944. Silencing of Endogenous Human á-Synuclein by a Lentiviral Vector Expressing Short Hairpin RNA

Abstract

Top of pageAbstract Synucleinopathies are a group of neurodegenerative disorders that include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Current experimental gene therapy approaches for these disorders include the use of viral vectors expressing neurotrophic factors, antiapoptotic genes and modulators of neurotransmission. However, recent human kindred and transgenic mouse studies have implicated a key role for human |[alpha]|-synuclein overexpression and toxic accumulation in the pathogenesis of synucleinopathies. RNA interference (RNAi), a recently discovered posttranscriptional gene silencing process has emerged as a powerful technology to 'turn off' or silence genes. Our approach is to develop viral vector-directed RNAi of human |[alpha]|-synuclein as a novel molecular therapeutic tool for synucleinopathies. We have previously reported a 21-nt sequence in the human |[alpha]|-synuclein gene, which constitutes an effective target for robust and selective silencing by RNAi. Following PCR-amplification of RNA-polymerase III H-1 promoter from human genomic DNA, we generated an expression plasmid for H-1 promoter-driven synthesis of short hairpin RNA (shRNA), which targets human |[alpha]|-synuclein for effective silencing. The H-1 promoter-based shRNA expression cassette was cloned into a lentivirus that also harbors a CMV promoter-driven enhanced green fluorescent protein (EGFP) as a marker gene. Infection of the human dopaminergic SH-SY5Y cell line with this lentivirus resulted in EGFP expression in about 89% of cells and approximately a 90% reduction in endogenous |[alpha]|-synuclein protein expression. This lentiviral vector-based siRNA system for highly effective silencing of human |[alpha]|-synuclein gene expression promises to be a novel therapeutic approach for diseases involving |[alpha]|-synuclein pathologies.