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Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease.

Alves S, Nascimento-Ferreira I, Auregan G, Hassig R, Dufour N, Brouillet E, Pedroso de Lima MC, Hantraye P, Pereira de Almeida L, Déglon N - PLoS ONE (2008)

Bottom Line: Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo.The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD.These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

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shRNA-expressing plasmids mediate the allele-specific silencing of endogenous wild-type ataxin-3 in transiently transfected human 293T cells.A) Western blot of human 293T cells transfected with different shRNA (shAtaxWT, shAtaxMUT and shGFP)-expressing plasmids (5 µg; 48 h post-transfection). The blot clearly indicates that shAtaxWT downregulates endogenous/wild-type human ATX3, whereas shAtaxMUT has no silencing effect. shGFP was used as a control vector and the protein tubulin was used as a loading control. This western blot is representative of three independent experiments.
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pone-0003341-g003: shRNA-expressing plasmids mediate the allele-specific silencing of endogenous wild-type ataxin-3 in transiently transfected human 293T cells.A) Western blot of human 293T cells transfected with different shRNA (shAtaxWT, shAtaxMUT and shGFP)-expressing plasmids (5 µg; 48 h post-transfection). The blot clearly indicates that shAtaxWT downregulates endogenous/wild-type human ATX3, whereas shAtaxMUT has no silencing effect. shGFP was used as a control vector and the protein tubulin was used as a loading control. This western blot is representative of three independent experiments.

Mentions: We further assessed the functionality and efficacy of our siRNA in a more physiological situation, by analyzing the silencing of endogenous ataxin-3 in human embryonic kidney 293T cells. We transfected human 293T cells with the shAtaxWT(G), shAtaxMUT(C) or shGFP plasmid. The level of endogenous wild-type ataxin-3 was determined by western blotting and found to be lowered by transfection with shAtaxWT(G), but not with shAtaxMUT(C) or shGFP (Fig. 3). These data demonstrate the efficacy and selectivity of allele-specific ataxin-3 silencing.


Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease.

Alves S, Nascimento-Ferreira I, Auregan G, Hassig R, Dufour N, Brouillet E, Pedroso de Lima MC, Hantraye P, Pereira de Almeida L, Déglon N - PLoS ONE (2008)

shRNA-expressing plasmids mediate the allele-specific silencing of endogenous wild-type ataxin-3 in transiently transfected human 293T cells.A) Western blot of human 293T cells transfected with different shRNA (shAtaxWT, shAtaxMUT and shGFP)-expressing plasmids (5 µg; 48 h post-transfection). The blot clearly indicates that shAtaxWT downregulates endogenous/wild-type human ATX3, whereas shAtaxMUT has no silencing effect. shGFP was used as a control vector and the protein tubulin was used as a loading control. This western blot is representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553199&req=5

pone-0003341-g003: shRNA-expressing plasmids mediate the allele-specific silencing of endogenous wild-type ataxin-3 in transiently transfected human 293T cells.A) Western blot of human 293T cells transfected with different shRNA (shAtaxWT, shAtaxMUT and shGFP)-expressing plasmids (5 µg; 48 h post-transfection). The blot clearly indicates that shAtaxWT downregulates endogenous/wild-type human ATX3, whereas shAtaxMUT has no silencing effect. shGFP was used as a control vector and the protein tubulin was used as a loading control. This western blot is representative of three independent experiments.
Mentions: We further assessed the functionality and efficacy of our siRNA in a more physiological situation, by analyzing the silencing of endogenous ataxin-3 in human embryonic kidney 293T cells. We transfected human 293T cells with the shAtaxWT(G), shAtaxMUT(C) or shGFP plasmid. The level of endogenous wild-type ataxin-3 was determined by western blotting and found to be lowered by transfection with shAtaxWT(G), but not with shAtaxMUT(C) or shGFP (Fig. 3). These data demonstrate the efficacy and selectivity of allele-specific ataxin-3 silencing.

Bottom Line: Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo.The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD.These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

Show MeSH
Related in: MedlinePlus