EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

Winkler, Johannes; Martin-Killias, Patricia; Plückthun, Andreas; Zangemeister-Wittke, Uwe (2009). EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins. Molecular cancer therapeutics, 8(9), pp. 2674-83. Philadelphia, Pa.: American Association for Cancer Research AACR 10.1158/1535-7163.MCT-09-0402

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Pharmacology

UniBE Contributor:

Zangemeister, Uwe

ISSN:

1535-7163

Publisher:

American Association for Cancer Research AACR

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:10

Last Modified:

04 May 2014 23:22

Publisher DOI:

10.1158/1535-7163.MCT-09-0402

PubMed ID:

19723880

Web of Science ID:

000269968900020

URI:

https://boris.unibe.ch/id/eprint/30684 (FactScience: 194953)

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