US20090228998A1 - Induction of exon skipping in eukaryotic cells - Google Patents
Induction of exon skipping in eukaryotic cells Download PDFInfo
- Publication number
- US20090228998A1 US20090228998A1 US12/383,897 US38389709A US2009228998A1 US 20090228998 A1 US20090228998 A1 US 20090228998A1 US 38389709 A US38389709 A US 38389709A US 2009228998 A1 US2009228998 A1 US 2009228998A1
- Authority
- US
- United States
- Prior art keywords
- exon
- mrna
- cell
- antisense
- dystrophin
- 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
-
- 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
-
- 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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- 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
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- 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
- C12N2310/30—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
- C12N2310/32—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
- C12N2310/34—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
- C12N2320/33—Alteration of splicing
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
Abstract
The present invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, by inducing so-called exon skipping in the cell. Exon-skipping results in mature mRNA that does not contain the skipped exon, which leads to an altered product of the exon codes for amino acids. Exon skipping is performed by providing a cell with an agent capable of specifically inhibiting an exon inclusion signal, for instance, an exon recognition sequence, of the exon. The exon inclusion signal can be interfered with by a nucleic acid comprising complementarity to a part of the exon. The nucleic acid, which is also herewith provided, can be used for the preparation of a medicament, for instance, for the treatment of an inherited disease.
Description
- This application is a continuation of co-pending U.S. patent application Ser. No. 10/395,031, filed Mar. 21, 2003, now U.S. Pat. No. ______, which is a continuation of International Application PCT/NL01/00697, filed Sep. 21, 2001, designating the United States, published in English Mar. 28, 2002, as WO 02/024906 A1 and subsequently published with corrections Jan. 23, 2003, as WO 02/024906 C2, the contents of the entirety of each of which are hereby incorporated herein by this reference.
- The invention relates to the field of gene therapy.
- Given the rapid advances of human genome research, professionals and the public expect that the near future will bring us, in addition to understanding of disease mechanisms and refined and reliable diagnostics, therapies for many devastating genetic diseases.
- While it is hoped that for some (e.g., metabolic) diseases, the improved insights will bring easily administrable small-molecule therapies, it is likely that in most cases one or another form of gene therapy will ultimately be required, i.e., the correction, addition or replacement of the defective gene product.
- In the past few years, research and development in this field have highlighted several technical difficulties which need to be overcome, e.g., related to the large size of many genes involved in genetic disease (limiting the choice of suitable systems to administer the therapeutic gene), the accessibility of the tissue in which the therapeutic gene should function (requiring the design of specific targeting techniques, either physically, by restricted injection, or biologically, by developing systems with tissue-specific affinities) and the safety to the patient of the administration system. These problems are to some extent interrelated, and it can be generally concluded that the smaller the therapeutic agent is, the easier it will become to develop efficient, targetable and safe administration systems.
- The present invention addresses this problem by inducing so-called exon-skipping in cells. Exon-skipping results in mature mRNA that does not contain the skipped exon and thus, when the exon codes for amino acids, can lead to the expression of an altered product. Technology for exon-skipping is currently directed toward the use of so-called “Anti-sense Oligonucleotides” (AONs).
- Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy (DMD). The mdx mouse, which carries a nonsense mutation in
exon 23 of the dystrophin gene, has been used as an animal model of Duchenne muscular dystrophy. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin-positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. - AONs directed to, respectively, the 3′ and 5′ splice sites of
introns 22 and 23 in dystrophin pre-mRNA have been shown to interfere with factors normally involved in removal ofintron 23 so thatexon 23 was also removed from the mRNA (Wilton, 1999). In a similar study, Dunckley et al. (1998) showed that exon skipping using AONs directed to 3′ and 5′ splice sites can have unexpected results. They observed skipping of not onlyexon 23 but also of exons 24-29, thus resulting in an mRNA containing an exon 22-exon 30 junction. - The underlying mechanism for the appearance of the unexpected 22-30 splicing variant is not known. It could be due to the fact that splice sites contain consensus sequences leading to promiscuous hybridization of the oligos used to direct the exon skipping. Hybridization of the oligos to other splice sites than the sites of the exon to be skipped of course could easily interfere with the accuracy of the splicing process. On the other hand, the accuracy could be lacking due to the fact that two oligos (for the 5′ and the 3′ splice site) need to be used. Pre-mRNA containing one but not the other oligo could be prone to unexpected splicing variants.
- To overcome these and other problems, the present invention provides a method for directing splicing of a pre-mRNA in a system capable of performing a splicing operation comprising contacting the pre-mRNA in the system with an agent capable of specifically inhibiting an exon inclusion signal of at least one exon in the pre-mRNA, the method further comprising allowing splicing of the pre-mRNA. Interfering with an exon inclusion signal (EIS) has the advantage that such elements are located within the exon. By providing an antisense oligo for the interior of the exon to be skipped, it is possible to interfere with the exon inclusion signal, thereby effectively masking the exon from the splicing apparatus. The failure of the splicing apparatus to recognize the exon to be skipped thus leads to exclusion of the exon from the final mRNA.
