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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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The single nucleotide polymorphism strategy used for the specific elimination of mutant or wild-type human ataxin-3 (ATX3) by RNA interference.A) Schematic representation of the lentiviral constructs encoding wild-type human ataxin-3 (27 CAG repeats) or mutant human ataxin-3 (72 CAG repeats) under control of the phosphoglycerate kinase-1 (PGK-1) promoter. Immediately after the last CAG repeat in the 3′ end, there is a linked single nucleotide polymorphism (SNP) (G987GG→C987GG) between wild-type and mutant human ataxin-3. B) Diagram of the shAtax vectors used to downregulate human ataxin-3: shRNA cassette under control of the H1 promoter (pol III) and a separate cassette containing the lacZ reporter gene under control of the PGK-1 promoter, making it possible to follow the expression of infected neurons. These shRNAs were designed to silence wild-type (shAtaxWT) or mutant human ataxin-3 (shAtaxMUT) selectively, making use of the (G987GG→C987GG) SNP.
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pone-0003341-g001: The single nucleotide polymorphism strategy used for the specific elimination of mutant or wild-type human ataxin-3 (ATX3) by RNA interference.A) Schematic representation of the lentiviral constructs encoding wild-type human ataxin-3 (27 CAG repeats) or mutant human ataxin-3 (72 CAG repeats) under control of the phosphoglycerate kinase-1 (PGK-1) promoter. Immediately after the last CAG repeat in the 3′ end, there is a linked single nucleotide polymorphism (SNP) (G987GG→C987GG) between wild-type and mutant human ataxin-3. B) Diagram of the shAtax vectors used to downregulate human ataxin-3: shRNA cassette under control of the H1 promoter (pol III) and a separate cassette containing the lacZ reporter gene under control of the PGK-1 promoter, making it possible to follow the expression of infected neurons. These shRNAs were designed to silence wild-type (shAtaxWT) or mutant human ataxin-3 (shAtaxMUT) selectively, making use of the (G987GG→C987GG) SNP.

Mentions: We developed two lentiviral vectors encoding siRNAs targeting an SNP (G987GG→C987GG) located at the 3′ end of the CAG expansion of the ataxin-3 gene for the specific silencing of mutant human ataxin-3 mRNA but not of the wild-type ataxin-3 mRNA. The wild-type ataxin-3 gene has a G at position 987, whereas the mutant ataxin-3 has a C at this position. We therefore designed two siRNAs corresponding to this SNP. The sequences are identical with the exception of a single nucleotide (G/C polymorphism) at the center of the shRNA molecule: shAtaxMUT(C) and shAtaxWT(G) (Fig. 1A). These shRNAs were inserted in the 3'LTR of a lentiviral vector containing the H1 promoter. The lacZ reporter gene was inserted into these constructs downstream from the internal mouse phosphoglycerate kinase 1 (PGK) promoter, to facilitate the identification of transduced cells (Fig. 1B).


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)

The single nucleotide polymorphism strategy used for the specific elimination of mutant or wild-type human ataxin-3 (ATX3) by RNA interference.A) Schematic representation of the lentiviral constructs encoding wild-type human ataxin-3 (27 CAG repeats) or mutant human ataxin-3 (72 CAG repeats) under control of the phosphoglycerate kinase-1 (PGK-1) promoter. Immediately after the last CAG repeat in the 3′ end, there is a linked single nucleotide polymorphism (SNP) (G987GG→C987GG) between wild-type and mutant human ataxin-3. B) Diagram of the shAtax vectors used to downregulate human ataxin-3: shRNA cassette under control of the H1 promoter (pol III) and a separate cassette containing the lacZ reporter gene under control of the PGK-1 promoter, making it possible to follow the expression of infected neurons. These shRNAs were designed to silence wild-type (shAtaxWT) or mutant human ataxin-3 (shAtaxMUT) selectively, making use of the (G987GG→C987GG) SNP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003341-g001: The single nucleotide polymorphism strategy used for the specific elimination of mutant or wild-type human ataxin-3 (ATX3) by RNA interference.A) Schematic representation of the lentiviral constructs encoding wild-type human ataxin-3 (27 CAG repeats) or mutant human ataxin-3 (72 CAG repeats) under control of the phosphoglycerate kinase-1 (PGK-1) promoter. Immediately after the last CAG repeat in the 3′ end, there is a linked single nucleotide polymorphism (SNP) (G987GG→C987GG) between wild-type and mutant human ataxin-3. B) Diagram of the shAtax vectors used to downregulate human ataxin-3: shRNA cassette under control of the H1 promoter (pol III) and a separate cassette containing the lacZ reporter gene under control of the PGK-1 promoter, making it possible to follow the expression of infected neurons. These shRNAs were designed to silence wild-type (shAtaxWT) or mutant human ataxin-3 (shAtaxMUT) selectively, making use of the (G987GG→C987GG) SNP.
Mentions: We developed two lentiviral vectors encoding siRNAs targeting an SNP (G987GG→C987GG) located at the 3′ end of the CAG expansion of the ataxin-3 gene for the specific silencing of mutant human ataxin-3 mRNA but not of the wild-type ataxin-3 mRNA. The wild-type ataxin-3 gene has a G at position 987, whereas the mutant ataxin-3 has a C at this position. We therefore designed two siRNAs corresponding to this SNP. The sequences are identical with the exception of a single nucleotide (G/C polymorphism) at the center of the shRNA molecule: shAtaxMUT(C) and shAtaxWT(G) (Fig. 1A). These shRNAs were inserted in the 3'LTR of a lentiviral vector containing the H1 promoter. The lacZ reporter gene was inserted into these constructs downstream from the internal mouse phosphoglycerate kinase 1 (PGK) promoter, to facilitate the identification of transduced cells (Fig. 1B).

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