SBIR-STTR Award

?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 Health'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. 490 BioTech proposes to expand the informational capacity of animal models by creating stem cell lines that self- generate bioluminescent light via expression of a 'humanized' 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 R&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 3D 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 490 BioTech's humanized bacterial luciferase technology to advance their in vivo experimental R&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 Statement:


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, 490 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.

NIH Spending Category:
Bioengineering; Biotechnology; Clinical Research; Regenerative Medicine; Stem Cell Research; Stem Cell Research - Nonembryonic - Human; Transplantation

Project Terms:
Address; Adipose tissue; Animal Experiments; Animal Model; Animal Welfare; Animals; Bacterial Luciferases; base; bioimaging; Biological Models; Bioluminescence; Businesses; Cell Line; Cell Lineage; Cell physiology; Cell Therapy; Cells; Chemicals; clinical practice; Clinical Trials; Color; Complement; consumer demand; Data; data acquisition; Data Collection; Dependence; Discrimination (Psychology); Disease; Electroporation; Engineering; Engraftment; Evaluation; expression vector; Fireflies; Firefly Luciferases; Fluorescence; Gene Delivery; Gene Transfer; Genes; Genome; Green Fluorescent Proteins; Healed; healing; Health; Human; human stem cells; Image; imaging platform; Imaging technology; Implant; implantation; improved; In Vitro; in vivo; in vivo imaging; Injection of therapeutic agent; innovation; instrumentation; Legal patent; Life; Light; light emission; Longevity; Luc Gene; Luciferases; Marketing; matrigel; Measurement; Mediating; Medical center; Mesenchymal Stem Cells; Methods; migration; Monitor; Morphologic artifacts; mouse model; Noise; novel; Organ; Osteogenesis; Outcome; Output; Performance; Phase; Phenotype; Physiological; Play; Procedures; Production; Protocols documentation; public health relevance; Reagent; Regenerative Medicine; Renilla; Reporter; Reporter Genes; Research; research and development; Research Personnel; response; Role; Safety; scaffold; self-renewal; Signal Transduction; Small Business Technology Transfer Research; stem cell fate; stem cell therapy; Stem cells; Subfamily lentivirinae; Surface; Techniques; Technology; Technology Transfer; Tennessee; Therapeutic; Therapeutic Effect; Time; Tissue Engineering; Tissues; Transduction Gene; Transfection; Transplantation; Transposase; treatment strategy; two-dimensional; United States National Institutes of Health; Universities; Variant

Expressing humanized bacterial luciferase in stem cells: Moving beyond firefly luciferase to expand the informational capacity of animal models for regenerative medicine Project Summary This Small Business Technology Transfer (STTR) Phase II project proposes to develop complementary autonomously bioluminescent (autobioluminescent) in vitro stem cell lines and in vivo small animal model systems that enable the continuous, reagent-free, and real-time bioimaging of mesenchymal stem cell (MSC) localization, differentiation into adipocyte, chondrocyte, and osteocyte lineages, and persistence post- differentiation at the site of activation. These models will specifically address the National Institutes of Health's request for new techniques for non-invasive, long-term tracking of stem cell survivability, engraftment, and migration following in vivo implantation. By addressing this critical need for new methods capable of elucidating the mechanisms underlying how stem cells identify areas of dysfunction within the body, differentiate into the relevant tissues required to correct the malady, and persist in synergy with existing tissue to enable long term functionality, these tools will significantly improve the transition of regenerative medicine studies towards translational and clinical practice outcomes. The autobioluminescent MSCs developed by 490 BioTech under our Phase I effort demonstrated the ability to track MSC localization in vitro and in vivo similarly to existing optical imaging approaches, but with significantly reduced cost and personnel effort. Furthermore, these models also negated the need for sample destruction or the stressful and potentially influential injection of an activating chemical concurrent with imaging while simultaneously providing an uninterrupted stream of visual data over the lifetime of the reporter cell as it interacts with its environment and undergoes differentiation. In partnership with the University of Tennessee Medical Center, this proposal will expand upon these accomplishments to develop fully self-contained autobioluminescent MSC-based cellular models capable of specifically reporting on their differentiation into adipocyte, chondrocyte, and osteocyte lineages, and complementary small animal models harboring native MSCs genetically programmed to autonomously enact their reporter functionality only following differentiation into adipocyte, chondrocyte, or osteocyte lineages in response to wounding or exogenous stimulation. These models will overcome the primary technical hurdles encountered with all existing bioluminescent and fluorescent stem cells currently on the market from companies such as PerkinElmer, ThermoFisher/Life Technologies, Promega, and ~30 smaller specialized business entities in the U.S. alone, which comprise an estimated market value of at least $2B, with a predicated annual growth rate of 16-40%. We believe that the products developed in this effort will be capable of significantly improving the throughput and effectiveness of regenerative medicine studies and advancing our understanding of stem cell-based treatment efficiency and efficacy to improve both public health and consumer safety. The functional demonstrations and data gathered in this effort will position these models to thrive within this market and produce an immediate and significant impact on the field of regenerative medicine that will benefit the population at large.

