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siRNA-based therapy for cerebral tauopathies

Final Report Summary - SITAU (siRNA-based therapy for cerebral tauopathies)

In order to deliver the siRNA in to cells several techniques has been suggested and tested with various successes. We have here investigated multiple transfection agents in vitro and polyethyleneImine(PEI-)-based and Accell-siRNA (Dharmacon)-based delivery in vivo. First, we evaluated siRNA interference efficacy of the enhanced green flourescent protein (eGFP) and the house-keeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using 'naked' siRNA molecules. We used several transfection agents including Hi-Perfect (Quigen), Dharmafect (Dharmacon), X-treme Gene (Roche), Saint-RED (Synvolux), Trans-TKO (Mirus) and Interferrin (Polyplus). We prepared and used primary cortical cultures from E15-17 Actin- eGFP transgenic and C57/B6 embryos, as well as human mesenchephalic cell culture (LuhMes). We found unfortunately that, in our hands, the transfection agents were not effective or toxic.

Further, we established collaboration with Prof. Culmsee at the Department of Pharmacology and Clinical Pharmacy, Philipps-University Marburg, where we started to work with lipofectamine 2000 (Invitrogen) as transfection agent, in which the Prof. Culmsee had experience. We used here both 'naked' and modified (ON-TARGETplus, Dharmacon) siRNA against the home-keeping genes GAPDH and Cyclophilin B (CycloB) in primary cortical cell cultures expressing P301S mutation, derived from our transgenic P301S mutant Tau mice as well as LuhMes cells and mouse neuroblastoma cells (HT22).

We also predicted good siRNA sequences for the human MAPT gene using the whiteheads siRNA prediction tool (see http://jura.wi.mit.edu/bioc/siRNAext/home.php online). We design siRNAs that targeted human mutant P301S tau, with a less affinity endogenous mouse tau, targeting the heterogene sequences between the human and the mouse MAPT genes. Four sequences were selected due to their poor overlap with the mouse tau gene and relative low overlap with any mouse genes. Although, using single or pools of GAPDH or CycloB, or the four Tau sequences alone or in a pool, we were not able to produce any consistent knockdown using lipofectamine 2000.

As the primary goal was to knockdown the protein level, we used western blot as the primary technique, rather than systematically looking at the RNA levels. As the transfection agent did not produce any reliable and consistent protein knock-down, we started to experimentally test the novel Accell-siRNA developed by Dharmacon. The Accell-siRNAs have been modified to enter the cell without any transfection agent, which also is more suitable for in vivo application. We tested Accell-siRNA in both primary neuronal cortical cultures as well as the LuhMes cells. Here, we were able to show that GAPDH and CycloB proteins could significant be knocked down in both cell types, as shown by western blot. The more potent protein knockdown, for both tested cells, was observed for CycloB.

Furthermore, we also tested pre-designed Accell-siRNA against Tau protein (smartPool of four sequence). Similar, we were here able to significantly knockdown the human and mouse Tau protein both in primary cortical neurons at concentrations low as 250 nM siRNA (60-95 %) and human mesencepahlic (LuhMes) cells at 125 nM (75-95 %). Substantial protein knockdown was observed already at 72 h post transfection in the LuhMes cells and at 96 to 144 h in the primary neurons.

Our data represent an important step forward in investigating the effect of siRNA interference in a clinical relevant model of human Tau-related disease. Moreover, applications in an animal's model of the disease (data in progress) will further shed light on the effect of siRNA interference and its potential application as a treatment for Tauopathies and related disorders.

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