RNA interference (RNAi) has emerged over the last 15 years as a specific and potent means of targeted gene silencing at the mRNA level. Since its initial discovery in C. elegans, researchers have sought to harness this mechanism, by way of short interfering RNAs (siRNA), as an efficient route to silencing disease-causing genes. However, inefficient cytosolic localization of delivered siRNA continues to be a key challenge in RNAi therapeutic development. The most widely used delivery strategy currently involves cationic polymers or lipid nanoparticles to encapsulate the siRNA cargo, deliver it to the cell, and facilitate endosomal release to the cytoplasm. However, the design of a single polymer or lipid formulation that can overcome the multiple biological barriers to delivery has proven difficult.
In this talk, we introduce a modular dual-delivery strategy that decouples cellular internalization from endosomal escape through the use of antibody conjugates. Cellular internalization of these conjugates will be dictated solely by the antibody while endosomal escape will be attained with an endosomal escape agent (EAA). Decoupling internalization from escape will enable us to screen EEAs that disrupt membranes for cytosolic translocation of siRNA independently of its endocytic pathway. Two bioconjugates, an antibody-siRNA and antibody-EEA, will be prepared using the same antibody to ensure compartmental co-localization within the cell, then co-delivered to initiate RNAi.
Toward this aim, I will introduce a novel method for the assembly of both RNA-transporting and EEA-transporting antibody conjugates. This method enables efficient and reagent free bioconjugate assembly at sub-micromolar concentrations, typical of commercial antibodies, without significant disruption of antibody binding affinity. The modularity of this system allows us to then explore the effect of entry pathway on gene silencing by substituting the targeting antibody. Furthermore, I will present in vitro data on novel biomaterials with lytic properties for use as EEAs. This novel dual-delivery approach will be used as a platform to elucidate structure-activity relationships of potent EEAs and effects of entry pathway on the efficient delivery of siRNAs.