title: 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.affiliation: Medical Genetics Branch, NHGRI, NIH, 10 Center Dr., MSC 1851, Bethesda, MD 20892, USA. mhuizing@mail.nih.gov.
authors.forename: Riko D
authors.initials: RD
authors.lastname: Klootwijk
authors.forename: Paul J M
authors.initials: PJ
authors.lastname: Savelkoul
authors.forename: Carla
authors.initials: C
authors.lastname: Ciccone
authors.forename: Irini
authors.initials: I
authors.lastname: Manoli
authors.forename: Natasha J
authors.initials: NJ
authors.lastname: Caplen
authors.forename: Donna M
authors.initials: DM
authors.lastname: Krasnewich
authors.forename: William A
authors.initials: WA
authors.lastname: Gahl
authors.forename: Marjan
authors.initials: M
authors.lastname: Huizing
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D002478:Cells, Cultured; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000494:pharmacology*; D005347:Fibroblasts / Q000201:enzymology*; D005799:Genes, Dominant; D006801:Humans; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D029461:Sialic Acid Storage Disease / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics
chemical_list: D009097:Multienzyme Complexes; D034741:RNA, Small Interfering; C482901:UDP-N-acetylglucosamine 2-epimerase - N-acetylmannosamine kinase; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid