title: EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins.
abstract: Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a designed ankyrin repeat protein (DARPin) specific for the epithelial cell adhesion molecule (EpCAM) as a carrier for small interfering RNA (siRNA) complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in Escherichia coli in soluble form, yet, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing four to five siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as shown on MCF-7 breast carcinoma cells. In contrast to unspecific LipofectAMINE transfection, down-regulation of antiapoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as shown by increased sensitivity to the anticancer agent doxorubicin.
authors.affiliation: Institute of Pharmacology, University of Bern, 3010 Bern, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Winkler
authors.forename: Patricia
authors.initials: P
authors.lastname: Martin-Killias
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.forename: Uwe
authors.initials: U
authors.lastname: Zangemeister-Wittke
mesh_terms: D017089:Ankyrin Repeat; D000951:Antigens, Neoplasm / Q000008:administration & dosage*; D001483:Base Sequence; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000008:administration & dosage*; D045744:Cell Line, Tumor; D017931:DNA Primers; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D018613:Microscopy, Confocal; D036103:Nanotechnology; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D017931:DNA Primers; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D034741:RNA, Small Interfering