Journal of RNAi and Gene Silencing (February 2006), 2(1), 154-160

New Methods and Technologies

Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles

Yusuke Ohnishi †‡, Katsushi Tokunaga ‡, Kiyotoshi Kaneko † and Hirohiko Hohjoh †*

ABSTRACT

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically suppressing the expression of alleles associated with disease. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital, but is also difficult. Here, we show ASP-RNAi against the Swedish- and London-type amyloid precursor protein (APP) variants related to familial Alzheimer’s disease using two reporter alleles encoding the Photinus and Renilla luciferase genes and carrying mutant and wild-type allelic sequences in their 3’-untranslated regions. We examined the effects of siRNA duplexes against the mutant alleles in allele-specific gene silencing and off-target silencing against the wild-type allele under heterozygous conditions, which were generated by cotransfecting the reporter alleles and siRNA duplexes into cultured human cells. Consistently, the siRNA duplexes determined to confer ASP-RNAi also inhibited the expression of the bona fide mutant APP and the production of either amyloid b 40- or 42-peptide in Cos-7 cells expressing both the full-length Swedish- and wild-type APP alleles. The present data suggest that the system with reporter alleles may permit the preclinical assessment of siRNA duplexes conferring ASP-RNAi, and thus contribute to the design and selection of the most suitable of such siRNA duplexes.

KEYWORDS: RNAi, allele-specific gene silencing, amyloid precursor protein, Swedish mutation, London mutation, reporter allele

† National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

‡ Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

*Correspondence to: Hirohiko Hohjoh, Email: hohjohh@ncnp.go.jp, Tel: +81 42 342 2711, ext. 5951 , Fax: +81 42 346 1755

© Copyright Yusuke Ohnishi et al

(Received 06 February 2006; Accepted 13 February 2006)