Commonly used antibody-siRNA conjugates, and many nanoparticle systems, utilize highly cationic peptides polymers both for non-covalent complexation with siRNA as well as endosomal disruption. However, cationic carriers can lead to toxic side effects, dissociation in serum, and rapid systemic clearance. Neutral endosome-disrupting agents can alleviate these effects, but cannot complex with RNA. Therefore, they must be covalently incorporated into the delivery system without inhibiting binding and internalization.
We propose here a co-delivery strategy utilizing two antibodies, covalently linked to either an siRNA or endosomal escape agent, to work together to facilitate efficient knockdown of a target gene. We have built a system using antibodies targeting either recycling or lysosomal targeting pathways, and a model endosomal escape agent, Listeriolysin O (LLO), connected via a reversibly attenuating linker for endosomal disruption with reduced outer membrane toxicity. We anticipate that the âplug-and-playâ nature of this method will allow for better understanding of the key variables, such as endocytic pathway, endosomal escape agent, and linker chemistry, in building an efficient siRNA delivery vehicle.