Identification of small molecules that facilitate therapeutic exon skipping

Abstract

This invention relates, e.g., to a method for enhancing exon skipping in a pre-mRNA of interest, comprising contacting the pre-mRNA with an effective amount of a small molecule selected from the compounds shown in Table 1, or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof, and, optionally, with an antisense oligonucleotide that is specific for a splicing sequence in the pre-mRNA Methods for treating Duchenne muscular dystrophy (DMD) are disclosed.

1 . A method for enhancing exon skipping in an mRNA of interest, comprising contacting the mRNA with an effective amount of a small molecule compound selected from one or more of furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, a RyCal, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,
 or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof. 
 
     
     
         2 . The method of  claim 1 , wherein an antisense oligonucleotide (AO) which is specific for a splicing sequence in the mRNA is administered in conjunction with the compound. 
     
     
         3 . The method of  claim 1 , wherein no AO is introduced in conjunction with the compound. 
     
     
         4 . The method of  claim 1 , wherein the mRNA is from the muscle dystrophin (DMD) gene, which encodes a muscle dystrophin protein. 
     
     
         5 . The method of  claim 4 , wherein the exon which is skipped is exon 23, 44, 45, 50, 51, 52 and/or 53 of the DMD gene. 
     
     
         6 . The method of  claim 1 , which is carried out in vitro. 
     
     
         7 . The method of  claim 1 , which is carried out in a subject that has Duchenne Muscular Dystrophy (DMD), is an animal model of DMD, or is another animal in which the exon skipping can be assayed. 
     
     
         8 . The method of  claim 7 , wherein the subject is human. 
     
     
         9 . The method of  claim 1 , wherein the compound is dantrolene or an active variant thereof. 
     
     
         10 . The method of  claim 9 , wherein the compound is dantrolene. 
     
     
         11 . A method for treating a subject that has Duchenne Muscular Dystrophy (DMD), or is a non-human model of DMD, comprising administering to the subject an effective amount of a small molecule compound selected from one or more of furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,
 or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof, 
 in conjunction with an AO which is specific for a splicing sequence of exon 23, 45, 44, 50, 51, 52 and/or 53 of the DMD gene. 
 
     
     
         12 . A method for identifying a small molecule compound that enhances exon skipping in an mRNA of interest, comprising testing small molecule candidates for their ability to enhance exon skipping in the mRNA, and selecting compounds which exhibit greater enhancement of exon skipping than one of the molecules in Table 1. 
     
     
         13 . The method of  claim 12 , wherein the small molecule candidates tested in conjunction with an AO specific for a splicing sequence of the exon that is to be skipped. 
     
     
         14 . The method of  claim 12 , wherein the small molecule candidate is a variant of furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,
 or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof. 
 
     
     
         15 . The method of  claim 13 , wherein the small molecule candidate is a variant of furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,
 or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof. 
 
     
     
         16 . A combination for enhancing exon skipping in an mRNA of interest, comprising
 a small molecule compound selected from one or more of furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,   or a a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof,   and an AO that is specific for an exon that is to be skipped,   and, optionally, a pharmaceutically acceptable carrier.   
     
     
         17 . A kit for enhancing exon skipping in an mRNA of interest, comprising a compound from Table 1, or a a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof, and an AO that is specific for an exon splicing sequence in the mRNA of interest, wherein the compound and the AO are optionally packaged in containers, separately or together. 
     
     
         18 . A kit for enhancing exon skipping in a muscle dystrophin mRNA in a subject that has Duchenne Muscular Dystrophy (DMD), or is an animal model of DMD, comprising a dosage form of a compound of Table 1, or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof, and of an AO that is specific for the exon which is to be skipped, and a pharmaceutically acceptable carrier, wherein the compound and the AO are optionally packaged in containers, separately or together,
 wherein the compound of Table 1 is one or more of Furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203×, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione,   or a pharmaceutically acceptable salt, hydrate, solvate, or isomer thereof.   
     
     
         19 . The method of  claim 1 , wherein the RyCal is Ryanodine or RyCal S 107.