US20110263682A1 - Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 - Google Patents
Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 Download PDFInfo
- Publication number
- US20110263682A1 US20110263682A1 US13/094,571 US201113094571A US2011263682A1 US 20110263682 A1 US20110263682 A1 US 20110263682A1 US 201113094571 A US201113094571 A US 201113094571A US 2011263682 A1 US2011263682 A1 US 2011263682A1
- Authority
- US
- United States
- Prior art keywords
- seq
- exon
- skipping
- molecule
- patient
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
- A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1719—Muscle proteins, e.g. myosin or actin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
- A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
- C12N2310/111—Antisense spanning the whole gene, or a large part of it
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/313—Phosphorodithioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/314—Phosphoramidates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N2310/3181—Peptide nucleic acid, PNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3233—Morpholino-type ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/31—Combination therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/33—Alteration of splicing
Definitions
- Myopathies are disorders that result in functional impairment of muscles.
- Muscular dystrophy refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles.
- Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
- DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction.
- DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies.
- DMD mutations in the DMD gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
- DMD Duchenne muscular dystrophy
- AONs antisense oligonucleotides interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol. Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet. 2003; 12(8):907-14.).
- In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23.
- Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat. Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle.
- In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol. Chem. 2008; 283: 5899-907).
- the present invention provides a method for inducing, and/or promoting skipping of at least one of exons 43, 46, 50-53 of the DMD pre-mRNA in a patient, preferably in an isolated cell of a patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon. It is to be understood that said method encompasses an in vitro, in vivo or ex vivo method.
- a method for inducing and/or promoting skipping of at least one of exons 43, 46, 50-53 of DMD pre-mRNA in a patient, preferably in an isolated cell of said patient, the method comprising providing said cell and/or said patient with a molecule that binds to a continuous stretch of at least 8 nucleotides within said exon.
- a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient.
- a patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background.
- an oligonucleotide used will preferably correct one mutation as present in the DMD gene of said patient and therefore will preferably create a DMD protein that will look like a BMD protein: said protein will preferably be a functional dystrophin as later defined herein.
- an oligonucleotide as used will preferably correct one mutation as present in the BMD gene of said patient and therefore will preferably create a dystrophin which will be more functional than the dystrophin which was originally present in said BMD patient.
- Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is performed by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with essential sequences such as for example the splice donor of splice acceptor sequence that required for splicing of said exon, or a molecule that is capable of interfering with an exon inclusion signal that is required for recognition of a stretch of nucleotides as an exon to be included in the mRNA.
- the term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
- inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.
- a method of the invention by inducing and/or promoting skipping of at least one of the following exons 43, 46, 50-53 of the DMD pre-mRNA in one or more (muscle) cells of a patient, provides said patient with a functional dystrophin protein and/or decreases the production of an aberrant dystrophin protein in said patient and/or increases the production of a functional dystrophin is said patient.
- Providing a patient with a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in said patient is typically applied in a DMD patient.
- Increasing the production of a functional dystrophin is typically applied in a BMD patient.
- a preferred method is a method, wherein a patient or one or more cells of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient is decreased and/or wherein the production of a functional dystrophin is increased in said patient, wherein the level of said aberrant or functional dystrophin is assessed by comparison to the level of said dystrophin in said patient at the onset of the method.
- Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR.
- An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein.
- a non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex.
- a non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
- Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR.
- 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.
- Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immuno fluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis.
- 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
- a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1.
- a functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person.
- the amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1.
- a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin.
- an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immuno fluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.
- DGC dystrophin-associated glycoprotein complex
- Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevent synthesis of the complete protein, i.e of a premature stop prevents the synthesis of the C-terminus.
- the DMD gene also comprises a mutation compared tot the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized.
- a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity.
- the genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144).
- Exon skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain.
- an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin.
- a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144).
- the central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144).
- a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
- a method of the invention may alleviate one or more characteristics of a myogenic or muscle cell of a patient or alleviate one or more symptoms of a DMD patient having a deletion including but not limited to exons 44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43 skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46 skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping), 13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52 (correctable by exon 51 skipping), exons 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60, (correctable by exon 52 skipping) and exons 10-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53
- a method of the invention may improve one or more characteristics of a muscle cell of a patient or alleviate one or more symptoms of a DMD patient having small mutations in, or single exon duplications of exon 43, 46, 50-53 in the DMD gene, occurring in a total of 36% of all DMD patients with a deletion (Aartsma-Rus et al, Hum. Mut. 2009)
- exon 46 and/or exon 50-53 is required to restore the open reading frame, including patients with specific deletions, small (point) mutations, or double or multiple exon duplications, such as (but not limited to) a deletion of exons 44-50 requiring the co-skipping of exons 43 and 51, with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, with a deletion of exons 44-52 requiring the co-skipping of exons 43 and 53, with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53, with a deletion of exons 51-54 requiring the co-skipping of exons 50 and 55, with a deletion of exons 53-54 requiring the co-skipping of exons 52 and 55, with a deletion of exons 53-56 requiring the co-skipping of exons 52 and 57,
- the skipping of exon 43 is induced, or the skipping of exon 46 is induced, or the skipping of exon 50 is induced or the skipping of exon 51 is induced or the skipping of exon 52 is induced or the skipping of exon 53 is induced.
- An induction of the skipping of two of these exons is also encompassed by a method of the invention. For example, preferably skipping of exons 50 and 51, or 52 and 53, or 43 and 51, or 43 and 53, or 51 and 52.
- the skilled person will know which combination of exons needs to be skipped in said patient.
- one or more symptom(s) of a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of one or more muscle cells from a DMD or a BMD patient is/are improved.
- symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.
- An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
- the improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic.
- a detectable decrease of creatine kinase in blood a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic
- a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic are known to the skilled person.
- Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).
- a detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD or BMD patient before treatment.
- a detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections.
- a detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD or BMD patient before treatment.
- a detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of a muscle fiber in a same DMD or BMD patient before treatment
- An alleviation of one or more symptoms may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life.
- assays are known to the skilled person.
- Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006).
- the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
- a treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more.
- Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro.
- the frequency of administration of a molecule or an oligonucleotide or a composition of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.
- a molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell.
- a solution that is compatible with the delivery method.
- the solution is a physiological salt solution.
- an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell.
- Preferred excipient are defined in the section entitled “pharmaceutical composition”.
- an additional molecule is used which is able to induce and/or promote skipping of another exon of the DMD pre-mRNA of a patient.
- the second exon is selected from: exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA of a patient.
- Molecules which can be used are depicted in any one of Table 1 to 7. This way, inclusion of two or more exons of a DMD pre-mRNA in mRNA produced from this pre-mRNA is prevented.
- This embodiment is further referred to as double- or multi-exon skipping (Aartsma-Rus A, Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet. 2004; 74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T, van Ommen G J, van Deutekom J C. Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Mol Ther 2006; 14(3):401-7). In most cases double-exon skipping results in the exclusion of only the two targeted exons from the DMD pre-mRNA.
- stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety.
- the at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.
- said compounds can be administered to an individual in any order.
- said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary.
- said compounds are administered sequentially.
- a molecule as defined herein is preferably an oligonucleotide or antisense oligonucleotide (AON).
- any of exon 43, 46, 50-53 is specifically skipped at a high frequency using a molecule that preferably binds to a continuous stretch of at least 8 nucleotides within said exon.
- this effect can be associated with a higher binding affinity of said molecule, compared to a molecule that binds to a continuous stretch of less than 8 nucleotides, there could be other intracellular parameters involved that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex.
- a molecule that binds to a continuous stretch of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides within said exon is used.
- a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of any of exon 43, 46, 50-53 of DMD pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% and most preferably up to 100%.
- “A part of said exon” preferably means a stretch of at least 8 nucleotides.
- an oligonucleotide of the invention consists of a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein.
- an oligonucleotide may comprise a sequence that is complementary to part of said exon DMD pre-mRNA as defined herein and additional flanking sequences.
- the length of said complementary part of said oligonucleotide is of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides.
- additional flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.
- a preferred molecule to be used in a method of the invention binds or is complementary to a continuous stretch of at least 8 nucleotides within one of the following nucleotide sequences selected from:
- the invention provides distinct molecules that can be used in a method for efficiently skipping of at least one of exon 43, exon 46 and/or exon 50-53.
- skipping effect can be addressed to the relatively high density of putative SR protein binding sites within said stretches, there could be other parameters involved that favor uptake of the molecule or other, intracellular parameters such as thermodynamic, kinetic, or structural characteristics of the hybrid duplex.
- a molecule that binds to a continuous stretch comprised within or consisting of any of SEQ ID NO 2-7 results in highly efficient skipping of exon 43, exon 46 and/or exon 50-53 respectively in a cell and/or in a patient provided with this molecule. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50 nucleotides within SEQ ID NO 2-7.
- the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences depicted in any of Tables 1 to 6.
- a molecule of the invention preferably comprises or consist of the antisense nucleotide sequence of SEQ ID NO 16, SEQ ID NO 65, SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO 127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ ID NO 299, SEQ ID NO:357.
- a preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 8 and 50 nucleotides or bases, more preferred between 10 and 50 nucleotides, more preferred between 20 and 40 nucleotides, more preferred between 20 and 30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleot
- a most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.
- a molecule of the invention is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the corresponding nucleotide sequence on a DMD pre-RNA molecule.
- Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein incorporated by reference.
- Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein incorporated by reference.
- a preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 2: 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAU AGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ which is present in exon 43 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 8 to SEQ ID NO 69.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 16 and/or SEQ ID NO 65.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 65. It was found that this molecule is very efficient in modulating splicing of exon 43 of the DMD pre-mRNA in a muscle cell and/or in a patient.
- Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 3: 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ which is present in exon 46 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70 to SEQ ID NO 122. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70, SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID NO 117.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 117. It was found that this molecule is very efficient in modulating splicing of exon 46 of the DMD pre-mRNA in a muscle cell or in a patient.
- Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 4: 5′-GGCGGTAAACCGUUUACUUCAAGAGCU GAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3′ which is present in exon 50 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127. It was found that this molecule is very efficient in modulating splicing of exon 50 of the DMD pre-mRNA in a muscle cell and/or in a patient.
- Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 5: 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′ which is present in exon 51 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 168 to SEQ ID NO 241.
- Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 6: 5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ which is present in exon 52 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 299. It was found that this molecule is very efficient in modulating splicing of exon 52 of the DMD pre-mRNA in a muscle cell and/or in a patient.
- Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 7: 5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ which is present in exon 53 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQ ID NO 358.
- the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 357. It was found that this molecule is very efficient in modulating splicing of exon 53 of the DMD pre-mRNA in a muscle cell and/or in a patient.
- a nucleotide sequence of a molecule of the invention may contain RNA residues, or one or more DNA residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.
- a molecule of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
- the nucleotide analogue or equivalent comprises a modified backbone.
- backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones.
- Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents.
- Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage.
- Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H.
- Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
- the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
- a preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition.
- the backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds.
- An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497).
- PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
- a further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring.
- a most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
- PMO phosphorodiamidate morpholino oligomer
- a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation.
- a preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
- a further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy.
- the sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof.
- Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
- LNA Locked Nucleic Acid
- a preferred LNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
- an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
- a preferred antisense oligonucleotide according to the invention comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
- RNA 2′-O-methyl modified ribose
- 2′-O-ethyl modified ribose 2′-O-propyl modified ribose
- substituted derivatives of these modifications such as halogenated derivatives.
- a most preferred antisense oligonucleotide according to the invention comprises of 2′-O-methyl phosphorothioate ribose.
- a functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent.
- an activity of said functional equivalent is inducing exon 43, 46, 50, 51, 52, or 53 skipping and providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by detection of exon 43, 46, 50, 51, 52, or 53 skipping and by quantifying the amount of functional dystrophin protein.
- a functional dystrophin is herein preferably defined as being a dystrophin able to bind actin and members of the DGC protein complex.
- the assessment of said activity of an oligonucleotide is preferably done by RT-PCR or by immunofluorescence or Western blot analyses. Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.
- distinct antisense oligonucleotides can be combined for efficiently skipping any of exon 43, exon 46, exon 50, exon 51, exon 52 and/or exon 53 of the human DMD pre-mRNA. It is encompassed by the present invention to use one, two, three, four, five or more oligonucleotides for skipping one of said exons (i.e. exon, 43, 46, 50, 51, 52, or 53). It is also encompassed to use at least two oligonucleotides for skipping at least two, of said exons. Preferably two of said exons are skipped. More preferably, these two exons are:
- An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably muscle cells.
- moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
- a preferred antisense oligonucleotide comprises a peptide-linked PMO.
- a preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery.
- systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells.
- a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for inducing skipping of exon 43, 46, 50, 51, 52, or 53 of the human DMD pre-mRNA.
- a cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of exon 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors.
- a viral-based expression vector comprising an expression cassette that drives expression of a molecule as identified herein. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter.
- a muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution.
- a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINETM 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof.
- AAV adenovirus associated virus
- a preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III).
- Pol III polymerase III-promoter
- a preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
- the invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of exon 43, exon 46, exon 50, exon 51, exon 52 or exon 53 of the human DMD pre-mRNA.
- a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.
- the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.
- a preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector as defined herein which is able to induce skipping of exon 6, 7, 11, 17, 19, 21, 43, 44, 45, 50, 51, 52, 53, 55, 57, 59, 62, 63, 65, 66, 69, or 75 of the DMD pre-mRNA.
- Preferred molecules able to induce skipping of any of these exon are identified in any one of Tables 1 to 7.
- Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art.
- Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT-18), LipofectinTM, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell.
- excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.
- Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release.
- DOTMA cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
- DOPE neutral lipid dioleoylphosphatidylethanolamine
- Another group of delivery systems are polymeric nanoparticles.
- Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.
- PBCA butylcyanoacrylate
- PHCA hexylcyanoacrylate
- the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids.
- This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide.
- the skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
- a compound as defined herein preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
- a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
- ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
- a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
- the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
- a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
- an in vitro concentration of a molecule or an oligonucleotide as defined herein which is ranged between 0.1 nM and 1 ⁇ M is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM.
- a molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added.
- oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses.
- concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
- a compound preferably an oligonucleotide to be used in the invention to prevent, treat DMD or BMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation.
- the delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
- a preferred oligonucleotide as defined herein optionally comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery.
- systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells.
- oligonucleotide comprising a specific nucleotide analog or equivalent
- an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting a different subset of muscle cells. Therefore, in this embodiment, it is preferred to use a combination of oligonucleotides comprising different nucleotide analogs or equivalents for modulating splicing of the DMD mRNA in at least one type of muscle cells.
- a molecule or a viral-based vector for use as a medicament, preferably for modulating splicing of the DMD pre-mRNA, more preferably for promoting or inducing skipping of any of exon 43, 46, 50-53 as identified herein.
- the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for modulating splicing of the DMD pre-mRNA.
- the splicing is preferably modulated in a human myogenic cell or muscle cell in vitro. More preferred is that splicing is modulated in a human muscle cell in vivo.
- the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for modulating splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD or BMD patient.
- the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
- the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
- reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
- the indefinite article “a” or “an” thus usually means “at least one”.
- AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software.
- AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
- Myotube cultures derived from a healthy individual (“human control”) (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet. 2003; 12(8): 907-14).
- myotube cultures were transfected with 50 nM and 150 nM (example 2), 200 nM and 500 nM (example 4) or 500 nM only (examples 1 and 3) of each AON.
- a series of AONs targeting sequences within exon 43 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 43 herein defined as SEQ ID NO 2, was indeed capable of inducing exon 43 skipping.
- PS237 SEQ ID NO: 65
- PS238 and PS240 are shown, inducing exon 43 skipping levels up to 13% and 36% respectively ( FIG. 1 ).
- the precise skipping of exon 43 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 43 skipping was observed in non-treated cells (NT).
- a series of AONs targeting sequences within exon 46 were designed and transfected in myotube cultures derived from a DMD patient carrying an exon 45 deletion in the DMD gene.
- antisense-induced exon 46 skipping would induce the synthesis of a novel, BMD-like dystrophin protein that may indeed alleviate one or more symptoms of the disease.
- Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 46 herein defined as SEQ ID NO 3, was indeed capable of inducing exon 46 skipping, even at relatively low AON concentrations of 50 nM.
- PS182 (SEQ ID NO: 117) reproducibly induced highest levels of exon 46 skipping (up to 50% at 50 nM and 74% at 150 nM), as shown in FIG. 2 .
- PS177, PS179, and PS181 are shown, inducing exon 46 skipping levels up to 55%, 58% and 42% respectively at 150 nM ( FIG. 2 ).
- the precise skipping of exon 46 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 46 skipping was observed in non-treated cells (NT).
- a series of AONs targeting sequences within exon 50 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 50 herein defined as SEQ ID NO 4, was indeed capable of inducing exon 50 skipping.
- PS248 SEQ ID NO: 127) reproducibly induced highest levels of exon 50 skipping (up to 35% at 500 nM), as shown in FIG. 3 .
- PS245, PS246, and PS247 are shown, inducing exon 50 skipping levels up to 14-16% at 500 nM ( FIG. 3 ).
- the precise skipping of exon 50 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 50 skipping was observed in non-treated cells (NT).
- a series of AONs targeting sequences within exon 51 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 51 herein defined as SEQ ID NO 5, was indeed capable of inducing exon 51 skipping.
- the AON with SEQ ID NO 180 reproducibly induced highest levels of exon 51 skipping (not shown).
- a series of AONs targeting sequences within exon 52 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 52 herein defined as SEQ ID NO 6, was indeed capable of inducing exon 52 skipping.
- PS236 SEQ ID NO: 299 reproducibly induced highest levels of exon 52 skipping (up to 88% at 200 nM and 91% at 500 nM), as shown in FIG. 4 .
- PS232 and AON 52-1 previously published by Aartsma-Rus et al.
- Oligonucleotides 2005 are shown, inducing exon 52 skipping at levels up to 59% and 10% respectively when applied at 500 nM ( FIG. 4 ).
- the precise skipping of exon 52 was confirmed by sequence analysis of the novel smaller transcript fragments. No exon 52 skipping was observed in non-treated cells (NT).
- a series of AONs targeting sequences within exon 53 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch within exon 53 herein defined as SEQ ID NO 7, was indeed capable of inducing exon 53 skipping.
- the AON with SEQ ID NO 328 reproducibly induced highest levels of exon 53 skipping (not shown).
- FIG. 1 In human control myotubes, a series of AONs (PS237, PS238, and PS240; SEQ ID NO 65, 66, 16 respectively) targeting exon 43 was tested at 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels of exon 43 skipping. (M: DNA size marker; NT: non-treated cells)
- FIG. 2 In myotubes from a DMD patient with an exon 45 deletion, a series of AONs (PS177, PS179, PS181, and PS182; SEQ ID NO 91, 70, 110, and 117 respectively) targeting exon 46 was tested at two different concentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproducibly induced highest levels of exon 46 skipping. (M: DNA size marker)
- FIG. 3 In human control myotubes, a series of AONs (PS245, PS246, PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively) targeting exon 50 was tested at 500 nM.
- PS248 SEQ ID NO 127) reproducibly induced highest levels of exon 50 skipping.
- M DNA size marker
- NT non-treated cells.
- FIG. 4 In human control myotubes, two novel AONs (PS232 and PS236; SEQ ID NO 246 and 299 respectively) targeting exon 52 were tested at two different concentrations (200 and 500 nM) and directly compared to a previously described AON (52-1).
- PS236 SEQ ID NO 299
- M DNA size marker
- NT non-treated cells
Abstract
Description
- This U.S. patent application is a continuation of PCT/NL2009/050113, filed on Mar. 11, 2009 which claims priority to PCT/NL2008/050673, filed on Oct. 27, 2008, which claims priority to European application no. 07119351.0, filed on Oct. 26, 2007, which claims the benefit of U.S. provisional patent application No. 61/000,670, filed on Oct. 26, 2007, the entirety of which is incorporated herein by reference. The invention relates to the field of genetics, more specifically human genetics. The invention in particular relates to modulation of splicing of the human Duchenne Muscular Dystrophy pre-mRNA.
- Myopathies are disorders that result in functional impairment of muscles. Muscular dystrophy (MD) refers to genetic diseases that are characterized by progressive weakness and degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood forms of muscular dystrophy. They are recessive disorders and because the gene responsible for DMD and BMD resides on the X-chromosome, mutations mainly affect males with an incidence of about 1 in 3500 boys.
- DMD and BMD are caused by genetic defects in the DMD gene encoding dystrophin, a muscle protein that is required for interactions between the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD is a severe, lethal neuromuscular disorder resulting in a dependency on wheelchair support before the age of 12 and DMD patients often die before the age of thirty due to respiratory- or heart failure. In contrast, BMD patients often remain ambulatory until later in life, and have near normal life expectancies. DMD mutations in the DMD gene are characterized by frame shifting insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations in general keep the reading frame intact, allowing synthesis of a partly functional dystrophin.
