Xu T.,Oak Ridge National Laboratory |
Ripp S.,490 BioTech, Inc |
Sayler G.S.,Oak Ridge National Laboratory |
Close D.M.,490 BioTech, Inc
PLoS ONE | Year: 2014
Background: Expression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines. Methodology/Principal Findings: The lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations. Conclusions/Significance: Our results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC50 values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows. © 2014 Xu et al.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 995.75K | Year: 2015
DESCRIPTION provided by applicant Expressing bacterial bioluminescence in human cell lines Engineering autobioluminescent reporter cells to screen for endocrine disruptor chemicals Project Summary This Small Business Innovation Research Phase II project proposes to build upon our successful Phase I demonstration of autonomous endocrine disruptor chemical EDC detection using human cell lines to engineer a novel Tier screening assay for the low cost high throughput detection of estrogenically and androgenically active compounds across multiple human tissue types to address the National Institute of Environmental Health Sciences NIEHS request for Improved Test Systems for Prioritization and Safety Evaluation Current Tier EDC screening approaches require the use of non human cell lines that can obscure bioavailability data employ radioactive materials that require dedicated use areas and specially trained personnel or rely upon the use of expensive analytical equipment that prevents high throughput testing Furthermore following this initial Tier screen those chemicals that putatively express endocrine disrupting activities must still be screened against Tier animal models to validate the results Currently there are over contract testing service companies in the U S alone that perform Tier style assays for the chemical pesticide and personal care products industries at an average cost of $ per assay However as the number of potential EDCs requiring testing expands and with many states now adopting legislation to begin screening all municipal water supplies for EDCs market growth within this sector is expected to increase at an annual rate of to approach $ billion by clearly demonstrating the need for new assays that can reduce screening costs increase screening throughput and provide more relevant human bioavailability data without necessitating the use of animal models The EDC responsive substrate free autobioluminescent reporter cell lines developed by BioTech under our Phase I efforts demonstrated the ability to detect EDCs similarly to existing Tier screening assays while employing a human cell culture based model that significantly reduced the cost and personnel effort of testing while simultaneously providing an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacted with and reacted to EDC treatment In this proposal we will leverage this technology to develop fully self contained human cell based high throughput screening assays that provide more accurate and realistic information in regards to EDC bioavailability and effects on human health With application of these assays reducing the cost of Tier EDC screening over fold to less than $ per compound we believe we possess a product capable of significantly impacting the EDC screening market and advancing our understanding of novel compound endocrine disruptor activity as it pertains to public health and consumer safety PUBLIC HEALTH RELEVANCE In response to public and scientific concerns that chemical exposure can interfere with the delicate balance of the endocrine system to trigger adverse health effects in humans and animals government agencies worldwide have implemented comprehensive screening programs to characterize tens of thousands of compounds for endocrine disruptor activity Unfortunately the complex nature and high cost of the screens currently used for these efforts have thus far only been able to generate data on a limited number of chemicals resulting in an extensive untested backlog that is hindering our understanding of how endocrine disruption impacts human and ecosystem health In this Phase II Randamp D effort BioTech proposes to implement a novel endocrine disruptor screening strategy based upon a `humanizedandapos bacterial luciferase genetic construct that links endocrine disruptor activity to autonomous bioluminescent light emission to provide increased data workflows at a greater speed and with less cost than any existing endocrine disruptor assay
Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.55K | Year: 2015
Dioxin and other dioxin-like chemicals are persistent environmental pollutants that threaten human and animal health due to their stability in the environment and their ability to accumulate in the food chain toward eventual human consumption. Incidents of dioxins being found in the food and animal feed chain have led to numerous recalls with consequent impacts on human and animal health as well as significant economic after-effects. The aim of this study is to design an improved assay for the detection and monitoring of dioxins in food and animal feeds. Using a unique assay strategy that consists of yeast cells that emit light upon contact with dioxin chemicals, it is anticipated that a sensitive, rapid, and cost-effective assay can be developed that outperforms the current inventory of dioxin assays and transitions screening toward assay formats that can be completed in several hours rather than several days and with no animal-based testing involved. Methods to accomplish this goal will include the application of synthetic biology to bioengineer the yeast cells to respond to the dioxin chemicals, with outputs focused on determining the sensitivity, speed, and economic savings associated with the assay. Use of this new assay technology will allow for expanded testing of food and feed commodities for dioxins without consequent increases in costs. This will permit government agencies and commercial manufacturers to test a greater percentage of the food/feed supply to provide a greater probability of early dioxin detection further up the farm-to-table pathway before products are released to the public. The resulting decrease in dioxin contamination incidents will ultimately reduce public health risks and improve the safety and security of the U.S. food supply.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 138.19K | Year: 2013
