SBIR-STTR Award

Human exposure to dangerous genotoxins is unavoidable, as DNA damaging agents are ubiquitous both in our environment and within our cells. DNA damaging agents and other genotoxins that arise from cellular metabolism, environmental sources or disease-related cellular defects contribute to cell death (e.g., neurodegeneration), gene mutations, gene rearrangements and in many cases, the onset of cancer, disease and aging phenotypes. In addition, many exogenous exposures such as chemotherapy and radiation treatment rely on the induction of tumor cell genotoxicity to mediate therapeutic response. Further, the ability to effectively and accurately repair spontaneous or induced DNA damage depends on the cellular DNA repair capacity. Therefore, the ability to quantify DNA damage and the rate of repair of the damage to the nuclear genome directly in human cells is critical in applications ranging from epidemiology to drug development. To address this technological need in the research community, to be better positioned to characterize the genotoxicity of newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair gene defects, we propose the development of the next generation in DNA damage detection and quantification technology. This proposal, to develop the 'DNA Repair on a Chip'technology, combines the use of agarose-based Microwell arrays, spatially- encoded cellular recognition, human tumor cell lines with genetically-defined DNA repair status and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays that will function as a tool to quantify DNA damage and measure cellular DNA Repair capacity at baseline and following genotoxin exposure on a single array or chip (DNA Repair on a Chip). The studies described in Aim 1 involve the development of a series of 24-well Spatially Encoded Microwell Arrays, with Microwells ranging from 10-50 5M in diameter and 20-50 5M in depth, suitable for gravity capture of a single cell of various sizes. Efficacy of the Microwell Arrays will be validated using radiation and small molecule inhibitors. Further, the sensitivity of the Microwell Arrays for analysis of cellular DNA Repair capacity will be evaluated using an isogenic panel of human tumor cell lines with defined defects in DNA Repair gene expression and following genotoxic stress. Iterative analysis and Microwell characterization will inform to finalize a set of 24-well Microwell Arrays for production and distribution. The studies described in Aim 2 involve additives to the Microwell Arrays that will enhance cell growth and attachment, providing optimal analysis of baseline DNA damage and most importantly, critical data on cellular capacity for in vivo repair post-damage. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses. Phase II of the project will be expanded to offer 96-well capability, end-user software for spatial recognition and quantitation plus micro-well additive options for specialized cell growth and attachment.

Public Health Relevance:
We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.

Thesaurus Terms:
Address;Adhesions;Agarose;Aging;Articulation;Assay;Bioassay;Biologic Assays;Biological Assay;Caliber;Cancers;Cell Attachment;Cell Death;Cell Line;Cell-Matrix Adhesions;Cell-Matrix Junction;Cellline;Cells;Cellular Expansion;Cellular Growth;Cellular Matrix;Comet Assay;Communities;Computer Software;Cytoskeletal System;Cytoskeleton;Dna Alteration;Dna Damage;Dna Damage Repair;Dna Injury;Dna Rearrangement;Dna Repair;Dna Repair Gene;Dna Mutation;Data;Defect;Detection;Development;Diameter;Disease;Disorder;Environment;Environmental Health;Environmental Health Science;Epidemiology;Exposure To;Extracellular Matrix Proteins;Force Of Gravity;Gel;Gene Alteration;Gene Expression;Gene Mutation;Gene Rearrangement;Generalized Growth;Genetic Toxicity Tests;Genetic Mutation;Genome;Genotoxic Stress;Genotoxicity Tests;Genotoxins;Glass;Gravities;Growth;Human;Intermediary Metabolism;Investigators;Joints;Life;Ligands;Malignant Neoplasms;Malignant Tumor;Man (Taxonomy);Measurement;Measures;Mediating;Metabolic Processes;Metabolism;Method Loinc Axis 6;Methodology;Modern Man;Mutagen Screening;Mutagenicity Tests;Mutagens;Nerve Degeneration;Neuron Degeneration;Nuclear;Outcome;Pharmaceutical Agent;Pharmaceuticals;Pharmacologic Substance;Pharmacological Substance;Phase;Phenotype;Plastics;Position;Positioning Attribute;Production;Proteins;Radiation;Research;Research Personnel;Researchers;Sepharose;Sequence Alteration;Series;Single-Cell Gel Electrophoresis;Software;Source;Strains Cell Lines;Surface;Technology;Therapeutic;Tissue Growth;Tumor Cell;Tumor Cell Line;Unscheduled Dna Synthesis;Base;Cell Growth;Cell Type;Chemotherapy;Computer Program/Software;Cultured Cell Line;Design;Designing;Developmental;Disease/Disorder;Drug Development;Drug Discovery;Epidemiologic;Epidemiological;Expectation;Exposed Human Population;Gene Product;Genotoxic Agent;Genotoxicity;Human Exposure;In Vivo;Inhibitor;Inhibitor/Antagonist;Intracellular Skeleton;Malignancy;Mutagen Testing;Necrocytosis;Neoplasm/Cancer;Neoplastic Cell;Neural Degeneration;Neurodegeneration;Neurodegenerative;Neuronal Degeneration;New Technology;Next Generation;Novel Technologies;Ontogeny;Ray (Radiation);Repair;Repaired;Response;Small Molecule;Tool

