Thesis Seminar: Janine Martin
Place: 2117 MERF
Developing RNAi Therapy for DYT1 Dystonia
DYT1 (OMIM) is the most common inherited dystonia, a disabling movement disorder with no effective treatment caused by a dominantly inherited mutation in the protein torsinA. Our group and others have previously demonstrated the therapeutic potential of RNAi for DYT1 dystonia by achieving potent shRNA-mediated silencing of mutant torsinA in cultured cells. In this project we had two main aims: one- to see if we could identify phenotypes that could be utilized to evaluate therapeutic efficacy in preclinical trials and two- to determine whether RNAi therapy could be successfully employed to treat DYT1 murine models. In an effort to identify novel phenotypes we examined whether the presence of mutant torsinA was sufficient to induce changes in transcriptional regulation, miRNA expression or produced an abnormal motor behavior in DYT1 murine models.
During our studies we found that abnormal accumulation of mutant torsinA at the nuclear envelope was not sufficient to induce transcriptional dysregulation in vitro or in vivo, despite its interaction with nuclear envelope proteins. We did find several miRNAs that had significantly altered expression in the cerebellum which may implicate a role for this brain region in DYT1 dystonia. And finally we found no gross motor abnormalities in heterozygous DYT1 knockin (KI) mice or in a novel DYT1 transgenic model indicating that these mice may better embody nonmanifesting DYT1 carriers.
Our preclinical trial to test the feasibility of employing RNAi therapy in DYT1 murine models presented some unexpected results. We found that intrastriatal injections of AAV2/1 vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise. Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA. Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than C57BL/6 mice, perhaps explained by the lower levels of exportin5 expression observed in 129/SvEv mice. In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity. Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.
In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.