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Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.
Pappadà, Mariangela; Bonuccelli, Ottavia; Buratto, Mattia; Fontana, Riccardo; Sicurella, Mariaconcetta; Caproni, Anna; Fuselli, Silvia; Benazzo, Andrea; Bertorelli, Roberto; De Sanctis, Veronica; Cavallerio, Paolo; Simioni, Valentina; Tugnoli, Valeria; Salvatori, Francesca; Marconi, Peggy.
Afiliação
  • Pappadà M; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
  • Bonuccelli O; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
  • Buratto M; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
  • Fontana R; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
  • Sicurella M; Department of Life Sciences and Biotechnology, University of Ferrara, via L. Borsari 46, 44121, Ferrara, Italy.
  • Caproni A; Department of Environmental Sciences and Prevention, University of Ferrara, via L. Borsari 46, 44121, Ferrara, Italy.
  • Fuselli S; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
  • Benazzo A; Department of Life Sciences and Biotechnology, University of Ferrara, via L. Borsari 46, 44121, Ferrara, Italy.
  • Bertorelli R; Department of Life Sciences and Biotechnology, University of Ferrara, via L. Borsari 46, 44121, Ferrara, Italy.
  • De Sanctis V; Next Generation Sequencing Core Facility, Department of Cellular, Computational and Integrative Biology-CIBIO, University of Trento, via Sommarive 9, 38123, Povo, Trento, Italy.
  • Cavallerio P; Next Generation Sequencing Core Facility, Department of Cellular, Computational and Integrative Biology-CIBIO, University of Trento, via Sommarive 9, 38123, Povo, Trento, Italy.
  • Simioni V; Next Generation Sequencing Core Facility, Department of Cellular, Computational and Integrative Biology-CIBIO, University of Trento, via Sommarive 9, 38123, Povo, Trento, Italy.
  • Tugnoli V; Division of Neurology, Department of Neuroscience and Rehabilitation, University Hospital of Ferrara, via A. Moro 8, 44100, Ferrara, Cona, Italy.
  • Salvatori F; Division of Neurology, Department of Neuroscience and Rehabilitation, University Hospital of Ferrara, via A. Moro 8, 44100, Ferrara, Cona, Italy.
  • Marconi P; Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy. francesca.salvatori@unife.it.
Sci Rep ; 12(1): 20285, 2022 11 24.
Article em En | MEDLINE | ID: mdl-36434031
ABSTRACT
SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Ataxias Espinocerebelares Limite: Animals País como assunto: Europa Idioma: En Ano de publicação: 2022 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Ataxias Espinocerebelares Limite: Animals País como assunto: Europa Idioma: En Ano de publicação: 2022 Tipo de documento: Article