- During the last decade, specific modification of splicing in order to restore the disrupted reading frame of the dystrophin transcript has emerged as a promising therapy for Duchenne muscular dystrophy (DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol. Ther. 2008; 10(2):140-9, Yokota, Duddy, Partidge, Acta Myol. 2007; 26(3):179-84, van Deutekom et al., N Engl J. Med. 2007; 357(26):2677-86).
- Using antisense oligonucleotides (AONs) interfering with splicing signals the skipping of specific exons can be induced in the DMD pre-mRNA, thus restoring the open reading frame and converting the severe DMD into a milder BMD phenotype (van Deutekom et al. Hum Mol. Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum Mol Genet. 2003; 12(8):907-14.). In vivo proof-of-concept was first obtained in the mdx mouse model, which is dystrophin-deficient due to a nonsense mutation in exon 23. Intramuscular and intravenous injections of AONs targeting the mutated exon 23 restored dystrophin expression for at least three months (Lu et al. Nat. Med. 2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci USA. 2005; 102(1):198-203). This was accompanied by restoration of dystrophin-associated proteins at the fiber membrane as well as functional improvement of the treated muscle. In vivo skipping of human exons has also been achieved in the hDMD mouse model, which contains a complete copy of the human DMD gene integrated in chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy. 2004; 10: 232-40; 't Hoen et al. J Biol. Chem. 2008; 283: 5899-907).
- Recently, a first-in-man study was successfully completed where an AON inducing the skipping of
exon 51 was injected into a small area of the tibialis anterior muscle of four DMD patients. Novel dystrophin expression was observed in the majority of muscle fibers in all four patients treated, and the AON was safe and well tolerated (van Deutekom et al. N Engl J. Med. 2007; 357: 2677-86). - In a first aspect, the present invention provides a method for inducing, and/or promoting skipping of at least one of
exons - Accordingly, a method is provided for inducing and/or promoting skipping of at least one of
exons - As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient.
- A patient is preferably intended to mean a patient having DMD or BMD as later defined herein or a patient susceptible to develop DMD or BMD due to his or her genetic background. In the case of a DMD patient, an oligonucleotide used will preferably correct one mutation as present in the DMD gene of said patient and therefore will preferably create a DMD protein that will look like a BMD protein: said protein will preferably be a functional dystrophin as later defined herein. In the case of a BMD patient, an oligonucleotide as used will preferably correct one mutation as present in the BMD gene of said patient and therefore will preferably create a dystrophin which will be more functional than the dystrophin which was originally present in said BMD patient.
- Exon skipping refers to the induction in a cell of a mature mRNA that does not contain a particular exon that is normally present therein. Exon skipping is performed by providing a cell expressing the pre-mRNA of said mRNA with a molecule capable of interfering with essential sequences such as for example the splice donor of splice acceptor sequence that required for splicing of said exon, or a molecule that is capable of interfering with an exon inclusion signal that is required for recognition of a stretch of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template in the cell nucleus by transcription.
- Within the context of the invention, inducing and/or promoting skipping of an exon as indicated herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the DMD mRNA in one or more (muscle) cells of a treated patient will not contain said exon. This is preferably assessed by PCR as described in the examples.
- Preferably, a method of the invention by inducing and/or promoting skipping of at least one of the following
exons - Providing a patient with a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in said patient is typically applied in a DMD patient. Increasing the production of a functional dystrophin is typically applied in a BMD patient.
- Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with a functional dystrophin protein and/or wherein the production of an aberrant dystrophin protein in said patient is decreased and/or wherein the production of a functional dystrophin is increased in said patient, wherein the level of said aberrant or functional dystrophin is assessed by comparison to the level of said dystrophin in said patient at the onset of the method.
- Decreasing the production of an aberrant dystrophin may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional dystrophin mRNA or protein. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.
- Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional dystrophin mRNA.
- Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immuno fluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.
- As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO:1. In other words, a functional dystrophin is a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a functional dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immuno fluorescence analysis of cross-sections, from a muscle biopsy, as known to the skilled person.
- Individuals or patients suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevent synthesis of the complete protein, i.e of a premature stop prevents the synthesis of the C-terminus. In Becker muscular dystrophy the DMD gene also comprises a mutation compared tot the wild type gene but the mutation does typically not induce a premature stop and the C-terminus is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as the wild type protein, not although not necessarily the same amount of activity. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). Exon skipping for the treatment of DMD is typically directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated by a method as defined herein will be provided a dystrophin which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: typically said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). The central rod-shaped domain of wild type dystrophin comprises 24 spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne Muscular Dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule, Muscle Nerve, 34: 135-144). For example, a central rod-shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC.
- A method of the invention may alleviate one or more characteristics of a myogenic or muscle cell of a patient or alleviate one or more symptoms of a DMD patient having a deletion including but not limited to exons 44, 44-46, 44-47, 44-48, 44-49, 44-51, 44-53 (correctable by exon 43 skipping), 19-45, 21-45, 43-45, 45, 47-54, 47-56 (correctable by exon 46 skipping), 51, 51-53, 51-55, 51-57 (correctable by exon 50 skipping), 13-50, 19-50, 29-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52 (correctable by exon 51 skipping), exons 8-51, 51, 53, 53-55, 53-57, 53-59, 53-60, (correctable by exon 52 skipping) and exons 10-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52 (correctable by exon 53 skipping) in the DMD gene, occurring in a total of 68% of all DMD patients with a deletion (Aartsma-Rus et al., Hum. Mut. 2009).
- Alternatively, a method of the invention may improve one or more characteristics of a muscle cell of a patient or alleviate one or more symptoms of a DMD patient having small mutations in, or single exon duplications of
exon - Furthermore, for some patients the simultaneous skipping of one of more exons in addition to exon 43, exon 46 and/or exon 50-53 is required to restore the open reading frame, including patients with specific deletions, small (point) mutations, or double or multiple exon duplications, such as (but not limited to) a deletion of exons 44-50 requiring the co-skipping of exons 43 and 51, with a deletion of exons 46-50 requiring the co-skipping of exons 45 and 51, with a deletion of exons 44-52 requiring the co-skipping of exons 43 and 53, with a deletion of exons 46-52 requiring the co-skipping of exons 45 and 53, with a deletion of exons 51-54 requiring the co-skipping of exons 50 and 55, with a deletion of exons 53-54 requiring the co-skipping of exons 52 and 55, with a deletion of exons 53-56 requiring the co-skipping of exons 52 and 57, with a nonsense mutation in exon 43 or exon 44 requiring the co-skipping of exon 43 and 44, with a nonsense mutation in exon 45 or exon 46 requiring the co-skipping of exon 45 and 46, with a nonsense mutation in exon 50 or exon 51 requiring the co-skipping of exon 50 and 51, with a nonsense mutation in exon 51 or exon 52 requiring the co-skipping of exon 51 and 52, with a nonsense mutation in exon 52 or exon 53 requiring the co-skipping of exon 52 and 53, or with a double or multiple exon duplication involving exons 43, 46, 50, 51, 52, and/or 53.
- In a preferred method, the skipping of
exon 43 is induced, or the skipping ofexon 46 is induced, or the skipping ofexon 50 is induced or the skipping ofexon 51 is induced or the skipping ofexon 52 is induced or the skipping ofexon 53 is induced. An induction of the skipping of two of these exons is also encompassed by a method of the invention. For example, preferably skipping ofexons - In a preferred method, one or more symptom(s) of a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of one or more muscle cells from a DMD or a BMD patient is/are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient self.
- An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.
- The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.
- Creatine kinase may be detected in blood as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same DMD or BMD patient before treatment.
- A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006) using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same DMD or BMD patient before treatment.
- A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). The increase is measured by comparison to the homogeneity of the diameter of a muscle fiber in a same DMD or BMD patient before treatment
- An alleviation of one or more symptoms may be assessed by any of the following assays on the patient self: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (Manzur A Y et al, (2008), Glucocorticoid corticosteroids for Duchenne muscular dystrophy (review), Wiley publishers, The Cochrane collaboration.) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a method of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular Disorders, 16: 591-602.2006). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
- A treatment in a method according to the invention may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more.
- Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of a molecule or an oligonucleotide or a composition of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (dose), the formulation of said molecule. The frequency may be ranged between at least once in a two weeks, or three weeks or four weeks or five weeks or a longer time period.
- A molecule or oligonucleotide or equivalent thereof can be delivered as is to a cell. When administering said molecule, oligonucleotide or equivalent thereof to an individual, it is preferred that it is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred for a method of the invention is the use of an excipient that will further enhance delivery of said molecule, oligonucleotide or functional equivalent thereof as defined herein, to a cell and into a cell, preferably a muscle cell. Preferred excipient are defined in the section entitled “pharmaceutical composition”.
- In a preferred method of the invention, an additional molecule is used which is able to induce and/or promote skipping of another exon of the DMD pre-mRNA of a patient. Preferably, the second exon is selected from:
exon exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connectexons - It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule.
- In case, more than one compounds or molecules are used in a method of the invention, said compounds can be administered to an individual in any order. In one embodiment, said compounds are administered simultaneously (meaning that said compounds are administered within 10 hours, preferably within one hour). This is however not necessary. In another embodiment, said compounds are administered sequentially.
- In a second aspect, there is provided a molecule for use in a method as described in the previous section entitled “Method”. A molecule as defined herein is preferably an oligonucleotide or antisense oligonucleotide (AON).
- It was found by the present investigators that any of
exon - In a preferred embodiment, a molecule or an oligonucleotide of the invention which comprises a sequence that is complementary to a part of any of
exon - A preferred molecule to be used in a method of the invention binds or is complementary to a continuous stretch of at least 8 nucleotides within one of the following nucleotide sequences selected from:
-
(SEQ ID NO: 2) 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAG CAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ for skipping of exon 43;(SEQ ID NO: 3) 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAA CCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ for skipping of exon 46;(SEQ ID NO: 4) 5′-GGCGGTAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUGA CCUAGC UCCUGGACUGACCACUAUUGG-3′ for skipping of exon 50;(SEQ ID NO: 5) 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUA AGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUG GAGGUAC-3′ for skipping of exon 51;(SEQ ID NO: 6) 5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUU ACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ for skipping of exon 52,and (SEQ ID NO: 7) 5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAG CUGAGCAGGUCUUAGGACAGGCCAGAG-3′ for skipping of exon 53. - Of the numerous molecules that theoretically can be prepared to bind to the continuous nucleotide stretches as defined by SEQ ID NO 2-7 within one of said exons, the invention provides distinct molecules that can be used in a method for efficiently skipping of at least one of
exon 43,exon 46 and/or exon 50-53. Although the skipping effect can be addressed to the relatively high density of putative SR protein binding sites within said stretches, there could be other parameters involved that favor uptake of the molecule or other, intracellular parameters such as thermodynamic, kinetic, or structural characteristics of the hybrid duplex. - It was found that a molecule that binds to a continuous stretch comprised within or consisting of any of SEQ ID NO 2-7 results in highly efficient skipping of
exon 43,exon 46 and/or exon 50-53 respectively in a cell and/or in a patient provided with this molecule. Therefore, in a preferred embodiment, a method is provided wherein a molecule binds to a continuous stretch of at least 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50 nucleotides within SEQ ID NO 2-7. - In a preferred embodiment for inducing and/or promoting the skipping of any of
exon 43,exon 46 and/or exon 50-53, the invention provides a molecule comprising or consisting of an antisense nucleotide sequence selected from the antisense nucleotide sequences depicted in any of Tables 1 to 6. A molecule of the invention preferably comprises or consist of the antisense nucleotide sequence ofSEQ ID NO 16,SEQ ID NO 65, SEQ ID NO 70,SEQ ID NO 91, SEQ ID NO 110, SEQ ID NO 117, SEQ ID NO 127, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 246, SEQ ID NO 299, SEQ ID NO:357. - A preferred molecule of the invention comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 8 and 50 nucleotides or bases, more preferred between 10 and 50 nucleotides, more preferred between 20 and 40 nucleotides, more preferred between 20 and 30 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides.
- A most preferred molecule of the invention comprises a nucleotide-based sequence of 25 nucleotides.
- Furthermore, none of the indicated sequences is derived from conserved parts of splice-junction sites. Therefore, said molecule is not likely to mediate differential splicing of other exons from the DMD pre-mRNA or exons from other genes.
- In one embodiment, a molecule of the invention is a compound molecule that binds to the specified sequence, or a protein such as an RNA-binding protein or a non-natural zinc-finger protein that has been modified to be able to bind to the corresponding nucleotide sequence on a DMD pre-RNA molecule. Methods for screening compound molecules that bind specific nucleotide sequences are, for example, disclosed in PCT/NL01/00697 and U.S. Pat. No. 6,875,736, which are herein incorporated by reference. Methods for designing RNA-binding Zinc-finger proteins that bind specific nucleotide sequences are disclosed by Friesen and Darby, Nature Structural Biology 5: 543-546 (1998) which is herein incorporated by reference.
- A preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 2: 5′-AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAU AGCAAGAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG-3′ which is present in
exon 43 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 8 to SEQ ID NO 69. - In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of
SEQ ID NO 16 and/orSEQ ID NO 65. - In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of
SEQ ID NO 65. It was found that this molecule is very efficient in modulating splicing ofexon 43 of the DMD pre-mRNA in a muscle cell and/or in a patient. - Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 3: 5′-UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUG AACCUGGAAAAGAGCAGCAACUAAAAGAAAAGC-3′ which is present in
exon 46 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70 to SEQ ID NO 122. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 70,SEQ ID NO 91, SEQ ID NO 110, and/or SEQ ID NO 117. - In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 117. It was found that this molecule is very efficient in modulating splicing of
exon 46 of the DMD pre-mRNA in a muscle cell or in a patient. - Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 4: 5′-GGCGGTAAACCGUUUACUUCAAGAGCU GAGGGCAAAGCAGCCUG ACCUAGCUCCUGGACUGACCACUAUUGG-3′ which is present in
exon 50 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 123 to SEQ ID NO 167 and/or SEQ ID NO 529 to SEQ ID NO 535. - In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127, or SEQ ID NO 165, or SEQ ID NO 166 and/or SEQ ID NO 167.
- In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 127. It was found that this molecule is very efficient in modulating splicing of
exon 50 of the DMD pre-mRNA in a muscle cell and/or in a patient. - Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 5: 5′-CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACU AAGGAAACUGCCAUC UCCAAACUAGAAAUGCCAUCUUCCUUGAUG UUGGAGGUAC-3′ which is present in
exon 51 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 168 to SEQ ID NO 241. - Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 6: 5′-AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAU UACCGCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCU-3′ which is present in
exon 52 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 242 to SEQ ID NO 310. In an even more preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 246 and/or SEQ ID NO 299. In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 299. It was found that this molecule is very efficient in modulating splicing ofexon 52 of the DMD pre-mRNA in a muscle cell and/or in a patient. - Another preferred molecule of the invention binds to at least part of the sequence of SEQ ID NO 7: 5′-AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAA GCUGAGCAGGUCUUAGGACAGGCCAGAG-3′ which is present in
exon 53 of the DMD gene. More preferably, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 311 to SEQ ID NO 358. - In a most preferred embodiment, the invention provides a molecule comprising or consisting of the antisense nucleotide sequence of SEQ ID NO 357. It was found that this molecule is very efficient in modulating splicing of
exon 53 of the DMD pre-mRNA in a muscle cell and/or in a patient. - A nucleotide sequence of a molecule of the invention may contain RNA residues, or one or more DNA residues, and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.
- It is preferred that a molecule of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense nucleotide for the target sequence. Therefore, in a preferred embodiment, the antisense nucleotide sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
- In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.
- It is further preferred that that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
- A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566-568).
- A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
- In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
- A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -aminoxy; methoxyethoxy; -dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Such preferred derivatized sugar moieties comprise Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-O,4′-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.
- It is understood by a skilled person that it is not necessary for all positions in an antisense oligonucleotide to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single antisense oligonucleotide or even at a single position within an antisense oligonucleotide. In certain embodiments, an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
- A preferred antisense oligonucleotide according to the invention comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
- A most preferred antisense oligonucleotide according to the invention comprises of 2′-O-methyl phosphorothioate ribose.
- A functional equivalent of a molecule of the invention may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is inducing
exon exon - It will be understood by a skilled person that distinct antisense oligonucleotides can be combined for efficiently skipping any of
exon 43,exon 46,exon 50,exon 51,exon 52 and/orexon 53 of the human DMD pre-mRNA. It is encompassed by the present invention to use one, two, three, four, five or more oligonucleotides for skipping one of said exons (i.e. exon, 43, 46, 50, 51, 52, or 53). It is also encompassed to use at least two oligonucleotides for skipping at least two, of said exons. Preferably two of said exons are skipped. More preferably, these two exons are: - 43 and 51, or
43 and 53, or
50 and 51, or
51 and 52, or
52 and 53. - The skilled person will know which combination of exons is preferred to be skipped depending on the type, the number and the location of the mutation present in a DMD or BMD patient.
- An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably muscle cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
- A preferred antisense oligonucleotide comprises a peptide-linked PMO.
- A preferred antisense oligonucleotide comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an antisense oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an antisense oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells. Therefore, in one embodiment it is preferred to use a combination of antisense oligonucleotides comprising different nucleotide analogs or equivalents for inducing skipping of
exon - A cell can be provided with a molecule capable of interfering with essential sequences that result in highly efficient skipping of
exon 43,exon 46,exon 50,exon 51,exon 52 orexon 53 of the human DMD pre-mRNA by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette that drives expression of a molecule as identified herein. Expression is preferably driven by a polymerase III promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or myogenic cell can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a plasmid can be provided by transfection using known transfection agentia such as, for example, LipofectAMINE™ 2000 (Invitrogen) or polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof. - One preferred antisense oligonucleotide expression system is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of small antisense nucleotide sequences for highly efficient skipping of
exon - A preferred AAV-based vector comprises an expression cassette that is driven by a polymerase III-promoter (Pol III). A preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA promoter.
- The invention therefore also provides a viral-based vector, comprising a Pol III-promoter driven expression cassette for expression of one or more antisense sequences of the invention for inducing skipping of
exon 43,exon 46,exon 50,exon 51,exon 52 orexon 53 of the human DMD pre-mRNA. - If required, a molecule or a vector expressing an antisense oligonucleotide of the invention can be incorporated into a pharmaceutically active mixture or composition by adding a pharmaceutically acceptable carrier.
- Therefore, in a further aspect, the invention provides a composition, preferably a pharmaceutical composition comprising a molecule comprising an antisense oligonucleotide according to the invention, and/or a viral-based vector expressing the antisense sequence(s) according to the invention and a pharmaceutically acceptable carrier.
- A preferred pharmaceutical composition comprises a molecule as defined herein and/or a vector as defined herein, and a pharmaceutical acceptable carrier or excipient, optionally combined with a molecule and/or a vector as defined herein which is able to induce skipping of
exon - Preferred excipients include excipients capable of forming complexes, vesicles and/or liposomes that deliver such a molecule as defined herein, preferably an oligonucleotide complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine and derivatives, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives,
ExGen 500, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such molecule, preferably an oligonucleotide as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver (oligonucleotide such as antisense) nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity. - Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.
- Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver a molecule or a compound as defined herein, preferably an oligonucleotide across cell membranes into cells.
- In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate a compound as defined herein, preferably an oligonucleotide as colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of a compound as defined herein, preferably an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver a compound as defined herein, preferably an oligonucleotide for use in the current invention to deliver said compound for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.
- In addition, a compound as defined herein, preferably an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an a compound as defined herein, preferably an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
- Therefore, in a preferred embodiment, a compound as defined herein, preferably an oligonucleotide are formulated in a medicament which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising a compound as defined herein, preferably an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said compound to a cell and/or enhancing its intracellular delivery.
- It is to be understood that a molecule or compound or oligonucleotide may not be formulated in one single composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each compound.
- In a preferred embodiment, an in vitro concentration of a molecule or an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 □M is used. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. If several molecules or oligonucleotides are used, these concentrations may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
- More preferably, a compound preferably an oligonucleotide to be used in the invention to prevent, treat DMD or BMD are synthetically produced and administered directly to a cell, a tissue, an organ and/or patients in formulated form in a pharmaceutically acceptable composition or preparation. The delivery of a pharmaceutical composition to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
- A preferred oligonucleotide as defined herein optionally comprising one or more nucleotide analogs or equivalents of the invention modulates splicing in one or more muscle cells, including heart muscle cells, upon systemic delivery. In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting of a different subset of muscle cells.