DESCRIPTION (provided by applicant): Expressing bacterial bioluminescence in human cell lines: Engineering autobioluminescent reporter cells to screen for endocrine disruptor chemicals Project Summary This Small Business Innovation Research Phase I projectproposes to engineer an autobioluminescent human cell line for improved screening of chemicals displaying endocrine disruptor activity to address the National Institute of Environmental Health Sciences (NIEHS) request for tools for improved exposure assessment related to endocrine active chemical exposures. Endocrine disruptor chemicals (EDCs) potentially originating from plastics, pesticides, and pharmaceutical and personal care products are under scrutiny due to implications that they cause adverse health effects in humans and wildlife, including increased cancer rates, lowered sperm counts, early puberty in females, and altered functions of reproductive organs. Chemical manufacturers and importers are being mandated to test their chemicals for endocrinetoxicity effects under the auspices of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). With 87,000 chemicals potentially requiring testing, the battery of Tier 1 EDC screening assays have been recognized as being too slow and too costly to meetcurrent demands, and the EPA has been tasked with transitioning its EDSP program towards faster and more cost-effective screening technologies. To address this need, 490 BioTech proposes to engineer a human cell line endowed with a bacterially derived bioluminescent reporter gene construct for improved monitoring of endocrine active chemicals. The current market of bioluminescent reporter cells being applied towards EDC screening rely upon a firefly luciferase gene construct that must be provided with a substrate to activate its light emission response, resulting in only marginally informative single time point snapshots of potential toxicological interactions. Our proposed substrate-free, autobioluminescent reporter cell line will emit light continuously and in real time in response to endocrine active compounds, thus providing an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacts and reacts to target chemical exposure. Further, using a human cell as the sensing platform provides more accurate and realistic information in regards to bioavailability and a chemical's true effect on individual human health. With over 500 contract laboratories performing Tier 1 screening assays at an average cost of 1,000/assay over a potential inventory of 87,00 chemicals, and with the EPA mandating the integration of faster/better/cheaper technologies into the Tier 1 screening scheme, we believe we possess a product capable of significantly impacting the chemical/drug screening market and, here in particular, advancing our understanding of endocrine active chemicals as they pertain to public health and consumer safety. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Certain hormonally active chemicals, collectively referred toas endocrine disruptor chemicals (EDCs), mimic or antagonize the actions of naturally occurring estrogens and are believed to result in adverse developmental, reproductive, neurological, and immune effects in both humans and wildlife. The chemical, pesticide, personal care products, plastics, and nutritional supplements industries have been tasked with screening their products for EDCs. Using a novel autobioluminescent human cell line, 490 BioTech proposes to advance current EDC screening assays with a reporter cell technology that surveys EDC activity continuously and in real time to deliver more data faster and more cost effectively for improved safeguarding of public health.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 149.96K | Year: 2015
DESCRIPTION provided by applicant This Small Business Technology Transfer STTR Phase I project proposes to develop autonomously bioluminescent human stem cells for continuous reagent free and real time bioimaging to address the National Institutes of Healthandapos s request for new techniques for non invasive long term tracking of stem cell survivability engraftment and migration following in vivo implantation The ability of stem cells to self renew and differentiate into other cell lineages has emerged as a valuable therapeutic approach to functionally heal previously irreparable tissues and organs However for the regenerative medicine field to effectively transition toward translational and clinical practice outcomes a strong dependence on animal models will be required to fully understand the capabilities and complexities of stem cells BioTech proposes to expand the informational capacity of animal models by creating stem cell lines that self generate bioluminescent light via expression of a andapos humanizedandapos bacterial luciferase thereby enabling stem cells to be continuously imaged throughout their lifetime as they physiologically function within their animal host This differs significantly from the current market of bioluminescent imaging technologies that rely on a firefly luciferase gene construct that must be provided with a chemical substrate to activate its light emission response resulting in only marginally informative single time point snapshots of cell function in tandem with repetitive animal injections that invoke unknown and potentially interfering interactions and adversely effects animal welfare In partnership with the University o Tennessee Medical Center the specific objectives of this Randamp D effort are to develop piggyBac transposition and lentiviral transduction methods for streamlined integration of the bioluminescent phenotype into adipose derived mesenchymal stem cell lines followed by performance evaluation in in vitro D scaffolds and in vivo mouse models to demonstrate proficiency toward uninterrupted imaging and enriched data flows that far exceed that of existing firefly luciferase methods With no change in instrumentation or fundamental bioluminescent protocols necessary researchers can seamlessly transition from firefly luciferase to BioTechandapos s humanized bacterial luciferase technology to advance their in vivo experimental Randamp D to more informative endpoints with fewer animals required The contribution of this innovative imaging platform to the field of regenerative medicine will provide more physiologically relevant and representative data critical to predicting the efficacy and safety of treatment strategies as they precede to clinical trials PUBLIC HEALTH RELEVANCE Regenerative medicine using stem cell therapies to replenish and restore tissues and organs has the potential to transform human health by curing and remedying previously unmanageable diseases Animal studies have played significant roles in deciphering the therapeutic capacity of stem cells but there exists a disparity between the results obtained from animal experiments and their transition to human clinical trials that are impeding advancements in the regenerative medicine field To increase the amount of experimental information obtainable from animal models BioTech proposes to create stem cell lines that continuously emit bioluminescent light thereby enabling implanted stem cells to be visualized and tracked throughout their lifetime for improved understanding of their therapeutic potential and limitations directly within living animal subjects
