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DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker

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Abstract

This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6 kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.

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References

  1. Padma VV (2015) An overview of targeted cancer therapy. BioMedicine 5:1–6. https://doi.org/10.7603/s40681-015-0019-4

    Article  Google Scholar 

  2. Baudino TA (2015) Targeted cancer therapy: the next generation of cancer treatment. Curr Drug Discov Technol 12:3–20. https://doi.org/10.2174/1570163812666150602144310

    Article  CAS  PubMed  Google Scholar 

  3. Eggel A, Buschor P, Baumann MJ et al (2011) Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein. Allergy 66:961–968. https://doi.org/10.1111/j.1398-9995.2011.02546.x

    Article  CAS  PubMed  Google Scholar 

  4. Hussain S, Plückthun A, Allen TM et al (2007) Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system. Mol Cancer Ther 6:3019–3027. https://doi.org/10.1158/1535-7163.MCT-07-0615

    Article  CAS  PubMed  Google Scholar 

  5. Winkler J, Martin-Killias P, Plückthun A et al (2009) EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins. Mol Cancer Ther 8:2674–2683. https://doi.org/10.1158/1535-7163.MCT-09-0402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Boersma YL, Pluckthun A (2011) DARPins and other repeat protein scaffolds: advances in engineering and applications. Curr Opin Biotechnol 22:849–857. https://doi.org/10.1016/j.copbio.2011.06.004

    Article  CAS  PubMed  Google Scholar 

  7. Stahl A, Stumpp MT, Schlegel A et al (2013) Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications. Angiogenesis 16:101–111. https://doi.org/10.1007/s10456-012-9302-0

    Article  CAS  PubMed  Google Scholar 

  8. Hosse RJ, Rothe A, Power BE (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci 15:14–27. https://doi.org/10.1110/ps.051817606.10.1093/protein/gzp011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Weidle UH, Auer J, Brinkmann U et al (2013) The emerging role of new protein scaffold-based agents for treatment of cancer. Cancer Genomics Proteomics 10:155–168

    CAS  PubMed  Google Scholar 

  10. Carter PJ (2011) Introduction to current and future protein therapeutics: a protein engineering perspective. Exp Cell Res 317:1261–1269. https://doi.org/10.1016/j.yexcr.2011.02.013

    Article  CAS  PubMed  Google Scholar 

  11. Stumpp MT, Amstutz P (2007) DARPins: a true alternative to antibodies. Curr Opin Drug Discov Devel 10:153–159

    CAS  PubMed  Google Scholar 

  12. Stumpp MT, Binz HK, Amstutz P (2008) DARPins: a new generation of protein therapeutics. Drug Discov Today 13:695–701. https://doi.org/10.1016/j.drudis.2008.04.013

    Article  CAS  PubMed  Google Scholar 

  13. Gronwall C, Stahl S (2009) Engineered affinity proteins generation and applications. J Biotechnol 140:254–269. https://doi.org/10.1016/j.jbiotec.2009.01.014

    Article  CAS  PubMed  Google Scholar 

  14. Stefan N, Martin-Killias P, Wyss-Stoeckle S et al (2011) DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J Mol Biol 413:826–843. https://doi.org/10.1016/j.jmb.2011.09.016

    Article  CAS  PubMed  Google Scholar 

  15. Kim WT, Ryu CJ (2017) Cancer stem cell surface markers on normal stem cells. BMB Rep 50:285–298. https://doi.org/10.5483/bmbrep.2017.50.6.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Abd El-Maqsoud NMR, Abd El-Rehim DM (2014) Clinicopathologic implications of EpCAM and Sox2 expression in breast cancer. Clin Breast Cancer 14:e1–9. https://doi.org/10.1016/j.clbc.2013.09.006

    Article  CAS  PubMed  Google Scholar 

  17. Bhindi R, Fahmy RG, Lowe HC et al (2007) Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies. Am J Pathol 171:1079–1088. https://doi.org/10.2353/ajpath.2007.070120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burnett JC, Rossi JJ (2012) RNA-based therapeutics: current progress and future prospects. Chem Biol 19:60–71. https://doi.org/10.1016/j.chembiol.2011.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jurk M, Vollmer J (2007) Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation. BioDrugs 21:387–401. https://doi.org/10.2165/00063030-200721060-00006

    Article  CAS  PubMed  Google Scholar 

  20. Lorenzer C, Dirin M, Winkler AM et al (2015) Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics. J Control Release 203:1–15. https://doi.org/10.1016/j.jconrel.2015.02.003

    Article  CAS  PubMed  Google Scholar 

  21. Choi YS, Lee JY, Suh JS et al (2010) The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 31:1429–1443. https://doi.org/10.1016/j.biomaterials.2009.11.001

    Article  CAS  PubMed  Google Scholar 

  22. Tai W, Gao X (2017) Functional peptides for siRNA delivery. Adv Drug Deliv Rev 110–111:157–168. https://doi.org/10.1016/j.addr.2016.08.004

    Article  CAS  PubMed  Google Scholar 

  23. Li H, Tsui TY, Ma W (2015) Intracellular delivery of molecular cargo using cell-penetrating peptides and the combination strategies. Int J Mol Sci 16:19518–19536. https://doi.org/10.3390/ijms160819518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. He H, Ye J, Liu E et al (2014) Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide. J Control Release 193:63–73. https://doi.org/10.1016/j.jconrel.2014.05.056

