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

Abstract

DYT1 is the most common inherited dystonia. Currently, there are no preventive or curative therapies for this dominantly inherited disease. DYT1 dystonia is caused by a common three-nucleotide deletion in the TOR1A gene that eliminates a glutamic acid residue from the protein torsinA. Recent studies suggest that torsinA carrying the disease-linked mutation, torsinA(⌬E) acts through a dominantnegative effect by recruiting wild-type torsinA [torsinA(wt)] into oligomeric structures in the nuclear envelope. Therefore, suppressing torsinA(⌬E) expression through RNA interference (RNAi) could restore the normal function of torsinA(wt), representing a potentially effective therapy regardless of the biological role of torsinA. Here, we have generated short hairpin RNAs (shRNAs) that mediate allele-specific suppression of torsinA(⌬E) and rescue cells from its dominant-negative effect, restoring the normal distribution of torsinA(wt). In addition, delivery of this shRNA by a recombinant feline immunodeficiency virus effectively silenced torsinA(⌬E) in a neural model of the disease. We further establish the feasibility of this viral-mediated RNAi approach by demonstrating significant suppression of endogenous torsinA in mammalian neurons. Finally, this silencing of torsinA is achieved without triggering an interferon response. These results support the potential use of viral-mediated RNAi as a therapy for DYT1 dystonia and establish the basis for preclinical testing in animal models of the disease.

Recent Advances in RNA Interference Therapeutics for CNS Diseases

“…Both viruses are minimally immunogenic and can transduce a number of CNS cell types. Recombinant lentiviruses are pseudotyped with various glycoproteins that can impart different tropisms after directed delivery into brain [47,48], and they have been used successfully in gain-of-function [49] and loss of function studies [50][51][52][53].One difference between lentivirus and AAV or adenovirus-based systems is the level of expression.This is due, in part, because LV-mediated transduction often results in low copy numbers of transgene/cell. Also, the placement of the expression cassette in the LV genome can affect expression levels [54].Most LV vectors integrate unless the integrase activity has been inactivated.As integrase-deficient vectors often have low titers compared with their integrase competent counterparts, their production for use for therapeutic applications is impractical.Integration competency for CNS applications may be less of an issue than in the setting of stem cell transduction (most cells in the CNS are not dividing), where integration and activation of an oncogenic gene provides a growth advantage for the transformed cell [55,56].…”

“…[21,53] SBMA CELF2 AAV9 Overexpression of naturally occurring miR-196a indirectly enhances decay of androgen receptor through silencing of CELF2 in vivo.…”

“…TorsinA is an ATPase associated with diverse cellular activities [136], whose mutant form (TorsinA(ΔE)) is thought to have a dominant negative effect [137,138]. Allele-specific TorsinA(ΔE) suppression was first achieved by shRNA delivery in vitro [53]. However, when AAV expressing the shRNAs were delivered into the striatum of transgenic DYT1 mice, the shRNAs induced lethal neurotoxicity [21].…”

Therapy for prion diseases: Insights from the use of RNA interference

“…A single treatment with focal injection of virus resulted in significantly prolonged survival time of treated 'spread' of tau or Alzheimer pathology in human brains over time and brain region is well recognized. 66 Stopping protein propagation in its tracks with localized treatments may be an option worth considering in other human neurodegenerative proteinopathies.…”

Experimental Therapeutics for Dystonia

“…One potential genetic approach involves the design of small interfering RNAs that target the product of the mutant allele to increase the proportion of normal RNAs. 42 Biochemical strategies could involve augmenting the functions of the normal protein product or blocking the functions of the mutant protein. For example, overexpression of normal torsinA may block the formation of deleterious protein aggregates associated with ␣-synuclein or polyglutamine-containing proteins.…”