Preserving genomic integrity is essential in order to suppress cancer, neurodegeneration, aging and other diseases. At odds with genomic preservation is DNA damage, which can drive mutations, sequence rearrangements and cellular toxicity. DNA damage is unavoidable, as DNA damaging agents are present in our environment and in our cells. To counteract the deleterious effects of DNA damage, we have evolved sophisticated DNA repair systems. It is now known that every major DNA repair pathway suppresses cancer. Furthermore, since cancer is often treated using DNA damaging agents, it is not surprising that the DNA repair capacity of tumors modulates sensitivity to chemotherapy. Despite its importance, measurements of DNA damage and repair are far from routine, primarily due to the lack of reliable and rapid DNA damage assays. Here, by bringing together convergent expertise among engineers, biologists and computer programmers, we propose to meet this need by developing a platform for rapid semi- automated single-cell DNA damage quantification that can be broadly distributed and readily applied by researchers in public health, academia, industry and medicine. As defined in the Phase I submission, we created and tested a prototype for a 96-well CometChip platform and have optimized the engineering design and a production apparatus to produce spatially encoded 20 and 96 well demonstrated that supplementation of the Microwell Comet gels with extracellular matrix proteins (EMPs) supports the growth of human cells for up to two weeks and the EMPs do not impact the formation of comets. To enable characterization of the genotoxicity of chemicals used commercially, those found in the environment or newly developed pharmaceuticals, and to quantify DNA repair capacity without the need to identify specific DNA Repair technology. This proposal, to develop the 'DNA Repair on a Chip' technology, combines the use of agarose based Microwell arrays, spatially encoded cellular recognition, automated data processing, and extra-cellular matrix proteins to optimize, validate and commercialize a series of Spatially Encoded Microwell Arrays. We will demonstrate that we have significantly advanced the manufacturing process (Aim1), have developed a macrowell former to produce 96-well and 384-welll CometChips (Aim 2), and propose the implementation of a graphical user interface for data analysis (Aim 3). Finally, we will rigorously validate this new technology by analyzing the genotoxic effects of a range of compounds from the NTP library for their impact on DNA damage and repair responses and to reveal inter-individual and inter-cell type variation in DNA damage responses (Aim 4). Through the integration of traditional methods in biology and engineering, the DNA Repair on a Chip platform described here represents a significant technological advance, providing high-throughput, objective, and quantitative measurements that have the potential to become a new standard in DNA damage analysis.

Public Health Relevance Statement:


Public Health Relevance:
We describe a new methodology that provides for robust, high-throughput DNA damage and repair analysis by exploiting gravity capture of single cells into a Microwell array. DNA damage levels are revealed morphologically by single-cell gel electrophoresis. The Microwell array enables fully automated DNA damage and DNA repair measurement of multiple experimental conditions simultaneously. This technological advance opens the door to new strategies for drug discovery, genotoxicity testing, and environmental health research through objective, quantitative analyses.

NIH Spending Category:
Bioengineering; Cancer; Genetic Testing; Genetics

Project Terms:
Academia; Aging; Area; Automatic Data Processing; base; Biological Assay; Biological Preservation; Biology; Caliber; cancer prevention; cell type; Cells; Chemicals; chemotherapy; clinical assay development; Comet Assay; Computer software; Computers; Cytoskeleton; Data; Data Analyses; Data Quality; Data Storage and Retrieval; design; Development; Disease; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA repair protein; DNA Sequence Rearrangement; Dose; drug development; drug discovery; Drug Industry; Engineering; engineering design; Ensure; Environment; Environmental Health; Epidemiologist; exposed human population; Exposure to; Extracellular Matrix Proteins; Feedback; Force of Gravity; Gel; Genomics; genotoxicity; Glass; Goals; graphical user interface; Growth; Hepatocyte; Human; Image; Image Analysis; In Vitro; in vivo; Individual; Industry; Institutes; Killings; Knowledge; Libraries; Mainstreaming (Education); Malignant Neoplasms; manufacturing process; Massachusetts; Measurement; Measures; Medicine; meetings; Methodology; Methods; Microscopy; Mutagenicity Tests; Mutagens; Mutation; neoplastic cell; Nerve Degeneration; new technology; novel; Output; Performance; Pharmacologic Substance; Phase; Play; Predisposition; prevent; Process; Production; programs; Proteins; prototype; public health medicine (field); public health relevance; Quality Control; Research; Research Personnel; response; Role; Sampling; Sepharose; Series; Services; Shipping; Ships; Site; Slide; Supplementation; System; Technology; Testing; Time; Toxic effect; tumor; University of Pittsburgh Cancer Institute; Validation; Variant