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

Abstract

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.

• Authors: Riko D Klootwijk, Paul J M Savelkoul, Carla Ciccone, Irini Manoli, Natasha J Caplen, Donna M Krasnewich, William A Gahl, Marjan Huizing
• Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology (FASEB J) Vol. 22 Issue 11 Pg. 3846-52 (Nov 2008) ISSN: 1530-6860 [Electronic] United States
• PMID: 18653764 (Publication Type: Journal Article, Research Support, N.I.H., Intramural)

Chemical References

• Multienzyme Complexes
• RNA, Small Interfering
• UDP-N-acetylglucosamine 2-epimerase - N-acetylmannosamine kinase
• Cytidine Monophosphate N-Acetylneuraminic Acid

Topics

• Alleles
• Amino Acid Substitution
• Cells, Cultured
• Cytidine Monophosphate N-Acetylneuraminic Acid (pharmacology)
• Fibroblasts (enzymology)
• Genes, Dominant
• Humans
• Multienzyme Complexes (antagonists & inhibitors, genetics, metabolism)
• Mutation, Missense
• RNA Interference
• RNA, Small Interfering (pharmacology)
• Sialic Acid Storage Disease (drug therapy, enzymology, genetics)