Title:
Mammalian RNA interference (RNAi) and its application: allele-specific silencing by RNAi

Hirohiko Hohjoh, Ph.D.
Section Chief
Department of Molecular Genetics, National Institute of Neuroscience, National Center of Neurology and Psychiatry

Abstract:
RNA interference (RNAi) is a powerful tool for suppressing the expression of a gene of interest, and its application is expanding to various fields of science. Allele-specific silencing by RNAi (allele-specific RNAi: ASP-RNAi) is an advanced application of RNAi technique, by which the expression of an allele of interest can be suppressed without inhibiting the expression of its corresponding allele. Thus, ASP-RNAi would be therapeutically useful, as it can specifically inhibit the expression of disease-causing alleles without suppressing the expression of corresponding wild-type alleles. To achieve such ASP-RNAi, it is vital to design and select small interfering RNAs (siRNAs) capable of allele discrimination; but, it is quite difficult. We have established an assay system using reporter alleles encoding the Photinus and Renilla luciferase genes to assess siRNAs conferring ASP-RNAi. The system allows for determination of competent siRNAs triggering allele-specific silencing against various disease-causing alleles. In our recent study, we have successfully designed siRNAs conferring allele-specific silencing against Huntington disease (a triplet repeat disease) -causing alleles [mutant Huntingtin (HTT) alleles] by using single nucleotide polymorphisms (SNPs) linked with the pathogenic aberrantly expanded CAG trinucleotide repeats as new ASP-RNAi targets. I would like to show and tell those data together with a new SNP typing procedure (pull-down method) for identification of SNP-haplotypes of the disease-causing, mutant HTT alleles.

References:
1. Takahashi M., Watanabe S., Murata M., Furuya H., Kanazawa I. , Wada K., and Hohjoh H. (2010) Tailor-made RNAi knockdown against triplet repeat disease-causing alleles. Proc Natl Acad Sci USA ., 107 : 21731-21736.

2. Hohjoh H. (2010) Allele-specific silencing by RNA interference. In RNA interference. Methods Mol. Biol. , 623 : 67-79.

3. Ohnishi Y., Tamura Y., Yoshida M., Tokunaga K., and Hohjoh H. (2008) Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi. PLoS ONE 3 (5): e2248.

4. Ohnishi Y., Tokunaga K., Kaneko K., and Hohjoh H. (2006) Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles. J. RNAi Gene silencing ,2 : 154-160.