siRNA has shown the potential in treating undruggable diseases such as cancer. Despite its potential, therapeutic application of siRNA has been greatly impeded by the lack of safe and effective delivery vectors. Viral vectors have safety concern and have been precluded for clinical application. Current most non-viral vectors including lipoplexes, polymers, inorganic nanoparticles, micelles, and cell penetrating peptide are positively charged and suffer from low serum stability, off-target effect, cytotoxicity and batch-to-batch variation. Additionally, inorganic nanoparticles and polymers have serious safety concerns due to non-degradable and poor clearance. Therefore, current carriers are unsuitable for systemic delivery of siRNA. In previous study, we have developed a non-cationic protein based aptamer-siRNA chimera carrier by adding 18 Histidine (His) peptide on human origin dsRNA binding domain (dsRBD) protein. DsRBD-18his does not rely on high positive charges to interact with RNA molecules; instead, it binds dsRNA via specific 3-D conformation. Histidine molecules have pKa value about 6, at neutral pH, they are uncharged, and charged in acidic condition such as endosome. 18His can confer sufficient buffering capacity to drive cargo endosomal escape. However, current siRNA vectors including dsRBD-18his lacking cell type specificity, and targeting molecules such as antibody, aptamer, or ligand have to be added into each cargo. That is a time consuming and costly process. RGD (Arg- Gly-Asp) peptide has been well characterized as a binding ligand of an integrin ?v?3, which is highly expressed on tumor neo-vasculature as well as some tumor cells, but not present in resting endothelial cells and normal organ systems. RGD peptide is a well validated tumor targeting molecule, and has been used for guiding imaging agents and drugs for tumor diagnosis and therapy in clinical setting. In this project, we will genetic engineer three tumor and tumor blood vessel targeted vectors by fusing RGD peptide into N-, C-, or both N-and C termini of dsRBD- 18His protein. New vectors will have three functions including siRNA binding, endosomal escape, and tumor targeting. New vectors will be uncharged, low toxicity, biodegradable, cell type specific, and ease of mass production.
The specific aims of this proposal are 1) cloning, expression, and characterization of RGD vectors, and 2) evaluation of tumor and tumor neo-vasculature targeting capabilities in cancer xenograft models. New vectors with load-to-go capability will simplify siRNA delivery in vivo, and any siRNAs can be loaded and targeting delivered to tumors or tumor blood vessels.
The goal of this project is to develop a wide applicable tumor-targeted siRNA vector. Current siRNA vectors suffer from toxicity, off-target and low serum stability. We will engineer a new vector which will be nontoxic, circulation stable, and targeting on tumor cells.