268456 All-Protein Vehicles for siRNA Delivery

Tuesday, October 30, 2012: 12:30 PM
Westmoreland West (Westin )
Nicole J. Yang, Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, Daniel G. Anderson, Chemical Engineering, Health Sciences & Technology, Massachusetts Institute of Technology, Cambridge, MA, Robert S. Langer, Massachusetts Institute of Technology, Cambridge, MA and K. Dane Wittrup, Chemical Engineering, Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

RNA interference (RNAi) is a naturally occurring gene-silencing mechanism which can be exploited to reduce the expression of disease-causing genes. Short-interfering RNA (siRNA) can artificially trigger RNAi, but cannot reach the cytoplasm of target cells efficiently on its own. As such, a protein-based delivery system for siRNA, composed of two separate agents, has been developed. The first construct binds to siRNA through a double-stranded RNA binding domain (dsRBD) and mediates internalization into cells through its targeting moiety against the epidermal growth factor receptor (EGFR). The second construct, a pore-forming protein taken from bacteria, allows endosomal release of siRNA. Administered together, this delivery system achieved efficient and specific silencing in vitro.

           Towards adapting this delivery system in vivo, we have optimized both the siRNA carrier and the pore-forming protein. First, we identified that the rapid dissociation of siRNA from the dsRBD is a major limitation for efficient tumor targeting, and improved the binding affinity of the dsRBD via yeast-surface display and directed evolution. The resulting dsRBD could bind to both unmodified and chemically-modified siRNA with higher affinity, independent of sequence. The accrued mutations displayed an overall increase in charge of the amino acid side chains, suggesting that interactions with the negatively-charged siRNA backbone were increased. Second, a reversible protein mask was engineered for the pore-former, resulting in a significant decrease in cytotoxicity without compromising efficacy. Consequently, the therapeutic window was increased by an order of magnitude in vitro. Ultimately, our objective is to achieve efficient gene silencing in an animal model to validate therapeutic applications of the current all-protein siRNA delivery system.

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