- The present invention does not interfere directly with the enzymatic process of the splicing machinery (the joining of the exons). It is thought that this allows the method to be more robust and reliable. It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In this concept, it is the particular spatial conformation that enables the splicing machinery to recognize the exon. However, the invention is certainly not limited to this model.
- It has been found that agents capable of binding to an exon can inhibit an EIS. Agents may specifically contact the exon at any point and still be able to specifically inhibit the EIS. The mRNA may be useful in itself. For instance, production of an undesired protein can be at least in part reduced by inhibiting inclusion of a required exon into the mRNA. A preferred method of the invention further comprises allowing translation of mRNA produced from splicing of the pre-mRNA. Preferably, the mRNA encodes a functional protein. In a preferred embodiment, the protein comprises two or more domains, wherein at least one of the domains is encoded by the mRNA as a result of skipping of at least part of an exon in the pre-mRNA.
- Exon skipping will typically, though not necessarily, be of relevance for proteins in the wild-type configuration, having at least two functional domains that each performs a function, wherein the domains are generated from distinct parts of the primary amino acid sequence. Examples are, for instance, transcription factors. Typically, these factors comprise a DNA binding domain and a domain that interacts with other proteins in the cell. Skipping of an exon that encodes a part of the primary amino acid sequence that lies between these two domains can lead to a shorter protein that comprises the same function, at least in part. Thus, detrimental mutations in this intermediary region (for instance, frame-shift or stop mutations) can be at least in part repaired by inducing exon skipping to allow synthesis of the shorter (partly) functional protein.
- Using a method of the invention, it is also possible to induce partial skipping of the exon. In this embodiment, the contacting results in activation of a cryptic splice site in a contacted exon. This embodiment broadens the potential for manipulation of the pre-mRNA leading to a functional protein. Preferably, the system comprises a cell. Preferably, the cell is cultured in vitro or in the organism in vivo. Typically, though not necessarily, the organism comprises a human or a mouse.
- In a preferred embodiment, the invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, the method comprising providing the cell with an agent capable of specifically inhibiting an exon inclusion signal of at least one of the exons, the method further comprising allowing translation of mRNA produced from splicing of the pre-mRNA.
- Any agent capable of specifically inhibiting an exon exclusion signal can be used for the present invention. Preferably, the agent comprises a nucleic acid or a functional equivalent thereof. Preferably, but not necessarily, the nucleic acid is in single-stranded form. Peptide nucleic acid and other molecules comprising the same nucleic acid binding characteristics in kind, but not necessarily in amount, are suitable equivalents. Nucleic acid or an equivalent may comprise modifications to provide additional functionality. For instance, 2′-O-methyl oligoribonucleotides can be used. These ribonucleotides are more resistant to RNAse action than conventional oligonucleotides.
- In a preferred embodiment of the invention, the exon inclusion signal is interfered with by an antisense nucleic acid directed to an exon recognition sequence (ERS). These sequences are relatively purine-rich and can be distinguished by scrutinizing the sequence information of the exon to be skipped (Tanaka et al., 1994, Mol. Cell. Biol. 14, p. 1347-1354). Exon recognition sequences are thought to aid inclusion into mRNA of so-called weak exons (Achsel et al., 1996, J. Biochem. 120, p. 53-60). These weak exons comprise, for instance, 5′ and or 3′ splice sites that are less efficiently recognized by the splicing machinery. In the present invention, it has been found that exon skipping can also be induced in so-called strong exons, i.e., exons which are normally efficiently recognized by the splicing machinery of the cell. From any given sequence, it is (almost) always possible to predict whether the sequence comprises putative exons and to determine whether these exons are strong or weak. Several algorithms for determining the strength of an exon exist. A useful algorithm can be found on the NetGene2 splice site prediction server (Brunak, et al., 1991, J. Mol. Biol. 220, p. 49-65). Exon skipping by a means of the invention can be induced in (almost) every exon, independent of whether the exon is a weak exon or a strong exon and also independent of whether the exon comprises an ERS. In a preferred embodiment, an exon that is targeted for skipping is a strong exon. In another preferred embodiment, an exon targeted for skipping does not comprise an ERS.
- Methods of the invention can be used in many ways. In one embodiment, a method of the invention is used to at least in part decrease the production of an aberrant protein. Such proteins can, for instance, be onco-proteins or viral proteins. In many tumors, not only the presence of an onco-protein but also its relative level of expression has been associated with the phenotype of the tumor cell. Similarly, not only the presence of viral proteins but also the amount of viral protein in a cell determines the virulence of a particular virus. Moreover, for efficient multiplication and spread of a virus, the timing of expression in the life cycle and the balance in the amount of certain viral proteins in a cell determines whether viruses are efficiently or inefficiently produced. Using a method of the invention, it is possible to lower the amount of aberrant protein in a cell such that, for instance, a tumor cell becomes less tumorigenic (metastatic) and/or a virus-infected cell produces less virus.
- In a preferred embodiment, a method of the invention is used to modify the aberrant protein into a functional protein. In one embodiment, the functional protein is capable of performing a function of a protein normally present in a cell but absent in the cells to be treated. Very often, even partial restoration of function results in significantly improved performance of the cell thus treated. Due to the better performance, such cells can also have a selective advantage over unmodified cells, thus aiding the efficacy of the treatment.
