(478e) High Efficiency siRNA-Mediated Knockdown Using High-Molecular-Weight Branched Polyethylenimine In Mammalian Cells

Authors: 
Pack, D. W. - Presenter, University of Illinois Urbana Champaign
Wong, L. - Presenter, University of Illinois


RNA interference has been widely used to down-regulate the expression of many targeted genes and is currently being explored for gene therapy. The main mediator of this phenomenon is short interfering RNA (siRNA). Commercially available high-molecular-weight (HMW) polyethylenimine (PEI) of 25-kDa in the branched-form was used to deliver siRNA in mammalian cells. siRNA polyplexes were characterized with regard to siRNA condensation and determined to be fully condensed at 0.4 μg PEI/μg siRNA by gel retardation and significant quenching of ethidium bromide measured at 0.2 μg PEI/μg siRNA. Sizes of all the polyplexes measured by dynamic light scattering were roughly 120 to 240 nm. Model cell lines, HEK293, NIH/3T3 and HeLa, were transiently co-transfected with luciferase-encoding plasmid (pGL3)-Lipofectamine 2000 lipoplexes and 5-50 nM luciferase-targeting siRNA (siGL3)-PEI polyplexes. The PEI/siGL3 was varied over a wide range from 4:1 to 40:1. The delivery efficiency of siRNA is highly dependent on the amount of PEI applied to the cells and not on the PEI/siGL3 ratio. At ≥1.33 μg/mL of PEI, a downregulation >80% is achieved with as little as 5 nM of siRNA (corresponding to PEI/siGL3 = 20:1 w:w). Polyplexes formed at high PEI concentration exhibit better cellular uptake and transfection efficiencies. The uptake of polyplexes formed at 2.67 μg/mL of PEI is ≥10-fold higher than polyplexes formed at 0.27 μg/mL of PEI and the knockdown rate improves by ≥30% at all siRNA concentrations.