Fluorescent proteins (FPs) are important life science research tools with a market size in excess of $200 million. FPs are used in many applications such as in vivo tissue imaging, cell tracing and intracellular protein tracking. However, current tools, mostly based on GFP-family templates (e.g. Monster Green(r) from Promega, tdTomato from Clontech), suffer several drawbacks including aggregation, bleaching, and toxicity. Most importantly, tissue autofluorescence and absorbance at the wavelengths at which these FPs operate (450-670 nm) creates high background and a low signal to noise ratio. This limits their use to minimal surface cross-sections with a depth limit on single cell imaging of 0.05 - 0.1 mm and large tumor imaging up to 10 mm. To overcome this limitation, neuroscientists must use highly invasive surgical methods to image deeper parts of the brain. Here we propose to develop a novel near-infrared (NIR) fluorescent protein, termed iRFP-Max which will overcome current issues and enable high resolution imaging of at least 10 mm in tissue depth for single cells and much deeper for tumor masses. This tool will enable a range of new applications and has the potential to become the industry standard for pre-clinical real-time in vivo imaging for drug development and neuroscience research. The benefit of FPs over other fluorescent reagents is the tight spatial and temporal control that can be achieved through genetic encoding. FP genes can be readily introduced into cells and provide tissue or neuron-specific expression. Importantly, FPs do not require addition of exogenous cofactors for activity. Due to these benefits, FPs are used in many different applications such as visualizing neural circuits (e.g. Brainbow), monitoring synaptic release (e.g. Synapto-pHluorin) and monitoring neural activity (e.g. GCaMP). FPs are also critical in the study of diseases such as brain cancer, PTSD, Alzheimer's and Parkinson's. In Phase I, we will create a new NIR FP by redesigning an iRFP template to have an optimal NIR spectral profile, brightness, and maturation time. Revolve is uniquely positioned to accomplish this goal due to our protein engineering expertise and proprietary DNA mutant library technology, PFunkel mutagenesis, which forms the core of our commercial library service. Our technology can be used to explore large swathes of protein sequence space in a rapid manner in conjunction with a robust fluorescence screen. In Phase 2, we will use iRFP-Max to generate validating imaging data in mammalian cells and animal models with our collaborators at JHMI. Revolve plans to develop a suite of new infrared tools based on iRFP-Max such as a NIR calcium-sensor for monitoring neuronal activity in deep brain tissue.
Understanding the inner workings and connections of neurons in the brain and nervous system is critical for advancing knowledge on mental health and developing new treatments for neurological diseases such as Alzheimer's and Parkinson's. We are developing a novel infrared fluorescent protein tool that will allow researchers to study and visualize cells functioning in vivo in real time at a tissue depth and clarity previously not possible.