- In this respect, systemic delivery of an oligonucleotide comprising a specific nucleotide analog or equivalent might result in targeting a subset of muscle cells, while an oligonucleotide comprising a distinct nucleotide analog or equivalent might result in targeting a different subset of muscle cells. Therefore, in this embodiment, it is preferred to use a combination of oligonucleotides comprising different nucleotide analogs or equivalents for modulating splicing of the DMD mRNA in at least one type of muscle cells.
- In a preferred embodiment, there is provided a molecule or a viral-based vector for use as a medicament, preferably for modulating splicing of the DMD pre-mRNA, more preferably for promoting or inducing skipping of any of
exon - In yet a further aspect, the invention provides the use of an antisense oligonucleotide or molecule according to the invention, and/or a viral-based vector that expresses one or more antisense sequences according to the invention and/or a pharmaceutical composition, for modulating splicing of the DMD pre-mRNA. The splicing is preferably modulated in a human myogenic cell or muscle cell in vitro. More preferred is that splicing is modulated in a human muscle cell in vivo. Accordingly, the invention further relates to the use of the molecule as defined herein and/or the vector as defined herein and/or or the pharmaceutical composition as defined herein for modulating splicing of the DMD pre-mRNA or for the preparation of a medicament for the treatment of a DMD or BMD patient.
- In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”. Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
- The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
- AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.
- Myotube cultures derived from a healthy individual (“human control”) (examples 1, 3, and 4;
exon exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet. 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 50 nM and 150 nM (example 2), 200 nM and 500 nM (example 4) or 500 nM only (examples 1 and 3) of each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 □l UNIFectylin per □g AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exons (43, 46, 50, 51, 52, or 53). PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChips Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA). - A series of AONs targeting sequences within
exon 43 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 43 herein defined as SEQ ID NO 2, was indeed capable of inducingexon 43 skipping. PS237 (SEQ ID NO: 65) reproducibly induced highest levels ofexon 43 skipping (up to 66%) at 500 nM, as shown inFIG. 1 . For comparison, also PS238 and PS240 are shown, inducingexon 43 skipping levels up to 13% and 36% respectively (FIG. 1 ). The precise skipping ofexon 43 was confirmed by sequence analysis of the novel smaller transcript fragments. Noexon 43 skipping was observed in non-treated cells (NT). - A series of AONs targeting sequences within
exon 46 were designed and transfected in myotube cultures derived from a DMD patient carrying an exon 45 deletion in the DMD gene. For patients with such mutation antisense-inducedexon 46 skipping would induce the synthesis of a novel, BMD-like dystrophin protein that may indeed alleviate one or more symptoms of the disease. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 46 herein defined as SEQ ID NO 3, was indeed capable of inducingexon 46 skipping, even at relatively low AON concentrations of 50 nM. PS182 (SEQ ID NO: 117) reproducibly induced highest levels ofexon 46 skipping (up to 50% at 50 nM and 74% at 150 nM), as shown inFIG. 2 . For comparison, also PS177, PS179, and PS181 are shown, inducingexon 46 skipping levels up to 55%, 58% and 42% respectively at 150 nM (FIG. 2 ). The precise skipping ofexon 46 was confirmed by sequence analysis of the novel smaller transcript fragments. Noexon 46 skipping was observed in non-treated cells (NT). - A series of AONs targeting sequences within
exon 50 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 50 herein defined as SEQ ID NO 4, was indeed capable of inducingexon 50 skipping. PS248 (SEQ ID NO: 127) reproducibly induced highest levels ofexon 50 skipping (up to 35% at 500 nM), as shown inFIG. 3 . For comparison, also PS245, PS246, and PS247 are shown, inducingexon 50 skipping levels up to 14-16% at 500 nM (FIG. 3 ). The precise skipping ofexon 50 was confirmed by sequence analysis of the novel smaller transcript fragments. Noexon 50 skipping was observed in non-treated cells (NT). - A series of AONs targeting sequences within
exon 51 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 51 herein defined as SEQ ID NO 5, was indeed capable of inducingexon 51 skipping. The AON with SEQ ID NO 180 reproducibly induced highest levels ofexon 51 skipping (not shown). - A series of AONs targeting sequences within
exon 52 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 52 herein defined as SEQ ID NO 6, was indeed capable of inducingexon 52 skipping. PS236 (SEQ ID NO: 299) reproducibly induced highest levels ofexon 52 skipping (up to 88% at 200 nM and 91% at 500 nM), as shown inFIG. 4 . For comparison, also PS232 and AON 52-1 (previously published by Aartsma-Rus et al. Oligonucleotides 2005) are shown, inducingexon 52 skipping at levels up to 59% and 10% respectively when applied at 500 nM (FIG. 4 ). The precise skipping ofexon 52 was confirmed by sequence analysis of the novel smaller transcript fragments. Noexon 52 skipping was observed in non-treated cells (NT). - A series of AONs targeting sequences within
exon 53 were designed and transfected in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that almost all AONs targeting a continuous nucleotide stretch withinexon 53 herein defined asSEQ ID NO 7, was indeed capable of inducingexon 53 skipping. The AON with SEQ ID NO 328 reproducibly induced highest levels ofexon 53 skipping (not shown). -
-
DMD gene amino acid sequence SEQ ID NO: 1: MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGR RLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGS TDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWV RQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWSVVCQQSAT QRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERT SSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMES EVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWEC LRVASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGP DLEDLKRQVQQHKVLQCDLEOEQVRVNSLTHMVVVVDESSGDHATAAL EEQLKVLGORVVANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLS EKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLK QDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTT QPSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEI RKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIER EKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFC QLLSERLNWLEYQNNIIAFYNQLQQLEOMTTTAENWLKIQPTTPSEPT AIKSQLKICKDEVNRLSGLQPQIERLKIQSIALKEKGQGPMFLDADFV AFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSET KLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLSTTVKEMSK KAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQNH IQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPS LNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEAL KGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPQELQKAVEEM KRAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQ WLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENIP GGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEEL ETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQ LAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRV LSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPAL ETKSVEQEVVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTE NPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLT EWLAATDMELTKRSAVEGMPSNLDSEVAWGKATQKEIEKOKVHLKSIT EVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYOKH METFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIR PKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRFAAISHRIKTGKA SIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNCGTV KELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALKDLRSQRRKKA LEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQ KKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRR LNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQ LLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQ SATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYD IKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRT LNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQ KNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQOLKEKLEQVKLLV EELPLRQCILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKV SRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEI YNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKR KLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTKIGASPTQTVTLVTQP WTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQR VMVGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQE ARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAE QVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVAN DLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHR LLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQ WQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFK WSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAP IGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGL EKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKI DETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEK VKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWK LLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYY INHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCL DLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLV NVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRY LFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRS CFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKC NICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCT PTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETP VTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLND SISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGE LERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSP RDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQ AEAKVNGTTVSSPSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPP QDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM SEQ ID NO 2 (exon 43): AGAUAGUCUACAACAAAGCUCAGGUCGGAUUGACAUUAUUCAUAGCAA GAAGACAGCAGCAUUGCAAAGUGCAACGCCUGUGG SEQ ID NO 3 (exon 46): UUAUGGUUGGAGGAAGCAGAUAACAUUGCUAGUAUCCCACUUGAACCU GGAAAAGAGCAGCAACUAAAAGAAAAGC SEQ ID NO 4 (exon 50): ′GGCGGTAAACCGUUUACUUCAAGAGCUGAGGGCAAAGCAGCCUG AC CUAGCUCCUGGACUGACCACUAUUGG SEQ ID NO 5 (exon 51): CUCCUACUCAGACUGUUACUCUGGUGACACAACCUGUGGUUACUAAGG AAACUGCCAUCUCCAAACUAGAAAUGCCAUCUUCCUUGAUGUUGGAGG UAC SEQ ID NO 6 (exon 52): AUGCAGGAUUUGGAACAGAGGCGUCCCCAGUUGGAAGAACUCAUUACC GCUGCCCAAAAUUUGAAAAA CAAGACCAGCAAUCAAGAGGCU SEQ ID NO 7 (exon 53): AAAUGUUAAAGGAUUCAACACAAUGGCUGGAAGCUAAGGAAGAAGCUG AGCAGGUCUUAGGACAGGCCAGAG -
TABLE 1 oligonucleotides for skipping DMD Gene Exon 43 SEQ ID NO 8 CCACAGGCGUUGCACUUUGCAAUGC SEQ ID NO 9 CACAGGCGUUGCACUUUGCAAUGCU SEQ ID NO 10 ACAGGCGUUGCACUUUGCAAUGCUG SEQ ID NO 11 CAGGCGUUGCACUUUGCAAUGCUGC SEQ ID NO 12 AGGCGUUGCACUUUGCAAUGCUGCU SEQ ID NO 13 GGCGUUGCACUUUGCAAUGCUGCUG SEQ ID NO 14 GCGUUGCACUUUGCAAUGCUGCUGU SEQ ID NO 15 CGUUGCACUUUGCAAUGCUGCUGUC SEQ ID NO 16 CGUUGCACUUUGCAAUGCUGCUG PS240 SEQ ID NO 17 GUUGCACUUUGCAAUGCUGCUGUCU SEQ ID NO 18 UUGCACUUUGCAAUGCUGCUGUCUU SEQ ID NO 19 UGCACUUUGCAAUGCUGCUGUCUUC SEQ ID NO 20 GCACUUUGCAAUGCUGCUGUCUUCU SEQ ID NO 21 CACUUUGCAAUGCUGCUGUCUUCUU SEQ ID NO 22 ACUUUGCAAUGCUGCUGUCUUCUUG SEQ ID NO 23 CUUUGCAAUGCUGCUGUCUUCUUGC SEQ ID NO 24 UUUGCAAUGCUGCUGUCUUCUUGCU SEQ ID NO 25 UUGCAAUGCUGCUGUCUUCUUGCUA SEQ ID NO 26 UGCAAUGCUGCUGUCUUCUUGCUAU SEQ ID NO 27 GCAAUGCUGCUGUCUUCUUGCUAUG SEQ ID NO 28 CAAUGCUGCUGUCUUCUUGCUAUGA SEQ ID NO 29 AAUGCUGCUGUCUUCUUGCUAUGAA SEQ ID NO 30 AUGCUGCUGUCUUCUUGCUAUGAAU SEQ ID NO 31 UGCUGCUGUCUUCUUGCUAUGAAUA SEQ ID NO 32 GCUGCUGUCUUCUUGCUAUGAAUAA SEQ ID NO 33 CUGCUGUCUUCUUGCUAUGAAUAAU SEQ ID NO 34 UGCUGUCUUCUUGCUAUGAAUAAUG SEQ ID NO 35 GCUGUCUUCUUGCUAUGAAUAAUGU SEQ ID NO 36 CUGUCUUCUUGCUAUGAAUAAUGUC SEQ ID NO 37 UGUCUUCUUGCUAUGAAUAAUGUCA SEQ ID NO 38 GUCUUCUUGCUAUGAAUAAUGUCAA SEQ ID NO 39 UCUUCUUGCUAUGAAUAAUGUCAAU SEQ ID NO 40 CUUCUUGCUAUGAAUAAUGUCAAUC SEQ ID NO 41 UUCUUGCUAUGAAUAAUGUCAAUCC SEQ ID NO 42 UCUUGCUAUGAAUAAUGUCAAUCCG SEQ ID NO 43 CUUGCUAUGAAUAAUGUCAAUCCGA SEQ ID NO 44 UUGCUAUGAAUAAUGUCAAUCCGAC SEQ ID NO 45 UGCUAUGAAUAAUGUCAAUCCGACC SEQ ID NO 46 GCUAUGAAUAAUGUCAAUCCGACCU SEQ ID NO 47 CUAUGAAUAAUGUCAAUCCGACCUG SEQ ID NO 48 UAUGAAUAAUGUCAAUCCGACCUGA SEQ ID NO 49 AUGAAUAAUGUCAAUCCGACCUGAG SEQ ID NO 50 UGAAUAAUGUCAAUCCGACCUGAGC SEQ ID NO 51 GAAUAAUGUCAAUCCGACCUGAGCU SEQ ID NO 52 AAUAAUGUCAAUCCGACCUGAGCUU SEQ ID NO 53 AUAAUGUCAAUCCGACCUGAGCUUU SEQ ID NO 54 UAAUGUCAAUCCGACCUGAGCUUUG SEQ ID NO 55 AAUGUCAAUCCGACCUGAGCUUUGU SEQ ID NO 56 AUGUCAAUCCGACCUGAGCUUUGUU SEQ ID NO 57 UGUCAAUCCGACCUGAGCUUUGUUG SEQ ID NO 58 GUCAAUCCGACCUGAGCUUUGUUGU SEQ ID NO 59 UCAAUCCGACCUGAGCUUUGUUGUA SEQ ID NO 60 CAAUCCGACCUGAGCUUUGUUGUAG SEQ ID NO 61 AAUCCGACCUGAGCUUUGUUGUAGA SEQ ID NO 62 AUCCGACCUGAGCUUUGUUGUAGAC SEQ ID NO 63 UCCGACCUGAGCUUUGUUGUAGACU SEQ ID NO 64 CCGACCUGAGCUUUGUUGUAGACUA SEQ ID NO 65 CGACCUGAGCUUUGUUGUAG PS237 SEQ ID NO 66 CGACCUGAGCUUUGUUGUAGACUAU PS238 SEQ ID NO 67 GACCUGAGCUUUGUUGUAGACUAUC SEQ ID NO 68 ACCUGAGCUUUGUUGUAGACUAUCA SEQ ID NO 69 CCUGA GCUUU GUUGU AGACU AUC -
TABLE 2 oligonucleotides for skipping DMD Gene Exon 46 SEQ ID NO 70 GCUUUUCUUUUAGUUGCUGCUCUUU PS179 SEQ ID NO 71 CUUUUCUUUUAGUUGCUGCUCUUUU SEQ ID NO 72 UUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID NO 73 UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO 74 UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID NO 75 UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NO 76 CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID NO 77 UUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID NO 78 UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO 79 UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NO 80 UAGUUGCUGCUCUUUUCCAGGUUCA SEQ ID NO 81 AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO 82 GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO 83 UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID NO 84 UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO 85 GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO 86 CUGCUCUUUUCCAGGUUCAAGUGGG SEQ ID NO 87 UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID NO 88 GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO 89 CUCUUUUCCAGGUUCAAGUGGGAUA SEQ ID NO 90 UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NO 91 UCUUUUCCAGGUUCAAGUGG PS177 SEQ ID NO 92 CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO 93 UUUUCCAGGUUCAAGUGGGAUACUA SEQ ID NO 94 UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID NO 95 UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID NO 96 UCCAGGUUCAAGUGGGAUACUAGCA SEQ ID NO 97 CCAGGUUCAAGUGGGAUACUAGCAA SEQ ID NO 98 CAGGUUCAAGUGGGAUACUAGCAAU SEQ ID NO 99 AGGUUCAAGUGGGAUACUAGCAAUG SEQ ID NO 100 GGUUCAAGUGGGAUACUAGCAAUGU SEQ ID NO 101 GUUCAAGUGGGAUACUAGCAAUGUU SEQ ID NO 102 UUCAAGUGGGAUACUAGCAAUGUUA SEQ ID NO 103 UCAAGUGGGAUACUAGCAAUGUUAU SEQ ID NO 104 CAAGUGGGAUACUAGCAAUGUUAUC SEQ ID NO 105 AAGUGGGAUACUAGCAAUGUUAUCU SEQ ID NO 106 AGUGGGAUACUAGCAAUGUUAUCUG SEQ ID NO 107 GUGGGAUACUAGCAAUGUUAUCUGC SEQ ID NO 108 UGGGAUACUAGCAAUGUUAUCUGCU SEQ ID NO 109 GGGAUACUAGCAAUGUUAUCUGCUU SEQ ID NO 110 GGAUACUAGCAAUGUUAUCUGCUUC PS181 SEQ ID NO 111 GAUACUAGCAAUGUUAUCUGCUUCC SEQ ID NO 112 AUACUAGCAAUGUUAUCUGCUUCCU SEQ ID NO 113 UACUAGCAAUGUUAUCUGCUUCCUC SEQ ID NO 114 ACUAGCAAUGUUAUCUGCUUCCUCC SEQ ID NO 115 CUAGCAAUGUUAUCUGCUUCCUCCA SEQ ID NO 116 UAGCAAUGUUAUCUGCUUCCUCCAA SEQ ID NO 117 AGCAAUGUUAUCUGCUUCCUCCAAC PS182 SEQ ID NO 118 GCAAUGUUAUCUGCUUCCUCCAACC SEQ ID NO 119 CAAUGUUAUCUGCUUCCUCCAACCA SEQ ID NO 120 AAUGUUAUCUGCUUCCUCCAACCAU SEQ ID NO 121 AUGUUAUCUGCUUCCUCCAACCAUA SEQ ID NO 122 UGUUAUCUGCUUCCUCCAACCAUAA -
TABLE 3 oligonucleotides for skipping DMD Gene Exon 50 SEQ ID NO 123 CCAAUAGUGGUCAGUCCAGGAGCUA SEQ ID NO 124 CAAUAGUGGUCAGUCCAGGAGCUAG SEQ ID NO 125 AAUAGUGGUCAGUCCAGGAGCUAGG SEQ ID NO 126 AUAGUGGUCAGUCCAGGAGCUAGGU SEQ ID NO 127 AUAGUGGUCAGUCCAGGAGCU PS248 SEQ ID NO 128 UAGUGGUCAGUCCAGGAGCUAGGUC SEQ ID NO 129 AGUGGUCAGUCCAGGAGCUAGGUCA SEQ ID NO 130 GUGGUCAGUCCAGGAGCUAGGUCAG SEQ ID NO 131 UGGUCAGUCCAGGAGCUAGGUCAGG SEQ ID NO 132 GGUCAGUCCAGGAGCUAGGUCAGGC SEQ ID NO 133 GUCAGUCCAGGAGCUAGGUCAGGCU SEQ ID NO 134 UCAGUCCAGGAGCUAGGUCAGGCUG SEQ ID NO 135 CAGUCCAGGAGCUAGGUCAGGCUGC SEQ ID NO 136 AGUCCAGGAGCUAGGUCAGGCUGCU SEQ ID NO 137 GUCCAGGAGCUAGGUCAGGCUGCUU SEQ ID NO 138 UCCAGGAGCUAGGUCAGGCUGCUUU SEQ ID NO 139 CCAGGAGCUAGGUCAGGCUGCUUUG SEQ ID NO 140 CAGGAGCUAGGUCAGGCUGCUUUGC SEQ ID NO 141 AGGAGCUAGGUCAGGCUGCUUUGCC SEQ ID NO 142 GGAGCUAGGUCAGGCUGCUUUGCCC SEQ ID NO 143 GAGCUAGGUCAGGCUGCUUUGCCCU SEQ ID NO 144 AGCUAGGUCAGGCUGCUUUGCCCUC SEQ ID NO 145 GCUAGGUCAGGCUGCUUUGCCCUCA SEQ ID NO 530 CUCAGCUCUUGAAGUAAACGGUUUA SEQ ID NO 532 CAGCUCUUGAAGUAAACGGUUUACC SEQ ID NO 534 GCUCUUGAAGUAAACGGUUUACCGC SEQ ID NO 146 CUAGGUCAGGCUGCUUUGCCCUCAG SEQ ID NO 147 UAGGUCAGGCUGCUUUGCCCUCAGC SEQ ID NO 148 AGGUCAGGCUGCUUUGCCCUCAGCU SEQ ID NO 149 GGUCAGGCUGCUUUGCCCUCAGCUC SEQ ID NO 150 GUCAGGCUGCUUUGCCCUCAGCUCU SEQ ID NO 151 UCAGGCUGCUUUGCCCUCAGCUCUU SEQ ID NO 152 CAGGCUGCUUUGCCCUCAGCUCUUG SEQ ID NO 153 AGGCUGCUUUGCCCUCAGCUCUUGA SEQ ID NO 154 GGCUGCUUUGCCCUCAGCUCUUGAA SEQ ID NO 155 GCUGCUUUGCCCUCAGCUCUUGAAG SEQ ID NO 156 CUGCUUUGCCCUCAGCUCUUGAAGU SEQ ID NO 157 UGCUUUGCCCUCAGCUCUUGAAGUA SEQ ID NO 158 GCUUUGCCCUCAGCUCUUGAAGUAA SEQ ID NO 159 CUUUGCCCUCAGCUCUUGAAGUAAA SEQ ID NO 160 UUUGCCCUCAGCUCUUGAAGUAAAC SEQ ID NO 161 UUGCCCUCAGCUCUUGAAGUAAACG SEQ ID NO 162 UGCCCUCAGCUCUUGAAGUAAACGG SEQ ID NO 163 GCCCUCAGCUCUUGAAGUAAACGGU SEQ ID NO 164 CCCUCAGCUCUUGAAGUAAACGGUU SEQ ID NO 165 CCUCAGCUCUUGAAGUAAAC PS246 SEQ ID NO 166 CCUCAGCUCUUGAAGUAAACG PS247 SEQ ID NO 167 CUCAGCUCUUGAAGUAAACG PS245 SEQ ID NO 529 CCUCAGCUCUUGAAGUAAACGGUUU SEQ ID NO 531 UCAGCUCUUGAAGUAAACGGUUUAC SEQ ID NO 533 AGCUCUUGAAGUAAACGGUUUACCG SEQ ID NO 535 CUCUUGAAGUAAACGGUUUACCGCC -