    Article  CAS  PubMed  Google Scholar 

  25. Kole R, Krainer AR, Altman S (2012) RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov 11:125–140. https://doi.org/10.1038/nrd3625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pärnaste L, Arukuusk P, Langel K et al (2017) The formation of nanoparticles between small interfering RNA and amphipathic cell-penetrating peptides. Mol Ther Nucleic Acids 7:1–10. https://doi.org/10.1016/j.omtn.2017.02.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bäumer S, Bäumer N, Appel N et al (2015) Antibody-mediated delivery of anti-KRAS-siRNA in vivo overcomes therapy resistance in colon cancer. Clin Cancer Res 21:1383–1394. https://doi.org/10.1158/1078-0432.CCR-13-2017

    Article  CAS  PubMed  Google Scholar 

  28. Yoo H, Mok H (2015) Evaluation of multimeric siRNA conjugates for efficient protamine-based delivery into breast cancer cells. Arch Pharm Res 38:129–136. https://doi.org/10.1007/s12272-014-0359-8

    Article  CAS  PubMed  Google Scholar 

  29. Zhang C, Ren W, Liu Q et al (2019) Transportan-derived cell-penetrating peptide delivers siRNA to inhibit replication of influenza virus in vivo. Drug Des Devel Ther 13:1059–1068. https://doi.org/10.2147/DDDT.S195481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu M, Feng B, Shi Y et al (2015) Protamine nanoparticles for improving shRNA-mediated anti-cancer effects. Nanoscale Res Lett 10:134. https://doi.org/10.1186/s11671-015-0845-z

    Article  PubMed  PubMed Central  Google Scholar 

  31. Chang LC, Lee HF, Yang Z et al (2001) Low molecular weight protamine (LMWP) as nontoxic heparin/low molecular weight heparin antidote (I): preparation and characterization. AAPS PharmSci 3:E17. https://doi.org/10.1208/ps030317

    Article  CAS  PubMed  Google Scholar 

  32. Shukla RS, Qin B, Cheng K (2014) Peptides used in the delivery of small noncoding RNA. Mol Pharm 11:3395–3408. https://doi.org/10.1021/mp500426r

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ye J, Liu E, Gong J et al (2017) High-yield synthesis of monomeric LMWP(CPP)-siRNA covalent conjugate for effective cytosolic delivery of siRNA. Theranostics 7:2495–2508. https://doi.org/10.7150/thno.19863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Boersma YL (2018) Advances in the application of designed ankyrin repeat proteins (DARPins) as research tools and protein therapeutics. Methods Mol Biol 1798:307–327. https://doi.org/10.1007/978-1-4939-7893-9-23

    Article  CAS  PubMed  Google Scholar 

  35. Milovnik P, Ferrari D, Sarkar CA et al (2009) Selection and characterization of DARPins specific for the neurotensin receptor 1. Protein Eng Des Sel 22:357–366

    Article  CAS  Google Scholar 

  36. Liang JF, Zhen L, Chang LC, Yang VC (2003) A less toxic heparin antagonist—low molecular weight protamine. Biochemistry (Moscow) 68:116–120

    Article  CAS  Google Scholar 

  37. Arabzadeh S, Tehranizadeh ZA, Haghighi HM et al (2019) Design, synthesis, and in vitro evaluation of low molecular weight protamine (LMWP)-based amphiphilic conjugates as gene delivery carriers. AAPS PharmSciTech. https://doi.org/10.1208/s12249-018-1235-5

    Article  PubMed  Google Scholar 

  38. Sokolova EA, Shilova ON, Kiseleva DV et al (2019) HER2-specific targeted toxin DARPin-LoPE: immunogenicity and antitumor effect on intraperitoneal ovarian cancer xenograft model. Int J Mol Sci 20:2399. https://doi.org/10.3390/ijms20102399

    Article  CAS  PubMed Central  Google Scholar 

  39. Shilova ON, Deyev SM (2019) Promising scaffolds for theranostics. Acta Nat 11:4–43

    Google Scholar 

Download references

Acknowledgements

The authors wish to express their deep gratitude to all who provided technical supports. This project was financially supported by Pasteur Institute of Iran.

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Author notes

  1. Yeganeh Talebkhan Garoosi and Morteza Karimipoor are co-correspondence authors

Authors and Affiliations

  1. Biotechnology Research Center, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran

    Nikta Babaee, Yeganeh Talebkhan Garoosi, Fatemeh Davami, Elham Bayat, Fereidoun Mahboudi & Farzaneh Barkhordari

  2. Biotechnology Research Center, Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran

    Nikta Babaee & Morteza Karimipoor

  3. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran

    Hossein Safarpour

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  1. Nikta Babaee
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Contributions

YT and MK were chief investigators. FD and MK and FM contributed to the design of the study. HS assisted in Molecular docking analysis. NB was responsible for the major experimental work and contributed in writing the main manuscript. FB and EB assisted in data collection. YT, MK and FD contributed revising the main manuscript.

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Correspondence to Yeganeh Talebkhan Garoosi or Morteza Karimipoor.

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Babaee, N., Talebkhan Garoosi, Y., Karimipoor, M. et al. DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker. Mol Biol Rep 47, 7323–7331 (2020). https://doi.org/10.1007/s11033-020-05752-5

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