Optogenetic strategies for stimulus-responsive viral gene delivery
Gomez, Eric Jordan
Doctor of Philosophy
Heightened interest in the field of gene therapy has led to the development of multitudes of gene vector candidates. Most, if not all, modern vector designs have focused on improving cell binding and entry in an effort to boost therapeutic potency. Cellular uptake is a critical first step to produce a viable gene therapy, but the numerous intracellular trafficking checkpoints--- including arguably the most important process of gene delivery, nuclear localization--- have been largely ignored. Engineering gene vectors that can more effectively navigate to the nucleus will lead to unequivocally better products with higher therapeutic efficacy, but it has remained challenging to control the intracellular processes affecting endocytosed vectors. Adeno-associated virus (AAV) is a leading gene vector that enters cells via clathrin-mediated endocytosis and escapes endosomes using a viral phospholipase motif. The natural AAV capsid has evolved to infiltrate a broad spectrum of cell types, but AAV's trajectory into the nucleus is significantly hampered by perinuclear sequestration. This situates AAV as a gene vector that is good at penetrating the cell endosomal membrane, but poor at penetrating the cell nuclei. The negative implications of this are three-fold: First, because AAV cannot effectively enter the nucleus, a high dose is required for only a small fraction of viruses to ultimately deliver a therapeutic transgene. Second, because AAV has a broad tropism the high dose requirement may lead to more viruses entering off-target tissues. Third, dose-dependent immune response can become a safety concern when more virus is needed. Thus, vectors engineered to overcome the nuclear uptake barrier may yield more effective and safer gene therapies. Furthermore, if this nuclear entry step can be made user-controllable, then the overall gene delivery process could be rendered more predictable both in space and time. To reach this goal, gene vectors can be built to be responsive to environmental cues. A variety of stimulus-responsive gene vectors, capable of sensing and responding to an environmental stimulus, have been developed, but mostly to address the problem of cellular uptake. Designing vectors capable of responding to a stimulus at a later step in intracellular trafficking, such as nuclear localization, may provide an extra degree of control over vector trajectory. However, it has proven challenging to develop a gene vector capable of improving nuclear localization in response to a stimulus in a dose-dependent and location specific-manner. Exogenous light stimulation could render gene delivery more quantitatively controllable both in space and time. Illumination of most wavelengths is relatively orthogonal to cell physiology and can be controlled in three dimensions: space, time, and brightness. By combining approaches from gene therapy with those from synthetic biology, a gene vector could be made light-responsive. For instance, by coupling an optogenetic sensor with an intracellular functional domain, such as a nuclear localization sequence, light could be harnessed to control the nuclear translocation of gene delivery vectors, leading to tunable gene expression. This thesis serves first to review the many barriers to gene delivery and the vectors designed to circumvent such barriers. I next make my case for using light as a stimulus to improve both nuclear localization and target cell specificity of gene vectors. I then describe my endeavors to engineer an AAV capable of participating in a light-activatable system that can simultaneously enhance viral nuclear localization and spatially define regions of interest for enhanced gene delivery. Finally, I reveal my light-activatable virus (LAV) prototype that is an all-in-one sensor-effector gene vector capable of tunable delivery via optically-induced conformational changes on the capsid. A panel of LAVs are characterized that are capable of adjusting gene delivery based on blue light flux. Light-activatable viral vectors could enable stronger, spatially-resolved gene gene expression in target cells, which would simultaneously mitigate off-target effects and immune reponse.