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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.

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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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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.

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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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