Mechanisms to investigate the nervous system : genetic engineering with adeno-associated viruses
Since the discovery of the adeno-associated virus (AAV) in 1965, AAVs have been harnessed as delivery vehicles for targeted transfer of DNA or RNA sequences for gene editing. Depending on the AAV cargo, the gene can systematically manipulate DNA, RNA, or amino acids affecting a cell's natural function adding to our fundamental understanding in systems such as the central and peripheral nervous systems. This research uses AAV delivery for both optogenetics and CRISPR-Cas to 1) engineer an in
... o model to stimulate the sympathetic and parasympathetic neurons connecting to heart muscle cells and 2) validate a technique to quantitatively measure the efficiency and tropism of in vivo central nervous system AAV delivery. This dissertation impacts the field by 1) simplifying and controlling the complex signaling of the ANS-cardiac connection and 2) implementing gene knock out to quantify AAV delivery efficiency. Finally, in the broader impact of my work, I share unorthodox communication experiences during my Ph.D. Optogenetics is a technique to control the cells and tissue regions of the nervous system with light. AAVs can deliver a messenger RNA sequence that codes for a light sensitive ion channel/pump, called an opsin, that allows researchers to control cell depolarization with light stimulation. Optogenetics can control sympathetic and parasympathetic neurons in the autonomic nervous system (ANS) that synapse the heart creating a systematic reaction measured by heartbeat. A simplified ANS-cardiac in vitro model with independent optogenetic control of sympathetic and parasympathetic neurons was engineered to isolate the neuronal contributions to heart muscle cells. An efficient and versatile gene-editing technique exploiting clustered regularly interspaced short palindromic repeats along with a Cas nuclease (CRISPR-Cas) can permanently edit a DNA or RNA sequence. AAVs can deliver CRISPR-Cas sequences targeting a specific gene to knock out resulting in a protein reduction. This protein reduction can be measured and correlated to the AAV delivery efficiency. Not only can this high throughput, quantitative efficiency screen has tissue type resolution but the potential iii for cell-type resolution if the gene codes for a specific cell marker protein. A screen was optimized to measure AAV transduction efficiency in neurons, astrocytes, and oligodendrocytes in the central nervous system.