Diseases caused by dominant, gain-of-function mutations develop in people bearing one mutant and one wild-type copy of the gene. Some of the best known examples of this class are neurodegenerative diseases, including Huntington's, a subset of amyotrophic lateral sclerosis (ALS), Alzheimer's and Parkinson's diseases. In all these diseases, the exact pathways whereby the mutant proteins cause cell degeneration are not entirely clear, but the origin of the cellular toxicity is known to be the mutant protein. Thus, selectively lowering or eliminating the mutant protein is a key step in developing effective therapies. Until recently, it was not clear how specific down-regulation of a wide variety of mutant proteins could be achieved. But now, new advances in RNA interference (RNAi) raise the possibility that RNAi can be developed and eventually applied as a therapeutic means for these neurodegenerative diseases. RNAi can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing short RNA duplexes (called small interfering RNAs or siRNAs) with sequence homologies to the target gene. Recent experiments indicate that small hairpin RNAs (shRNAs) transcribed in vivo can trigger degradation of corresponding mRNAs similar to siRNA. These developments raise the possibility that siRNA duplexes or vectors expressing shRNAs may be used to block the expression of a toxic mutant gene. This proposal investigates in vivo efficacy of RNAi therapy using transgenic technology in a mouse model for ALS that is caused by mutations in Cu, Zn superoxide dismutase (SOD1). To determine the potential of RNAi therapy, we will express shRNAs targeting specifically the mutant mRNAs in transgenic mice. We will test how effective and how specific these shRNAs are in suppressing the mutant protein expression and alleviating the disease. To determine in which cell types the suppression of the mutant expression is most crucial, we will express shRNAs in selected cell types using Cre-lox recombination system, We will determine in which cell type suppression of mutant SOD1 expression has the largest impact in alleviating disease. To determine the optimal time for therapy, we will use the Tamoxifeninducible Cre recombinant system to determine at what stage of the disease induction of shRNA to suppress mutant SOD1expresion is most effective.
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