(681d) Light-Induced Gene Silencing for Applications in Regenerative Medicine

The dynamic modulation of gene expression to illuminate and manipulate cell growth and signaling is an essential aspect in tissue engineering technologies, yet presents a particular challenge in complex tissues that must control phenotypes of multiple types of cells in a region-specific fashion. Specifically, methods to control the intracellular regenerative responses within complex tissue scaffolds using gene therapy would provide an essential complement to current biomaterial strategies employed to manipulate cell behavior via extracellular signals. Strategies employing stimuli-responsive nanomaterials have shown tremendous promise in generating cellular responses with some degree of spatiotemporal control; however, such approaches often suffer from off-target effects due to an inability to maintain target dormancy prior to the application of the stimulus. To address these challenges, we have developed novel and tailorable mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) [mPEG-b-P(APNBMA)]-based block copolymers (BCP)s with proven biocompatibility, stable and tunable nucleic acid binding, and light-triggered side chain cleavage leading to rapid nucleic acid release. Herein, we report the ability to tune gene silencing efficiency over a range of approximately 0 – 85%, on the basis of varied light irradiation and varied polyplex composition. Using fluorescence correlation spectroscopy and fluorescence resonance energy transfer, we demonstrate that the variations in silencing efficiency are directly controlled by light-induced changes in polyplex structure leading to siRNA release within the cytoplasm, and we show that the polyplexes remain entirely dormant in the absence of illumination. Through development and application of mass action kinetic modeling, we show that the maximal silencing efficiency is defined by a pseudo-steady state balancing the rates of mRNA production and siRNA/RISC-mediated mRNA cleavage. This work establishes the framework for addressing a key challenge in regenerative medicine while also exploring the fundamental mechanisms of nanomaterial intracellular stability and delivery barriers.