Genetically Manipulating Protein Expression to Probe 3D Cell Behavior using Ultrasound-Responsive Biomaterials

项目摘要 蛋白质在时间和空间上的可控呈现对于生物过程的协调是必不可少的 无论是健康的还是患病的组织。非常需要重新创建和操纵这些复杂的动态 3D支架内的过程，以更好地了解它们的生物学作用，并为再生治疗提供信息。 在3D支架内的特定时间和位置诱导基因表达和基因编辑将使 研究人员控制必要的蛋白质表达以研究细胞行为的基本机制和影响 细胞间的相互作用。然而，在3D支架中对细胞进行基因操作仍然是一个挑战 具有空间和时间控制的非侵入性。此外，很少有方法允许单独协调多个 一种材料内的基因操纵，使得很难复制在 组织成熟、血管化和伤口愈合。为了应对这些挑战，我的实验室正在开发一种新的 一类被称为SonoScaffold的生物材料，用于受控的、超声波介导的细胞遗传操作 3D文化。我们的总体愿景是利用这些新的3D生物材料来研究细胞行为、疾病模型 状态和促进组织修复。在我们的创新方法中，聚焦超声与集成的 生物材料内的回声颗粒将核酸局部输送到细胞以操纵其蛋白质 表达和分泌。聚焦超声可以在以下位置创建用户定义的3D转染图 受控制的时间。我们将使用这个平台来解决有关空间呈现和 蛋白质表达的时间影响3D中的细胞行为，如定向迁移和趋化。至 要做到这一点，我的研究计划将设计出超声波中介的策略，以实现两项基本能力 对于支架中的遗传操作：基因过度表达的时空控制，以及对 基于CRISPR的基因编辑。我的程序的第二个主题将生成一个集成了脚手架的库 回声颗粒，每个颗粒对不同的超声参数作出反应，从而使多重DNA成为可能 具有时空控制的投递。虽然我们的方法是通用的和细胞类型不可知的，但我们将首先测试 SonoScaffold平台在内皮细胞共培养系统中用于操纵细胞间通讯 超声波定义的图案。我们将使用这个系统来帮助指导内皮细胞的迁移 趋化性的研究。这将包括超声介导的表达和多重生长的CRISPR敲除 单独因素和组合因素，深入了解各个因素如何协调3D单元格 行为。总之，这项工作将产生一种变革性的3D可编程细胞培养材料 这使得嵌入细胞和多细胞的非侵入性、时空定义的基因操作成为可能 结构，并提供了对协调细胞过程的新见解。这项计划将立即产生效果。 对社会，使通过基因操作细胞来调节细胞行为的新的和创新的研究成为可能 在3D环境中的过程，以促进我们的理解和提供新的治疗策略。

Project Summary Controlled presentation of proteins in time and space is essential for the coordination of biological processes in both healthy and diseased tissue. There is a significant need to recreate and manipulate these complex dynamic processes within 3D scaffolds to better understand their biological roles and inform regenerative therapies. Inducing gene expression and gene editing at specific times and locations within 3D scaffolds will enable researchers to control protein expression necessary to study basic mechanisms of cell behavior and influence cell interactions. However, it remains a challenge to genetically manipulate cells within 3D scaffolds noninvasively with spatial and temporal control. Further, few methods allow individual coordination of multiple genetic manipulations within a material, rendering it difficult to replicate the complex processes observed in tissue maturation, vascularization and wound healing. To address these challenges, my lab is developing a new class of biomaterials, called SonoScaffolds, for controlled, ultrasound-mediated genetic manipulation of cells in 3D culture. Our overall vision is to leverage these new 3D biomaterials to study cell behavior, model disease states and facilitate tissue repair. In our innovative approach, focused ultrasound interacts with integrated echogenic particles within the biomaterials to locally deliver nucleic acids to cells to manipulate their protein expression and secretion. The focused ultrasound can create user-defined 3D patterns of transfection at controlled times. We will use this platform to address key questions regarding how spatial presentation and timing of protein expression affects cellular behaviors in 3D, such as directed migration and chemotaxis. To achieve this, my research program will devise ultrasound-mediated strategies to enable two essential capabilities for genetic manipulation in scaffolds: spatiotemporal control of gene overexpression, and precise control over CRISPR-based gene editing. A second theme of my program will generate a library of scaffold-integrated echogenic particles that each respond to distinct ultrasound parameters thereby enabling multiplexed DNA delivery with spatiotemporal control. While our approach is versatile and cell-type agnostic, we will first test the SonoScaffold platform in an endothelial co-culture system for manipulation of intercellular communication in ultrasound-defined patterns. We will use this system to facilitate guidance of endothelial cell migration for the study of chemotaxis. This will include ultrasound-mediated expression and CRISPR knockout of multiple growth factors both individually and in combination, providing insight into how individual factors coordinate 3D cell behavior. Together, this work will generate a transformative new class of 3D-programmable cell culture materials that enable noninvasive, spatiotemporally-defined genetic manipulation of embedded cells and multicellular structures, and provide new insights into coordinated cell processes. This program will have immediate benefits to society, enabling new and innovative studies into regulation of cell behavior by genetically manipulating cell processes in the 3D context to advance our understanding and inform new therapeutic strategies.