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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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Allele-specific silencing of mutant human ataxin-3 in rat brain.A) Laser confocal microscopy, showing neuronal transduction 2 months after injection in the rat striatum with recombinant lentiviral vectors encoding shAtaxMUT (n = 7), shAtaxWT (n = 8) or shGFP (n = 4) and mutant human ataxin-3 (MUT ATX3). The viral vectors also contained a separate PGK-LacZ cassette encoding β-galactosidase, to facilitate the detection of infected neurons (B, H, N and E, K, Q, high magnification). In adult rats expressing MUT ATX3 and shAtaxMUT (n = 7), the number of neurons containing MUT ATX3-positive aggregates was much smaller (M) and the high magnification merged image (R) indicates that the few cells positive for MUT ATX3 did not express the lacZ reporter gene present in the shAtaxMUT vector. These cells were therefore not transduced with the vectors encoding the silencing sequences. By contrast, in animals expressing MUT ATX3 and shGFP (n = 4) or the control shAtaxWT (n = 8) (A and G, respectively) high magnification merged images show many MUT ATX3-positive cells simultaneously expressing the lacZ reporter gene present in both shAtaxWT or shGFP (F and L, respectively). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.
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pone-0003341-g005: Allele-specific silencing of mutant human ataxin-3 in rat brain.A) Laser confocal microscopy, showing neuronal transduction 2 months after injection in the rat striatum with recombinant lentiviral vectors encoding shAtaxMUT (n = 7), shAtaxWT (n = 8) or shGFP (n = 4) and mutant human ataxin-3 (MUT ATX3). The viral vectors also contained a separate PGK-LacZ cassette encoding β-galactosidase, to facilitate the detection of infected neurons (B, H, N and E, K, Q, high magnification). In adult rats expressing MUT ATX3 and shAtaxMUT (n = 7), the number of neurons containing MUT ATX3-positive aggregates was much smaller (M) and the high magnification merged image (R) indicates that the few cells positive for MUT ATX3 did not express the lacZ reporter gene present in the shAtaxMUT vector. These cells were therefore not transduced with the vectors encoding the silencing sequences. By contrast, in animals expressing MUT ATX3 and shGFP (n = 4) or the control shAtaxWT (n = 8) (A and G, respectively) high magnification merged images show many MUT ATX3-positive cells simultaneously expressing the lacZ reporter gene present in both shAtaxWT or shGFP (F and L, respectively). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.

Mentions: In animals infected with lentiviral vectors encoding mutant ataxin-3(C) and the non specific shAtaxWT(G) or the shGFP control, immunohistochemical analysis of coronal rat brain sections with anti-ataxin-3 (1H9) and β-galactosidase antibodies showed that many neurons expressed both transgenes (Fig. 4A–C, Fig 5A–L). In animals expressing mutant ataxin-3 and shAtaxMUT(C), far fewer neurons produced the pathogenic protein (Fig. 4E, Fig. 5M). The merged images (Fig. 4F, Fig. 5O) indicate that only a few mutant ataxin-3-positive-cells did not express the lacZ reporter gene present in the shAtaxMUT(C) vector (high magnification Fig. 5R). These cells corresponded to neurons not co-infected with both vectors, which were therefore not treated with the siRNA.


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)

Allele-specific silencing of mutant human ataxin-3 in rat brain.A) Laser confocal microscopy, showing neuronal transduction 2 months after injection in the rat striatum with recombinant lentiviral vectors encoding shAtaxMUT (n = 7), shAtaxWT (n = 8) or shGFP (n = 4) and mutant human ataxin-3 (MUT ATX3). The viral vectors also contained a separate PGK-LacZ cassette encoding β-galactosidase, to facilitate the detection of infected neurons (B, H, N and E, K, Q, high magnification). In adult rats expressing MUT ATX3 and shAtaxMUT (n = 7), the number of neurons containing MUT ATX3-positive aggregates was much smaller (M) and the high magnification merged image (R) indicates that the few cells positive for MUT ATX3 did not express the lacZ reporter gene present in the shAtaxMUT vector. These cells were therefore not transduced with the vectors encoding the silencing sequences. By contrast, in animals expressing MUT ATX3 and shGFP (n = 4) or the control shAtaxWT (n = 8) (A and G, respectively) high magnification merged images show many MUT ATX3-positive cells simultaneously expressing the lacZ reporter gene present in both shAtaxWT or shGFP (F and L, respectively). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553199&req=5

pone-0003341-g005: Allele-specific silencing of mutant human ataxin-3 in rat brain.A) Laser confocal microscopy, showing neuronal transduction 2 months after injection in the rat striatum with recombinant lentiviral vectors encoding shAtaxMUT (n = 7), shAtaxWT (n = 8) or shGFP (n = 4) and mutant human ataxin-3 (MUT ATX3). The viral vectors also contained a separate PGK-LacZ cassette encoding β-galactosidase, to facilitate the detection of infected neurons (B, H, N and E, K, Q, high magnification). In adult rats expressing MUT ATX3 and shAtaxMUT (n = 7), the number of neurons containing MUT ATX3-positive aggregates was much smaller (M) and the high magnification merged image (R) indicates that the few cells positive for MUT ATX3 did not express the lacZ reporter gene present in the shAtaxMUT vector. These cells were therefore not transduced with the vectors encoding the silencing sequences. By contrast, in animals expressing MUT ATX3 and shGFP (n = 4) or the control shAtaxWT (n = 8) (A and G, respectively) high magnification merged images show many MUT ATX3-positive cells simultaneously expressing the lacZ reporter gene present in both shAtaxWT or shGFP (F and L, respectively). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.
Mentions: In animals infected with lentiviral vectors encoding mutant ataxin-3(C) and the non specific shAtaxWT(G) or the shGFP control, immunohistochemical analysis of coronal rat brain sections with anti-ataxin-3 (1H9) and β-galactosidase antibodies showed that many neurons expressed both transgenes (Fig. 4A–C, Fig 5A–L). In animals expressing mutant ataxin-3 and shAtaxMUT(C), far fewer neurons produced the pathogenic protein (Fig. 4E, Fig. 5M). The merged images (Fig. 4F, Fig. 5O) indicate that only a few mutant ataxin-3-positive-cells did not express the lacZ reporter gene present in the shAtaxMUT(C) vector (high magnification Fig. 5R). These cells corresponded to neurons not co-infected with both vectors, which were therefore not treated with the siRNA.

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