Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells

Agnes L Nishimura, Carole Shum, Emma L Scotter, Amr Abdelgany, Valentina Sardone, Jamie Wright, Younbok Lee, Han-Jou Chen, Bilada Bilican, Monica Carrasco, Tom Maniatis, Siddharthan Chandran, Boris Rogelj, Jean-Marc Gallo, Christopher E Shaw

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

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.
Original languageEnglish
Article numbere91269
Number of pages9
JournalPLoS ONE
Volume9
Issue number3
DOIs
Publication statusPublished - 20 Mar 2014

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