TABLE 4 oligonucleotides for skipping DMD Gene Exon 51 SEQ ID NO 168 GUACCUCCAACAUCAAGGAAGAUGG SEQ ID NO 169 UACCUCCAACAUCAAGGAAGAUGGC SEQ ID NO 170 ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NO 171 CCUCCAACAUCAAGGAAGAUGGCAU SEQ ID NO 172 CUCCAACAUCAAGGAAGAUGGCAUU SEQ ID NO 173 UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID NO 174 CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID NO 175 CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID NO 176 AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID NO 177 ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 178 CAUCAAGGAAGAUGGCAUUUCUAGU SEQ ID NO 179 AUCAAGGAAGAUGGCAUUUCUAGUU SEQ ID NO 180 UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO 181 CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID NO 182 AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 183 AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID NO 184 GGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID NO 185 GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID NO 186 AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NO 187 AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID NO 188 GAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID NO 189 AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID NO 190 UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID NO 191 GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID NO 192 GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO 193 CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID NO 194 AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID NO 195 UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID NO 196 UUCUAGUUUGGAGAUGGCAGUUUCC SEQ ID NO 197 UCUAGUUUGGAGAUGGCAGUUUCCU SEQ ID NO 198 CUAGUUUGGAGAUGGCAGUUUCCUU SEQ ID NO 199 UAGUUUGGAGAUGGCAGUUUCCUUA SEQ ID NO 200 AGUUUGGAGAUGGCAGUUUCCUUAG SEQ ID NO 201 GUUUGGAGAUGGCAGUUUCCUUAGU SEQ ID NO 202 UUUGGAGAUGGCAGUUUCCUUAGUA SEQ ID NO 203 UUGGAGAUGGCAGUUUCCUUAGUAA SEQ ID NO 204 UGGAGAUGGCAGUUUCCUUAGUAAC SEQ ID NO 205 GAGAUGGCAGUUUCCUUAGUAACCA SEQ ID NO 206 AGAUGGCAGUUUCCUUAGUAACCAC SEQ ID NO 207 GAUGGCAGUUUCCUUAGUAACCACA SEQ ID NO 208 AUGGCAGUUUCCUUAGUAACCACAG SEQ ID NO 209 UGGCAGUUUCCUUAGUAACCACAGG SEQ ID NO 210 GGCAGUUUCCUUAGUAACCACAGGU SEQ ID NO 211 GCAGUUUCCUUAGUAACCACAGGUU SEQ ID NO 212 CAGUUUCCUUAGUAACCACAGGUUG SEQ ID NO 213 AGUUUCCUUAGUAACCACAGGUUGU SEQ ID NO 214 GUUUCCUUAGUAACCACAGGUUGUG SEQ ID NO 215 UUUCCUUAGUAACCACAGGUUGUGU SEQ ID NO 216 UUCCUUAGUAACCACAGGUUGUGUC SEQ ID NO 217 UCCUUAGUAACCACAGGUUGUGUCA SEQ ID NO 218 CCUUAGUAACCACAGGUUGUGUCAC SEQ ID NO 219 CUUAGUAACCACAGGUUGUGUCACC SEQ ID NO 220 UUAGUAACCACAGGUUGUGUCACCA SEQ ID NO 221 UAGUAACCACAGGUUGUGUCACCAG SEQ ID NO 222 AGUAACCACAGGUUGUGUCACCAGA SEQ ID NO 223 GUAACCACAGGUUGUGUCACCAGAG SEQ ID NO 224 UAACCACAGGUUGUGUCACCAGAGU SEQ ID NO 225 AACCACAGGUUGUGUCACCAGAGUA SEQ ID NO 226 ACCACAGGUUGUGUCACCAGAGUAA SEQ ID NO 227 CCACAGGUUGUGUCACCAGAGUAAC SEQ ID NO 228 CACAGGUUGUGUCACCAGAGUAACA SEQ ID NO 229 ACAGGUUGUGUCACCAGAGUAACAG SEQ ID NO 230 CAGGUUGUGUCACCAGAGUAACAGU SEQ ID NO 231 AGGUUGUGUCACCAGAGUAACAGUC SEQ ID NO 232 GGUUGUGUCACCAGAGUAACAGUCU SEQ ID NO 233 GUUGUGUCACCAGAGUAACAGUCUG SEQ ID NO 234 UUGUGUCACCAGAGUAACAGUCUGA SEQ ID NO 235 UGUGUCACCAGAGUAACAGUCUGAG SEQ ID NO 236 GUGUCACCAGAGUAACAGUCUGAGU SEQ ID NO 237 UGUCACCAGAGUAACAGUCUGAGUA SEQ ID NO 238 GUCACCAGAGUAACAGUCUGAGUAG SEQ ID NO 239 UCACCAGAGUAACAGUCUGAGUAGG SEQ ID NO 240 CACCAGAGUAACAGUCUGAGUAGGA SEQ ID NO 241 ACCAGAGUAACAGUCUGAGUAGGAG -
TABLE 5 oligonucleotides for skipping DMD Gene Exon 52 SEQ ID NO 242 AGCCUCUUGAUUGCUGGUCUUGUUU SEQ ID NO 243 GCCUCUUGAUUGCUGGUCUUGUUUU SEQ ID NO 244 CCUCUUGAUUGCUGGUCUUGUUUUU SEQ ID NO 245 CCUCUUGAUUGCUGGUCUUG SEQ ID NO 246 CUCUUGAUUGCUGGUCUUGUUUUUC PS232 SEQ ID NO 247 UCUUGAUUGCUGGUCUUGUUUUUCA SEQ ID NO 248 CUUGAUUGCUGGUCUUGUUUUUCAA SEQ ID NO 249 UUGAUUGCUGGUCUUGUUUUUCAAA SEQ ID NO 250 UGAUUGCUGGUCUUGUUUUUCAAAU SEQ ID NO 251 GAUUGCUGGUCUUGUUUUUCAAAUU SEQ ID NO 252 GAUUGCUGGUCUUGUUUUUC SEQ ID NO 253 AUUGCUGGUCUUGUUUUUCAAAUUU SEQ ID NO 254 UUGCUGGUCUUGUUUUUCAAAUUUU SEQ ID NO 255 UGCUGGUCUUGUUUUUCAAAUUUUG SEQ ID NO 256 GCUGGUCUUGUUUUUCAAAUUUUGG SEQ ID NO 257 CUGGUCUUGUUUUUCAAAUUUUGGG SEQ ID NO 258 UGGUCUUGUUUUUCAAAUUUUGGGC SEQ ID NO 259 GGUCUUGUUUUUCAAAUUUUGGGCA SEQ ID NO 260 GUCUUGUUUUUCAAAUUUUGGGCAG SEQ ID NO 261 UCUUGUUUUUCAAAUUUUGGGCAGC SEQ ID NO 262 CUUGUUUUUCAAAUUUUGGGCAGCG SEQ ID NO 263 UUGUUUUUCAAAUUUUGGGCAGCGG SEQ ID NO 264 UGUUUUUCAAAUUUUGGGCAGCGGU SEQ ID NO 265 GUUUUUCAAAUUUUGGGCAGCGGUA SEQ ID NO 266 UUUUUCAAAUUUUGGGCAGCGGUAA SEQ ID NO 267 UUUUCAAAUUUUGGGCAGCGGUAAU SEQ ID NO 268 UUUCAAAUUUUGGGCAGCGGUAAUG SEQ ID NO 269 UUCAAAUUUUGGGCAGCGGUAAUGA SEQ ID NO 270 UCAAAUUUUGGGCAGCGGUAAUGAG SEQ ID NO 271 CAAAUUUUGGGCAGCGGUAAUGAGU SEQ ID NO 272 AAAUUUUGGGCAGCGGUAAUGAGUU SEQ ID NO 273 AAUUUUGGGCAGCGGUAAUGAGUUC SEQ ID NO 274 AUUUUGGGCAGCGGUAAUGAGUUCU SEQ ID NO 275 UUUUGGGCAGCGGUAAUGAGUUCU SEQ ID NO 276 UUUGGGCAGCGGUAAUGAGUUCUUC SEQ ID NO 277 UUGGGCAGCGGUAAUGAGUUCUUCC SEQ ID NO 278 UGGGCAGCGGUAAUGAGUUCUUCCA SEQ ID NO 279 GGGCAGCGGUAAUGAGUUCUUCCAA SEQ ID NO 280 GGCAGCGGUAAUGAGUUCUUCCAAC SEQ ID NO 281 GCAGCGGUAAUGAGUUCUUCCAACU SEQ ID NO 282 CAGCGGUAAUGAGUUCUUCCAACUG SEQ ID NO 283 AGCGGUAAUGAGUUCUUCCAACUGG SEQ ID NO 284 GCGGUAAUGAGUUCUUCCAACUGGG SEQ ID NO 285 CGGUAAUGAGUUCUUCCAACUGGGG SEQ ID NO 286 GGUAAUGAGUUCUUCCAACUGGGGA SEQ ID NO 287 GGUAAUGAGUUCUUCCAACUGG SEQ ID NO 288 GUAAUGAGUUCUUCCAACUGGGGAC SEQ ID NO 289 UAAUGAGUUCUUCCAACUGGGGACG SEQ ID NO 290 AAUGAGUUCUUCCAACUGGGGACGC SEQ ID NO 291 AUGAGUUCUUCCAACUGGGGACGCC SEQ ID NO 292 UGAGUUCUUCCAACUGGGGACGCCU SEQ ID NO 293 GAGUUCUUCCAACUGGGGACGCCUC SEQ ID NO 294 AGUUCUUCCAACUGGGGACGCCUCU SEQ ID NO 295 GUUCUUCCAACUGGGGACGCCUCUG SEQ ID NO 296 UUCUUCCAACUGGGGACGCCUCUGU SEQ ID NO 297 UCUUCCAACUGGGGACGCCUCUGUU SEQ ID NO 298 CUUCCAACUGGGGACGCCUCUGUUC SEQ ID NO 299 UUCCAACUGGGGACGCCUCUGUUCC PS236 SEQ ID NO 300 UCCAACUGGGGACGCCUCUGUUCCA SEQ ID NO 301 CCAACUGGGGACGCCUCUGUUCCAA SEQ ID NO 302 CAACUGGGGACGCCUCUGUUCCAAA SEQ ID NO 303 AACUGGGGACGCCUCUGUUCCAAAU SEQ ID NO 304 ACUGGGGACGCCUCUGUUCCAAAUC SEQ ID NO 305 CUGGGGACGCCUCUGUUCCAAAUCC SEQ ID NO 306 UGGGGACGCCUCUGUUCCAAAUCCU SEQ ID NO 307 GGGGACGCCUCUGUUCCAAAUCCUG SEQ ID NO 308 GGGACGCCUCUGUUCCAAAUCCUGC SEQ ID NO 309 GGACGCCUCUGUUCCAAAUCCUGCA SEQ ID NO 310 GACGCCUCUGUUCCAAAUCCUGCAU -
TABLE 6 oligonucleotides for skipping DMD Gene Exon 53SEQ ID NO 311 CUCUGGCCUGUCCUAAGACCUGCUC SEQ ID NO 312 UCUGGCCUGUCCUAAGACCUGCUCA SEQ ID NO 313 CUGGCCUGUCCUAAGACCUGCUCAG SEQ ID NO 314 UGGCCUGUCCUAAGACCUGCUCAGC SEQ ID NO 315 GGCCUGUCCUAAGACCUGCUCAGCU SEQ ID NO 316 GCCUGUCCUAAGACCUGCUCAGCUU SEQ ID NO 317 CCUGUCCUAAGACCUGCUCAGCUUC SEQ ID NO 318 CUGUCCUAAGACCUGCUCAGCUUCU SEQ ID NO 319 UGUCCUAAGACCUGCUCAGCUUCUU SEQ ID NO 320 GUCCUAAGACCUGCUCAGCUUCUUC SEQ ID NO 321 UCCUAAGACCUGCUCAGCUUCUUCC SEQ ID NO 322 CCUAAGACCUGCUCAGCUUCUUCCU SEQ ID NO 323 CUAAGACCUGCUCAGCUUCUUCCUU SEQ ID NO 324 UAAGACCUGCUCAGCUUCUUCCUUA SEQ ID NO 325 AAGACCUGCUCAGCUUCUUCCUUAG SEQ ID NO 326 AGACCUGCUCAGCUUCUUCCUUAGC SEQ ID NO 327 GACCUGCUCAGCUUCUUCCUUAGCU SEQ ID NO 328 ACCUGCUCAGCUUCUUCCUUAGCUU SEQ ID NO 329 CCUGCUCAGCUUCUUCCUUAGCUUC SEQ ID NO 330 CUGCUCAGCUUCUUCCUUAGCUUCC SEQ ID NO 331 UGCUCAGCUUCUUCCUUAGCUUCCA SEQ ID NO 332 GCUCAGCUUCUUCCUUAGCUUCCAG SEQ ID NO 333 CUCAGCUUCUUCCUUAGCUUCCAGC SEQ ID NO 334 UCAGCUUCUUCCUUAGCUUCCAGCC SEQ ID NO 335 CAGCUUCUUCCUUAGCUUCCAGCCA SEQ ID NO 336 AGCUUCUUCCUUAGCUUCCAGCCAU SEQ ID NO 337 GCUUCUUCCUUAGCUUCCAGCCAUU SEQ ID NO 338 CUUCUUCCUUAGCUUCCAGCCAUUG SEQ ID NO 339 UUCUUCCUUAGCUUCCAGCCAUUGU SEQ ID NO 340 UCUUCCUUAGCUUCCAGCCAUUGUG SEQ ID NO 341 CUUCCUUAGCUUCCAGCCAUUGUGU SEQ ID NO 342 UUCCUUAGCUUCCAGCCAUUGUGUU SEQ ID NO 343 UCCUUAGCUUCCAGCCAUUGUGUUG SEQ ID NO 344 CCUUAGCUUCCAGCCAUUGUGUUGA SEQ ID NO 345 CUUAGCUUCCAGCCAUUGUGUUGAA SEQ ID NO 346 UUAGCUUCCAGCCAUUGUGUUGAAU SEQ ID NO 347 UAGCUUCCAGCCAUUGUGUUGAAUC SEQ ID NO 348 AGCUUCCAGCCAUUGUGUUGAAUCC SEQ ID NO 349 GCUUCCAGCCAUUGUGUUGAAUCCU SEQ ID NO 350 CUUCCAGCCAUUGUGUUGAAUCCUU SEQ ID NO 351 UUCCAGCCAUUGUGUUGAAUCCUUU SEQ ID NO 352 UCCAGCCAUUGUGUUGAAUCCUUUA SEQ ID NO 353 CCAGCCAUUGUGUUGAAUCCUUUAA SEQ ID NO 354 CAGCCAUUGUGUUGAAUCCUUUAAC SEQ ID NO 355 AGCCAUUGUGUUGAAUCCUUUAACA SEQ ID NO 356 GCCAUUGUGUUGAAUCCUUUAACAU SEQ ID NO 357 CCAUUGUGUUGAAUCCUUUAACAUU SEQ ID NO 358 CAUUGUGUUGAAUCCUUUAACAUUU -
TABLE 7 oligonucleotides for skipping other exons of the DMD gene as identified DMD Gene Exon 6 SEQ ID NO 359 CAUUUUUGACCUACAUGUGG SEQ ID NO 360 UUUGACCUACAUGUGGAAAG SEQ ID NO 361 UACAUUUUUGACCUACAUGUGGAAA G SEQ ID NO 362 GGUCUCCUUACCUAUGA SEQ ID NO 363 UCUUACCUAUGACUAUGGAUGAGA SEQ ID NO 364 AUUUUUGACCUACAUGGGAAAG SEQ ID NO 365 UACGAGUUGAUUGUCGGACCCAG SEQ ID NO 366 GUGGUCUCCUUACCUAUGACUGUGG SEQ ID NO 367 UGUCUCAGUAAUCUUCUUACCUAU DMD Gene Exon 7 SEQ ID NO 368 UGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 369 CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 370 AUUUACCAACCUUCAGGAUCGAGUA SEQ ID NO 371 GGCCUAAAACACAUACACAUA DMD Gene Exon 11 SEQ ID NO 372 CCCUGAGGCAUUCCCAUCUUGAAU SEQ ID NO 373 AGGACUUACUUGCUUUGUUU SEQ ID NO 374 CUUGAAUUUAGGAGAUUCAUCUG SEQ ID NO 375 CAUCUUCUGAUAAUUUUCCUGUU DMD Gene Exon 17 SEQ ID NO 376 CCAUUACAGUUGUCUGUGUU SEQ ID NO 377 UGACAGCCUGUGAAAUCUGUGAG SEQ ID NO 378 UAAUCUGCCUCUUCUUUUGG DMD Gene Exon 19 SEQ ID NO 379 CAGCAGUAGUUGUCAUCUGC SEQ ID NO 380 GCCUGAGCUGAUCUGCUGGCAUCUUGC SEQ ID NO 381 GCCUGAGCUGAUCUGCUGGCAUCUUGCAGUU SEQ ID NO 382 UCUGCUGGCAUCUUGC DMD Gene Exon 21 SEQ ID NO 383 GCCGGUUGACUUCAUCCUGUGC SEQ ID NO 384 GUCUGCAUCCAGGAACAUGGGUC SEQ ID NO 385 UACUUACUGUCUGUAGCUCUUUCU SEQ ID NO 386 CUGCAUCCAGGAACAUGGGUCC SEQ ID NO 387 GUUGAAGAUCUGAUAGCCGGUUGA DMD Gene Exon 44 SEQ ID NO 388 UCAGCUUCUGUUAGCCACUG SEQ ID NO 389 UUCAGCUUCUGUUAGCCACU SEQ ID NO 390 UUCAGCUUCUGUUAGCCACUG SEQ ID NO 391 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 392 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 393 UCAGCUUCUGUUAGCCACUGA SEQ ID NO 394 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 395 UCAGCUUCUGUUAGCCACUGAU SEQ ID NO 396 UUCAGCUUCUGUUAGCCACUGAU SEQ ID NO 397 UCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 398 UUCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 399 UCAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 400 UUCAGCUUCUGUUAGCCACUGAUA SEQ ID NO 401 UCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 402 UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 403 UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 404 UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 405 CAGCUUCUGUUAGCCACUG SEQ ID NO 406 CAGCUUCUGUUAGCCACUGAU SEQ ID NO 407 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 408 CAGCUUCUGUUAGCCACUGAUU SEQ ID NO 409 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 410 CAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 411 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 412 CAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 413 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 414 CAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 415 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 416 AGCUUCUGUUAGCCACUGAU SEQ ID NO 417 GCUUCUGUUAGCCACUGAUU SEQ ID NO 418 AGCUUCUGUUAGCCACUGAUU SEQ ID NO 419 GCUUCUGUUAGCCACUGAUUA SEQ ID NO 420 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 421 GCUUCUGUUAGCCACUGAUUAA SEQ ID NO 422 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 423 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 424 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 425 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 426 CCAUUUGUAUUUAGCAUGUUCCC SEQ ID NO 427 AGAUACCAUUUGUAUUUAGC SEQ ID NO 428 GCCAUUUCUCAACAGAUCU SEQ ID NO 429 GCCAUUUCUCAACAGAUCUGUCA SEQ ID NO 430 AUUCUCAGGAAUUUGUGUCUUUC SEQ ID NO 431 UCUCAGGAAUUUGUGUCUUUC SEQ ID NO 432 GUUCAGCUUCUGUUAGCC SEQ ID NO 433 CUGAUUAAAUAUCUUUAUAU C SEQ ID NO 434 GCCGCCAUUUCUCAACAG SEQ ID NO 435 GUAUUUAGCAUGUUCCCA SEQ ID NO 436 CAGGAAUUUGUGUCUUUC DMD Gene Exon 45 SEQ ID NO 437 UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 438 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 439 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 440 CAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 441 AGUUGCAUUCAAUGUUCUGA SEQ ID NO 442 GAUUGCUGAAUUAUUUCUUCC SEQ ID NO 443 GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID NO 444 AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID NO 445 UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID NO 446 UGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID NO 447 GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID NO 448 CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID NO 449 UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID NO 450 GAAUUAUUUCUUCCCCAGUUGCAUU SEQ ID NO 451 AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID NO 452 AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID NO 453 UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID NO 454 UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID NO 455 AUUUCUUCCCCAGUUGCAUUCAAUG SEQ ID NO 456 UUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID NO 457 UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID NO 458 UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID NO 459 CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID NO 460 UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID NO 461 UCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID NO 462 CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 463 CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID NO 464 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 465 CAGUUGCAUUCAAUGUUCUGACAAC SEQ ID NO 466 AGUUGCAUUCAAUGUUCUGACAACA SEQ ID NO 467 UCC UGU AGA AUA CUG GCA UC SEQ ID NO 468 UGCAGACCUCCUGCCACCGCAGAUUCA SEQ ID NO 469 UUGCAGACCUCCUGCCACCGCAGAUUCAGGCUUC SEQ ID NO 470 GUUGCAUUCAAUGUUCUGACAACAG SEQ ID NO 471 UUGCAUUCAAUGUUCUGACAACAGU SEQ ID NO 472 UGCAUUCAAUGUUCUGACAACAGUU SEQ ID NO 473 GCAUUCAAUGUUCUGACAACAGUUU SEQ ID NO 474 CAUUCAAUGUUCUGACAACAGUUUG SEQ ID NO 475 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 476 UCAAUGUUCUGACAACAGUUUGCCG SEQ ID NO 477 CAAUGUUCUGACAACAGUUUGCCGC SEQ ID NO 478 AAUGUUCUGACAACAGUUUGCCGCU SEQ ID NO 479 AUGUUCUGACAACAGUUUGCCGCUG SEQ ID NO 480 UGUUCUGACAACAGUUUGCCGCUGC SEQ ID NO 481 GUUCUGACAACAGUUUGCCGCUGCC SEQ ID NO 482 UUCUGACAACAGUUUGCCGCUGCCC SEQ ID NO 483 UCUGACAACAGUUUGCCGCUGCCCA SEQ ID NO 484 CUGACAACAGUUUGCCGCUGCCCAA SEQ ID NO 485 UGACAACAGUUUGCCGCUGCCCAAU SEQ ID NO 486 GACAACAGUUUGCCGCUGCCCAAUG SEQ ID NO 487 ACAACAGUUUGCCGCUGCCCAAUGC SEQ ID NO 488 CAACAGUUUGCCGCUGCCCAAUGCC SEQ ID NO 489 AACAGUUUGCCGCUGCCCAAUGCCA SEQ ID NO 490 ACAGUUUGCCGCUGCCCAAUGCCAU SEQ ID NO 491 CAGUUUGCCGCUGCCCAAUGCCAUC SEQ ID NO 492 AGUUUGCCGCUGCCCAAUGCCAUCC SEQ ID NO 493 GUUUGCCGCUGCCCAAUGCCAUCCU SEQ ID NO 494 UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 495 UUGCCGCUGCCCAAUGCCAUCCUGG SEQ ID NO 496 UGCCGCUGCCCAAUGCCAUCCUGGA SEQ ID NO 497 GCCGCUGCCCAAUGCCAUCCUGGAG SEQ ID NO 498 CCGCUGCCCAAUGCCAUCCUGGAGU SEQ ID NO 499 CGCUGCCCAAUGCCAUCCUGGAGUU SEQ ID NO 500 UGUUUUUGAGGAUUGCUGAA SEQ ID NO 501 UGUUCUGACAACAGUUUGCCGCUGCCCAAUGCCA UCCUGG DMD Gene Exon 55 SEQ ID NO 502 CUGUUGCAGUAAUCUAUGAG SEQ ID NO 503 UGCAGUAAUCUAUGAGUUUC SEQ ID NO 504 GAGUCUUCUAGGAGCCUU SEQ ID NO 505 UGCCAUUGUUUCAUCAGCUCUUU SEQ ID NO 506 UCCUGUAGGACAUUGGCAGU SEQ ID NO 507 CUUGGAGUCUUCUAGGAGCC DMD Gene Exon 57 SEQ ID NO 508 UAGGUGCCUGCCGGCUU SEQ ID NO 509 UUCAGCUGUAGCCACACC SEQ ID NO 510 CUGAACUGCUGGAAAGUCGCC SEQ ID NO 511 CUGGCUUCCAAAUGGGACCUGAAAAAGAAC DMD Gene Exon 59 SEQ ID NO 512 CAAUUUUUCCCACUCAGUAUU SEQ ID NO 513 UUGAAGUUCCUGGAGUCUU SEQ ID NO 514 UCCUCAGGAGGCAGCUCUAAAU DMD Gene Exon 62 SEQ ID NO 515 UGGCUCUCUCCCAGGG SEQ ID NO 516 GAGAUGGCUCUCUCCCAGGGACCCUGG SEQ ID NO 517 GGGCACUUUGUUUGGCG DMD Gene Exon 63 SEQ ID NO 518 GGUCCCAGCAAGUUGUUUG SEQ ID NO 519 UGGGAUGGUCCCAGCAAGUUGUUUG SEQ ID NO 520 GUAGAGCUCUGUCAUUUUGGG DMD Gene Exon 65 SEQ ID NO 521 GCUCAAGAGAUCCACUGCAAAAAAC SEQ ID NO 522 GCCAUACGUACGUAUCAUAAACAUUC SEQ ID NO 523 UCUGCAGGAUAUCCAUGGGCUGGUC DMD Gene Exon 66 SEQ ID NO 524 GAUCCUCCCUGUUCGUCCCCUAUUAUG DMD Gene Exon 69 SEQ ID NO 525 UGCUUUAGACUCCUGUACCUGAUA DMD Gene Exon 75 SEQ ID NO 526 GGCGGCCUUUGUGUUGAC SEQ ID NO 527 GGACAGGCCUUUAUGUUCGUGCUGC SEQ ID NO 528 CCUUUAUGUUCGUGCUGCU -
FIG. 1 . In human control myotubes, a series of AONs (PS237, PS238, and PS240;SEQ ID NO exon 43 was tested at 500 nM. PS237 (SEQ ID NO 65) reproducibly induced highest levels ofexon 43 skipping. (M: DNA size marker; NT: non-treated cells) -
