Human Molecular Genetics Advance Access originally published online on February 7, 2008
Human Molecular Genetics 2008 17(10):1436-1445; doi:10.1093/hmg/ddn032
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siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells


1 Molecular Neurogenetics Unit, Department of Neurology 2 Center for Molecular Imaging Research 3 Department of Radiology, Massachusetts General Hospital 4 Program in Neuroscience, Harvard Medical School, Boston, MA 02114, USA 5 Alnylam Pharmaceuticals, Cambridge, MA 02142, USA
* To whom correspondence should be addressed at: Molecular Neurogenetics Unit, Massachusetts General Hospital-East, 13th Street, Building 149, Charlestown, MA 02129, USA. Tel: +1 6177265728; Fax: +1 6177241537; Email: breakefield{at}hms.harvard.edu
Received December 6, 2007; Accepted January 29, 2008
Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinA
E). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinA
E when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinA
E mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinA
E message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinA
E acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinA
E to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.
These authors are considered as co-second authors.