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

使用超声响应生物材料对蛋白质表达进行基因操作以探测 3D 细胞行为

• 批准号: 10712639
• 负责人: Carolyn Schutt Ibsen
• 金额: $ 38.31万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10712639
• 关键词: 3-Dimensional; Address; Affect; Biocompatible Materials; Biological; Biological Process; Cell Communication; Cell Culture Techniques; Cell physiology; Cells; Cellular Structures; Chemotaxis; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Communication; Complex; DNA delivery; Disease; Disease model; Endothelial Cells; Endothelium; Focused Ultrasound; Gene Expression; Genes; Growth Factor; Individual; Knock-out; Libraries; Location; Mediating; Methods; Natural regeneration; Nucleic Acids; Pattern; Process; Protein Secretion; Proteins; Regulation; Research; Research Personnel; Role; Societies; System; Testing; Time; Tissues; Transfection; Vascularization; Vision; Work; bioscaffold; cell behavior; cell motility; cell type; genetic manipulation; innovation; insight; intercellular communication; migration; novel therapeutic intervention; overexpression; particle; programs; protein expression; regenerative therapy; scaffold; spatiotemporal; three dimensional cell culture; tissue repair; ultrasound; wound healing

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.

项目概要 蛋白质在时间和空间上的受控表达对于生物过程的协调至关重要 健康组织和患病组织。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，以更好地了解其生物学作用并为再生疗法提供信息。 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能够 研究人员控制研究细胞行为和影响的基本机制所必需的蛋白质表达 细胞相互作用。然而，在 3D 支架内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，很少有方法允许多个个体的单独协调 材料内的基因操作，使得很难复制在材料中观察到的复杂过程 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 一类生物材料，称为 SonoScaffolds，用于受控、超声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 状态并促进组织修复。在我们的创新方法中，聚焦超声波与集成的相互作用 生物材料内的回声颗粒将核酸局部递送至细胞以操纵其蛋白质 表达和分泌。聚焦超声可以创建用户定义的 3D 转染模式 受控时间。我们将使用这个平台来解决有关如何空间呈现和 蛋白质表达的时间会影响 3D 中的细胞行为，例如定向迁移和趋化性。到 为了实现这一目标，我的研究计划将设计超声波介导的策略来实现两项基本功能 用于支架中的基因操作：基因过表达的时空控制，以及基因过表达的精确控制 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脚手架的库 回声粒子各自响应不同的超声参数，从而实现多重 DNA 时空控制的交付。虽然我们的方法是通用的并且与细胞类型无关，但我们将首先测试 内皮共培养系统中的 SonoScaffold 平台用于操纵细胞间通讯 超声波定义的模式。我们将使用该系统来促进内皮细胞迁移的指导 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 单独和组合因素，深入了解各个因素如何协调 3D 细胞 行为。总之，这项工作将产生一种革命性的新型 3D 可编程细胞培养材料 能够对嵌入细胞和多细胞进行非侵入性、时空定义的遗传操作 结构，并为协调细胞过程提供新的见解。该计划将带来立竿见影的好处 为社会带来通过基因操纵细胞来调节细胞行为的新的和创新的研究 3D 背景下的过程，以增进我们的理解并为新的治疗策略提供信息。