Research Grant Award

Luis Pereira de Almeida, PD, MSc, PhD

Center for Neurosciences of Coimbra, University of Coimbra
Lentiviral vector-based silencing of ataxia-3

Spinocerebellar ataxia 3 or Machado-Joseph disease (SCA3/MJD) is a genetic neurodegenerative disorder originally described in people of Portuguese descent, and now established as the most common of the dominantly inherited ataxias worldwide. The disease is associated with the expansion of a (CAG) tract within the coding region of the causative gene-MJD1. The abnormal over-repetition of the CAG trinucleotide is translated into an expanded polyglutamine (poly Q) tract within the protein ataxin-3 (ATX3), resulting in severe clinical features and leading inevitably to death. No treatment is available. Although A TX3 has been recently established as a poly-ubiquitin-binding protein, with ubiquitin protease activity its function remains largely unknown, partly because there are no reports of a knock out animal. In this project we intend to develop lentiviral vectors (LV) for silencing the endogenous MJD1 gene to gain insights into the role of ATX3 in cells and assess the importance of ATX3 expression in the adult animal.

We previously demonstrated that LV is particularly suited for gene therapy of the GNS as shown by efficient transduction of neurons in the rat brain and long-term expression of transgenes. LV have already been used to model a poly Q disorder in the rat (Huntington's disease), to express a therapeutic protein or to down-regulate a reporter gene expression. In the present proposal, we will extend the use of L V for gene silencing of endogenous ataxin-3. For this purpose, LV encoding double stranded RNAs (ds RNAs) complementary to the MJD1 rat gene will be developed. By inserting their genes into the genome of neurons, transduction mediated by LV will lead to long-term expression of ds RNAs and permanent inhibition of the expression of ATX3 through a mechanism of RNA interference. The effects resulting from endogenous ATX3 suppression will be evaluated following transduction of cell lines with L V targeting the ATX3 mRNA. These studies will then be transposed to the rat brain, where injection of LV encoding the silencing ds RNAs will allow an evaluation, in the adult animal, of potential toxic effects resulting from ATX3 silencing. This study is important to select the best strategy for therapy of MJD, particularly to evaluate the need for selective inhibition of mutant ATX3 in therapeutic approaches based on RNA interference.