title: Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells.
abstract: TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Agnes L
authors.initials: AL
authors.lastname: Nishimura
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Carole
authors.initials: C
authors.lastname: Shum
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Scotter
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Amr
authors.initials: A
authors.lastname: Abdelgany
authors.affiliation: Department of Public Health, Neuroscience, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Sardone
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Jamie
authors.initials: J
authors.lastname: Wright
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Youn-Bok
authors.initials: YB
authors.lastname: Lee
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Han-Jou
authors.initials: HJ
authors.lastname: Chen
authors.affiliation: MRC Centre for Regenerative Medicine and Centre for Neurodegeneration, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Bilada
authors.initials: B
authors.lastname: Bilican
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York, United States of America.
authors.forename: Monica
authors.initials: M
authors.lastname: Carrasco
authors.affiliation: Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York, United States of America.
authors.forename: Tom
authors.initials: T
authors.lastname: Maniatis
authors.affiliation: MRC Centre for Regenerative Medicine and Centre for Neurodegeneration, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Siddharthan
authors.initials: S
authors.lastname: Chandran
authors.affiliation: Department of Biotechnology, Jozef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Boris
authors.initials: B
authors.lastname: Rogelj
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Gallo
authors.affiliation: Department of Clinical Neuroscience, King's College London, London, United Kingdom.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Shaw
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000473:pathology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D058953:Neural Stem Cells / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering