Htt548-GFP Fusion and DsRed-miRNA Vectors To Study mi-RNA-Mediated Down-Regulation of htt Expression

0328716 - ÚŽFG 2010 US eng A - Abstrakt
Phan, A. T. - Hammond, D. - Strnádel, Ján - Nejime, T. - Hruška-Plocháň, Marian - Usvald, Dušan - Marsala, S. - Motlík, Jan - Miyanohara, A.
Htt548-GFP Fusion and DsRed-miRNA Vectors To Study mi-RNA-Mediated Down-Regulation of htt Expression.
12th Annual Meeting of American society for gene therapy-Abstracts. Milwaukee: Marathon Multimedia, 2009. s. 1-1.
[American society for gene therapy. 27.05.2009-30.05.2009, San Diego]
Výzkumný záměr: CEZ:AV0Z50450515
Klíčová slova: Huntington´s disease * Neurodegenerative disease
Kód oboru RIV: FH - Neurologie, neurochirurgie, neurovědy

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat encoding poly-glutamine (polyQ) in the first exon of the gene encoding the protein huntingtin (htt). HD is an autosomal dominant disorder and feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms has been reported. We have previously developed and characterized lentivirus vectors expressing truncated forms (548aa, 1213aa, or 548aa+GFP fusion) and full-length htts containing either 23Q or 145Q as normal or mutant gene, respectively. We have demonstrated efficient packaging of these lentivirus vectors and expression of htts from human HD promoter in cultured HEK293 cells and rat cortical primary cultures by Western blot and immunohistological analyses. Some of these vectors were injected into striatum of SD rats and appearance of htt-aggregates formed only by mutant (145Q) but not by normal (23Q) htt was confirmed. In the case of the administration of HD-145Q548-GFP fusion construct, aggregate formation and neuronal degenerations were well characterized by GFP and by loss of DARPP-32 immunoreactivity. In the present study, we developed a vector expressing miRNA targeted to htt and the effect of the miRNA was assessed by GFP expression using htt548-GFP fusion construct. Since the fluorescence expressed by htt promoter-driven htt548-GFP was very weak, we replaced the htt promoter with hCMV promoter for this assay. The miRNA sequence was designed to target downstream of the polyQ and inserted into the 3' non-coding region of the htt promoter-driven DsRed gene to enable evaluation of the expression of the miRNA indirectly by DsRed expression. After co-transfection of HEK293 cells with the htt548-GFP vector and DsRed-miRNA vector, significant reduction of GFP expression was observed, whereas control miRNA vector did not show any effect on the GFP expression. Co-infection experiments of HEK293 cells and rat cortical primary cultures with HIV1 vectors expressing the htt548-GFP and DsRed-miRNA are currently underway. Since we have previously demonstrated the usefulness of the HIV1-htt145Q548-GFP vector for in vivo study of htt-aggregate formation and neuronal degeneration, utilization of the current DsRed-miRNA vector will provide an effective system to study the feasibility of RNAi-mediated treatment in an acute intrastriatal vector-mediated model of HD and in transgenic HD animal models. Keywords
Trvalý link: http://hdl.handle.net/11104/0174961