FIG. 2 . In myotubes from a DMD patient with an exon 45 deletion, a series of AONs (PS177, PS179, PS181, and PS182;SEQ ID NO 91, 70, 110, and 117 respectively) targetingexon 46 was tested at two different concentrations (50 and 150 nM). PS182 (SEQ ID NO 117) reproducibly induced highest levels ofexon 46 skipping. (M: DNA size marker) -
FIG. 3 . In human control myotubes, a series of AONs (PS245, PS246, PS247, and PS248; SEQ ID NO 167, 165, 166, and 127 respectively) targetingexon 50 was tested at 500 nM. PS248 (SEQ ID NO 127) reproducibly induced highest levels ofexon 50 skipping. (M: DNA size marker; NT: non-treated cells). -
FIG. 4 . In human control myotubes, two novel AONs (PS232 and PS236; SEQ ID NO 246 and 299 respectively) targetingexon 52 were tested at two different concentrations (200 and 500 nM) and directly compared to a previously described AON (52-1). PS236 (SEQ ID NO 299) reproducibly induced highest levels ofexon 52 skipping. (M: DNA size marker; NT: non-treated cells).
Claims (11)
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/631,686 US9499818B2 (en) | 2007-10-26 | 2015-02-25 | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
US15/289,053 US20170044534A1 (en) | 2007-10-26 | 2016-10-07 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US16/024,558 US10876114B2 (en) | 2007-10-26 | 2018-06-29 | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53 |
US17/129,117 US20210139904A1 (en) | 2007-10-26 | 2020-12-21 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP07119351.0 | 2007-10-26 | ||
EP07119351 | 2007-10-26 | ||
PCT/NL2008/050673 WO2009054725A2 (en) | 2007-10-26 | 2008-10-27 | Means and methods for counteracting muscle disorders |
NLPCT/NL2008/050673 | 2008-10-27 | ||
PCT/NL2009/050113 WO2010050802A2 (en) | 2008-10-27 | 2009-03-11 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50- 53. |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2009/050113 Continuation WO2010050802A2 (en) | 2007-10-26 | 2009-03-11 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50- 53. |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/631,686 Continuation US9499818B2 (en) | 2007-10-26 | 2015-02-25 | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
Publications (1)
Publication Number | Publication Date |
---|---|
US20110263682A1 true US20110263682A1 (en) | 2011-10-27 |
Family
ID=39045623
Family Applications (18)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/767,702 Ceased US9243245B2 (en) | 2007-10-26 | 2010-04-26 | Means and methods for counteracting muscle disorders |
US13/094,571 Abandoned US20110263682A1 (en) | 2007-10-26 | 2011-04-26 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US13/094,548 Active US9926557B2 (en) | 2007-10-26 | 2011-04-26 | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US14/097,210 Abandoned US20140113955A1 (en) | 2007-10-26 | 2013-12-04 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/134,971 Abandoned US20140128592A1 (en) | 2007-10-26 | 2013-12-19 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/200,251 Abandoned US20140221458A1 (en) | 2007-10-26 | 2014-03-07 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/542,183 Active US9528109B2 (en) | 2007-10-26 | 2014-11-14 | Methods and means for efficient skipping of exon 45 in duchenne muscular dystrophy pre-mRNA |
US14/631,686 Active US9499818B2 (en) | 2007-10-26 | 2015-02-25 | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
US14/990,712 Abandoned US20160304864A1 (en) | 2007-10-26 | 2016-01-07 | Means and methods for counteracting muscle disorders |
US15/289,053 Abandoned US20170044534A1 (en) | 2007-10-26 | 2016-10-07 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US15/390,836 Abandoned US20170107512A1 (en) | 2007-10-26 | 2016-12-27 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/024,558 Active US10876114B2 (en) | 2007-10-26 | 2018-06-29 | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53 |
US16/229,534 Abandoned US20190112604A1 (en) | 2007-10-26 | 2018-12-21 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/229,821 Abandoned US20190119679A1 (en) | 2007-10-26 | 2018-12-21 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/283,458 Abandoned US20190177725A1 (en) | 2007-10-26 | 2019-02-22 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/584,115 Active US11427820B2 (en) | 2007-10-26 | 2019-09-26 | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US17/129,117 Pending US20210139904A1 (en) | 2007-10-26 | 2020-12-21 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US17/814,781 Pending US20230151362A1 (en) | 2007-10-26 | 2022-07-25 | Methods and means for efficient dkipping of exon 45 in duchenne muscular dystrophy pre-mrna |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/767,702 Ceased US9243245B2 (en) | 2007-10-26 | 2010-04-26 | Means and methods for counteracting muscle disorders |
Family Applications After (16)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/094,548 Active US9926557B2 (en) | 2007-10-26 | 2011-04-26 | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US14/097,210 Abandoned US20140113955A1 (en) | 2007-10-26 | 2013-12-04 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/134,971 Abandoned US20140128592A1 (en) | 2007-10-26 | 2013-12-19 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/200,251 Abandoned US20140221458A1 (en) | 2007-10-26 | 2014-03-07 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US14/542,183 Active US9528109B2 (en) | 2007-10-26 | 2014-11-14 | Methods and means for efficient skipping of exon 45 in duchenne muscular dystrophy pre-mRNA |
US14/631,686 Active US9499818B2 (en) | 2007-10-26 | 2015-02-25 | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
US14/990,712 Abandoned US20160304864A1 (en) | 2007-10-26 | 2016-01-07 | Means and methods for counteracting muscle disorders |
US15/289,053 Abandoned US20170044534A1 (en) | 2007-10-26 | 2016-10-07 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US15/390,836 Abandoned US20170107512A1 (en) | 2007-10-26 | 2016-12-27 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/024,558 Active US10876114B2 (en) | 2007-10-26 | 2018-06-29 | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53 |
US16/229,534 Abandoned US20190112604A1 (en) | 2007-10-26 | 2018-12-21 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/229,821 Abandoned US20190119679A1 (en) | 2007-10-26 | 2018-12-21 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/283,458 Abandoned US20190177725A1 (en) | 2007-10-26 | 2019-02-22 | METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-mRNA |
US16/584,115 Active US11427820B2 (en) | 2007-10-26 | 2019-09-26 | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US17/129,117 Pending US20210139904A1 (en) | 2007-10-26 | 2020-12-21 | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50-53 |
US17/814,781 Pending US20230151362A1 (en) | 2007-10-26 | 2022-07-25 | Methods and means for efficient dkipping of exon 45 in duchenne muscular dystrophy pre-mrna |
Country Status (17)
Country | Link |
---|---|
US (18) | US9243245B2 (en) |
EP (4) | EP3238737B1 (en) |
JP (6) | JP5600064B2 (en) |
CN (5) | CN105641700B (en) |
AU (1) | AU2008317566B2 (en) |
CA (1) | CA2704049A1 (en) |
CY (2) | CY1117286T1 (en) |
DK (1) | DK2203173T3 (en) |
ES (5) | ES2639852T3 (en) |
HK (2) | HK1185098A1 (en) |
HR (1) | HRP20160025T1 (en) |
HU (2) | HUE028662T2 (en) |
IL (4) | IL205322A (en) |
NZ (2) | NZ584793A (en) |
PL (1) | PL2203173T3 (en) |
PT (1) | PT2203173E (en) |
WO (1) | WO2009054725A2 (en) |
Cited By (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100130591A1 (en) * | 2008-10-24 | 2010-05-27 | Peter Sazani | Multiple exon skipping compositions for dmd |
US20110015258A1 (en) * | 2004-06-28 | 2011-01-20 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
WO2013112053A1 (en) * | 2012-01-27 | 2013-08-01 | Prosensa Technologies B.V. | Rna modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy |
US8637483B2 (en) | 2009-11-12 | 2014-01-28 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US20140057964A1 (en) * | 2008-09-11 | 2014-02-27 | Royal Holloway, University Of London | Oligomers |
US9139828B2 (en) | 2008-05-14 | 2015-09-22 | Prosensa Technologies B.V. | Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means |
US9217148B2 (en) | 2013-03-14 | 2015-12-22 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
US20160201089A1 (en) * | 2013-06-05 | 2016-07-14 | Duke University | Rna-guided gene editing and gene regulation |
US9499818B2 (en) | 2007-10-26 | 2016-11-22 | BioMarin Technologies, B.V. | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
US9506058B2 (en) | 2013-03-15 | 2016-11-29 | Sarepta Therapeutics, Inc. | Compositions for treating muscular dystrophy |
US9512424B2 (en) | 2011-12-28 | 2016-12-06 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US9890379B2 (en) | 2006-08-11 | 2018-02-13 | Biomarin Technologies B.V. | Treatment of genetic disorders associated with DNA repeat instability |
US9988629B2 (en) | 2014-03-12 | 2018-06-05 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US10450568B2 (en) | 2015-10-09 | 2019-10-22 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US10676735B2 (en) | 2015-07-22 | 2020-06-09 | Duke University | High-throughput screening of regulatory element function with epigenome editing technologies |
US10676726B2 (en) | 2015-02-09 | 2020-06-09 | Duke University | Compositions and methods for epigenome editing |
US10711256B2 (en) | 2012-04-27 | 2020-07-14 | Duke University | Genetic correction of mutated genes |
US10851373B2 (en) | 2015-09-15 | 2020-12-01 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US11034956B2 (en) * | 2009-04-24 | 2021-06-15 | Biomarin Technologies B.V. | Oligonucleotide comprising an inosine for treating DMD |
USRE48960E1 (en) | 2004-06-28 | 2022-03-08 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20220186217A1 (en) * | 2018-12-06 | 2022-06-16 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US11427817B2 (en) | 2015-08-25 | 2022-08-30 | Duke University | Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases |
WO2022241408A1 (en) | 2021-05-10 | 2022-11-17 | Entrada Therapeutics, Inc. | Compositions and methods for modulating tissue distribution of intracellular therapeutics |
WO2022271818A1 (en) | 2021-06-23 | 2022-12-29 | Entrada Therapeutics, Inc. | Antisense compounds and methods for targeting cug repeats |
US20230045002A1 (en) * | 2021-07-09 | 2023-02-09 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11633496B2 (en) | 2018-08-02 | 2023-04-25 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11679161B2 (en) | 2021-07-09 | 2023-06-20 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy |
US11787869B2 (en) | 2018-08-02 | 2023-10-17 | Dyne Therapeutics, Inc. | Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness |
Families Citing this family (70)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1191097A1 (en) * | 2000-09-21 | 2002-03-27 | Leids Universitair Medisch Centrum | Induction of exon skipping in eukaryotic cells |
CA2524255C (en) | 2003-03-21 | 2014-02-11 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mrna by interfering with the secondary rna structure |
EP3103800B1 (en) | 2003-04-11 | 2018-06-13 | PTC Therapeutics, Inc. | 1,2,4-oxadiazole benzoic acid compound and its use for nonsense suppression and the treatment of disease |
WO2006086667A2 (en) | 2005-02-09 | 2006-08-17 | Avi Bio Pharma, Inc. | Antisense composition and method for treating muscle atrophy |
AU2009310557B2 (en) | 2008-10-27 | 2014-09-11 | Academisch Ziekenhuis Leiden | Methods and means for efficient skipping of exon 45 in Duchenne Muscular Dystrophy pre-mRNA |
US20120270930A1 (en) | 2009-10-29 | 2012-10-25 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Methods and compositions for dysferlin exon-skipping |
CA2807187C (en) | 2010-08-05 | 2019-06-11 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins. |
TWI541024B (en) | 2010-09-01 | 2016-07-11 | 日本新藥股份有限公司 | Antisense nucleic acid |
BR112013020273A2 (en) | 2011-02-08 | 2016-10-18 | Charlotte Mecklenburg Hospital | antisense oligonucleotides |
WO2012138223A2 (en) | 2011-04-05 | 2012-10-11 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Compounds and methods for altering activin receptor-like kinase signalling |
JP5850519B2 (en) | 2011-05-09 | 2016-02-03 | ネッパジーン株式会社 | A therapeutic agent for muscular dystrophy containing morpholino-loaded bubble liposomes as active ingredients |
US9765398B2 (en) * | 2011-07-28 | 2017-09-19 | The Regents Of The University Of California | Exonic splicing enhancers and exonic splicing silencers |
US20130085139A1 (en) | 2011-10-04 | 2013-04-04 | Royal Holloway And Bedford New College | Oligomers |
DE102012103041A1 (en) | 2012-04-10 | 2013-10-10 | Eberhard-Karls-Universität Tübingen Universitätsklinikum | New isolated antisense-oligonucleotide comprising sequence that is hybridized to messenger RNA-splicing-sequence of mutation-bearing exons of pre-messenger RNA of titin-gene and induces skipping of exons, used to treat heart disease |
EP2870246B1 (en) * | 2012-07-03 | 2019-09-11 | BioMarin Technologies B.V. | Oligonucleotide for the treatment of muscular dystrophy patients |
US9849066B2 (en) | 2013-04-24 | 2017-12-26 | Corning Incorporated | Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients |
TWI636978B (en) | 2014-03-06 | 2018-10-01 | 美商Ptc治療公司 | Pharmaceutical compositions and salts of a 1,2,4-oxadiazole benzoic acid |
GB201410693D0 (en) | 2014-06-16 | 2014-07-30 | Univ Southampton | Splicing modulation |
AU2015277924B2 (en) | 2014-06-17 | 2021-02-25 | National Center Of Neurology And Psychiatry | Antisense nucleic acids |
WO2016025469A1 (en) * | 2014-08-11 | 2016-02-18 | The Board Of Regents Of The University Of Texas System | Prevention of muscular dystrophy by crispr/cas9-mediated gene editing |
WO2016054615A2 (en) | 2014-10-03 | 2016-04-07 | Cold Spring Harbor Laboratory | Targeted augmentation of nuclear gene output |
MA41795A (en) | 2015-03-18 | 2018-01-23 | Sarepta Therapeutics Inc | EXCLUSION OF AN EXON INDUCED BY ANTISENSE COMPOUNDS IN MYOSTATIN |
CN107667174B (en) * | 2015-05-21 | 2021-10-26 | 翼治疗有限公司 | Antisense oligonucleotides for treating dystrophic epidermolysis bullosa |
WO2016198676A1 (en) * | 2015-06-10 | 2016-12-15 | Association Institut De Myologie | Combined therapy for duchenne muscular dystrophy |
WO2017062835A2 (en) | 2015-10-09 | 2017-04-13 | Sarepta Therapeutics, Inc. | Compositions and methods for treating duchenne muscular dystrophy and related disorders |
SG11201802870RA (en) | 2015-10-09 | 2018-05-30 | Univ Southampton | Modulation of gene expression and screening for deregulated protein expression |
EP3368042A4 (en) | 2015-10-30 | 2019-06-26 | PTC Therapeutics, Inc. | Methods for treating epilepsy |
WO2017106377A1 (en) | 2015-12-14 | 2017-06-22 | Cold Spring Harbor Laboratory | Antisense oligomers for treatment of autosomal dominant mental retardation-5 and dravet syndrome |
US11096956B2 (en) | 2015-12-14 | 2021-08-24 | Stoke Therapeutics, Inc. | Antisense oligomers and uses thereof |
GB2574525B (en) * | 2015-12-21 | 2020-09-02 | Sutura Therapeutics Ltd | Improved drug delivery by conjugating oligonucleotides to stitched/stapled peptides |
GB2545898B (en) | 2015-12-21 | 2019-10-09 | Sutura Therapeutics Ltd | Improved drug delivery by conjugating oligonucleotides to stitched/stapled peptides |
MA45328A (en) | 2016-04-01 | 2019-02-06 | Avidity Biosciences Llc | NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF |
AU2017250756B2 (en) | 2016-04-15 | 2021-04-08 | Research Institute At Nationwide Children's Hospital | Adeno-associated virus vector delivery of B-sarcoglycan and microRNA-29 and the treatment of muscular dystrophy |
MA45290A (en) * | 2016-05-04 | 2019-03-13 | Wave Life Sciences Ltd | PROCESSES AND COMPOSITIONS OF BIOLOGICALLY ACTIVE AGENTS |
EP3478697A1 (en) | 2016-06-30 | 2019-05-08 | Sarepta Therapeutics, Inc. | Exon skipping oligomers for muscular dystrophy |
JP7125940B2 (en) | 2016-12-19 | 2022-08-25 | サレプタ セラピューティクス, インコーポレイテッド | Exon-skipping oligomeric conjugates for muscular dystrophy |
CA3047010A1 (en) | 2016-12-19 | 2018-06-28 | Sarepta Therapeutics, Inc. | Exon skipping oligomer conjugates for muscular dystrophy |
PL3554553T3 (en) | 2016-12-19 | 2022-11-07 | Sarepta Therapeutics, Inc. | Exon skipping oligomer conjugates for muscular dystrophy |
CA3049424A1 (en) | 2017-01-06 | 2018-07-12 | Avidity Biosciences Llc | Nucleic acid-polypeptide compositions and methods of inducing exon skipping |
PL3596222T3 (en) * | 2017-03-17 | 2023-10-09 | Research Institute At Nationwide Children's Hospital | Adeno-associated virus vector delivery of muscle specific micro-dystrophin to treat muscular dystrophy |
JP2020517613A (en) * | 2017-04-20 | 2020-06-18 | シンセナ アーゲー | Modified oligomeric compounds containing tricyclo DNA nucleosides and uses thereof |
GB201711809D0 (en) * | 2017-07-21 | 2017-09-06 | Governors Of The Univ Of Alberta | Antisense oligonucleotide |
CA3072205A1 (en) | 2017-08-04 | 2019-02-07 | Skyhawk Therapeutics, Inc. | Methods and compositions for modulating splicing |
GB2599884B (en) | 2017-08-25 | 2022-08-31 | Stoke Therapeutics Inc | Antisense oligomers for treatment of conditions and diseases |
EA201991450A1 (en) | 2017-09-22 | 2019-12-30 | Сарепта Терапьютикс, Инк. | OLIGOMER CONJUGATES FOR EXONISM SKIP IN MUSCULAR DYSTROPHY |