- This aspect of the invention is particularly suited for the restoration of expression of defective genes. This is achieved by causing the specific skipping of targeted exons, thus bypassing or correcting deleterious mutations (typically stop-mutations or frame-shifting point mutations, single- or multi-exon deletions or insertions leading to translation termination).
- Compared to gene-introduction strategies, this novel form of splice-modulation gene therapy requires the administration of much smaller therapeutic reagents, typically, but not limited to, 14-40 nucleotides. In a preferred embodiment, molecules of 14-25 nucleotides are used since these molecules are easier to produce and enter the cell more effectively. The methods of the invention allow much more flexibility in the subsequent design of effective and safe administration systems. An important additional advantage of this aspect of the invention is that it restores (at least some of) the activity of the endogenous gene, which still possesses most or all of its gene-regulatory circuitry, thus ensuring proper expression levels and the synthesis of tissue-specific isoforms.
- This aspect of the invention can in principle be applied to any genetic disease or genetic predisposition to disease in which targeted skipping of specific exons would restore the translational reading frame when this has been disrupted by the original mutation, provided that translation of an internally slightly shorter protein is still fully or partly functional. Preferred embodiments for which this application can be of therapeutic value are: predisposition to second hit mutations in tumor suppressor genes, e.g., those involved in breast cancer, colon cancer, tuberous sclerosis, neurofibromatosis etc., where (partial) restoration of activity would preclude the manifestation of nullosomy by second hit mutations and thus would protect against tumorigenesis. Another preferred embodiment involves the (partial) restoration of defective gene products which have a direct disease causing effect, e.g., hemophilia A (clotting factor VIII deficiency), some forms of congenital hypothyroidism (due to thyroglobulin synthesis deficiency) and Duchenne muscular dystrophy (DMD), in which frame-shifting deletions, duplications and stop mutations in the X-linked dystrophin gene cause severe, progressive muscle degradation. DMD is typically lethal in late adolescence or early adulthood, while non-frame-shifting deletions or duplications in the same gene cause the much milder Becker muscular dystrophy (BMD), compatible with a life expectancy between 35-40 years to normal. In the embodiment as applied to DMD, the present invention enables exon skipping to extend an existing deletion (or alter the mRNA product of an existing duplication) by as many adjacent exons as required to restore the reading frame and generate an internally slightly shortened, but still functional, protein. Based on the much milder clinical symptoms of BMD patients with the equivalent of this induced deletion, the disease in the DMD patients would have a much milder course after AON-therapy.
- Many different mutations in the dystrophin gene can lead to a dysfunctional protein. (For a comprehensive inventory see www.dmd.nl, the internationally accepted database for DMD and related disorders.) The precise exon to be skipped to generate a functional dystrophin protein varies from mutation to mutation. Table 1 comprises a non-limiting list of exons that can be skipped and lists for the mentioned exons some of the more frequently occurring dystrophin gene mutations that have been observed in humans and that can be treated with a method of the invention. Skipping of the mentioned exon leads to a mutant dystrophin protein comprising at least the functionality of a Becker mutant. Thus, in one embodiment, the invention provides a method of the invention wherein the exon inclusion signal is present in
exon numbers exon 46 with a means or a method of the invention, approximately 7% of DMD-deletion containing patients can be treated, resulting in the patients to comprise dystrophin-positive muscle fibers. By inducing skipping ofexon 51, approximately 15% of DMD-deletion containing patients can be treated with a means or method of the invention. Such treatment will result in the patient having at least some dystrophin-positive fibers. Thus, with either skipping ofexon exon 46 orexon 51. In a particularly preferred embodiment, the agent comprises a nucleic acid sequence according tohAON# 4,hAON# 6,hAON# 8,hAON# 9,hAON# 11 and/or one or more of hAON#21-30 or a functional part, derivative and/or analogue of the hAON. A functional part, derivative and/or analogue of the hAON comprises the same exon skipping activity in kind, but not necessarily in amount, in a method of the invention. -
TABLE 1 Therapeutic for DMD-deletions Frequency in Exon to be skipped (exons) www.dmd.nl (%) 2 3-7 2 8 3-7 4 4-7 5-7 6-7 43 44 5 44-47 44 35-43 8 45 45-54 45 18-44 13 46-47 44 46-48 46-49 46-51 46-53 46 45 7 50 51 5 51-55 51 50 15 45-50 48-50 49-50 52 52-63 52 51 3 53 53-55 53 45-52 9 48-52 49-52 50-52 52 - It can be advantageous to induce exon skipping of more than one exon in the pre-mRNA. For instance, considering the wide variety of mutations and the fixed nature of exon lengths and amino acid sequence flanking such mutations, the situation can occur that for restoration of function more than one exon needs to be skipped. A preferred but non-limiting example of such a case in the DMD deletion database is a 46-50 deletion. Patients comprising a 46-50 deletion do not produce functional dystrophin. However, an at least partially functional dystrophin can be generated by inducing skipping of both
exon 45 andexon 51. Another preferred but non-limiting example is patients comprising a duplication ofexon 2. By providing one agent capable of inhibiting an EIS ofexon 2, it is possible to partly skip either one or bothexons 2, thereby regenerating the wild-type protein next to the truncated ordouble exon 2 skipped protein. Another preferred but non-limiting example is the skipping ofexons 45 through 50. This generates an in-frame Becker-like variant. This Becker-like variant can be generated to cure any mutation localized inexons - In another aspect, the invention provides a method for selecting the suitable agents for splice-therapy and their validation as specific exon-skipping agents in pilot experiments. A method is provided for determining whether an agent is capable of specifically inhibiting an exon inclusion signal of an exon, comprising providing a cell having a pre-mRNA containing the exon with the agent, culturing the cell to allow the formation of an mRNA from the pre-mRNA and determining whether the exon is absent the mRNA. In a preferred embodiment, the agent comprises a nucleic acid or a functional equivalent thereof, the nucleic acid comprising complementarity to a part of the exon. Agents capable of inducing specific exon skipping can be identified with a method of the invention. It is possible to include a prescreen for agents by first identifying whether the agent is capable of binding with a relatively high affinity to an exon containing nucleic acid, preferably RNA. To this end, a method for determining whether an agent is capable of specifically inhibiting an exon inclusion signal of an exon is provided, further comprising first determining in vitro the relative binding affinity of the nucleic acid or functional equivalent thereof to an RNA molecule comprising the exon.
