Toward therapy for DYT1 dystonia: Allele‐specific silencing of mutant TorsinA

Abstract

A three-nucleotide (GAG) deletion in the TOR1A gene is the most common cause of inherited dystonia, DYT1. Because the mutant protein, TorsinA (TA), is thought to act in a dominant manner to cause disease, inhibiting expression from the mutant gene represents a potentially powerful therapeutic strategy. In an effort to develop therapy for this disease, we tested whether small interfering RNA (siRNA) could selectively silence expression of mutant TA. Exploiting the three-base pair difference between wild-type and mutant alleles, we designed siRNAs to silence expression of mutant, wild-type, or both forms of TA. In transfected cells, siRNA successfully suppressed wild-type or mutant TA in an allele-specific manner: for example, mutant-specific siRNA reduced the levels of mutant TA to less than 1% of controls with minimal effect on wild-type TA expression. In cells expressing both alleles, thus simulating the heterozygous state, siRNA-mediated suppression remained robust and allele specific. Our siRNA studies demonstrate allele-specific targeting of a dominant neurogenetic disease gene and suggest the broad therapeutic potential of siRNA for DYT1 dystonia and other dominantly inherited neurological diseases.

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

“…The potency and selectivity of this approach has recently been documented for several dominant disease genes (27,28), including SOD1. Our findings confirm and extend the recent report of Ding et al (29), which described RNAi-mediated, allele-specific silencing of the SOD1 G85R and SOD1 G93A alleles.…”

“…In recent months, several studies have reported RNAi-mediated inactivation of dominant disease alleles differing from their wild-type counterparts by a single nucleotide (27,28), and one report demonstrated specific inhibition of mutant SOD1 alleles (29). The present study confirms and extends these observations and identifies previously uncharacterized siRNA sequences for selective silencing of FALS-linked mutant SOD1.…”

Silencing Primary Dystonia: Lentiviral-Mediated RNA Interference Therapy for DYT1 Dystonia

“…The various torsinA expression plasmids used for this study have been described previously (Gonzalez-Alegre et al, 2003). To generate the U6shRNA constructs, we used a PCR-based approach described previously (Harper et al, 2005), with primers designed to generate the different hairpins shown in Figure 1: five hairpins targeting torsinA(⌬E) (U6shTAmut1-5) and three control hairpins, one targeting torsinA(wt) (U6shTAwt), one targeting a sequence common for both for human and murine torsinA (U6shTAcom), and a mismatched sequence not targeting either allele as shown previously (U6shTAmis) (Gonzalez-Alegre et al, 2003). The PCR product was ligated into the pCR2.1 vector using the TOPO-TA Cloning kit (Invitrogen, San Diego, CA) and the product verified by sequencing.…”

“…The PCR product was then inserted into the multiple cloning site of pcDNA3.1 (Invitrogen). Additional plasmids included GFP-Q19 (GFP fused to a stretch of 19 glutamines), torsinA(wt)-GFP (a kind gift from Dr. P. Hanson, Washington University, St. Louis, MO) (Naismith et al, 2004), and HA-torsinA(⌬E) (Gonzalez-Alegre et al, 2003). Feline immunodeficiency virus (FIV) pseudotyped with VSV-G (vesicular stomatitis virus G) were generated by the Gene Transfer Vector Core of the University of Iowa (www.uiowa.edu/ϳgene/) as described previously (Brooks et al, 2002).…”

“…First, because DYT1 likely results from a dominant-negative effect, a reduction in torsinA(⌬E) would restore the normal function of torsinA(wt). Second, using in vitrosynthesized short interfering RNA (siRNA), we have previously demonstrated that the 3 nt difference between wt and mutant alleles is sufficient to permit specific silencing of the mutant gene (Gonzalez-Alegre et al, 2003). Third, because DYT1 dystonia is caused by a single common mutation, this therapeutic approach would be applicable to all patients.…”