Autonomously-activating bioluminescent reporters to enable continuous, real-time, non-invasive brain cell imaging

Autonomously activating bioluminescent reporters to enable continuousreal timenoninvasive brain cell imaging Project Summary This Small Business Innovation ResearchSBIRPhase I project proposes to develop a set of genetically encodedself exciting bioluminescentautobioluminescentoptical imaging reporters that will enable continuous neuron and astrocyte specific imaging without perturbing endogenous cellular metabolismwhile using common laboratory equipmentThe treatment and management of brain disorders imposes enormous financial and social costsThe NIH therefore launched the BRAIN initiative to develop a new generation of innovative research tools and therapies that enable brain cell specific imagingspatiotemporal trackingand continuousnon invasive cell monitoring to facilitate new methods for evaluating brain cell physiology and new means to identify relative connectivityThe current generation of brain cell imaging toolswhich rely on fluorescent and luciferin luciferase bioluminescent chemistriesare incapable of achieving the NIH BRAIN initiative s goalsFluorescent imaging modalities require an excitation light to trigger their emission signalThis excitation is difficult to deliver to the brain and causes high levels of background autofluorescence that restricts signal discriminationBioluminescent imagingwhich has a greater signal to noise ratio due to a lack of background bioluminescence in tissueis similarly limited in that it can only produce an emission signal in the presence of an externally supplied substrateluciferinRepeated application of this substrate results in discontinuous signaling and substrate distribution kinetics that are challenging to replicateThereforeto meet the needs of the NIH BRAIN initiative and overcome the limitations restricting existing brain cell imaging toolsBioTech will reengineer our synthetic autobioluminescent genetic operon to create a new type of reporting technology that requires no external stimulatione glight or luciferin substrateto provide continuousnon invasivebrain cell specific optical monitoringThe genetic topology of the synthetic autobioluminescent operon will be systematically optimized to maximize signal output and minimize its effects on the host brain cell s metabolismUsing this technologyneuron and astrocyte specific autobioluminescent differentiation reporter platforms will be developed that will enable researchers to track the onset of brain cell differentiation and the subsequent fate of descendant cellsThese autobioluminescent brain cell specific technologies will be validated usingD cell culture to demonstrate their utility and generate proof of principle dataAt the conclusion of this project we will deliver a set of genetic constructs endowing brain cell specificcontinuously autobioluminescent phenotypes that researchers can utilize to continuously and non invasively label and track cellular locationphysiologyand connectivity Project Narrative Brain disorders result from a variety of causes that each require different diagnosistreatmentand palliative care approaches that impose enormous financial and social costsResearch aimed at lowering these costs and improving outcomes by investigators from across diverse fieldssuch as genetic engineeringstem cell culture differentiationregenerative medicineand tissue engineeringis hindered by the unavailability of methods to continuously track specific brain cellsor groups of brain cellsto discern their function in healthy and diseased statesTo overcome these limitations and address the needs of the NIH BRAIN initiativeBioTech will develop a set of self excitingcontinuously bioluminescent optical imaging reporters thatunlike existing systemsare pre engineered to support genetically encodedautonomousmetabolically neutralneuron or astrocyte specific bioluminescent expression that can be monitored with common laboratory equipment