fixed tissues). For instance, the ability to access archived post mortem samples will permit the response of host cells not accessible from living patients to be studied in depth. This Phase I will also demonstrate that the assay has the reproducibility to reliably measure changes in gene expression <50%, ideally <25%, and to enable whole genome dose response data to be obtained from which precise EC50 values can be obtained for every regulated gene, permitting genes to be clustered in a novel manner which the investigators hypothesize reflects mechanism of action and functionally different response phenotypes. The Phase I will demonstrate this using a set of [1,600] genes (matching the Agilent feature count of (1,600) genes/array for their 8 arrays/slide format) and model systems of PMA-differentiated Thp-1 cells stimulated with bacterial LPS as a model of host cell response, matched frozen and fixed tissue, and whole blood.