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EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins
Johannes Winkler1, Patricia Martin-Killias, Andreas Plückthun
1Institute of Pharmacology, University of Bern, 3010 Bern, Switzerland.
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.
Insights
Researchers developed a novel protein carrier for targeted cancer gene therapy. This system delivers small interfering RNA (siRNA) to tumors, effectively down-regulating bcl-2 and enhancing cancer cell apoptosis.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Therapy
Background:
- Targeted delivery of nucleic acids for cancer therapy faces challenges in tumor specificity and cellular uptake.
- Epithelial cell adhesion molecule (EpCAM) is a potential target for cancer cell-specific delivery systems.
Purpose of the Study:
- To develop a novel protein-based delivery system for small interfering RNA (siRNA) targeting bcl-2 mRNA in cancer cells.
- To enhance the specificity and efficiency of siRNA delivery using a designed ankyrin repeat protein (DARPin) fused to a protamine sequence.
Main Methods:
- Engineered fusion proteins combining an EpCAM-specific DARPin with a truncated human protamine-1 sequence for siRNA complexation.
- Generated DARPin dimers to improve cell binding affinity and siRNA delivery efficiency.
- Expressed and purified fusion proteins under denaturing conditions, followed by refolding.
- Assessed siRNA binding capacity, EpCAM-specific binding to MCF-7 cells, and bcl-2 mRNA down-regulation via siRNA delivery.
Main Results:
- Fusion proteins effectively complexed 4-5 siRNA molecules per protamine unit.
- The fusion proteins demonstrated specific binding to EpCAM on MCF-7 breast carcinoma cells.
- siRNA delivery mediated by the fusion proteins resulted in EpCAM-dependent down-regulation of antiapoptotic bcl-2 mRNA.
- Inhibition of bcl-2 expression increased tumor cell apoptosis and sensitivity to doxorubicin.
Conclusions:
- EpCAM-specific DARPin-protamine fusion proteins serve as an effective delivery system for siRNA in cancer gene therapy.
- This targeted approach enables specific down-regulation of bcl-2, promoting cancer cell apoptosis and enhancing chemosensitivity.
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