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

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Abstract
The delivery to primary cells is a major challenge in the application of siRNAs in biological research. Using a fusion protein consisting of a double-stranded RNA binding domain and a protein transduction domain, Eguchi et al. are able to transfect siRNAs into a wide variety of cells with very high efficiency without cytotoxicity. 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.