Journal Article

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

Amr Abdelgany, Matthew Wood and David Beeson

in Human Molecular Genetics

Volume 12, issue 20, pages 2637-2644
Published in print October 2003 | ISSN: 0964-6906
Published online October 2003 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/ddg280
Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference

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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-α-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of αS226F 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.

Journal Article.  4837 words.  Illustrated.

Subjects: Genetics and Genomics

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