EP3684376A4 (en) * | 2017-09-22 | 2021-10-20 | Avidity Biosciences, Inc. | Nucleic acid-polypeptide compositions and methods of inducing exon skipping |
US20200254002A1 (en) | 2017-09-28 | 2020-08-13 | Sarepta Therapeutics, Inc. | Combination therapies for treating muscular dystrophy |
JP2020536060A (en) | 2017-09-28 | 2020-12-10 | サレプタ セラピューティクス, インコーポレイテッド | Combination therapy to treat muscular dystrophy |
EP3687577A1 (en) | 2017-09-28 | 2020-08-05 | Sarepta Therapeutics, Inc. | Combination therapies for treating muscular dystrophy |
KR102527941B1 (en) | 2017-12-06 | 2023-05-02 | 어비디티 바이오사이언시스 인크. | Compositions and methods of treating muscle atrophy and myotonic dystrophy |
EP3755352A4 (en) * | 2018-02-20 | 2021-12-22 | Edgewise Therapeutics, Inc. | Methods and compositions for treating movement disorders |
BR112020020670A2 (en) * | 2018-04-12 | 2021-03-02 | Wave Life Sciences Ltd. | oligonucleotide composition, pharmaceutical composition, method of altering the splicing of a target transcript, method of treating muscular dystrophy, method of preparing an oligonucleotide or an oligonucleotide composition thereof and oligonucleotide |
US10758629B2 (en) | 2018-05-29 | 2020-09-01 | Sarepta Therapeutics, Inc. | Exon skipping oligomer conjugates for muscular dystrophy |
US11168141B2 (en) | 2018-08-02 | 2021-11-09 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
CN109486813B (en) * | 2018-10-10 | 2022-01-18 | 广州医科大学附属第二医院 | U1-snRNA for repairing abnormal splicing of PremRNA of TPP1 gene and application thereof |
WO2020163405A1 (en) | 2019-02-05 | 2020-08-13 | Skyhawk Therapeutics, Inc. | Methods and compositions for modulating splicing |
MX2021012428A (en) * | 2019-04-10 | 2022-01-19 | Ptc Therapeutics Inc | Method for treating nonsense mutation mediated duchenne muscular dystrophy in pediatric patients. |
CN110288555B (en) * | 2019-07-02 | 2022-08-02 | 桂林电子科技大学 | Low-illumination enhancement method based on improved capsule network |
CA3149488A1 (en) * | 2019-08-02 | 2021-02-11 | Research Institute At Nationwide Children's Hospital | Exon 44-targeted nucleic acids and recombinant adeno-associated virus comprising said nucleic acids for treatment of dystrophin-based myopathies |
WO2021055011A1 (en) * | 2019-09-19 | 2021-03-25 | Sudhir Agrawal | Compounds and methods useful for modulating gene splicing |
CN115210376A (en) | 2020-02-28 | 2022-10-18 | 日本新药株式会社 | Antisense nucleic acids inducing skipping of exon 51 |
WO2021188390A1 (en) | 2020-03-19 | 2021-09-23 | Avidity Biosciences, Inc. | Compositions and methods of treating facioscapulohumeral muscular dystrophy |
US20230287410A1 (en) | 2020-05-11 | 2023-09-14 | Stoke Therapeutics, Inc. | Opa1 antisense oligomers for treatment of conditions and diseases |
KR20240009393A (en) | 2021-03-31 | 2024-01-22 | 엔트라다 테라퓨틱스, 인크. | Cyclic cell penetrating peptide |
EP4088722A1 (en) * | 2021-05-12 | 2022-11-16 | Justus-Liebig-Universität Gießen | Pharmaceutical compositions, uses thereof and methods for treatment of genetic diseases caused by intronic splice-acceptor site mutations |
AU2022307934A1 (en) * | 2021-07-09 | 2024-01-25 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
CA3231330A1 (en) | 2021-09-16 | 2023-03-23 | Avidity Biosciences, Inc. | Compositions and methods of treating facioscapulohumeral muscular dystrophy |
EP4215614A1 (en) | 2022-01-24 | 2023-07-26 | Dynacure | Combination therapy for dystrophin-related diseases |
WO2023168427A1 (en) | 2022-03-03 | 2023-09-07 | Yale University | Compositions and methods for delivering therapeutic polynucleotides for exon skipping |
WO2024069229A2 (en) | 2022-08-03 | 2024-04-04 | Sutura Therapeutics Ltd | Biologically active compounds |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060024715A1 (en) * | 2004-07-02 | 2006-02-02 | Affymetrix, Inc. | Methods for genotyping polymorphisms in humans |
US20060099612A1 (en) * | 2004-09-02 | 2006-05-11 | Suntory Limited | Method for analyzing genes of industrial yeasts |
US20060160121A1 (en) * | 2004-10-05 | 2006-07-20 | Wyeth | Probe arrays for detecting multiple strains of different species |
US20070134655A1 (en) * | 2002-11-14 | 2007-06-14 | Itzhak Bentwich | Bioinformatically detectable group of novel regulatory genes and uses thereof |
US20100130591A1 (en) * | 2008-10-24 | 2010-05-27 | Peter Sazani | Multiple exon skipping compositions for dmd |
Family Cites Families (215)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2752581C2 (en) | 1977-11-25 | 1979-12-13 | Wolfgang 4044 Kaarst Keil | Device for the complete demineralisation of water |
US5034506A (en) | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US5541308A (en) | 1986-11-24 | 1996-07-30 | Gen-Probe Incorporated | Nucleic acid probes for detection and/or quantitation of non-viral organisms |
US5766847A (en) | 1988-10-11 | 1998-06-16 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Process for analyzing length polymorphisms in DNA regions |
DE3834636A1 (en) | 1988-10-11 | 1990-04-19 | Max Planck Gesellschaft | METHOD FOR ANALYZING LENGTH POLYMORPHISMS IN DNA AREAS |
US6867195B1 (en) | 1989-03-21 | 2005-03-15 | Vical Incorporated | Lipid-mediated polynucleotide administration to reduce likelihood of subject's becoming infected |
US5681941A (en) | 1990-01-11 | 1997-10-28 | Isis Pharmaceuticals, Inc. | Substituted purines and oligonucleotide cross-linking |
US5608046A (en) | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
FR2675803B1 (en) | 1991-04-25 | 1996-09-06 | Genset Sa | CLOSED, ANTISENSE AND SENSE OLIGONUCLEOTIDES AND THEIR APPLICATIONS. |
CA2082411A1 (en) | 1991-06-28 | 1992-12-29 | Robert D. Rosenberg | Localized oligonucleotide therapy |
AU659482B2 (en) | 1991-06-28 | 1995-05-18 | Massachusetts Institute Of Technology | Localized oligonucleotide therapy |
US6200747B1 (en) | 1992-01-28 | 2001-03-13 | North Shore University Hospital Research Corp. | Method and kits for detection of fragile X specific, GC-rich DNA sequences |
US5869252A (en) | 1992-03-31 | 1999-02-09 | Abbott Laboratories | Method of multiplex ligase chain reaction |
US6172208B1 (en) | 1992-07-06 | 2001-01-09 | Genzyme Corporation | Oligonucleotides modified with conjugate groups |
US5418139A (en) | 1993-02-10 | 1995-05-23 | University Of Iowa Research Foundation | Method for screening for cardiomyopathy |
CA2116280A1 (en) | 1993-03-05 | 1994-09-06 | Marcy E. Macdonald | Huntingtin dna, protein and uses thereof |
CA2162361C (en) | 1993-05-11 | 2008-10-21 | Ryszard Kole | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
US5695933A (en) | 1993-05-28 | 1997-12-09 | Massachusetts Institute Of Technology | Direct detection of expanded nucleotide repeats in the human genome |
US5741645A (en) | 1993-06-29 | 1998-04-21 | Regents Of The University Of Minnesota | Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis |
US5624803A (en) | 1993-10-14 | 1997-04-29 | The Regents Of The University Of California | In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom |
US5627263A (en) | 1993-11-24 | 1997-05-06 | La Jolla Cancer Research Foundation | Integrin-binding peptides |
DE4342605A1 (en) | 1993-12-14 | 1995-06-22 | Buna Gmbh | Functionalized olefin homo- and copolymers |
US5962332A (en) | 1994-03-17 | 1999-10-05 | University Of Massachusetts | Detection of trinucleotide repeats by in situ hybridization |
DE69503126T2 (en) | 1994-05-05 | 1998-11-12 | Beckman Instruments Inc | REPETITIVE OLIGONUCLEOTIDE MATRIX |
US5968909A (en) | 1995-08-04 | 1999-10-19 | Hybridon, Inc. | Method of modulating gene expression with reduced immunostimulatory response |
US5854223A (en) | 1995-10-06 | 1998-12-29 | The Trustees Of Columbia University In The City Of New York | S-DC28 as an antirestenosis agent after balloon injury |
US20070173465A9 (en) | 1995-10-11 | 2007-07-26 | Monahan Sean D | Expression of zeta negative and zeta positive nucleic acids using a dystrophin gene |
US7034009B2 (en) | 1995-10-26 | 2006-04-25 | Sirna Therapeutics, Inc. | Enzymatic nucleic acid-mediated treatment of ocular diseases or conditions related to levels of vascular endothelial growth factor receptor (VEGF-R) |
US6300060B1 (en) | 1995-11-09 | 2001-10-09 | Dana-Farber Cancer Institute, Inc. | Method for predicting the risk of prostate cancer morbidity and mortality |
DE69729179T2 (en) | 1996-02-14 | 2004-12-30 | Isis Pharmaceuticals, Inc., Carlsbad | Patchy sugar-modified oligonucleotides |
EP0869186A4 (en) | 1996-07-18 | 2002-04-03 | Srl Inc | Method for the diagnosis of spinocerebellar ataxia type 2 and primers therefor |
WO1998018920A1 (en) | 1996-10-30 | 1998-05-07 | Srl, Inc. | cDNA FRAGMENTS OF GENE CAUSATIVE OF SPINOCEREBELLAR ATAXIA TYPE 2 |
US5853995A (en) | 1997-01-07 | 1998-12-29 | Research Development Foundation | Large scale genotyping of diseases and a diagnostic test for spinocerebellar ataxia type 6 |
AU6591798A (en) | 1997-03-31 | 1998-10-22 | Yale University | Nucleic acid catalysts |
US20020137890A1 (en) | 1997-03-31 | 2002-09-26 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
AU7265298A (en) | 1997-04-29 | 1998-11-24 | Trustees Of Boston University | Methods and compositions for targeted dna differential display |
US6329501B1 (en) | 1997-05-29 | 2001-12-11 | Auburn University | Methods and compositions for targeting compounds to muscle |
US6514755B1 (en) | 1998-08-18 | 2003-02-04 | Regents Of The University Of Minnesota | SCA7 gene and methods of use |
US6280938B1 (en) | 1997-08-19 | 2001-08-28 | Regents Of The University Of Minnesota | SCA7 gene and method of use |
US6794499B2 (en) | 1997-09-12 | 2004-09-21 | Exiqon A/S | Oligonucleotide analogues |
AU750947C (en) | 1997-09-22 | 2003-05-22 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Nucleic acid catalysts with endonuclease activity |
US6130207A (en) | 1997-11-05 | 2000-10-10 | South Alabama Medical Science Foundation | Cell-specific molecule and method for importing DNA into a nucleus |
JP3012923B2 (en) | 1998-01-26 | 2000-02-28 | 新潟大学長 | Drug for treating CAG repeat disease |
KR100280219B1 (en) | 1998-02-26 | 2001-04-02 | 이수빈 | Diagnostic Method and Diagnostic Reagent of Neuropsychiatric Disease Using Trinucleic Acid Repeat Sequence |
US6322978B1 (en) | 1998-04-20 | 2001-11-27 | Joslin Diabetes Center, Inc. | Repeat polymorphism in the frataxin gene and uses therefore |
CA2330574A1 (en) | 1998-04-29 | 1999-11-04 | Ribozyme Pharmaceuticals, Inc. | Nucleoside triphosphates and their incorporation into ribozymes |
EP1089764B1 (en) | 1998-06-10 | 2004-09-01 | Biognostik Gesellschaft für biomolekulare Diagnostik mbH | Stimulating the immune system |
US6924355B2 (en) | 1998-09-01 | 2005-08-02 | Genentech, Inc. | PRO1343 polypeptides |
CA2343934A1 (en) | 1998-09-25 | 2000-04-06 | The Children's Medical Center Corporation | Short peptides which selectively modulate the activity of protein kinases |
US6210892B1 (en) | 1998-10-07 | 2001-04-03 | Isis Pharmaceuticals, Inc. | Alteration of cellular behavior by antisense modulation of mRNA processing |
US6172216B1 (en) | 1998-10-07 | 2001-01-09 | Isis Pharmaceuticals Inc. | Antisense modulation of BCL-X expression |
EP1124950B1 (en) * | 1998-10-26 | 2006-07-12 | Avi Biopharma, Inc. | Morpholino based p53 antisense oligonucleotide, and uses thereof |
US6399575B1 (en) | 1998-11-10 | 2002-06-04 | Auburn University | Methods and compositions for targeting compounds to the central nervous system |
US6133031A (en) | 1999-08-19 | 2000-10-17 | Isis Pharmaceuticals Inc. | Antisense inhibition of focal adhesion kinase expression |
US20040226056A1 (en) | 1998-12-22 | 2004-11-11 | Myriad Genetics, Incorporated | Compositions and methods for treating neurological disorders and diseases |
US20020049173A1 (en) | 1999-03-26 | 2002-04-25 | Bennett C. Frank | Alteration of cellular behavior by antisense modulation of mRNA processing |
US6379698B1 (en) | 1999-04-06 | 2002-04-30 | Isis Pharmaceuticals, Inc. | Fusogenic lipids and vesicles |
KR20020013519A (en) | 1999-04-08 | 2002-02-20 | 추후제출 | Antisense Oligonucleotides Comprising Universal and/or Degenerate Bases |
JP2000325085A (en) | 1999-05-21 | 2000-11-28 | Masafumi Matsuo | Pharmaceutical composition for treatment of duchenne muscular dystrophy |
US20030236214A1 (en) | 1999-06-09 | 2003-12-25 | Wolff Jon A. | Charge reversal of polyion complexes and treatment of peripheral occlusive disease |
US6656730B1 (en) | 1999-06-15 | 2003-12-02 | Isis Pharmaceuticals, Inc. | Oligonucleotides conjugated to protein-binding drugs |
US6355481B1 (en) | 1999-06-18 | 2002-03-12 | Emory University | Hybridoma cell line and monoclonal antibody for huntingtin protein |
CA2403243A1 (en) | 1999-08-31 | 2001-03-08 | Ribozyme Pharmaceuticals, Inc. | Nucleic acid based modulators of gene expression |
AU7856600A (en) | 1999-10-04 | 2001-05-10 | University Of Medicine And Dentistry Of New Jersey | Novel carbamates and ureas |
US6165786A (en) | 1999-11-03 | 2000-12-26 | Isis Pharmaceuticals, Inc. | Antisense modulation of nucleolin expression |
WO2001059102A2 (en) | 2000-02-08 | 2001-08-16 | Ribozyme Pharmaceuticals, Inc. | Nucleozymes with endonuclease activity |
EP1133993A1 (en) | 2000-03-10 | 2001-09-19 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Substances for the treatment of spinal muscular atrophy |
US20020187931A1 (en) | 2000-04-13 | 2002-12-12 | Michael Hayden | Modulating cell survival by modulating huntingtin function |
US6653467B1 (en) | 2000-04-26 | 2003-11-25 | Jcr Pharmaceutical Co., Ltd. | Medicament for treatment of Duchenne muscular dystrophy |
AU2001261063A1 (en) | 2000-04-28 | 2001-11-12 | Xiao Xiao | Dna sequences encoding dystrophin minigenes and methods of use thereof |
ES2238044T3 (en) | 2000-05-01 | 2005-08-16 | Hybridon, Inc. | MODULATION OF IMMUNOLOGICAL STIMULATION MEDIATED BY THE CPG OLIGONUCLEOTIDE BY POSITIONAL MODIFICATION OF NUCLEOSIDS. |
US20020013287A1 (en) | 2000-05-09 | 2002-01-31 | Reliable Biopharmaceuticals, Inc. St Louis Missouri | Polymeric compounds useful as prodrugs |
IL153200A0 (en) | 2000-05-31 | 2003-07-06 | Genset Sa | PHARMACEUTICAL COMPOSITIONS COMPRISING A gOBG3 |
CN1326990A (en) | 2000-06-07 | 2001-12-19 | 上海博德基因开发有限公司 | New polypeptide-human DNA-like CGG repeative conjugated protein 16.17 and polynucleotide for encoding such polypeptide |
US20030124523A1 (en) | 2000-06-22 | 2003-07-03 | Asselbergs Fredericus Alphonsus Maria | Organic compounds |
US6794192B2 (en) | 2000-06-29 | 2004-09-21 | Pfizer Inc. | Target |
RU2165149C1 (en) | 2000-07-03 | 2001-04-20 | Шапошников Валерий Геннадьевич | "sugar wool" products forming and packaging method |
US6727355B2 (en) * | 2000-08-25 | 2004-04-27 | Jcr Pharmaceuticals Co., Ltd. | Pharmaceutical composition for treatment of Duchenne muscular dystrophy |
JP4836366B2 (en) | 2000-08-25 | 2011-12-14 | 雅文 松尾 | Duchenne muscular dystrophy treatment |
EP1191097A1 (en) | 2000-09-21 | 2002-03-27 | Leids Universitair Medisch Centrum | Induction of exon skipping in eukaryotic cells |
AU3922802A (en) | 2000-10-02 | 2002-05-27 | Keck Graduate Inst | Methods for identifying nucleotides at defined positions in target nucleic acidsusing fluorescence polarization |
ATE400656T1 (en) | 2000-10-06 | 2008-07-15 | Univ Michigan | MINI-DYSTROPHIN NUCLEIC ACID AND PEPTIDE SEQUENCES |
US6623927B1 (en) | 2000-11-08 | 2003-09-23 | Council Of Scientific And Industrial Research | Method of detection of allelic variants of SCA2 gene |
AU2002236499A8 (en) | 2000-11-30 | 2009-12-03 | Uab Research Foundation | Receptor-mediated uptake of peptides that bind the human transferrin receptor |
US7001994B2 (en) | 2001-01-18 | 2006-02-21 | Genzyme Corporation | Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins |
TWI329129B (en) | 2001-02-08 | 2010-08-21 | Wyeth Corp | Modified and stabilized gdf propeptides and uses thereof |
KR100408053B1 (en) | 2001-02-13 | 2003-12-01 | 엘지전자 주식회사 | Torque ripple reduction method for srm |
US20070021360A1 (en) * | 2001-04-24 | 2007-01-25 | Nyce Jonathan W | Compositions, formulations and kit with anti-sense oligonucleotide and anti-inflammatory steroid and/or obiquinone for treatment of respiratory and lung disesase |
CA2414782C (en) | 2001-05-11 | 2012-10-09 | Regents Of The University Of Minnesota | Intron associated with myotonic dystrophy type 2 and methods of use |
US20050014172A1 (en) | 2002-02-20 | 2005-01-20 | Ivan Richards | RNA interference mediated inhibition of muscarinic cholinergic receptor gene expression using short interfering nucleic acid (siNA) |
AU2004266311B2 (en) | 2001-05-18 | 2009-07-23 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA) |
US20050282188A1 (en) | 2001-05-18 | 2005-12-22 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA) |
US20050277133A1 (en) | 2001-05-18 | 2005-12-15 | Sirna Therapeutics, Inc. | RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA) |
IL143379A (en) | 2001-05-24 | 2013-11-28 | Yissum Res Dev Co | Antisense oligonucleotide against the r isophorm of human ache and uses thereof |
EP1406875B1 (en) | 2001-06-26 | 2013-07-31 | Bristol-Myers Squibb Company | N-heterocyclic inhibitors of tnf-alpha expression |
JP4903984B2 (en) | 2001-07-06 | 2012-03-28 | トピジェン・ファーマシューティカルズ・インコーポレーテッド | Methods for increasing the efficiency of oligonucleotides in vivo and inhibiting inflammation in mammals |
US20030109476A1 (en) | 2001-08-07 | 2003-06-12 | Kmiec Eric B. | Compositions and methods for the prevention and treatment of Huntington's disease |
US20070264194A1 (en) | 2001-08-10 | 2007-11-15 | The Scripps Research Institute | Peptides That Bind To Atherosclerotic Lesions |
