July 27, 2017
... washed with PBS, trypsinized, neutralized with cold PBSA containing 2% FBS and analyzed on an iQue Screener (IntelliCyt). All liquid handling was performed using an EL406 plate washer (BioTek) and a Freedom EVO 150 liquid handling system (Tecan) to minimize variability. Background from untreated cells were subtracted from all measurements, which were then converted to number of fluorophores using ... ed with cold PBSA containing 2% FBS and analyzed on an iQue Screener (IntelliCyt). All liquid handling was performed using an EL406 plate washer (BioTek) and a Freedom EVO 150 liquid handling system (Tecan) to minimize variability. Background from untreated cells were subtracted from all measurements, which were then converted to number of fluorophores using Quantum Alexa Fluor 647 MESF beads foll
Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids. Š The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Yang, NJ; Kauke, MJ; Sun, F; Yang, LF; Maass, KF; Traxlmayr, MW; Yu, Y; Xu, Y; Langer, RS; Anderson, DG; Wittrup, KD;
Journal: Nucleic Acids Res. Pages: 7602-7614