Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown

Abstract

Knockdown of genes by RNA interference (RNAi) in vitro requires methods of transfection or transduction, both of which have limited impact in vivo. As a virus-free approach, we chemically coupled cell surface receptors internalizing antibodies to the short interfering RNA (siRNA) carrier peptide protamine using the bispecific cross-linker sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone to carry siRNA. This complex can efficiently find, bind and internalize into receptor-positive cells in vitro and in vivo, which can be checked by flow cytometry, fluorescence microscopy and western blotting. This method obtains results similar to those of siRNA targeting molecules engineered by genetic fusions between receptor-binding and siRNA carrier units, with the advantage of using readily available purified proteins without the need for engineering, expression and purification of respective constructs. The procedure for coupling the complex takes 2 d, and the functional assays take 2 weeks.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Composition of the antibody-siRNA complex.
Figure 3: Quality control assays for functional antibody-siRNA complexes.
Figure 4: Quality control, binding and cellular uptake.
Figure 5: mRNA target knockdown.
Figure 6: The presence of antibody-siRNA complexes in xenograft-transplanted cells in vivo after intraperitoneal injection.
Figure 7: Antibody–siRNA complex formation can be applied to IGF1R targeting.
Figure 2: Flowchart illustrating the protocol to form and use the CSP–siRNA complexes.

References

  1. Bäumer, S. et al. Antibody-mediated delivery of anti-KRAS-siRNA in vivo overcomes therapy resistance in colon cancer. Clin. Cancer Res. 21, 1383–1394 (2015).

    Article  Google Scholar 

  2. Choi, Y.S. et al. The systemic delivery of siRNAs by a cell penetrating peptide, low-molecular-weight protamine. Biomaterials 31, 1429–1443 (2009).

    Article  Google Scholar 

  3. El-Andaloussi, S. et al. Exosome-mediated delivery of siRNA in vitro and in vivo. Nat. Protoc. 7, 2112–2126 (2012).

    CAS  Article  Google Scholar 

  4. Liu, B. Exploring cell type-specific internalizing antibodies for targeted delivery of siRNA. Brief. Funct. Genomics Proteomics 6, 112–119 (2007).

    CAS  Article  Google Scholar 

  5. Hsu, C.Y. & Uludag, H. A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine. Nat. Protoc. 7, 935–945 (2012).

    CAS  Article  Google Scholar 

  6. Di Paolo, D. et al. Selective therapeutic targeting of the anaplastic lymphoma kinase with liposomal siRNA induces apoptosis and inhibits angiogenesis in neuroblastoma. Mol. Ther. 19, 2201–2212 (2011).

    CAS  Article  Google Scholar 

  7. Casi, G. & Neri, D. Antibody-drug conjugates: basic concepts, examples and future perspectives. J. Control. Release 161, 422–428 (2012).

    CAS  Article  Google Scholar 

  8. Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717 (2005).

    CAS  Article  Google Scholar 

  9. Kies, M.S. & Harari, P.M. Cetuximab (Imclone/Merck/Bristol-Myers Squibb). Curr. Opin. Investig. Drugs 3, 1092–1100 (2002).

    CAS  PubMed  Google Scholar 

  10. Hauser, P.V. et al. Novel siRNA delivery system to target podocytes in vivo. PLoS ONE 5, e9463 (2010).

    Article  Google Scholar 

  11. Yao, Y.D. et al. Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis. Sci. Transl. Med. 4, 130ra48 (2012).

    Article  Google Scholar 

  12. Peer, D., Zhu, P., Carman, C.V., Lieberman, J. & Shimaoka, M. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc. Natl. Acad. Sci. USA 104, 4095–4100 (2007).

    CAS  Article  Google Scholar 

  13. Kittler, R. et al. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat. Methods 4, 337–344 (2007).

    CAS  Article  Google Scholar 

  14. Tap, W.D. et al. Phase II study of ganitumab, a fully human anti-type-1 insulin-like growth factor receptor antibody, in patients with metastatic Ewing family tumors or desmoplastic small round cell tumors. J. Clin. Oncol. 30, 1849–1856 (2012).