US20060074034A1 (en) | 2001-09-17 | 2006-04-06 | Collins Douglas A | Cobalamin mediated delivery of nucleic acids, analogs and derivatives thereof |
KR20030035047A (en) | 2001-10-29 | 2003-05-09 | (주)바이오코돈 | Use of BMP-4 gene and its gene product for treatment and diagnosis of Lichen Planus |
WO2003037172A2 (en) | 2001-11-01 | 2003-05-08 | Gpc Biotech Inc. | Endothelial-cell binding peptides for diagnosis and therapy |
US20030134790A1 (en) | 2002-01-11 | 2003-07-17 | University Of Medicine And Dentistry Of New Jersey | Bone Morphogenetic Protein-2 And Bone Morphogenetic Protein-4 In The Treatment And Diagnosis Of Cancer |
US20030203356A1 (en) | 2002-01-22 | 2003-10-30 | The Cleveland Clinic Foundation | RNase L activator-antisense complexes |
WO2003069330A1 (en) | 2002-02-11 | 2003-08-21 | The Trustees Of Columbia University In The City Of New York | System and method for identifying proteins involved in force-initiated signal transduction |
US20050096284A1 (en) | 2002-02-20 | 2005-05-05 | Sirna Therapeutics, Inc. | RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA) |
MXPA04008419A (en) | 2002-03-01 | 2004-11-26 | Univ Tulane | Conjugates of therapeutic or cytotoxic agents and biologically active peptides. |
US20040101852A1 (en) | 2002-11-21 | 2004-05-27 | Isis Pharmaceuticals Inc. | Modulation of CGG triplet repeat binding protein 1 expression |
ITRM20020253A1 (en) | 2002-05-08 | 2003-11-10 | Univ Roma | SNRNA CHEMICAL MOLECULES WITH ANTISENSE SEQUENCES FOR SPLICING JUNCTIONS OF THE DYSTROPHINE GENE AND THERAPEUTIC APPLICATIONS. |
US20040102395A1 (en) | 2002-11-22 | 2004-05-27 | Isis Pharmaceuticals Inc. | Modulation of IAP-like expression |
EP1380644A1 (en) | 2002-07-08 | 2004-01-14 | Kylix B.V. | The use of specified TCF target genes to identify drugs for the treatment of cancer, in particular colorectal cancer, in which TCF/beta-catenin/WNT signalling plays a central role |
WO2004011060A2 (en) | 2002-07-26 | 2004-02-05 | Mirus Corporation | Delivery of molecules and complexes to mammalian cells in vivo |
US20050255086A1 (en) | 2002-08-05 | 2005-11-17 | Davidson Beverly L | Nucleic acid silencing of Huntington's Disease gene |
AU2003282078A1 (en) | 2002-08-12 | 2004-02-25 | Universite De Sherbrooke | Methods to reprogram splice site selection in pre-messenger rnas |
GB0219143D0 (en) | 2002-08-16 | 2002-09-25 | Univ Leicester | Modified tailed oligonucleotides |
US20040219565A1 (en) | 2002-10-21 | 2004-11-04 | Sakari Kauppinen | Oligonucleotides useful for detecting and analyzing nucleic acids of interest |
DE60219215T2 (en) | 2002-10-23 | 2008-01-03 | Centre For Research And Technology Hellas/Intitute Of Agrobiotechnology In.A, Thermi | PRION-BINDING PEPTIDE SEQUENCES |
US7892793B2 (en) | 2002-11-04 | 2011-02-22 | University Of Massachusetts | Allele-specific RNA interference |
ES2440284T3 (en) | 2002-11-14 | 2014-01-28 | Thermo Fisher Scientific Biosciences Inc. | SiRNA directed to tp53 |
US7655785B1 (en) | 2002-11-14 | 2010-02-02 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof |
EP2135948B1 (en) | 2002-11-25 | 2014-09-17 | Masafumi Matsuo | ENA nucleic acid drugs modifying splicing in mRNA precursor |
GB0228079D0 (en) | 2002-12-02 | 2003-01-08 | Laxdale Ltd | Huntington's Disease |
ATE479752T1 (en) | 2003-03-07 | 2010-09-15 | Alnylam Pharmaceuticals Inc | THERAPEUTIC COMPOSITIONS |
CA2524255C (en) * | 2003-03-21 | 2014-02-11 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mrna by interfering with the secondary rna structure |
US7514551B2 (en) | 2003-04-03 | 2009-04-07 | Enzo Life Sciences, Inc. | Multisignal labeling reagents, and processes and uses therefor |
WO2004101787A1 (en) | 2003-05-14 | 2004-11-25 | Japan Science And Technology Agency | Inhibition of the expression of huntington gene |
KR20060026860A (en) | 2003-06-02 | 2006-03-24 | 와이어쓰 | Use of myostatin (gdf8) inhibitors in conjunction with corticosteroids for treating neuromuscular disorders |
ES2302898T3 (en) | 2003-07-11 | 2008-08-01 | Lbr Medbiotech B.V. | TRANSFER OF GENES TO MUSCLE CELLS MEDIATED BY THE MANOSA-6-PHOSPHATE RECEIVER. |
US20050048495A1 (en) | 2003-08-29 | 2005-03-03 | Baker Brenda F. | Isoform-specific targeting of splice variants |
US20050054752A1 (en) | 2003-09-08 | 2005-03-10 | O'brien John P. | Peptide-based diblock and triblock dispersants and diblock polymers |
US7355018B2 (en) | 2003-09-30 | 2008-04-08 | Regeneron Pharmaceuticals, Inc. | Modified IGF1 polypeptides with increased stability and potency |
US20050222009A1 (en) | 2003-10-14 | 2005-10-06 | Itschak Lamensdorf | Dual phase - PNA conjugates for the delivery of PNA through the blood brain barrier |
US20050191636A1 (en) | 2004-03-01 | 2005-09-01 | Biocept, Inc. | Detection of STRP, such as fragile X syndrome |
US20080207538A1 (en) | 2004-03-11 | 2008-08-28 | Lawrence David S | Enhanced Production of Functional Proteins From Defective Genes |
WO2005105995A2 (en) | 2004-04-14 | 2005-11-10 | Sirna Therapeutics, Inc. | RNA INTERFERENCE MEDIATED TREATMENT OF POLYGLUTAMINE (POLYQ) REPEAT EXPANSION DISEASES USING SHORT INTERFERING NUCLEIC ACID (siNA) |
EP1752536A4 (en) | 2004-05-11 | 2008-04-16 | Alphagen Co Ltd | Polynucleotide causing rna interfere and method of regulating gene expression with the use of the same |
US20050288246A1 (en) | 2004-05-24 | 2005-12-29 | Iversen Patrick L | Peptide conjugated, inosine-substituted antisense oligomer compound and method |
CA2567810A1 (en) | 2004-05-27 | 2005-12-08 | Acceleron Pharma Inc. | Cerberus/coco derivatives and uses thereof |
DE602005026386D1 (en) | 2004-06-28 | 2011-03-31 | Univ Western Australia | ANTISENSE OLIGONUCLEOTIDES FOR THE INDUCTION OF EXON-SKIPPING AND METHOD OF USE THEREOF |
EP1618881A1 (en) * | 2004-07-20 | 2006-01-25 | Santhera Pharmaceuticals (Schweiz) GmbH | Use of non-glucocorticoid steroids for the treatment of muscular dystrophy |
US20110046200A1 (en) | 2004-08-03 | 2011-02-24 | Michael T Howard | Use of antisense oligonucleotides to effect translation modulation |
ITRM20040568A1 (en) | 2004-11-18 | 2005-02-18 | Uni Degli Studi Di Roma Tor Vergata | USE OF THE "PHAGE DISPLAY" TECHNIQUE FOR THE IDENTIFICATION OF PEPTIDES WITH CAPACITY OF STAMIN CELLS / PROGENITOR, PEPTIDES SO OBTAINED AND THEIR USES. |
US7838657B2 (en) | 2004-12-03 | 2010-11-23 | University Of Massachusetts | Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences |
KR100663277B1 (en) | 2004-12-20 | 2007-01-02 | 삼성전자주식회사 | Device and?method for processing system-related event in wireless terminal |
US20060148740A1 (en) | 2005-01-05 | 2006-07-06 | Prosensa B.V. | Mannose-6-phosphate receptor mediated gene transfer into muscle cells |
US20120122801A1 (en) | 2005-01-05 | 2012-05-17 | Prosensa B.V. | Mannose-6-phosphate receptor mediated gene transfer into muscle cells |
WO2006083800A2 (en) | 2005-01-31 | 2006-08-10 | University Of Iowa Research Foundation | Nucleic acid silencing of huntington's disease gene |
EP1877099B1 (en) | 2005-04-06 | 2012-09-19 | Genzyme Corporation | Therapeutic conjugates comprising a lysosomal enzyme, polysialic acid and a targeting moiety |
KR20080031164A (en) | 2005-04-22 | 2008-04-08 | 아카데미슈 지켄후이스 라이덴 | Modulation of exon recognition in pre-mrna by interfering with the binding of sr proteins and by interfering with seconcary rna structure |
WO2006121960A2 (en) | 2005-05-06 | 2006-11-16 | Medtronic, Inc. | Methods and sequences to suppress primate huntington gene expression |
US7902352B2 (en) | 2005-05-06 | 2011-03-08 | Medtronic, Inc. | Isolated nucleic acid duplex for reducing huntington gene expression |
WO2006121277A1 (en) | 2005-05-09 | 2006-11-16 | Korea Research Institute Of Standards And Science | Fret probes for fluorescent detection of the epsps gene |
ES2702531T3 (en) | 2005-06-23 | 2019-03-01 | Biogen Ma Inc | Compositions and procedures for SMN2 splicing modulation |
EP2062980B1 (en) | 2005-06-28 | 2011-08-31 | Medtronic, Inc. | Methods and sequences to preferentially suppress expression of mutated huntingtin gene. |
DK2179737T3 (en) * | 2005-07-01 | 2013-11-11 | Index Pharmaceuticals Ab | MODULE RESPONSE ON STEROIDS |
JP5111385B2 (en) | 2005-10-28 | 2013-01-09 | アルナイラム ファーマシューティカルズ, インコーポレイテッド | Composition and method for suppressing expression of huntingtin gene |
US7906617B2 (en) | 2005-12-15 | 2011-03-15 | E. I. Du Pont De Nemours And Company | Polyethylene binding peptides and methods of use |
EP2422819B1 (en) | 2006-01-26 | 2017-03-01 | Ionis Pharmaceuticals, Inc. | Compositions and their uses directed to Huntingtin |
WO2007123391A1 (en) | 2006-04-20 | 2007-11-01 | Academisch Ziekenhuis Leiden | Therapeutic intervention in a genetic disease in an individual by modifying expression of an aberrantly expressed gene. |
EP1857548A1 (en) * | 2006-05-19 | 2007-11-21 | Academisch Ziekenhuis Leiden | Means and method for inducing exon-skipping |
US7855053B2 (en) | 2006-07-19 | 2010-12-21 | The Regents Of The University Of California | Methods for detecting the presence of expanded CGG repeats in the FMR1 gene 5′ untranslated region |
CN101501193B (en) | 2006-08-11 | 2013-07-03 | 普罗森那技术公司 | Methods and means for treating DNA repeat instability associated genetic disorders |
ES2373246T3 (en) | 2006-08-11 | 2012-02-01 | Prosensa Technologies B.V. | COMPLEMENTARY MONOCATENARY OLIGONUCLEOTIDES OF REPETITIVE ELEMENTS FOR THE TREATMENT OF GENETIC DISORDERS ASSOCIATED WITH THE INSTABILITY OF DNA REPETITIONS. |
AU2007300529A1 (en) | 2006-09-27 | 2008-04-03 | Merck Sharp & Dohme Corp. | Acylated piperidine derivatives as melanocortin-4 receptor modulators |
EP2087110A2 (en) | 2006-10-11 | 2009-08-12 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Influenza targets |
WO2008103755A1 (en) | 2007-02-20 | 2008-08-28 | Mayo Foundation For Medical Education And Research | Treating cancer with viral nucleic acid |
PL381824A1 (en) | 2007-02-22 | 2008-09-01 | Instytut Chemii Bioorganicznej Pan W Poznaniu | The sequence of siRNA, vector, molecular target for siRNA reagents and vectors introduced to cells and tissues, the manner of assessment of specifity of silencing of a mutated transcript, the manner of testing of influences of RNA interference route of enz |
WO2009005793A2 (en) | 2007-06-29 | 2009-01-08 | Avi Biopharma, Inc. | Tissue specific peptide conjugates and methods |
NZ582521A (en) | 2007-07-12 | 2011-09-30 | Prosensa Technologies Bv | A conjugate comprising the amino acid sequence LGAQSNF for targeting compounds to muscle tissue |
JP2010533170A (en) | 2007-07-12 | 2010-10-21 | プロセンサ テクノロジーズ ビー.ブイ. | Molecules for targeting compounds to various selected organs, tissues or tumor cells |
US9212205B2 (en) | 2007-07-26 | 2015-12-15 | University Of Rochester | Nucleic acid binding compounds and methods of use |
US8088904B2 (en) | 2007-08-15 | 2012-01-03 | Isis Pharmaceuticals, Inc. | Tetrahydropyran nucleic acid analogs |
NZ584793A (en) | 2007-10-26 | 2012-05-25 | Academisch Ziekenhuis Leiden | Means and methods for counteracting muscle disorders |
EP2249874A1 (en) | 2008-02-08 | 2010-11-17 | ProSensa Holding BV | Methods and means for treating dna repeat instability associated genetic disorders |
WO2009101399A1 (en) | 2008-02-12 | 2009-08-20 | Isis Innovation Limited | Treatment of muscular dystrophy using peptide nucleic acid ( pna) |
EP2105145A1 (en) | 2008-03-27 | 2009-09-30 | ETH Zürich | Method for muscle-specific delivery lipid-conjugated oligonucleotides |
EP2297341A4 (en) | 2008-05-09 | 2013-01-09 | Univ British Columbia | Methods and compositions for the treatment of huntington's disease |
EP2119783A1 (en) | 2008-05-14 | 2009-11-18 | Prosensa Technologies B.V. | Method for efficient exon (44) skipping in Duchenne Muscular Dystrophy and associated means |
WO2009144481A2 (en) | 2008-05-30 | 2009-12-03 | Isis Innovation Limited | Conjugates for delivery of biologically active compounds |
US20110152352A1 (en) | 2008-06-10 | 2011-06-23 | Tufts University | Smad proteins control drosha-mediated mirna maturation |
GB2457965B8 (en) | 2008-07-01 | 2011-02-16 | Renovo Ltd | Methods and systems for determining efficacy of medicaments. |
US20110218334A1 (en) | 2008-07-11 | 2011-09-08 | Alnylam Pharmaceuticals, Inc. | PHOSPHOROTHIOATE OLIGONUCLEOTIDES AND NON-NUCLEOSIDIC PHOSPHOROTHIOATES AS DELIVERY AGENTS FOR iRNA AGENTS |
AU2009276763B2 (en) | 2008-07-29 | 2015-07-16 | The Board Of Regents Of The University Of Texas Sytem | Selective inhibition of polyglutamine protein expression |
US8084601B2 (en) * | 2008-09-11 | 2011-12-27 | Royal Holloway And Bedford New College Royal Holloway, University Of London | Oligomers |
WO2010044894A1 (en) | 2008-10-15 | 2010-04-22 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotonic dystrophy |
AU2009310557B2 (en) | 2008-10-27 | 2014-09-11 | Academisch Ziekenhuis Leiden | Methods and means for efficient skipping of exon 45 in Duchenne Muscular Dystrophy pre-mRNA |
US20100248239A1 (en) | 2009-03-24 | 2010-09-30 | Mayo Foundation For Medical Education And Research | Methods and materials for detecting fragile x mutations |
EP2411532B1 (en) | 2009-03-24 | 2017-04-05 | Asuragen, Inc. | Pcr methods for characterizing the 5' untranslated region of the fmr1 and fmr2 genes |
JP2012523225A (en) | 2009-04-10 | 2012-10-04 | アソシアシオン・アンスティテュ・ドゥ・ミオロジー | Tricyclo-DNA antisense oligonucleotides, compositions and methods for treatment of disease |
EP2421971B1 (en) | 2009-04-24 | 2016-07-06 | BioMarin Technologies B.V. | Oligonucleotide comprising an inosine for treating dmd |
EP2440566A4 (en) | 2009-06-08 | 2013-10-16 | Miragen Therapeutics | CHEMICAL MODIFICATION MOTIFS FOR miRNA INHIBITORS AND MIMETICS |
EP3626823A1 (en) | 2009-09-11 | 2020-03-25 | Ionis Pharmaceuticals, Inc. | Modulation of huntingtin expression |
RS58079B1 (en) | 2009-11-12 | 2019-02-28 | Univ Western Australia | Antisense molecules and methods for treating pathologies |
US20110166081A1 (en) | 2009-12-03 | 2011-07-07 | University Of Iowa Research Foundation | Alpha-dystroglycan as a Protein Therapeutic |
WO2011078797A2 (en) | 2009-12-22 | 2011-06-30 | Singapore Health Services Pte. Ltd | Antisense oligonucleotides and uses threreof |
CA2785451C (en) | 2009-12-24 | 2019-01-22 | Prosensa Technologies B.V. | Molecule for treating an inflammatory disorder |
EP2536738A4 (en) | 2010-02-08 | 2014-09-17 | Isis Pharmaceuticals Inc | Methods and compositions useful in treatment of diseases or conditions related to repeat expansion |
WO2011097614A1 (en) | 2010-02-08 | 2011-08-11 | Isis Pharmaceuticals, Inc. | Mehods and compositions useful in diseases or conditions related to repeat expansion |
US20130237585A1 (en) | 2010-07-19 | 2013-09-12 | University Of Rochester | Modulation of dystrophia myotonica-protein kinase (dmpk) expression |
SG187165A1 (en) | 2010-08-20 | 2013-02-28 | Replicor Inc | Oligonucleotide chelate complexes |
TWI541024B (en) | 2010-09-01 | 2016-07-11 | 日本新藥股份有限公司 | Antisense nucleic acid |
BR112013020273A2 (en) | 2011-02-08 | 2016-10-18 | Charlotte Mecklenburg Hospital | antisense oligonucleotides |
EP3067421B1 (en) | 2011-02-08 | 2018-10-10 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
EP2699269A1 (en) | 2011-04-22 | 2014-02-26 | Prosensa Technologies B.V. | New compounds for treating, delaying and/or preventing a human genetic disorder such as myotonic dystrophy type 1 (dm1) |
CA3092114A1 (en) | 2011-05-05 | 2012-11-08 | Sarepta Therapeutics, Inc. | Peptide oligonucleotide conjugates |
US20140298496A1 (en) | 2011-06-23 | 2014-10-02 | Cold Spring Harbor Laboratory | Phenocopy model of disease |
JP6317675B2 (en) | 2011-11-30 | 2018-04-25 | サレプタ セラピューティクス, インコーポレイテッド | Oligonucleotides for treating prolonged repeat disease |
WO2013082578A1 (en) | 2011-12-03 | 2013-06-06 | Thomas Krupenkin | Method and apparatus for mechanical energy harvesting using combined magnetic and microfluidic energy generation |
WO2013090457A2 (en) | 2011-12-12 | 2013-06-20 | Oncoimmunin Inc. | In vivo delivery of oligonucleotides |
CN110055244A (en) | 2011-12-28 | 2019-07-26 | 日本新药株式会社 | Antisense nucleic acid |
AU2013212758A1 (en) | 2012-01-27 | 2014-08-14 | Biomarin Technologies B.V. | RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy |
WO2013120003A1 (en) | 2012-02-08 | 2013-08-15 | Isis Pharmaceuticals, Inc. | Modulation of rna by repeat targeting |
CN110025628B (en) | 2012-04-23 | 2023-03-31 | 维科医疗有限公司 | RNA-regulatory oligonucleotides with improved properties for the treatment of neuromuscular disorders |
AR091065A1 (en) | 2012-05-18 | 2014-12-30 | Replicor Inc | A PHARMACEUTICAL FORMULATION THAT INCLUDES AN ANTIVIRAL OLIGONUCLEOTIDE CHELATE FOR THE TREATMENT OF AN ANTI-VIRAL INFECTION |
EP2870246B1 (en) | 2012-07-03 | 2019-09-11 | BioMarin Technologies B.V. | Oligonucleotide for the treatment of muscular dystrophy patients |
HUE042218T2 (en) * | 2013-03-14 | 2019-06-28 | Sarepta Therapeutics Inc | Exon skipping compositions for treating muscular dystrophy |
-
2008
- 2008-10-27 NZ NZ584793A patent/NZ584793A/en unknown
- 2008-10-27 DK DK08842559.0T patent/DK2203173T3/en active
- 2008-10-27 JP JP2010530946A patent/JP5600064B2/en active Active
- 2008-10-27 ES ES13160310.2T patent/ES2639852T3/en active Active
- 2008-10-27 PL PL08842559T patent/PL2203173T3/en unknown
- 2008-10-27 CN CN201610053552.8A patent/CN105641700B/en active Active
- 2008-10-27 ES ES17171389T patent/ES2914775T3/en active Active