- In yet another aspect, an agent is provided that is obtainable by a method of the invention. In a preferred embodiment, the agent comprises a nucleic acid or a functional equivalent thereof. Preferably the agent, when used to induce exon skipping in a cell, is capable of at least in part reducing the amount of aberrant protein in the cell. More preferably, the exon skipping results in an mRNA encoding a protein that is capable of performing a function in the cell. In a particularly preferred embodiment, the pre-mRNA is derived from a dystrophin gene. Preferably, the functional protein comprises a mutant or normal dystrophin protein. Preferably, the mutant dystrophin protein comprises at least the functionality of a dystrophin protein in a Becker patient. In a particularly preferred embodiment, the agent comprises the nucleic acid sequence of
hAON# 4,hAON# 6,hAON# 8,hAON# 9,hAON# 11 and/or one or more of hAON#21-30 or a functional part, derivative and/or analogue of the hAON. A functional part, derivative and/or analogue of the hAON comprises the same exon skipping activity in kind, but not necessarily in amount, in a method of the invention. - The art describes many ways to deliver agents to cells. Particularly, nucleic acid delivery methods have been widely developed. The artisan is well capable of determining whether a method of delivery is suitable for performing the present invention. In a non-limiting example, the method includes the packaging of an agent of the invention into liposomes, the liposomes being provided to cells comprising a target pre-mRNA. Liposomes are particularly suited for delivery of nucleic acid to cells. Antisense molecules capable of inducing exon skipping can be produced in a cell upon delivery of nucleic acid containing a transcription unit to produce antisense RNA. Non-limiting examples of suitable transcription units are small nuclear RNA (SNRP) or tRNA transcription units. The invention, therefore, further provides a nucleic acid delivery vehicle comprising a nucleic acid or functional equivalent thereof of the invention capable of inhibiting an exon inclusion signal. In one embodiment, the delivery vehicle is capable of expressing the nucleic acid of the invention. Of course, in case, for instance, single-stranded viruses are used as a vehicle, it is entirely within the scope of the invention when such a virus comprises only the antisense sequence of an agent of the invention. In another embodiment of single strand viruses, AONs of the invention are encoded by small nuclear RNA or tRNA transcription units on viral nucleic encapsulated by the virus as vehicle. A preferred single-stranded virus is adeno-associated virus.
- In yet another embodiment, the invention provides the use of a nucleic acid or a nucleic acid delivery vehicle of the invention for the preparation of a medicament. In a preferred embodiment, the medicament is used for the treatment of an inherited disease. More preferably, the medicament is used for the treatment of Duchenne Muscular Dystrophy.
-
FIG. 1 . Deletion ofexon 45 is one of the most frequent DMD-mutations. Due to this deletion,exon 44 is spliced toexon 46, the translational reading frame is interrupted, and a stop codon is created inexon 46 leading to a dystrophin deficiency. Our aim is to artificially induce the skipping of an additional exon,exon 46, in order to reestablish the reading frame and restore the synthesis of a slightly shorter, but largely functional, dystrophin protein as found in the much milder affected Becker muscular dystrophy patients affected by a deletion of bothexons -
FIG. 2 .Exon 46 contains a purine-rich region that is hypothesized to have a potential role in the regulation of its splicing in the pre-mRNA. A series of overlapping 2′O-methyl phosphorothioate antisense oligoribonucleotides (AONs) was designed directed at this purine-rich region inmouse dystrophin exon 46. The AONs differ both in length and sequence. The chemical modifications render the AONs resistant to endonucleases and RNaseH inside the muscle cells. To determine the transfection efficiency in our in vitro studies, the AONs contained a 5′ fluorescein group which allowed identification of AON-positive cells. -
FIG. 3 . To determine the binding affinity of the different AONs to thetarget exon 46 RNA, we performed gel mobility shift assays. In this figure, the five mAONs (mAON# exon 46, did not generate a band shift. -
FIGS. 4A and 4B . The mouse- and human-specific AONs which showed the highest binding affinity in the gel mobility shift assays were transfected into mouse and human myotube cultures. -
FIG. 4A . RT-PCR analysis showed a truncated product, of which the size corresponded toexon 45 directly spliced toexon 47, in the mouse cell cultures upon transfection with thedifferent mAONs# exon 46 skipping was detected following transfection with a random AON. -
FIG. 4B . RT-PCR analysis in the human muscle cell cultures derived from one unaffected individual (C) and two unrelated DMD patients (P1 and P2) revealed truncated products upon transfection withhAON# 4 andhAON# 8. In the control, this product corresponded toexon 45 spliced toexon 47, while in the patients, the fragment size corresponded toexon 44 spliced toexon 47. Noexon 46 skipping was detected in the non-transfected cell cultures or following transfection with a random hAON.Highest exon 46 skipping efficiencies were obtained withhAON# 8. -
FIG. 5 . Sequence data from the RT-PCR products obtained from patient DL279.1 (corresponding to P1 inFIG. 4 ), which confirmed the deletion ofexon 45 in this patient (upper panel), and the additional skipping ofexon 46 following transfection with hAON#8 (lower panel). The skipping ofexon 46 was specific, andexon 44 was exactly spliced toexon 47, which reestablishes the translational reading frame. -
FIG. 6 . Immunohistochemical analysis of the muscle cell culture from patient DL279.1 upon transfection withhAON# 8. Cells were subject to two different dystrophin antibodies raised against different regions of the protein, located proximally (ManDys-1, ex. 31-32) and distally (Dys-2, ex. 77-79) from the targetedexon 46. The lower panel shows the absence of a dystrophin protein in the myotubes, whereas the hAON#8-induced skipping ofexon 46 clearly restored the synthesis of a dystrophin protein as detected by both antibodies (upper panel). -
FIG. 7A . RT-PCR analysis of RNA isolated from human control muscle cell cultures treated withhAON# 23, #24, #27, #28, or #29. An additional aberrant splicing product was obtained in cells treated withhAON# 28 and #29. Sequence analysis revealed the utilization of an in-frame cryptic splice site withinexon 51 that is used at a low frequency upon AON treatment. The product generated included apartial exon 51, which also had a restored reading frame, thereby confirming further the therapeutic value. -
FIG. 7B . A truncated product, with a size corresponding to exon 50 spliced toexon 52, was detected in cells treated withhAON# 23 and #28. Sequence analysis of these products confirmed the precise skipping ofexon 51. -
FIG. 8A . Gel mobility shift assays were performed to determine the binding affinity of the different h29AON#'s for theexon 29 target RNA. When compared to non-hybridized RNA (none),h29AON# 1, #2, #4, #6, #9, #10, and #11 generated complexes with lower gel mobilities, indicating their binding to the RNA. A random AON derived fromdystrophin exon 19 did not generate a complex. -
FIG. 8B . RT-PCR analysis of RNA isolated from human control muscle cell cultures treated withh29AON# 1, #2, #4, #6, #9, #10, or #11 revealed a truncated product of which the size corresponded toexon 28 spliced toexon 30. These results indicate thatexon 29 can specifically be skipped using AONs directed to sequences either within (h29AON# 1, #2, #4, or #6) or outside (h29AON# 9, #10, or #11) the hypothesized ERS inexon 29. An additional aberrant splicing product was observed that resulted from skipping of bothexon 28 and exon 29 (confirmed by sequence data not shown). Although this product was also present in non-treated cells, suggesting that this alternative skipping event may occur naturally, it was enhanced by the AON-treatment.AON 19, derived fromdystrophin exon 19, did not induceexon 29 skipping. -
FIG. 8C . The specific skipping ofexon 29 was confirmed by sequence data from the truncated RT-PCR fragments. Shown here is the sequence obtained from theexon 29 skipping product in cells treated withh29AON# 1. -
FIG. 9A . RT-PCR analysis of RNA isolated from mouse gastrocnemius muscles two days post-injection of 5, 10, or 20 μg of eithermAON# 4, #6, or #11. Truncated products, with a size corresponding to exon 45 spliced toexon 47, were detected in all treated muscles. The samples -RT, -RNA, AD-1, and AD-2 were analyzed as negative controls for the RT-PCR reactions. -
FIG. 9B . Sequence analysis of the truncated products generated bymAON# 4 and #6 (and #11, not shown) confirmed the precise skipping ofexon 46. - Since
exon 45 is one of the most frequently deleted exons in DMD, we initially aimed at inducing the specific skipping of exon 46 (FIG. 1 ). This would produce the shorter, largely functional dystrophin found in BMD patients carrying a deletion ofexons exon 45. - Design of mAONs and hAONs
- A series of mouse- and human-specific AONs (mAONs and hAONs) was designed, directed at an internal part of
exon 46 that contains a stretch of purine-rich sequences and is hypothesized to have a putative regulatory role in the splicing process of exon 46 (FIG. 2 ). For the initial screening of the AONs in the gel mobility shift assays (see below), we used non-modified DNA-oligonucleotides (synthesized by EuroGentec, Belgium). For the actual transfection experiments in muscle cells, we used 2′-O-methyl-phosphorothioate oligoribonucleotides (also synthesized by EuroGentec, Belgium). These modified RNA oligonucleotides are known to be resistant to endonucleases and RNaseH, and to bind to RNA with high affinity. The sequences of those AONs that were eventually effective and applied in muscle cells in vitro are shown below. The corresponding mouse and human-specific AONs are highly homologous but not completely identical. - The listing below refers to the deoxy-form used for testing, in the finally used 2-O-methyl ribonucleotides all T's should be read as U's.
-
mAON#2: 5′ GCAATTGTTATCTGCTT (SEQ ID NO: 1) mAON#3: 5′ GTTATCTGCTTCTTCC (SEQ ID NO: 2) mAON#4: 5′ CTGCTTCTTCCAGCC (SEQ ID NO: 3) mAON#5: 5′ TCTGCTTCTTCCAGC (SEQ ID NO: 4) mAON#6: 5′ GTTATCTGCTTCTTCCAGCC (SEQ ID NO: 5) mAON#7: 5′ CTTTTAGCTGCTGCTC (SEQ ID NO: 6) mAON#8: 5′ GTTGTTCTTTTAGCTGCTGC (SEQ ID NO: 7) mAON#9: 5′ TTAGCTGCTGCTCAT (SEQ ID NO: 8) mAON#10: 5′ TTTAGCTGCTGCTCATCTCC (SEQ ID NO: 9) mAON#11: 5′ CTGCTGCTCATCTCC (SEQ ID NO: 10) hAON#4: 5′ CTGCTTCCTCCAACC (SEQ ID NO: 11) hAON#6: 5′ GTTATCTGCTTCCTCCAACC (SEQ ID NO: 12) hAON#8: 5′ GCTTTTCTTTTAGTTGCTGC (SEQ ID NO: 13) hAON#9: 5′ TTAGTTGCTGCTCTT (SEQ ID NO: 14) hAON#11: 5′ TTGCTGCTCTTTTCC (SEQ ID NO: 15) - The efficacy of the AONs is determined by their binding affinity for the target sequence. Notwithstanding recent improvements in computer simulation programs for the prediction of RNA-folding, it is difficult to speculate which of the designed AONs would be capable of binding the target sequence with a relatively high affinity. Therefore, we performed gel mobility shift assays (according to protocols described by Bruice et al., 1997). The
exon 46 target RNA fragment was generated by in vitro T7-transcription from a PCR fragment (amplified from either murine or human muscle mRNA using a sense primer that contains the T7 promoter sequence) in the presence of 32P-CTP. The binding affinity of the individual AONs (0.5 pmol) for the target transcript fragments was determined by hybridization at 37° C. for 30 minutes and subsequent polyacrylamide (8%) gel electrophoresis. We performed these assays for the screening of both the mouse and human-specific AONs (FIG. 3 ). At least 5 different mouse-specific AONs (mAON# hAON# - Transfection into Muscle Cell Cultures
- The exon 46-specific AONs which showed the highest target binding affinity in gel mobility shift assays were selected for analysis of their efficacy in inducing the skipping in muscle cells in vitro. In all transfection experiments, we included a non-specific AON as a negative control for the specific skipping of
exon 46. As mentioned, the system was first set up in mouse muscle cells. We used both proliferating myoblasts and post-mitotic myotube cultures (expressing higher levels of dystrophin) derived from the mouse muscle cell line C2C12. For the subsequent experiments in human-derived muscle cell cultures, we used primary muscle cell cultures isolated from muscle biopsies from one unaffected individual and two unrelated DMD patients carrying a deletion ofexon 45. These heterogeneous cultures contained approximately 20-40% myogenic cells. The different AONs (at a concentration of 1 μM) were transfected into the cells using the cationic polymer PEI (MBI Fermentas) at a ratio-equivalent of 3. The AONs transfected in these experiments contained a 5′ fluorescein group which allowed us to determine the transfection efficiencies by counting the number of fluorescent nuclei. Typically, more than 60% of cells showed specific nuclear uptake of the AONs. To facilitate RT-PCR analysis, RNA was isolated 24 hours post-transfection using RNAzol B (CamPro Scientific, The Netherlands). - RNA was reverse transcribed using C. therm. polymerase (Roche) and an exon 48-specific reverse primer. To facilitate the detection of skipping of
dystrophin exon 46, the cDNA was amplified by two rounds of PCR, including a nested amplification using primers inexons 44 and 47 (for the human system), orexons 45 and 47 (for the mouse system). In the mouse myoblast and myotube cell cultures, we detected a truncated product of which the size corresponded toexon 45 directly spliced to exon 47 (FIG. 4 ). Subsequent sequence analysis confirmed the specific skipping ofexon 46 from these mouse dystrophin transcripts. The efficiency of exon skipping was different for the individual AONs, withmAON# 4 and #11 showing the highest efficiencies. Following these promising results, we focused on inducing a similar modulation of dystrophin splicing in the human-derived muscle cell cultures. Accordingly, we detected a truncated product in the control muscle cells, corresponding toexon 45 spliced toexon 47. Interestingly, in the patient-derived muscle cells, a shorter fragment was detected, which consisted ofexon 44 spliced toexon 47. The specific skipping ofexon 46 from the human dystrophin transcripts was confirmed by sequence data. This splicing modulation of both the mouse and human dystrophin transcript was neither observed in non-transfected cell cultures nor in cultures transfected with a non-specific AON. - We intended to induce the skipping of
exon 46 in muscle cells from patients carrying anexon 45 deletion in order to restore the translation and synthesis of a dystrophin protein. To detect a dystrophin product upon transfection withhAON# 8, the two patient-derived muscle cell cultures were subject to immunocytochemistry using two different dystrophin monoclonal antibodies (Mandys-1 and Dys-2) raised against domains of the dystrophin protein located proximal and distal of the targeted region respectively. Fluorescent analysis revealed restoration of dystrophin synthesis in both patient-derived cell cultures (FIG. 5 ). Approximately at least 80% of the fibers stained positive for dystrophin in the treated samples. - Our results show, for the first time, the restoration of dystrophin synthesis from the endogenous DMD gene in muscle cells from DMD patients. This is a proof of principle of the feasibility of targeted modulation of dystrophin pre-mRNA splicing for therapeutic purposes.
