Abstract
Molecular therapy using small interfering RNA (siRNA) shows great promise in the development of novel therapeutics for cancer. Although various approaches have been developed for in vivo delivery of siRNAs into tumors, stability of siRNA in blood circulation, and low efficiency of siRNA delivery into tumor cells are the major obstacles for further translation into cancer therapeutics. In this protocol, we describe methods of the production of shRNA expressing DNA nanocassettes by PCR amplification of double-stranded DNA fragments containing a U6 promoter and a shRNA gene. Those DNA nanocassettes can be conjugated to the polymer coating of nanoparticles that are targeted to cellular receptors highly expressed in tumor cells, such as urokinase plasminogen activator receptor (uPAR), for targeted delivery and receptor mediated internalization of shRNA expressing DNA nanocassettes. Methods for in vitro and in vivo evaluation of target specificity and gene-knockdown effect are also provided.