    CAS  Article  Google Scholar 

  15. Mayeenuddin, L.H., Yu, Y., Kang, Z., Helman, L.J. & Cao, L. Insulin-like growth factor 1 receptor antibody induces rhabdomyosarcoma cell death via a process involving AKT and bcl-x(L). Oncogene 29, 6367–6377 (2010).

    CAS  Article  Google Scholar 

  16. Steele-Perkins, G. & Roth, R.A. Monoclonal antibody alpha IR-3 inhibits the ability of insulin-like growth factor II to stimulate a signal from the type I receptor without inhibiting its binding. Biochem. Biophys. Res. Commun. 171, 1244–1251 (1990).

    CAS  Article  Google Scholar 

  17. Zia, F. et al. Monoclonal antibody alpha IR-3 inhibits non-small cell lung cancer growth in vitro and in vivo. J. Cell. Biochem. Suppl. 24, 269–275 (1996).

    CAS  Article  Google Scholar 

  18. Gargiulo, G., Serresi, M., Cesaroni, M., Hulsman, D. & van Lohuizen, M. In vivo shRNA screens in solid tumors. Nat. Protoc. 9, 2880–2902 (2014).

    CAS  Article  Google Scholar 

  19. Burnett, J.C., Rossi, J.J. & Tiemann, K. Current progress of siRNA/shRNA therapeutics in clinical trials. Biotechnol. J. 6, 1130–1146 (2011).

    CAS  Article  Google Scholar 

  20. Tabernero, J. et al. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 3, 406–417 (2013).

    CAS  Article  Google Scholar 

  21. Kumar, P. et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134, 577–586 (2008).

    CAS  Article  Google Scholar 

  22. Surendranath, V., Theis, M., Habermann, B.H. & Buchholz, F. Designing efficient and specific endoribonuclease-prepared siRNAs. Methods Mol. Biol. 942, 193–204 (2013).

    CAS  Article  Google Scholar 

  23. Deacon, R.M. Housing, husbandry and handling of rodents for behavioral experiments. Nat. Protoc. 1, 936–946 (2006).

    Article  Google Scholar 

  24. Schmidt, L.H. et al. The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J. Thorac. Oncol. 6, 1984–1992 (2011).

    Article  Google Scholar 

  25. Bäumer, N. et al. A limited role for the cell cycle regulator cyclin A1 in murine leukemogenesis. PLoS ONE 10, e0129147 (2015).

    Article  Google Scholar 

  26. Bäumer, N. et al. Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6. Development 130, 2903–2915 (2003).

    Article  Google Scholar 

  27. Di Scipio, F., Raimondo, S., Tos, P. & Geuna, S. A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation. Microsc. Res. Tech. 71, 497–502 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Wilhelm Sander Stiftung grant nos. 2009.041.2 (to S.B. and C.M.-T.), 2014.054.1 (to W.E.B. and S.B.) and 2010.015.1 (to W.E.B.); by Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion, Cluster of Excellence (to W.E.B.); and by Innovative Medizinische Forschung (IMF) grant nos. 111418 (to S.B.) and 121314 (to N.B.) by the Medical Faculty, University of Münster.

Author information

Authors and Affiliations

Authors

Contributions

N.B., N.A. and S.B. wrote most of the protocol. N.B., N.A., L.T., F.B., C.R. and S.B. performed the experiments and analyzed the data. S.B., C.M.-T. and W.E.B. initially designed the protocol and supervised the study. All authors contributed to writing the paper.

Corresponding author

Correspondence to Sebastian Bäumer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bäumer, N., Appel, N., Terheyden, L. et al. Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown. Nat Protoc 11, 22–36 (2016). https://doi.org/10.1038/nprot.2015.137

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.137

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing