Project Summary Blood tests are among the most common clinical tools due to their low cost, simplicity, and ability to observe many markers at once. However, currently blood tests can only monitor a fraction of physiological processes that happen to have a serum marker. What if we made synthetic serum markers that can track other physiological processes? The PI has recently developed such markers called Released Markers of Activity or RMAs. RMAs are expressed in the brain in response to a physiological process, such as neuronal activity, but then exit into blood for simple detection. In our preliminary data, RMAs could monitor expression in as few as 10s-100s of cells in the brain and show signal levels up to 39,000-fold over baseline when monitoring a single brain region. The PI was able to use RMAs to measure neuronal (c-Fos) activity in specific brain regions with a simple blood test. Since this discovery, our laboratory has been working on the new paradigm of ‘imaging’ the brain with blood sampling. This approach has many advantages. First, typical reporters need to be measured within the brain where access is difficult and the signal is compromised by surrounding tissue. Our key innovation is to keep record of the RMA’s location in the brain, but access it with a simple blood test that is not compromised by the skull, tissue scattering, or high background from surrounding cells. Second, there is a large number of genes in each cell and tens of distinct brain regions, but available reporters can at most represent only a few signals (e.g. a few colors of fluorescent proteins). RMAs use biochemical detection and thus can be massively multiplexed similarly to how thousands of proteins can be detected in blood simultaneously using mass spectrometry. Third, RMAs can surveil large brain regions, unlike invasive locally-implanted devices. Finally, RMAs are inexpensive and easy to use - they are genetically encoded proteins and can use simple equipment such as a plate reader. The PI has a track record of developing innovative technologies that blend interfaces of multiple disciplines, such as genetically-encoded MRI contrast agents, Acoustically Targeted Chemogenetics (ATAC) for noninvasive neu- romodulation, or acoustically-targeted viral vectors (AAV.FUS). In this proposal, the PI will use cutting-edge single-molecule protein detection to increase sensitivity of RMAs from 10s-100s of cells to single neurons. Then we will develop ‘fast’ RMAs that can measure more rapid changes in gene expression. Finally, we will enable massively-multiplexed readout of RMAs using peptide barcoding to track gene expression in multiple brain re- gions at once, and, if feasible, in single cells in whole brain regions. Using RMAs we will enable a previously inaccessible study. We will monitor activity of neurons encoding a fear memory and measure whether activation of these neurons in safe environment is responsible for fear renewal, a finding that would have broad implications for treatment of anxiety disorders. New reporters, such as fluorescent proteins historically enabled broad discov- ery. RMAs too have the potential to transform broad areas of science as they can be adapted to noninvasively track different in vivo systems and tissues with single-cell sensitivity, massive multiplexity, and low cost.

Project narrative Blood tests are among the most common clinical tools due to their low cost, simplicity, and ability to observe many markers at once. However, currently blood tests can only monitor a fraction of physiological processes that happen to have a serum marker. Here, we are developing synthetic markers that can exit from the brain into blood to and in the process measure critical aspects of brain physiology with a simple blood test.