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Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein

Nature Biotechnology volume 27, pages 567–571 (2009)Cite this article

Abstract

RNA interference (RNAi) induced by short interfering RNA (siRNA) allows for discovery research and large-scale screening1,2,3,4,5; however, owing to their size and anionic charge, siRNAs do not readily enter cells4,5. Current approaches do not deliver siRNAs into a high percentage of primary cells without cytotoxicity. Here we report an efficient siRNA delivery approach that uses a peptide transduction domain–double-stranded RNA-binding domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA's negative charge and allowing PTD-mediated cellular uptake. PTD-DRBD–delivered siRNA induced rapid RNAi in a large percentage of various primary and transformed cells, including T cells, human umbilical vein endothelial cells and human embryonic stem cells. We observed no cytotoxicity, minimal off-target transcriptional changes and no induction of innate immune responses. Thus, PTD-DRBD–mediated siRNA delivery allows efficient gene silencing in difficult-to-transfect primary cell types.

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Figure 1: PTD-DRBD–mediated siRNA delivery.
Figure 2: PTD-DRBD siRNA delivery into T cells and HUVECs.
Figure 3: PTD-DRBD–mediated RNAi responses.

References

  1. de Fougerolles, A., Vornlocher, H.P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6, 443–453 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kim, D.H. & Rossi, J.J. Strategies for silencing human disease using RNA interference. Nat. Rev. Genet. 8, 173–184 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Bumcrot, D., Manoharan, M., Koteliansky, V. & Sah, D.W. RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711–719 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Whitehead, K.A., Langer, R. & Anderson, D.G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Behlke, M.A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 18, 305–320 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Gump, J.M. & Dowdy, S.F. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol. Med. 13, 443–448 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Nakase, I. et al. Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther. 10, 1011–1022 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Wadia, J.S., Stan, R.V. & Dowdy, S.F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Turner, J.J. et al. RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. Blood Cells Mol. Dis. 38, 1–7 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Meade, B.R. & Dowdy, S.F. Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Adv. Drug Deliv. Rev. 60, 530–536 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Bevilacqua, P.C. & Cech, T.R. Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR. Biochemistry 35, 9983–9994 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Tian, B., Bevilacqua, P.C., Diegelman-Parente, A. & Mathews, M.B. The doublestranded-RNA-binding motif: interference and much more. Nat. Rev. Mol. Cell Biol. 5, 1013–1023 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Ryter, J.M. & Schultz, S.C. Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. EMBO J. 17, 7505–7513 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Henderson, J.K. et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells 20, 329–337 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Hay, D.C., Sutherland, L., Clark, J. & Burdon, T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 22, 225–235 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Zhang, W. et al. Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nat. Med. 11, 56–62 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Judge, A.D. et al. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. Mol. Ther. 13, 494–505 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Safran, M. et al. Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination. Mol. Imaging 2, 297–302 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Miyoshi, H. et al. Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank V. Nizet (UCSD) for PBMCs. The hES cell line HUES9 was kindly provided by D. Melton (HHMI, Harvard University). A.E. was funded by a Japan Society for the Promotion of Science Research Fellowships for Young Scientists. This work was supported by a Specialized Center of Research grant from the Leukemia & Lymphoma Society (S.F.D.), the Elsa U. Pardee Foundation (S.F.D.), the Howard Hughes Medical Institute (S.F.D.) and the California Institute for Regenerative Medicine (S.F.D.).

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Authors and Affiliations

  1. Howard Hughes Medical Institute, La Jolla, California, USA

    Akiko Eguchi, Bryan R Meade & Steven F Dowdy

  2. Department of Cellular & Molecular Medicine, UCSD School of Medicine, La Jolla, California, USA

    Akiko Eguchi, Bryan R Meade, Craig T Fredrickson, Karl Willert & Steven F Dowdy

  3. Japan Society for the Promotion of Science, Tokyo, Japan

    Akiko Eguchi

  4. Department of Pediatrics, UCSD School of Medicine, La Jolla, California, USA

    Yung-Chi Chang

  5. Life Technologies, Austin, Texas, USA

    Nitin Puri

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  1. Akiko Eguchi
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Contributions

A.E. and B.R.M. designed, purified PTD-DRBD and performed RNAi experiments. Y.-C.C performed PBMC experiments. C.T.F. performed hES cell culture. K.W. supervised hES cell culture. N.P. provided siRNAs reagents. S.F.D. supervised and analyzed data. A.E. and S.F.D. contributed to writing the manuscript, and all authors discussed and refined the manuscript.

Corresponding author

Correspondence to Steven F Dowdy.

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Competing interests

A.E., B.R.M. and S.F.D. are co-inventors on a patent application related to the method described in this publication, which has been licensed to Traversa Therapeutics. S.F.D. is the scientific founder of Traversa Therapeutics.

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Eguchi, A., Meade, B., Chang, YC. et al. Efficient siRNA delivery into primary cells by a peptide transduction domain–dsRNA binding domain fusion protein. Nat Biotechnol 27, 567–571 (2009). https://doi.org/10.1038/nbt.1541

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