Noninvasive site-specific measurement of gene expression in deep tissues with secreted reporters

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Project Summary Monitoring gene delivery in deep tissues is critical for in vivo studies and translation of gene therapies, but available methods for achieving such measurements have limited capabilities. Ideally, levels of gene expression could be noninvasively measured anywhere in the body of both small and large animals. Ideally, multiple genes could be measured at once, without the need for expensive equipment that would put such a method out of financial reach for academic research groups. However, no such method exists as of now. In this proposal, we will develop technologies for monitoring gene expression that, when fully developed, will be: 1) noninvasive, 2) site-specific, 3) could view multiple types of molecules at once (high-multiplexity of imaging), 4) are not impacted by the depth of penetration, skull thickness, or organ size; 5) will use inexpensive equipment allowing democratized access. To achieve this, we will attempt a drastically different approach – instead of imaging reporters within the deep tissue, we will design reporters that can be easily transported from a known region of the brain into the blood, where they can be easily measured. We will develop two independent methods for achieving this goal. First, will express gene expression reporters that exit the cells into brain interstitial space. Then we will temporarily enhance vascular permeability in selected ~millimeter-sized brain regions to allow for free diffusion of these reporters from the brain into the blood. In the second method, we will develop a new class of gene expression reporters that can cross through an intact brain vasculature to continuously report on gene expression within the transduced brain cells. Spatial precision could be provided for this method using noninvasive gene delivery that genetically labels cells with site-specificity. With each of these methods, we will be able to measure gene expression in a specific brain region with a simple blood draw. Our technologies will be particularly beneficial in the studies of large animal species, where monitoring gene expression in the brain, in most cases, is not feasible without euthanasia. While initially focused on the brain as a test-case with a high need for such methods, the same concepts can be applied to other tissues.