Ji Wan and Pavitra Narasimha will present their work at student seminar in 2-501 BSB on 25th August 2011
Background on Pavitra Narasimha’s talk :
RNAi therapy for Spinocerebellar ataxia 7 (SCA7)
We are investigating RNA interference (RNAi) therapy for the autosomal dominant polyglutamine (polyQ) expansion disorder spinocerebellar ataxia type 7 (SCA7). Normal individuals have ~10 CAG repeats in ATXN7, while an expansion of >37 CAG repeats causes the disease. SCA7 is characterized by Purkinje cell (PC) degeneration and retinal cone-rod dystrophy (>59 CAG repeats). Using our prior experience in developing artificial miRNAs for directed gene silencing (Boudreau et al., 2009), we designed miR-based vectors for silencing mouse and human ataxin-7 (ATXN7). RNAi triggers were designed for allele-specific and non-allele specific silencing (miS1-S4). For allele-specific silencing, artificial miRNAs were designed targeting a SNP (miSNP) common among South African patients. The miR vector candidates and a scrambled control were tested for knockdown of human-ATXN7-92Q (myc-tagged human ATXN7 with a pathogenic repeat of 92 polyQs) in vitro in HEK293 cells. Significant knockdown of human ATXN7 protein was seen with miS4 and miSNP by western blot. To test knockdown of mouse Atxn7, N2A (neuroblastoma) cells were used. MiS4 showed a significant (P<0.05) ~40% knockdown of Atxn7 mRNA, while miSNP vector did not, demonstrating its allele-specificity. The ATXN7 targeting vectors (miR.Sca7S4 and miR.Sca7SNP) are being tested in vivo for SCA7 therapy.
We have successfully transduced cerebellar PCs by stereotactic injection into the wildtype mouse cerebellum using AAV2/1 viral vectors expressing eGFP. Thus we can use AAV2/1 to target cerebellar PCs for SCA7 therapy.To test for transduction of retinal photoreceptors, we performed sub-retinal injections in wildtype mice eyes with AAV2/1 and AAV2/2 viral vectors expressing eGFP. We were able to successfully transduce the photoreceptor cells of the retina, as seen by eGFP expression. To determine the mRNA expression pattern of Atxn7 in the retina, RNA in situ hybridization was initially performed on the mouse wildtype retina. In situ hybridization is now being done on human retinal sections to confirm ATXN7 mRNA expression. These studies will determine the cellular target for SCA7 RNAi therapy in the retina.
Background on Ji Wan’s talk
Analysis of molecular dynamics of HD disease by mRNA and microRNA profiling of BA4 cortex samples