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.92K | Year: 2015
DESCRIPTION provided by applicant Expanding the throughput of real time toxicological screening of cardiac differentiation by expressing a synthetic luciferase luciferin genetic pathway in iPSCs Project Summary This Small Business Innovation Research SBIR Phase I project proposes to develop autonomously bioluminescent induced pluripotent stem cells iPSCs for continuous reagent free and real time toxicological screening to address the National Institute of Environmental Health Sciences NIEHS request for novel high throughput assays to evaluate the effects of chemical compounds on the differentiation of pluripotent stem cells as their ability to differentiate along well defined lineage pathways offers a powerful approach to understanding how chemical perturbations disrupt metabolic and regulatory functions along those pathways The autobioluminescent iPSCs developed here will significantly contribute towards NIEHSandapos s mission to andapos discover how the environment affects people in order to promote healthier livesandapos by expanding the knowledge base of chemical exposure toxicological effects This is especially important given that the commercial marketplace maintains an inventory of tens of thousands of chemicals the majority of which are poorly understood in terms of their risks and hazards to human health and currently require animal based testing approaches that are expensive time consuming and ethically contentious to determine their human health effects As an alternative stem cell based assays such as the one developed here may mimic human disease states more reliably than animal models while providing valuable information towards understanding how chemical exposures influence cancer risks developmental defects and other adverse health outcomes Therefore there exists significant impetus for the integration of stem cells in chemical screening programs such as Tox and ToxCast but under the mandate that they function under high throughput conditions While this goal is not obtainable using existing bioluminescent reporter technologies such as firefly luciferase that must be provided with a chemical substrate to activate their light emission responses resulting in only marginally informative single time point snapshots of potential toxicological interactions BioTechandapos s synthetic luciferase technology enables reporter cells to emit light continuously and in real time thereby providing an uninterrupted stream of visual data over the lifetime of the cell as it interacts and reacts to chemical perturbations The goal of this research effort is o express our synthetic luciferase system in iPSCs and demonstrate real time continuous visualization of iPSC to cardiomyocyte differentiation under chemical toxicity exposure pressures Our specific aims will focus on assay development and optimization benchmark comparisons against existing commercial assay systems and assay validation against a chemical subset of the Tox K library PUBLIC HEALTH RELEVANCE Humans interact with a vast landscape of chemicals on a daily basis yet the health effects of many of these chemicals are poorly understood Numerous U S and international multiagency sponsored programs have been established to test chemicals for toxicity effects to better ensure consumer safety but the process of testing tens of thousands of chemicals is challenging and is currently limited by assays that do not provide data fast enough nor with sufficient informational content that directly relates to human health impacts To assist in creating assays that will better predict chemical risks and hazards BioTech proposes to create human stem cell lines capable of autonomously emitting bioluminescent light in response to chemical exposure events thereby enabling chemical toxicity screening to occur throughout the lifetime of the cell to yield an expanded informational database of toxicological effects with direct relevance to human health
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 149.05K | Year: 2015
DESCRIPTION provided by applicant Development of a high throughput screen to detect the effects of both pre and post biotransformed compounds for enhanced content drug discovery workflows Project Summary This Small Business Innovation Research Phase I project proposes to engineer a panel of autonomously bioluminescent human cell lines for the simultaneous high throughput detection of both pre and post biotransformed cytotoxicity onsets resulting from drug treatment across multiple tissue types to address the National Institute of General Medical Sciences NIGMS request for novel in vivo and in vitro methods for predicting the safety and toxicities of pharmacologic agents With an average of novel molecules that must be screened for each new lead compound developed and an average of to years of research and development at a cost of up to $ B to manufacture one new drug pharmaceutical companies must develop new testing regimens that provide more data at a lower cost in order to achieve the economics necessary to remain profitable Up to of failures for these new compounds at the clinical level are related to cytotoxicity which often onl manifests during the costly and time consuming process of whole animal testing This problem could be significantly mitigated by coordinately screening multiple tissue types simultaneously in a high throughput fashion during upstream tier screening However with existing bioluminescent reporter technology this is simply not possible because the current market of bioluminescent reporter cells being applied toward toxicology screening relies upon a firefly luciferase gene construct that must be provided with a chemical substrate to activate its light emission response