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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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.).
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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