title: Targeted Delivery with Imaging Assessment of siRNA Expressing Nanocassettes into Cancer.
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.
authors.affiliation: Department of Surgery, Emory University School of Medicine, Clinic C, Room C-4088, 1365 C Clifton Road, NE, Atlanta, GA, 30322, USA.|Department of Gastrointestinal Surgery, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Surgery, Emory University School of Medicine, Clinic C, Room C-4088, 1365 C Clifton Road, NE, Atlanta, GA, 30322, USA. Lyang02@emory.edu.
authors.forename: Lily
authors.initials: L
authors.lastname: Yang
mesh_terms: D051696:Activating Transcription Factors / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D015870:Gene Expression; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000628:therapy; D010957:Plasmids / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: D051696:Activating Transcription Factors; D034741:RNA, Small Interfering
keywords: Cancer therapy; Imaging; In vivo siRNA delivery; Targeted nanoparticle; shRNA expressing nanoparticle