- The targeted skipping of
exon 51. We demonstrated the feasibility of AON-mediated modulation ofdystrophin exon 46 splicing, in mouse and human muscle cells in vitro. These findings warranted further studies to evaluate AONs as therapeutic agents for DMD. Since most DMD-causing deletions are clustered in two mutation hot spots, the targeted skipping of one particular exon can restore the reading frame in series of patients with different mutations (see Table 1).Exon 51 is an interesting target exon. The skipping of this exon is therapeutically applicable in patients carryingdeletions spanning exon 50, exons 45-50, exons 48-50, exons 49-50,exon 52, and exons 52-63, which includes a total of 15% of patients from our Leiden database. - We designed a series of ten human-specific AONs (hAON#21-30, see below) directed at different purine-rich regions within
dystrophin exon 51. These purine-rich stretches suggested the presence of a putative exon splicing regulatory element that we aimed to block in order to induce the elimination of that exon during the splicing process. All experiments were performed according to protocols as described for the skipping of exon 46 (see above). Gel mobility shift assays were performed to identify those hAONs with high binding affinity for the target RNA. We selected the five hAONs that showed the highest affinity. These hAONs were transfected into human control muscle cell cultures in order to test the feasibility of skippingexon 51 in vitro. RNA was isolated 24 hours post-transfection, and cDNA was generated using an exon 53- or 65-specific reverse primer. PCR-amplification of the targeted region was performed using different primercombinations flanking exon 51. The RT-PCR and sequence analysis revealed that we were able to induce the specific skipping ofexon 51 from the human dystrophin transcript. We subsequently transfected two hAONs (#23 and #29) shown to be capable of inducing skipping of the exon into six different muscle cell cultures derived from DMD-patients carrying one of the mutations mentioned above. The skipping ofexon 51 in these cultures was confirmed by RT-PCR and sequence analysis (FIG. 7 ). More importantly, immunohistochemical analysis, using multiple antibodies raised against different parts of the dystrophin protein, showed in all cases that, due to the skipping ofexon 51, the synthesis of a dystrophin protein was restored. - Exon 51-specific hAONs:
-
hAON#21: 5′ CCACAGGTTGTGTCACCAG (SEQ ID NO: 16) hAON#22: 5′ TTTCCTTAGTAACCACAGGTT (SEQ ID NO: 17) hAON#23: 5′ TGGCATTTCTAGTTTGG (SEQ ID NO: 18) hAON#24: 5′ CCAGAGCAGGTACCTCCAACATC (SEQ ID NO: 19) hAON#25: 5′ GGTAAGTTCTGTCCAAGCCC (SEQ ID NO: 20) hAON#26: 5′ TCACCCTCTGTGATTTTAT (SEQ ID NO: 21) hAON#27: 5′ CCCTCTGTGATTTT (SEQ ID NO: 22) hAON#28: 5′ TCACCCACCATCACCCT (SEQ ID NO: 23) hAON#29: 5′ TGATATCCTCAAGGTCACCC (SEQ ID NO: 24) hAON#30: 5′ CTGCTTGATGATCATCTCGTT (SEQ ID NO: 25) - The skipping of one additional exon, such as
exon 46 orexon 51, restores the reading frame for a considerable number of different DMD mutations. The range of mutations for which this strategy is applicable can be enlarged by the simultaneous skipping of more than one exon. For instance, in DMD patients with a deletion ofexon 46 toexon 50, only the skipping of both the deletion-flankingexons - A mutation in
exon 29 leads to the skipping of this exon in two Becker muscular dystrophy patients (Ginjaar at al., 2000, EJHG, vol. 8, p. 793-796). We studied the feasibility of directing the skipping ofexon 29 through targeting the site of mutation by AONs. The mutation is located in a purine-rich stretch that could be associated with ERS activity. We designed a series of AONs (see below) directed to sequences both within (h29AON# 1 to h29AON#6) and outside (h29AON# 7 to h29AON#11) the hypothesized ERS. Gel mobility shift assays were performed (as described) to identify those AONs with highest affinity for the target RNA (FIG. 8 ). Subsequently,h29AON# 1, #2, #4, #6, #9, #10, and #11 were transfected into human control myotube cultures using the PEI transfection reagent. RNA was isolated 24 hrs post-transfection, and