Résumé :
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Communication n° 720. It is only three years since the initial demonstration that short interference RNA (siRNA) functions as a gene silencing mechanism in mammalian cells, a finding that opened up enormous perspectives for analysing gene function and also for therapeutic uses. This demonstration came soon after the discovery of neural stem cells (NSC) in the adult mammalian brain, a discovery that again led to predictions of multiple therapeutic uses, particularly for neurodegenerative diseases. As a team with a central focus on non- viral gene delivery in vivo, we have previously established i) that a cationic polymer (polyethylenime, PEI) can be used to target plasmid DNA to the NSC of the mouse brain (Lemkine, Levi Demeneix, AFM/CNRS patent 2001, Lemkine et al, 2002) and more recently that cationic lipid formulations provide the most efficient vectors for delivery of siRNA into the mouse brain (Hassani et al. 2004). In the past year we have furthered our knowledge of siRNA delivery in vivo by comparing exogenous delivery of 23 mer siRNA and endogenous production of siRNA from a plasmid encoding a small hairpin construction (shRNA) delivered to the target tissue. Having evaluated different promoters to drive shRNA production at multiple time points in various situations we conclude that cationic lipid vectorisation of siRNA is by far the most efficient (>80% target gene inhibition) delivery method. Progress towards therapeutic endpoints implicating NSC is hindered by the absence of knowledge of the basic biology of the stem cell population in the brain. Having spent two years in a thorough investigation of the best means of optimising siRNA to NSC in vivo we are now using the technology developed to increase our knowledge of NSC function by analysing key signalling pathways controlling NSC division and differentiation in the mouse brain. The overall objective is to exploit our recently optimised techniques for siRNA vectorisation into the NSC population in vivo to determine 1) the precise population(s) of NSC targeted (the NSC population includes pluripotent progenitors, the bona fide stem cells and their immediate, multipotent progeny, the rapidly dividing progenitor population). Both populations are candidates for targeting and have therapeutic potential. 2) the role of thyroid hormone (TH) signalling on NSC proliferation and differentiation. 3) the cascades of regulation and the target genes implicated in TH control of NSC cycling.
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