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.

journal_name

Mol Cancer Ther

authors

Winkler J,Martin-Killias P,Plückthun A,Zangemeister-Wittke U

doi

10.1158/1535-7163.MCT-09-0402

subject

Has Abstract

pub_date

2009-09-01 00:00:00

pages

2674-83

issue

9

eissn

1535-7163

issn

1538-8514

pii

1535-7163.MCT-09-0402

journal_volume

8

pub_type

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