METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA

• US 20170107512A1
• Filed: 12/27/2016
• Published: 04/20/2017
• Est. Priority Date: 10/26/2007
• Status: Abandoned Application

• Current Assignee: Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.
• Original Assignee: Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.
• Inventors: RUS, Adriana Marie, De KIMPE, Josephus Johannes, PLATENBURG, Gerard Johannes, Van DEUTEKOM, Judith Christina Theodora, Van OMMEN, Garrit-Jan Boudewijn
• Application Number:
  US15/390,836
• Publication Number:
  US 20170107512A1
• Time in Patent Office: Days
• 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 isolate muscle cell with a molecule that binds to a continuous stretch of at least 21 nucleotides within said exon. The invention further relates to such molecule used in the method.

Claims

1. An antisense oligonucleotide of 25-29 nucleotides in length, comprising the base sequence of 5′

-UUUGCCGCUGCCCAAUGCCAUCCUG-3′

(SEQ ID;

NO;

     3) or a DNA oligonucleotide thereof, said oligonucleotide comprising a modification and is complementary along its entire length to a sequence within exon 45 of a human dystrophin pre-mRNA, said oligonucleotide being capable of inducing skipping of exon 45 by at least 50%.

2. The antisense oligonucleotide of claim 1, said oligonucleotide consisting of said base sequence or consisting of the DNA sequence thereof.

3. The antisense oligonucleotide of claim 1 wherein said modification comprises a modified sugar, a modified base and/or a modified a modified internucleoside linkage.

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

-O-alkyl substituted ribose moiety.

5. The antisense oligonucleotide of claim 3, 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.

6. The antisense oligonucleotide of claim 5, wherein the ribose is a 2′

-O-substituted ribose.

7. The antisense oligonucleotide of claim 6, wherein the ribose is a 2′

-O-methyl ribose.

8. The oligonucleotide of claim 3, wherein all internucleoside linkages of said oligonucleotide are modified.

9. The oligonucleotide of claim 8, said internucleoside linkages comprising phosphorothioate.

10. The oligonucleotide of claim 8, said internucleoside linkages comprising a phosphorodiamidate morpholino oligomer (PMO).

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

12. The oligonucleotide of claim 8, wherein said linkages are 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.

13. The oligonucleotide of claim 1, wherein said oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

14. A method for directing splicing of a Duchenne Muscular Dystrophy (DMD) gene pre-mRNA in muscle cells of a DMD patient to direct exclusion of exon 45 during splicing of said DMD gene pre-mRNA to a DMD gene mRNA, the method comprising:

providing said cells with an isolated antisense RNA or DNA oligonucleotide 25-29 nucleotides in length whose sequence is fully complementary to exon 45 of a DMD gene pre-mRNA, said RNA oligonucleotide comprising the base sequence 5′

-UUUGCCGCUGCCCAAUGCCAUCCUG-3′

(SEQ ID;

NO;

     3) and said DNA oligonucleotide comprising the DNA base sequence thereof, said oligonucleotide comprising a modification, such that said antisense oligonucleotide functions to produce an in-frame DMD gene mRNA lacking exon 45, thereby directing exclusion of exon 45 during splicing.

15. The method of claim 14, wherein said mRNA encodes a functional shortened dystrophin protein.

16. The method of claim 15, wherein expression of said shortened dystrophin protein is detected at the muscle cell membrane by immunohistochemical and/or western blot analysis

17. The method of claim 14, wherein said RNA or DNA oligonucleotide consists of the nucleotide base sequence 5′

-UUUGCCGCUGCCCAAUGCCAUCCUG 3′

(SEQ ID NO;

     3) or the DNA sequence thereof, respectively.

19. The method of claim 14, wherein said modification is selected from the group consisting of:

a modified base, a modified sugar, a non-natural internucleoside linkage, and a modified backbone.

20. The method of claim 19, wherein the modified 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.

21. The method of claim 14, wherein said oligonucleotide is a phosphorothioate oligonucleotide.

22. The method of claim 14, wherein said oligonucleotide is a 2′

-O-methyl phosphorothioate oligonucleotide.

23. The method of claim 14, wherein said oligonucleotide is a phosphorodiamidate oligonucleotideage.

24. The method of claim 14, wherein said modification is selected from a group consisting of 2′

-O-Methyl phosphorothioate, phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid, and locked nucleic acid.