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Allele‐specific silencing of the dominant disease allele in sialuria by RNA interference
Author(s) -
Klootwijk Riko D.,
Savelkoul Paul J. M.,
Ciccone Carla,
Manoli Irini,
Caplen Natasha J.,
Krasnewich Donna M.,
Gahl William A.,
Huizing Marjan
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.08-110890
Subject(s) - gene silencing , allele , sialic acid , mutant , small interfering rna , biology , rna interference , gene , genetics , microbiology and biotechnology , rna
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. FASEB J. 22, 3846–3852 (2008)