IDENTIFICATION OF SMALL MOLECULES THAT FACILITATE THERAPEUTIC EXON SKIPPING

• US 20160220538A1
• Filed: 03/30/2016
• Published: 08/04/2016
• Est. Priority Date: 08/30/2011
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Miceli, M. Carrie, Nelson, Stanley F., Moran, Miriana
• Granted Patent: US 10,188,633 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/122 having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
• CPC Class Codes: A61K 31/138 Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine atenolol A61K31/165; pindolol A61K31/404; timolol A61K31/5377
• CPC Class Codes: A61K 31/166 having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
• CPC Class Codes: A61K 31/277 having a ring, e.g. verapamil
• CPC Class Codes: A61K 31/4025 not condensed and containing further heterocyclic rings, e.g. cromakalim
• CPC Class Codes: A61K 31/404 Indoles, e.g. pindolol
• CPC Class Codes: A61K 31/407 condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
• CPC Class Codes: A61K 31/4178 not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
• CPC Class Codes: A61K 31/4184 condensed with carbocyclic rings, e.g. benzimidazoles
• CPC Class Codes: A61K 31/454 containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• CPC Class Codes: A61K 31/475 having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine vincamine A61K31/4375
• CPC Class Codes: A61K 31/5377 not condensed and containing further heterocyclic rings, e.g. timolol
• CPC Class Codes: A61K 31/5415 ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
• CPC Class Codes: A61K 31/549 having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
• CPC Class Codes: A61K 31/554 having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
• CPC Class Codes: A61K 31/609 Amides, e.g. salicylamide labetalol, metoclopramide A61K31/166
• CPC Class Codes: A61K 31/7064 containing condensed or non-condensed pyrimidines
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 : Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 : Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: C12N 15/113 : Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/113 : targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: C12N 2310/3233 : Morpholino-type ring
• CPC Class Codes: C12N 2320/31 : Combination therapy
• CPC Class Codes: C12N 2320/33 : Alteration of splicing
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6897 : involving reporter genes operably linked to promoters

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.

Claims

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 109203X, 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.

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, wherein the method is carried out in vitro.

7. The method of claim 1, wherein the method 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 109203X, 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 furaltadone hydrochloride, 5-iodotubericidin, bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220, dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF 109203X, halofantrine, niclosamide, pimozide, reserpine, syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid, or menadione.

13. The method of claim 12, wherein the small molecule candidates are 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 109203X, 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 109203X, 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. -18. (canceled)