Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression

Citation

Duan, Mengtong (Tom) (2024) Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/avgk-yc71. https://resolver.caltech.edu/CaltechTHESIS:06032024-011201674

Abstract

This thesis presents a comprehensive exploration of the next generation of mammalian Acoustic Reporter Genes (mARGs), unveiling a novel approach for non-invasive, real-time imaging of cellular processes and gene expression within live animals1. Building on the foundational work of first-generation ARGs2,3, which introduced the groundbreaking concept of using gas vesicle (GV) genes as genetically encoded ultrasound contrast agents, this research tackles the inherent limitations of these pioneering systems. The first segment details the development and characterization of the second-generation mARGs which significantly improve upon their predecessors by offering robust expression without the need for monoclonal screening, dynamic non-destructive imaging capabilities, and customizable acoustic properties through gene and protein level modifications. This advancement not only enhances the utility of mARGs in biomedical imaging but also paves the way for their application in novel therapeutic monitoring strategies, as exemplified by real-time tracking of tumor development and ultrasound-guided tumor biopsies that leverage gene expression information.

Further, the thesis delves into the structural, genetic, and biochemical principles underpinning GV assembly, addressing a critical knowledge gap that has persisted despite the utility of GVs in ultrasound imaging. Understanding these assembly mechanisms is crucial for the engineering of improved ARGs.

The exploration then extends into innovative bioengineering methodologies, specifically Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER), a synthetic biology breakthrough enabling the expression of multiple proteins at precise stoichiometries from single, compact transcripts.4 SEMPER represents a strategic advancement in the field, facilitating efficient formation of multi-protein complexes, minimizing cellular toxicity, and broadening the scope of potential applications in genetic engineering, including the creation of enhanced cell lines and circuits for research and therapeutic purposes.

Collectively, this work not only advances our understanding of GV-based ultrasound imaging and gene expression tracking but also introduces versatile genetic tools for the manipulation of cellular machinery. These achievements mark significant strides in the fields of synthetic biology and molecular imaging, setting the stage for future innovations in non-invasive diagnostics, cellular therapy, and cancer monitoring research. Through the integration of improved acoustic reporter genes, insights into gas vesicle assembly, and the SEMPER method for gene expression, this thesis embodies a holistic approach to overcoming current challenges and unlocking new potentials in biomedical engineering and synthetic biology.

Thesis Files