Methods and means for efficient skipping of exon 45 in duchenne muscular dystrophy pre-mRNA

• US 9,528,109 B2
• Filed: 11/14/2014
• Issued: 12/27/2016
• Est. Priority Date: 10/26/2007
• Status: Active Grant

• Current Assignee: Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.
• Original Assignee: Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.
• Inventors: De Kimpe, Josephus Johannes, Rus, Adriana Marie, Platenburg, Gerard Johannes, Van Deutekom, Judith Christina Theodora, Van Ommen, Garrit-Jan Boudewijn
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US14/542,183
• Publication Number:
  US 20150073037A1
• Time in Patent Office: 774 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 31/56 Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
• CPC Class Codes: A61K 31/57 substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
• CPC Class Codes: A61K 31/573 substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• CPC Class Codes: A61K 31/58 containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin digitoxin A61K31/7048
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 38/1719 Muscle proteins, e.g. myosin or actin
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/14 for calcium homeostasis vitamin D A61P3/02; parathyroid hormones A61P5/18; calcitonin A61P5/22; osteoporosis A61P19/10; bone metastasis A61P35/04
• CPC Class Codes: A61P 39/06 Free radical scavengers or antioxidants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/31 : of the backbone
• CPC Class Codes: C12N 2310/313 : Phosphorodithioates
• CPC Class Codes: C12N 2310/314 : Phosphoramidates
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/3181 : Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 : having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3233 : Morpholino-type ring
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2320/31 : Combination therapy
• CPC Class Codes: C12N 2320/33 : Alteration of splicing

Abstract

The invention relates to a method for inducing or promoting skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular Dystrophy patient, preferably in an isolated (muscle) cell, the method comprising providing an isolated muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within the exon. The invention further relates to such molecule used in the method.

Claims

1. An antisense oligonucleotide whose base sequence consists of the base sequence of 5 ′

UUUGCCGCUGCCCAAUGCCAUCCUG-3′

(SEQ ID;

NO;

     3), said oligonucleotide comprising a modification.

2. The oligonucleotide of claim 1, comprising a phosphorodiamidate morpholino oligomer (PMO).

3. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a locked nucleic acid (LNA).

4. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified base.

5. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified sugar moiety.

6. The antisense oligonucleotide of claim 1 wherein the oligonucleotide comprises a modified internucleoside linkage.

7. The oligonucleotide of claim 1 wherein said oligonucleotide comprises a phosphorothioate internucleoside linkage and a 2′

-O-alkyl substituted ribose moiety.

8. The oligonucleotide of claim 5, wherein the modified sugar moiety is selected from the group consisting of:

a ribose that is mono- or di-substituted at the 2′

, 3′

, and/or 5′

position.

9. The oligonucleotide of claim 8, wherein the ribose is a 2′

-O-substituted ribose.

10. The oligonucleotide of claim 9, wherein the ribose is a 2′

-O-methyl ribose.

11. The oligonucleotide of claim 6, said oligonucleotide comprising a modified backbone such that all of internucleoside linkages of said oligonucleotide are modified.

12. The oligonucleotide of claim 11, said internucleoside linkages comprising phosphorothioate.

13. The oligonucleotide of claim 11, said internucleoside linkages comprising a phosphorodiamidate morpholino oligomer (PMO).

14. The oligonucleotide of claim 1 said modification comprising a peptide nucleic acid, and/or locked nucleic acid.

15. The oligonucleotide of claim 11, wherein said backbone is selected from the group consisting of a morpholino backbone, a carbamate backbone, a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl backbone, a thioformacetyl backbone, a methyleneformacetyl backbone, a riboacetyl backbone, an alkene containing backbone, a sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a methyleneimino backbone, a methylenehydrazino backbone and an amide backbone.

16. The antisense oligonucleotide of claim 1 wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 50%.

17. The antisense oligonucleotide of claim 16, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 60%.

18. The antisense oligonucleotide of claim 17, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 70%.

19. The antisense oligonucleotide of claim 18, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 80%.

20. The antisense oligonucleotide of claim 19, wherein said oligonucleotide is capable of inducing skipping of exon 45 by at least 90%.

21. A viral-based vector comprising an expression cassette comprising a nucleotide sequence encoding the oligonucleotide of claim 1.

22. A pharmaceutical composition comprising the oligonucleotide of claim 1, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 1, further comprising an antisense oligonucleotide which induces or promotes skipping of exon 7, 44, 46, 51, 53, 59, or 67 of dystrophin pre-mRNA of a patient.