Single-cell Encoded Photoporation for High-throughput Combinatorial siRNA Screening

Since the discovery of RNAi and the demonstration of siRNA in post-transcriptional gene silencing, there has been tremendous interests and efforts in harnessing siRNAs for biomedical research and drug development. Thus far, the success has been limited. One of the major issues is that effective gene silencing by siRNA is often hindered by the presence of complementary signal transduction pathways, which may counteract the action of a single siRNA and substantially offset the therapeutic silencing effects. Alternatively, the use of cocktail siRNAs has been suggested as a feasible and promising drug discovery route. However, the identification of the most safe and efficient siRNA combination remains challenging and a crucial bottleneck. This proposal aims to address the unmet needs by developing a high-throughput technology for screening siRNA-based cocktail therapeutics. Technically, as one of the key innovations, we propose a multilayer functionalization of hierarchical assembled nano-/micro composite carriers (Knock-beads) with responsiveness to near infrared light and magnetic field, to facilitate photoporation-induced cellular transfection of RNAs with single cell encoding capability. So that randomized gene silencing by different siRNA variants in a large number of individual cells could be performed, recorded, and analyzed in a highly parallel manner. Depends on the operation scale, using this system, hundreds or even thousands of siRNA cocktail formulations could be assayed in just a single well of cell culture, which would be tremendously useful for accelerating the testing of siRNA combinations. Such benefits are not just limited to reducing the time and cost associated with siRNA screening by power of magnitude, but also rely on the capability to answer fundamental siRNA questions that are not addressable by existing methods. In addition to the technical advancement, as a proof-of- concept, we will further perform a small of screen of six cell apoptosis or proliferation related siRNAs to test all the possible 64 combinations in seeking for the most effective cocktail for potential inhibition of cancer cell growth. We believe that the success of this project will lead to a systematic and high-throughput platform technology that is greatly useful to accelerate further development of siRNA-based therapeutics and enhance their clinical adoption.