Patent ID: 9650684
Date: 2017-05-16
CPC Classifications: C12Q

Claim:
1. A method of identifying a candidate general or alternative splicing inhibitor of RNA splicing comprising a two step assay, said method comprising the steps of: a) a first screening step comprising assessing the effect of a candidate general or alternative splicing inhibitor on expression, function, or activity of a first reporter construct expressed by a first recombinant mammalian cell by exposing said candidate general or alternative splicing inhibitor to said first recombinant cell for less than 4 hours, said first reporter construct comprising an open reading frame (ORF) interrupted by a chimeric β-globin intron, wherein the chimeric β-globin intron comprises multiple internal stop codons, wherein protein translated from unspliced transcripts is truncated, further wherein said first reporter construct comprises a protein and RNA destabilizing sequence and has a half-life of less than 4 hours, wherein the candidate alternative splice inhibitor is selected for a subsequent counter screen when the candidate inhibits the expression, activity, or function of the reporter protein relative to control; b) after the first screening step, a second counter-screening step comprising assessing the effect of the selected candidate inhibitor from the first screen on the expression, function, or activity of a second reporter construct expressed by a second recombinant mammalian cell by exposing the selected candidate inhibitor from the first screen to said second recombinant cell for less than 4 hours, said second reporter construct comprising an intronless ORF, further wherein said second reporter construct comprises a protein and RNA destabilizing sequence and has a half-life of less than 4 hours, wherein when the selected candidate inhibitor has a different effect on the expression, function, or activity of the intronless reporter construct of the first screening step as compared to the intron containing reporter construct, then the selected candidate compound is identified as an inhibitor of constitutive or alternative splicing.