Block copolymers for siRNA delivery

The discovery of the RNA interference (RNAi) pathway in plants in the early 90's and the subsequent demonstration of its presence in mammalian cells in 2001 were a major breakthrough in biological research. Indeed it was quickly realized that this highly specific mechanism of gene silencing could be harnessed to study genomics or interfere with disease causing genes. Because of the numerous potential applications, small interfering RNAs (siRNAs) which are able to induce the RNAi pathway became one of the most promising tools for molecular biology and started to be intensively studied. However the potential of siRNAs has not yet been fully exploited, mainly because of a lack of effective carrier capable to deliver them in all cell-types. In this project Poly(ethyleneglycol)-Poly(propylenesulfide)-Poly(ethyleneimine) (PEG-b-PPS-b-PEI), a block-co-polymer already developed by our research group for the delivery of DNA, has been used to deliver siRNA in different cell-types. Because the size of polyplexes, resulting from the complexation of polymer with siRNA, is supposed to be an important parameter for the transfection efficiency, a blend of PEG-b-PPS and PPS-b-PEI was also tested as it exhibits very small aggregates sizes. Block copolymers have been first synthesized and complexed with siRNA. The Physicochemical properties of the resulting polyplexes, such as the surface charge, the size, or the complexation efficiency were then assessed. Inhibition of Lamin A/C in HeLa and mesenchymal stem cells (MSCs) in culture was tried with Lipofectamine 2000 used as a positive control. Specific gene downregulation was determined by quantitative PCR (qPCR). PEG-b-PPS-b-PEI-siRNA and PEG-b-PPS/PPS-b-PEI-siRNA complexes exhibited a downregulation much weaker than that of Lipofectamine 2000 for both cell-types. Moreover the presence of tri-block or blend of di-blocks in the culture media induced Lamin A/C overexpression. Another siRNA model has been tested which targets the green fluorescent protein (GFP) in HeLa cells previously transfected with GFPcoding DNA. Finally the capacity of the polyplexes to protect siRNA against digestion by RNases and their stability in presence of heparin were also tested

Hubbell, Jeffrey A.
Laboratory for Regenerative Medicine & Pharmacobiology, IBI, SV, EPFL

 Record created 2009-06-26, last modified 2018-01-28

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