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Allele-specific silencing of the dominant disease allele in sialuria by RNA interference

Riko D. Klootwijk^*,1,2, Paul J. M. Savelkoul^*,1, Carla Ciccone^*, Irini Manoli^*, Natasha J. Caplen, Donna M. Krasnewich^*, William A. Gahl^*, and Marjan Huizing^*,3

^* Medical Genetics Branch, National Human Genome Research Institute,

Gene Silencing Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, and

Office of Rare Diseases, Intramural Program, Office of the Director, National Institutes of Health, Bethesda, Maryland, USA.

³Correspondence: Medical Genetics Branch, NHGRI, NIH, 10 Center Dr., MSC 1851, Bethesda, MD 20892, USA. E-mail: mhuizing{at}mail.nih.gov

Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.—Klootwijk, R. D., Savelkoul, P. J. M., Ciccone, C., Manoli, I., Caplen, N. J., Krasnewich, D. M., Gahl, W. A., Huizing, M. Allele-specific silencing of the dominant disease allele in sialuria by RNA interference.

Key Words: real-time PCR • feedback inhibition • sialic acid • UDP-GlcNAc 2-epimerase/ManNAc kinase • siRNA • allosteric site