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

Abstract

SUMMARYShort interfering RNA (siRNA) induced RNA interference (RNAi) responses allow for discovery research and performing large scale screening1-5; however, due to their size and anionic charge, siRNAs have no bioavailability to enter cells4,5. Current approaches fail to deliver siRNAs into a high percentage of primary cells in a non-cytotoxic fashion. Here we report an efficient siRNA delivery approach that utilizes a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA negative charge and allow for PTD-mediated cellular uptake. PTD-DRBD delivered siRNAs induced rapid RNAi responses in a non-cytotoxic manner in the entire cell population of primary and transformed cells, including T cells, HUVECs and hESCs. Whole genome microarray analysis showed minimal transcriptional changes by PTD-DRBD and we did not detect any innate immune responses in PBMCs. Thus, PTD-DRBD mediated siRNA delivery allows efficient RNAi manipulation of difficult primary cell types.

Identification of a Novel Cell-Penetrating Peptide from Human Phosphatidate Phosphatase LpIN3

“…Polyarginine (11R)-conjugated VIVIT peptide, which is a specific NFAT inhibitory peptide, was able to inhibit graft rejection of allogeneic islet transplantation in mice (Noguchi et al, 2004). More recently, TAT-CPP-conjugated RNA binding successfully delivered siRNA for efficient gene silencing in various cell types including primary T cells (Eguchi et al, 2009). Although extensive studies have suggested the possible use of those CPPs as drug delivery systems for human disease, clinical trials have been ongoing since the middle of the 2000s (van den Berg and Dowdy, 2011).…”

A specific aptamer-cell penetrating peptides complex delivered siRNA efficiently and suppressed prostate tumor growthin vivo

“…Despite various approaches have been explored, such as osmotic methods, lipid conjugation and virus vectors, most of them are limited for research and perform the best on adherent tumor cells. CPPs have demonstrated strong transfection ability by delivery siRNA into some acknowledged hard-transfected primary cells like T cells, HUVECs and hESCs 16 and even the blood-brain barrier, which indicates its potential use in vivo. 33,34 In this study, we used 3TAT-DRBD to bind sur-siRNA and delivered it to the cytoplasm.…”

“…To construct the A10-STD-(sur-siRNA) complex 10 mL 5 mM sur-siRNA in water was mixed with 10 mL 20-50 mM STD in PBS with 10% glycerol on ice for 30 min, then mixed with 10 mL 20 mM biotinylated A10 in PBS (pH 7.4) for another 30 min. 16 To confirm the binding between STD and sur-siRNA or A10, 10 mL sur-siRNA (diluted to 1/32 mM) and 10 mL A10 (diluted to 1/4 mM) were incubated with STD fusion protein in PBS with 10% glycerol on ice for 2 hours respectively. STD fusion protein (20 mg) was 2-folds diluted up to 1/128.…”

“…15 For siRNAs delivery, cationic CPPs and anionic siRNAs will aggregate due to charge neutralization, which may affect biological functions of siRNA. Eguchi A et al [16][17][18] solved this problem by construction of a fusion protein of CPPs with double strand RNA binding domain (DRBD) to mask siRNA's charges, which can efficiently deliver siRNA into primary cells. For specific delivery of siRNA, there is an increasing interest in aptamers.…”

Importance of the physicochemical properties of fluorescent dyes for obtaining target-specific in vivo images by membrane-permeable macromolecular imaging probes

“…Recent studies demonstrate that protein transduction domains (PTDs), also known as cell-penetrating peptides, can deliver conjugated macromolecular cargos by penetrating the cell membrane, and they have been utilized as transport vectors in biomedical applications [5][6][7]. At present, in vivo studies of PTDs are being conducted to deliver proteins, oligonucleotides, siRNA, liposomes, and nanoparticles inside cells where they target disease-specific molecules [8][9][10][11][12]. Therefore, PTD-conjugated macromolecules are promising materials targeting intracellular molecules.…”