resulting in only marginally informative single time point snapshots of potential toxicological interactions In contrast our substrate free autobioluminescent reporter cell lines emit light continuously and can modulate the output level of this signal in real time in response to cytotoxic interactions This provides an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacts and reacts to compound exposure at both its pre and post biotransformed states Furthermore by leveraging human cells as the sensing platform our assay provides more accurate and realistic information in regards to bioavailability and a chemicalandapos s true effect on individual human health than does the employment of small animal models With current in vitro screening assays now representing a $ B market with a predicted annual growth rate we believe we possess a product capable of significantly impacting the chemical drug screening market and here in particular advancing our understanding of cytotoxic chemical biotransformations as they pertain to public health and consumer safety PUBLIC HEALTH RELEVANCE The drug discovery process requires that upwards of molecules be screened for each new lead compound developed and relies upon an expensive and time consuming combination of in vitro cell culture based and in vivo whole animal based models to identify validate and ensure the safety of any resulting potential therapeutic agents This process is frustratingly exacerbated by an unfortunate dichotomy whereby the inexpensive in vitro cell culture systems used for tier screening contribute to failures due to their inabiliy to model the complexity and parallel systems interaction inherent in whole animal models which are responsible for up to of new compound failures at the clinical level and because they are not capable of demonstrating species specific effects To overcome these detractions and develop an improved tier screening system that reduces the cost and time required for new compound evaluation BioTech proposes to develop a panel of multiple continuously bioluminescent human cell lines that will permit the simultaneous monitoring of each line to ascertain both the individual effects of compound treatment as well as the downstream effects of a compoundandapos s biotransformed metabolic breakdown products in real time
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE I | Award Amount: 150.00K | Year: 2014
This Small Business Innovation Research (SBIR) Phase I project proposes to develop self-generated bioluminescence in zebrafish to create improved models for new drug discovery, therapeutic testing, and biomedical disease imaging. Zebrafish share many common anatomical, developmental, and physiological features with humans, including disease pathologies and drug metabolism effects, and serve as ideal models for understanding how drugs and other chemicals facilitate disease management as well as cause harm through unidentified side-effects. The objectives of this research effort are to express the bacterial luciferase genes in zebrafish to create autonomously bioluminescent zebrafish models capable of delineating the biological and metabolic effects of human/drug interactions. The self-generation of bioluminescence in zebrafish is anticipated to enable higher-throughput screening assays to generate data in real time, faster, and in larger quantities than previously possible, thereby accelerating the pace of new drug discovery while reducing associated costs.
The broader impact/commercial potential of this project, if successful, will be to improve pharmaceutical drug discovery and efficacy testing, and to facilitate better human and animal disease management. Small and rapidly growing zebrafish function to reduce space and maintenance requirements, thus lowering the costs associated with pharmaceutical research and new drug discovery while decreasing the industrys reliance on animals for laboratory testing. Scientific and technological understanding in the biomedical fields will be enhanced due to massively data-rich higher-throughput screening for drug action and disease therapies. Ancillary to these applications will be similar zebrafish screening strategies relevant to the detection and surveillance of chemical contaminants in the environment for the assurance of clean and safe water to better meet growing societal needs.
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2014
This Small Business Innovation Research (SBIR) Phase I project proposes to develop self-generated bioluminescence in zebrafish to create improved models for new drug discovery, therapeutic testing, and biomedical disease imaging. Zebrafish share many common anatomical, developmental, and physiological features with humans, including disease pathologies and drug metabolism effects, and serve as ideal models for understanding how drugs and other chemicals facilitate disease management as well as cause harm through unidentified side-effects. The objectives of this research effort are to express the bacterial luciferase genes in zebrafish to create autonomously bioluminescent zebrafish models capable of delineating the biological and metabolic effects of human/drug interactions. The self-generation of bioluminescence in zebrafish is anticipated to enable higher-throughput screening assays to generate data in real time, faster, and in larger quantities than previously possible, thereby accelerating the pace of new drug discovery while reducing associated costs. The broader impact/commercial potential of this project, if successful, will be to improve pharmaceutical drug discovery and efficacy testing, and to facilitate better human and animal disease management. Small and rapidly growing zebrafish function to reduce space and maintenance requirements, thus lowering the costs associated with pharmaceutical research and new drug discovery while decreasing the industry's reliance on animals for laboratory testing. Scientific and technological understanding in the biomedical fields will be enhanced due to massively data-rich higher-throughput screening for drug action and disease therapies. Ancillary to these applications will be similar zebrafish screening strategies relevant to the detection and surveillance of chemical contaminants in the environment for the assurance of clean and safe water to better meet growing societal needs.
490 BioTech, Inc | Date: 2016-02-22
Biochemicals, namely, polynucleotides for in vitro and in vivo scientific and research purposes; Cells for scientific, laboratory or medical research.