Public Health Relevance Statement:
Project Narrative Stem cell-based regenerative medicine approaches have the potential to transform human health by improving wound healing efficiency, repairing previously irreversible losses of physical function, and curing or mitigating currently unmanageable diseases. At the core of this field are animal studies that attempt to unlock the mechanisms underlying how these cells identify areas of dysfunction within the body, differentiate into the relevant tissues required to correct the malady, and persist in synergy with existing tissue to enable long term functionality. To address the current lack of suitable models for longitudinally tracking stem cell homing and differentiation, this Phase II R&D effort will develop complementary in vitro cellular and in vivo animal model toolsets endowed with 490 BioTech's unique, substrate-free, autonomously bioluminescent light emission gene cassette to enable the continuous, non-invasive monitoring of mesenchymal stem cell localization, differentiation into adipocyte, chondrocyte, and osteocyte lineages, and post-differentiated tissue persistence to provide investigators with the data needed to understand stem cell functionality and develop new regenerative medicine treatment approaches.

Project Terms:
Address; Adipocytes; Adipose tissue; Animal Model; Animal Welfare; Animals; Architecture; Area; Bacterial Luciferases; base; Behavior; bioimaging; Biological Models; Bioluminescence; Biotechnology; Bone Tissue; Businesses; Cartilage; Cell Differentiation process; Cell Line; Cell Lineage; Cell model; Cell physiology; Cell Therapy; Cells; Chemicals; Chondrocytes; clinical practice; Color; cost; Data; Data Collection; Deposition; Detection; Discrimination; Disease; DNA cassette; Effectiveness; Engraftment; Ensure; Environment; Equipment; Fireflies; Firefly Luciferases; Fluorescence; fluorescence imaging; Functional disorder; Generations; Genetic; Green Fluorescent Proteins; Growth; healing; Health; Homing; Human; Human Resources; Image; imaging approach; Imaging technology; implantation; improved; In Vitro; in vivo; in vivo Model; Influentials; Injection of therapeutic agent; interest; Kinetics; Life; Light; light emission; Luciferases; Medical center; Mesenchymal Stem Cells; Methods; migration; Modeling; Morphologic artifacts; Noise; non-invasive monitor; novel; optical imaging; Osteocytes; Outcome; Output; Phase; Physical Function; Physiological; Population; Positioning Attribute; Procedures; Process; Production; Promega; promoter; Protocols documentation; Public Health; Publishing; Reagent; Regenerative Medicine; Renilla; repaired; Reporter; Reporter Genes; Reporting; research and development; Research Personnel; response; Safety; Sampling; Signal Transduction; Site; Stem cells; Stream; synergism; System; Techniques; Technology; Technology Transfer; temporal measurement; Tennessee; Therapeutic Effect; Time; Tissue Differentiation; Tissue Engineering; tissue repair; Tissues; tool; Transplantation; United States National Institutes of Health; Universities; Validation; Variant; Visual; Work; wound; Wound Healing