Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-Cell Phenotyping in Whole Organs

Abstract

A central question in biology is how different cell types interact with each other and their native environment to form complex functional systems and networks. Although our ability to investigate this question has considerably expanded from the development of genetically encoded tools, some limitations still persist. For instance, we are limited in our ability to visualize the native three dimensional environments of whole organs. Additionally, it is challenging to efficiently deliver transgenes into difficult-to-target areas through directinjections, such as the cardiac ganglia, or broadly distributed networks, such as the myenteric nervous system, which limits our ability to extensively study these areas. Therefore, tools and methods that overcome these limitations are needed. Towards this end, my thesis work has been focused on developing tools for single-cell resolution phenotyping in whole organs. I have been developing tissue clearing technologies to render whole organs transparent for optical interrogation and characterizing viral capsids and engineering viral vectors for noninvasive widespread gene delivery to the central and peripheral nervous system.

Tissue clearing techniques for three dimensional optical interrogation were invented over a century ago. However, these earlier methods used harsh organic chemicals and failed to retain the tissue’s native fluorescence or epitopes. These earlier methods eventually became incompatible to the hundreds of newly generated transgenic mouse lines that allowed for cell type-specific expression of fluorescent transgenes or to fluorescent labeling techniques, such as immunohistochemistry (IHC). The first part of my dissertation is aimed at addressing these limitations by further developing and standardizing a tissue clearing method that utilizes the vasculature to perfuse clearing reagents. This technique, called perfusion assisted agent release in situ (PARS) enables (i) whole organ clearing of soft tissue, (ii) preservation of native fluorescence, and (iii) preservation of epitopes compatible with IHC.

Although PARS allows us to optically investigate whole soft tissue organs, it is unsuitable for clearing bone tissue. The clearing of bone is important as it may provide optical access to delicate environments, such as the lymphatic vessels lining the dural sinuses beneath the skull that would otherwise be damaged through traditional methods. However, clearing bone tissue is challenging since it is composed of both soft (bone marrow) and hard (mineral) tissue. To overcome this challenge, I developed a clearing method that rendered intact bone tissue transparent by using EDTA to decalcify bones and by constructing a convective flow chamber to efficiently clear bones. This method, called Bone CLARITY, is able to preserve native fluorescence and epitopes. In order to demonstrate the utility of Bone CLARITY, I collaborated with colleagues to quantitatively access a rare and non-uniformly distributed population of osteoprogenitor cells in their native three dimensional environment. Bone CLARITY in conjunction with light-sheet microscope enabled the early detection of an increase to this osteoprogenitor population after administration of a novel anabolic drug, which may have been undetected with traditional techniques.

Towards my second goal, I have been working on characterizing adeno-associated viruses (AAVs) for non-invasive widespread gene delivery across the central or peripheral nervous system. Through systemic delivery, these novel AAVs are able to efficiently deliver transgenes to (i) difficult-to-target areas, such as the dorsal root ganglia; (ii) cellular populations that are widely distributed across the mouse body, such as neurons in the myenteric nervous system, and (iii) through highly selective barriers, such as the blood-brain barrier.