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

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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.