Acoustic Reporter Genes for Noninvasive Imaging of Cellular Function

The study of cellular function within the context of intact living organisms is a grand challenge in biological research. Addressing this challenge requires imaging tools that can visualize cells inside the body. If successful, this would greatly increase our ability to study a battery of processes from brain development to tumorigenesis, to monitoring cell-based therapeutics. To date, most common methods for imaging cellular processes such as gene expression have relied on optical reporters, such as fluorescent or luminescent proteins, which provide high molecular precision for studies in petri dishes and transparent organisms, but have limited performance in large animals due to the poor penetration of light in biological tissue. Conversely, magnetic resonance imaging (MRI) and ultrasound can image tissues at depth with high spatial and temporal resolution, but they lack molecular reporters analogous to the green fluorescent protein (GFP). As a result, they have made limited impact on biological research. To address this, we focus on developing biomolecular reporters for MRI and ultrasound — based on a unique class of air-filled protein nanostructures called gas vesicles — using them to image the location and function of cells deep inside the body. This thesis begins with a brief review of genetically encoded materials for noninvasive imaging, highlighting key advances over the past two decades and providing context for the work below. We discuss the development of increasingly sophisticated tools starting from early efforts to engineer single molecule reporters to recent work on multi-component genetic machinery (including gas vesicles) with multi-modality capabilities. In Chapter 2, we present a platform for engineering the surface of gas vesicles to modulate their acoustic, surface charge, and molecular- targeting properties as injectable acoustic biomolecules. In Chapter 3, we present the recombinant expression of gas vesicles as injectable contrast agents in common lab strain bacteria to facilitate the genetic engineering of the entire gas vesicle gene cluster and to assist this technology’s adoption by other (non-specialist) research groups. This work characterized the ultrasound and hyperpolarized 129Xenon-MRI contrast of gas vesicles as nanoscale contrast agents. In a parallel effort, we developed a hybrid gene cluster that when introduced to microbes enables the imaging of their gene expression using ultrasound. These bacterial acoustic reporter genes were used to image the location of probiotic cells inside the gastrointestinal tract of mice. However, the ability for these genes to be expressed in mammalian cells had not been demonstrated and presented a major challenge in synthetic biology. In Chapter 4, we addressed this by introducing the first mammalian acoustic reporter genes — a genetic program whose introduction to mammalian cells resulted in the expression of gas vesicles that can be visualized by ultrasound. These mammalian acoustic reporter genes will enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo. In Chapter 5, we explore a new paradigm in MRI by taking advantage of the acousto-magnetic property of gas vesicles. Here, we present background-free MRI to address a longstanding challenge in untangling the signal of exogenous contrast agents from the endogenous MRI contrast produced by biological tissues. Chapter 6 explores the optical properties of gas vesicles as genetically encodable phase contrast agents in digital holographic imaging. Chapter 7 is a brief discussion of the potential future directions for this work. The data presented in this thesis lays the ground for exciting new research on developing noninvasive biomolecular tools that will enable the discovery of novel biological processes.