- 2008-10-27 PT PT88425590T patent/PT2203173E/en unknown
- 2008-10-27 EP EP17171389.4A patent/EP3238737B1/en active Active
- 2008-10-27 AU AU2008317566A patent/AU2008317566B2/en active Active
- 2008-10-27 CA CA2704049A patent/CA2704049A1/en not_active Abandoned
- 2008-10-27 WO PCT/NL2008/050673 patent/WO2009054725A2/en active Application Filing
- 2008-10-27 EP EP08842559.0A patent/EP2203173B1/en not_active Revoked
- 2008-10-27 EP EP13160310.2A patent/EP2614827B1/en active Active
- 2008-10-27 HU HUE08842559A patent/HUE028662T2/en unknown
- 2008-10-27 ES ES08842559.0T patent/ES2564563T3/en active Active
- 2008-10-27 CN CN200880120198XA patent/CN101896186A/en active Pending
-
2009
- 2009-01-13 NZ NZ592446A patent/NZ592446A/en unknown
- 2009-01-13 CN CN200980152464.1A patent/CN102264903B/en active Active
- 2009-01-13 HU HUE13160338A patent/HUE027124T2/en unknown
- 2009-03-11 ES ES09788170.0T patent/ES2692886T3/en active Active
- 2009-03-11 ES ES18180952T patent/ES2936464T3/en active Active
- 2009-03-11 EP EP22203933.1A patent/EP4183399A1/en active Pending
- 2009-03-11 CN CN2009801519198A patent/CN102256606A/en active Pending
- 2009-03-11 CN CN201510825064.XA patent/CN105647921A/en active Pending
-
2010
- 2010-04-25 IL IL205322A patent/IL205322A/en active IP Right Grant
- 2010-04-26 US US12/767,702 patent/US9243245B2/en not_active Ceased
-
2011
- 2011-04-26 US US13/094,571 patent/US20110263682A1/en not_active Abandoned
- 2011-04-26 US US13/094,548 patent/US9926557B2/en active Active
-
2012
- 2012-01-16 HK HK13112417.5A patent/HK1185098A1/en unknown
-
2013
- 2013-12-04 US US14/097,210 patent/US20140113955A1/en not_active Abandoned
- 2013-12-19 US US14/134,971 patent/US20140128592A1/en not_active Abandoned
-
2014
- 2014-02-07 JP JP2014022084A patent/JP5879374B2/en active Active
- 2014-03-07 US US14/200,251 patent/US20140221458A1/en not_active Abandoned
- 2014-11-14 US US14/542,183 patent/US9528109B2/en active Active
-
2015
- 2015-02-25 US US14/631,686 patent/US9499818B2/en active Active
- 2015-10-06 IL IL241928A patent/IL241928B/en active IP Right Grant
- 2015-11-09 JP JP2015219928A patent/JP2016033140A/en active Pending
-
2016
- 2016-01-07 US US14/990,712 patent/US20160304864A1/en not_active Abandoned
- 2016-01-11 HR HRP20160025TT patent/HRP20160025T1/en unknown
- 2016-03-11 CY CY20161100214T patent/CY1117286T1/en unknown
- 2016-04-04 CY CY20161100268T patent/CY1117454T1/en unknown
- 2016-10-07 US US15/289,053 patent/US20170044534A1/en not_active Abandoned
- 2016-12-27 US US15/390,836 patent/US20170107512A1/en not_active Abandoned
-
2017
- 2017-05-12 JP JP2017095804A patent/JP6579629B2/en active Active
-
2018
- 2018-04-26 HK HK18105446.9A patent/HK1245670A1/en unknown
- 2018-06-29 US US16/024,558 patent/US10876114B2/en active Active
- 2018-08-13 IL IL261127A patent/IL261127B/en active IP Right Grant
- 2018-12-21 US US16/229,534 patent/US20190112604A1/en not_active Abandoned
- 2018-12-21 US US16/229,821 patent/US20190119679A1/en not_active Abandoned
-
2019
- 2019-02-22 US US16/283,458 patent/US20190177725A1/en not_active Abandoned
- 2019-04-25 JP JP2019084505A patent/JP6885620B2/en active Active
- 2019-09-26 US US16/584,115 patent/US11427820B2/en active Active
-
2020
- 2020-12-21 US US17/129,117 patent/US20210139904A1/en active Pending
-
2021
- 2021-05-06 JP JP2021078561A patent/JP7107622B2/en active Active
- 2021-06-23 IL IL284321A patent/IL284321B/en unknown
-
2022
- 2022-07-25 US US17/814,781 patent/US20230151362A1/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070134655A1 (en) * | 2002-11-14 | 2007-06-14 | Itzhak Bentwich | Bioinformatically detectable group of novel regulatory genes and uses thereof |
US20060024715A1 (en) * | 2004-07-02 | 2006-02-02 | Affymetrix, Inc. | Methods for genotyping polymorphisms in humans |
US20060099612A1 (en) * | 2004-09-02 | 2006-05-11 | Suntory Limited | Method for analyzing genes of industrial yeasts |
US20060160121A1 (en) * | 2004-10-05 | 2006-07-20 | Wyeth | Probe arrays for detecting multiple strains of different species |
US20100130591A1 (en) * | 2008-10-24 | 2010-05-27 | Peter Sazani | Multiple exon skipping compositions for dmd |
Non-Patent Citations (1)
Title |
---|
McClorey et al., Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD, 2006, Gene Therapy, volume 13, pages 1373-1381. * |
Cited By (99)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10421966B2 (en) | 2004-06-28 | 2019-09-24 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8455635B2 (en) | 2004-06-28 | 2013-06-04 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8450474B2 (en) | 2004-06-28 | 2013-05-28 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9035040B2 (en) | 2004-06-28 | 2015-05-19 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8455634B2 (en) | 2004-06-28 | 2013-06-04 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10781451B2 (en) | 2004-06-28 | 2020-09-22 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8476423B2 (en) | 2004-06-28 | 2013-07-02 | The University of Western Austrailia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8486907B2 (en) | 2004-06-28 | 2013-07-16 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9024007B2 (en) | 2004-06-28 | 2015-05-05 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8524880B2 (en) | 2004-06-28 | 2013-09-03 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9447415B2 (en) | 2004-06-28 | 2016-09-20 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
USRE48960E1 (en) | 2004-06-28 | 2022-03-08 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10995337B2 (en) | 2004-06-28 | 2021-05-04 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10968450B2 (en) | 2004-06-28 | 2021-04-06 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9994851B2 (en) | 2004-06-28 | 2018-06-12 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9018368B2 (en) | 2004-06-28 | 2015-04-28 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9441229B2 (en) | 2004-06-28 | 2016-09-13 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20110015258A1 (en) * | 2004-06-28 | 2011-01-20 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8455636B2 (en) | 2004-06-28 | 2013-06-04 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9175286B2 (en) | 2004-06-28 | 2015-11-03 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10227590B2 (en) | 2004-06-28 | 2019-03-12 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
USRE47769E1 (en) | 2004-06-28 | 2019-12-17 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
USRE47751E1 (en) | 2004-06-28 | 2019-12-03 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
USRE47691E1 (en) | 2004-06-28 | 2019-11-05 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9605262B2 (en) | 2004-06-28 | 2017-03-28 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9249416B2 (en) | 2004-06-28 | 2016-02-02 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10266827B2 (en) | 2004-06-28 | 2019-04-23 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US9422555B2 (en) | 2004-06-28 | 2016-08-23 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US10689646B2 (en) | 2006-08-11 | 2020-06-23 | Biomarin Technologies B.V. | Treatment of genetic disorders associated with DNA repeat instability |
US9890379B2 (en) | 2006-08-11 | 2018-02-13 | Biomarin Technologies B.V. | Treatment of genetic disorders associated with DNA repeat instability |
US11274299B2 (en) | 2006-08-11 | 2022-03-15 | Vico Therapeutics B.V. | Methods and means for treating DNA repeat instability associated genetic disorders |
US10876114B2 (en) | 2007-10-26 | 2020-12-29 | Biomarin Technologies B.V. | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53 |
US9926557B2 (en) | 2007-10-26 | 2018-03-27 | Biomarin Technologies B.V. | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US11427820B2 (en) | 2007-10-26 | 2022-08-30 | Biomarin Technologies B.V. | Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA |
US9499818B2 (en) | 2007-10-26 | 2016-11-22 | BioMarin Technologies, B.V. | Methods and means for efficient skipping of at least one of the exons 51-53, 55, 57 and 59 of the human duchenne muscular dystrophy gene |
US10246707B2 (en) | 2008-05-14 | 2019-04-02 | Biomarin Technologies B.V. | Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means |
US9139828B2 (en) | 2008-05-14 | 2015-09-22 | Prosensa Technologies B.V. | Method for efficient exon (44) skipping in duchenne muscular dystrophy and associated means |
US20140057964A1 (en) * | 2008-09-11 | 2014-02-27 | Royal Holloway, University Of London | Oligomers |
US11697811B2 (en) | 2008-09-11 | 2023-07-11 | Royal Holloway, University Of London | Oligomers |
US9650632B2 (en) | 2008-09-11 | 2017-05-16 | Royal Holloway, University Of London | Oligomers |
US9243252B2 (en) * | 2008-09-11 | 2016-01-26 | Royal Holloway, University Of London | Oligomers |
US10457944B2 (en) | 2008-09-11 | 2019-10-29 | Royal Holloway, University Of London | Oligomers |
US9243251B2 (en) | 2008-09-11 | 2016-01-26 | Royal Holloway, University Of London | Oligomers |
US9447417B2 (en) | 2008-10-24 | 2016-09-20 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US9234198B1 (en) | 2008-10-24 | 2016-01-12 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US8871918B2 (en) | 2008-10-24 | 2014-10-28 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US9434948B2 (en) | 2008-10-24 | 2016-09-06 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US20100130591A1 (en) * | 2008-10-24 | 2010-05-27 | Peter Sazani | Multiple exon skipping compositions for dmd |
US8865883B2 (en) | 2008-10-24 | 2014-10-21 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US9447416B2 (en) | 2008-10-24 | 2016-09-20 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US9453225B2 (en) | 2008-10-24 | 2016-09-27 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US11634714B2 (en) | 2009-04-24 | 2023-04-25 | Biomarin Technologies B.V. | Oligonucleotide comprising an inosine for treating DMD |
US11034956B2 (en) * | 2009-04-24 | 2021-06-15 | Biomarin Technologies B.V. | Oligonucleotide comprising an inosine for treating DMD |
US8637483B2 (en) | 2009-11-12 | 2014-01-28 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US9758783B2 (en) | 2009-11-12 | 2017-09-12 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US10287586B2 (en) | 2009-11-12 | 2019-05-14 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US9228187B2 (en) | 2009-11-12 | 2016-01-05 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US11447776B2 (en) | 2009-11-12 | 2022-09-20 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US10781450B2 (en) | 2009-11-12 | 2020-09-22 | Sarepta Therapeutics, Inc. | Antisense molecules and methods for treating pathologies |
US9890381B2 (en) | 2011-12-28 | 2018-02-13 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US9512424B2 (en) | 2011-12-28 | 2016-12-06 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US10781448B2 (en) | 2011-12-28 | 2020-09-22 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
WO2013112053A1 (en) * | 2012-01-27 | 2013-08-01 | Prosensa Technologies B.V. | Rna modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy |
US10913946B2 (en) | 2012-01-27 | 2021-02-09 | Biomarin Technologies B.V. | RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy |
US10179912B2 (en) | 2012-01-27 | 2019-01-15 | Biomarin Technologies B.V. | RNA modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy |
EP4043039A1 (en) * | 2012-01-27 | 2022-08-17 | BioMarin Technologies B.V. | Rna modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy |
CN104203289A (en) * | 2012-01-27 | 2014-12-10 | 普罗森萨科技有限公司 | RNA modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy |
US10711256B2 (en) | 2012-04-27 | 2020-07-14 | Duke University | Genetic correction of mutated genes |
US11898176B2 (en) | 2012-04-27 | 2024-02-13 | Duke University | Genetic correction of mutated genes |
US11932851B2 (en) | 2013-03-14 | 2024-03-19 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
US10907154B2 (en) | 2013-03-14 | 2021-02-02 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
US9217148B2 (en) | 2013-03-14 | 2015-12-22 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
US9506058B2 (en) | 2013-03-15 | 2016-11-29 | Sarepta Therapeutics, Inc. | Compositions for treating muscular dystrophy |
US10364431B2 (en) | 2013-03-15 | 2019-07-30 | Sarepta Therapeutics, Inc. | Compositions for treating muscular dystrophy |
US10337003B2 (en) | 2013-03-15 | 2019-07-02 | Sarepta Therapeutics, Inc. | Compositions for treating muscular dystrophy |
US20160201089A1 (en) * | 2013-06-05 | 2016-07-14 | Duke University | Rna-guided gene editing and gene regulation |
US10745714B2 (en) | 2013-06-05 | 2020-08-18 | Duke University | RNA-guided gene editing and gene regulation |
US10704060B2 (en) * | 2013-06-05 | 2020-07-07 | Duke University | RNA-guided gene editing and gene regulation |
US11053497B2 (en) | 2014-03-12 | 2021-07-06 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US9988629B2 (en) | 2014-03-12 | 2018-06-05 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
US10676726B2 (en) | 2015-02-09 | 2020-06-09 | Duke University | Compositions and methods for epigenome editing |
US11155796B2 (en) | 2015-02-09 | 2021-10-26 | Duke University | Compositions and methods for epigenome editing |
US10676735B2 (en) | 2015-07-22 | 2020-06-09 | Duke University | High-throughput screening of regulatory element function with epigenome editing technologies |
US11427817B2 (en) | 2015-08-25 | 2022-08-30 | Duke University | Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases |
US10851373B2 (en) | 2015-09-15 | 2020-12-01 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
CN113913426A (en) * | 2015-09-15 | 2022-01-11 | 日本新药株式会社 | Antisense nucleic acid |
US10450568B2 (en) | 2015-10-09 | 2019-10-22 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US11787869B2 (en) | 2018-08-02 | 2023-10-17 | Dyne Therapeutics, Inc. | Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness |
US11833217B2 (en) | 2018-08-02 | 2023-12-05 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11633496B2 (en) | 2018-08-02 | 2023-04-25 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11795234B2 (en) | 2018-08-02 | 2023-10-24 | Dyne Therapeutics, Inc. | Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide |
US11795233B2 (en) | 2018-08-02 | 2023-10-24 | Dyne Therapeutics, Inc. | Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide |
US20220186217A1 (en) * | 2018-12-06 | 2022-06-16 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
WO2022241408A1 (en) | 2021-05-10 | 2022-11-17 | Entrada Therapeutics, Inc. | Compositions and methods for modulating tissue distribution of intracellular therapeutics |
WO2022271818A1 (en) | 2021-06-23 | 2022-12-29 | Entrada Therapeutics, Inc. | Antisense compounds and methods for targeting cug repeats |
US11771776B2 (en) * | 2021-07-09 | 2023-10-03 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11679161B2 (en) | 2021-07-09 | 2023-06-20 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy |
US20230045002A1 (en) * | 2021-07-09 | 2023-02-09 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11844843B2 (en) | 2021-07-09 | 2023-12-19 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10876114B2 (en) | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53 | |
EP2349287B1 (en) | Methods and means for efficient skipping of at least one of the following exons of the human duchenne muscular dystrophy gene: 43, 46, 50- 53. | |
AU2009310558B8 (en) | Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50- 53. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: PROSENSA HOLDING B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DE KIMPE, JOSEPHUS JOHANNES;PLATENBURG, GERARDUS JOHANNES;VAN DEUTEKOM, JUDITH CHRISTINA THEODORA;AND OTHERS;REEL/FRAME:026556/0428 Effective date: 20110511 Owner name: PROSENSA TECHNOLOGIES B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DE KIMPE, JOSEPHUS JOHANNES;PLATENBURG, GERARDUS JOHANNES;VAN DEUTEKOM, JUDITH CHRISTINA THEODORA;AND OTHERS;REEL/FRAME:026556/0428 Effective date: 20110511 Owner name: PROSENSA B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DE KIMPE, JOSEPHUS JOHANNES;PLATENBURG, GERARDUS JOHANNES;VAN DEUTEKOM, JUDITH CHRISTINA THEODORA;AND OTHERS;REEL/FRAME:026556/0428 Effective date: 20110511 Owner name: ACADEMISCH ZIEKENHUIS LEIDEN, NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DE KIMPE, JOSEPHUS JOHANNES;PLATENBURG, GERARDUS JOHANNES;VAN DEUTEKOM, JUDITH CHRISTINA THEODORA;AND OTHERS;REEL/FRAME:026556/0428 Effective date: 20110511 |
|
AS | Assignment |
Owner name: PROSENSA HOLDING N.V., NETHERLANDS Free format text: CHANGE OF NAME;ASSIGNOR:PROSENSA HOLDING B.V.;REEL/FRAME:034115/0965 Effective date: 20140828 Owner name: PROSENSA HOLDING B.V., NETHERLANDS Free format text: CHANGE OF NAME;ASSIGNOR:PROSENSA B.V.;REEL/FRAME:034115/0916 Effective date: 20140828 Owner name: PROSENSA TECHNOLOGIES B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PROSENSA HOLDING N.V.;REEL/FRAME:034115/0924 Effective date: 20140828 |
|
AS | Assignment |
Owner name: PROSENSA TECHNOLOGIES B.V., NETHERLANDS Free format text: CHANGE OF ADDRESS OF ASSIGNEE;ASSIGNOR:PROSENSA TECHNOLOGIES B.V.;REEL/FRAME:034916/0132 Effective date: 20110704 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: BIOMARIN TECHNOLOGIES B.V., NETHERLANDS Free format text: CHANGE OF NAME;ASSIGNOR:PROSENSA TECHNOLOGIES B.V.;REEL/FRAME:036732/0042 Effective date: 20150908 |