Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference.

Journal article

Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders.

Authors: Abdelgany, A; Wood, M; Beeson, D

• Publication status: Published
• Journal: Human molecular genetics
• Volume: 12
• Issue: 20
• Pages: 2637-2644
• Publication date: 2003-10-01
• DOI: 10.1093/hmg/ddg280
• EISSN: 1460-2083
• ISSN: 0964-6906
• Language: English
• Keywords: RNA Interference; Synapses; Down-Regulation; Molecular Sequence Data; DNA; Base Pair Mismatch; Microscopy, Fluorescence; Receptors, Cholinergic; Alleles; RNA, Small Interfering; Cell Line; Transcription, Genetic; Base Sequence; Oligonucleotides; Humans; Gene Silencing; RNA; Animals; Mutation; Promoter Regions, Genetic; Transfection