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

项目摘要 监测深部组织中的基因递送对于体内研究和基因疗法的翻译至关重要，但 用于实现这种测量的可用方法具有有限的能力。理想情况下，基因表达水平 可以在小型和大型动物体内的任何地方进行非侵入性测量。理想情况下，多个基因 可以立即测量，而不需要昂贵的设备，将这种方法排除在外。 为学术研究团体提供资金支持。但是，目前还没有这样的方法。在本提案中，我们 将开发用于监测基因表达的技术，当完全开发时，将是：1）非侵入性的，2） 位点特异性，3）可以同时查看多种类型的分子（成像的高度多重性），4）不受影响 通过穿透深度，头骨厚度或器官大小; 5）将使用廉价的设备， 民主化的访问。为了实现这一点，我们将尝试一种截然不同的方法-而不是成像 报告员在深层组织，我们将设计的报告员，可以很容易地从一个已知的区域运输， 大脑进入血液，在那里它们可以很容易地被测量。我们将开发两种独立的方法， 实现这一目标。首先，将表达基因表达报告基因，其离开细胞进入脑间质空间。 然后，我们将暂时增强选定的~毫米大小的大脑区域的血管渗透性， 这些报告分子从脑自由扩散到血液中。在第二种方法中，我们将开发一个新类 基因表达报告者可以穿过完整的脑血管系统， 在转导的脑细胞内表达。空间精度可以提供这种方法使用 非侵入性的基因传递，基因标记细胞的位点特异性。通过这些方法，我们将 能够通过简单的抽血来测量特定大脑区域的基因表达。我们的技术将 在大型动物物种的研究中特别有益，其中监测大脑中的基因表达， 在大多数情况下，如果没有安乐死，是不可行的。虽然最初将大脑作为一个测试案例，但具有很高的 由于需要这种方法，相同的概念可以应用于其他组织。

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.