cDNA was generated using an exon 31-specific reverse primer. PCR-amplification of the targeted region was performed using different primercombinations flanking exon 29. This RT-PCR and subsequent sequence analysis (FIGS. 8B and 8C ) revealed that we were able to induce the skipping ofexon 29 from the human dystrophin transcript. However, the AONs that facilitated this skipping were directed to sequences both within and outside the hypothesized ERS (h29AON# 1, #2, #4, #6, #9, and #11). These results suggest that skipping ofexon 29 occurs independent of whether or notexon 29 contains an ERS and that, therefore, the binding of the AONs to exon 29 more likely inactivated an exon inclusion signal rather than an ERS. This proof of ERS-independent exon skipping may extend the overall applicability of this therapy to exons without ERS's. -
h29AON#1: 5′ TATCCTCTGAATGTCGCATC (SEQ ID NO: 26) h29AON#2: 5′ GGTTATCCTCTGAATGTCGC (SEQ ID NO: 27) h29AON#3: 5′ TCTGTTAGGGTCTGTGCC (SEQ ID NO: 28) h29AON#4: 5′ CCATCTGTTAGGGTCTGTG (SEQ ID NO: 29) h29AON#5: 5′ GTCTGTGCCAATATGCG (SEQ ID NO: 30) h29AON#6: 5′ TCTGTGCCAATATGCGAATC (SEQ ID NO: 31) h29AON#7: 5′ TGTCTCAAGTTCCTC (SEQ ID NO: 32) h29AON#8: 5′ GAATTAAATGTCTCAAGTTC (SEQ ID NO: 33) h29AON#9: 5′ TTAAATGTCTCAAGTTCC (SEQ ID NO: 34) h29AON#10: 5′ GTAGTTCCCTCCAACG (SEQ ID NO: 35) h29AON#11: 5′ CATGTAGTTCCCTCC (SEQ ID NO: 36) - Following the promising results in cultured muscle cells, we tested the different mouse dystrophin exon 46-specific AONs in vivo by injecting them, linked to polyethylenimine (PEI), into the gastrocnemius muscles of control mice. With
mAON# 4, #6, and #11, previously shown to be effective in mouse muscle cells in vitro, we were able to induce the skipping ofexon 46 in muscle tissue in vivo as determined by both RT-PCR and sequence analysis (FIG. 9 ). The invivo exon 46 skipping was dose-dependent with highest efficiencies (up to 10%) following injection of 20 μg per muscle per day for two subsequent days. -
- Achsel et al., 1996, J. Biochem. 120, pp. 53-60.
- Bruice T. W. and Lima, W. F., 1997, Biochemistry 36(16): pp. 5004-5019.
- Brunak at al., 1991, J. Mol. Biol. 220, pp. 49-65.
- Dunckley, M. G. et al., 1998, Human
molecular genetics 7, pp. 1083-1090. - Ginjaar et al., 2000, EJHG, vol. 8, pp. 793-796.
- Mann et al., 2001, PNAS vol. 98, pp. 42-47.
- Tanaka et al., 1994 Mol. Cell. Biol. 14, pp. 1347-1354.
- Wilton, S. D., et al., 1999,
Neuromuscular disorders 9, pp. 330-338. - Details and background on Duchenne Muscular Dystrophy and related diseases can be found on website http://www.dmd.nl
Claims (49)
1-24. (canceled)
25. A method for directing splicing of a dystrophin pre-mRNA in a cell having dystrophin pre-mRNA, the method comprising:
contacting the dystrophin pre-mRNA in the cell with an antisense-oligonucleotide having between 14-40 nucleotides, capable of specifically inhibiting an exon inclusion signal of exon number 2, 8, 29, 43, 44, 45; 46, 50, 51, 52, or 53 in the pre-mRNA, and
allowing splicing of the pre-mRNA.
26. The method according to claim 25 , wherein the cell is a cell from an individual suffering from Duchenne Muscular Dystrophy (DMD).
27. The method according to claim 26 , wherein the dystrophin pre-mRNA comprises a DMD deletion of one or more exons selected from the groups consisting of exons 3-7, 4-7, 5-7, 6-7, 18-44, 35-43, 44, 44-47, 45, 45-54, 45-52, 50, 50-52, 45-50, 46-47, 46-48, 46 49, 46-51, 46-53, 48-52, 48-50, 49-50, 49-52, 52, 52-63, 51, 51-55, 53, and 53-55.
28. The method according to claim 25 , wherein the antisense-oligonucleotide exhibiting the specific inhibition of an exon inclusion signal is obtainable by a method comprising:
providing a second cell with the antisense-oligonucleotide, the cell having pre-mRNA containing the exon,
culturing the second cell to form mRNA in the second cell from the pre-mRNA in the second cell, and
determining whether the exon is absent from the thus formed mRNA in the second cell.
29. The method according to claim 25 , further comprising allowing translation of an mRNA produced from splicing of the dystrophin pre-mRNA.
30. The method according to claim 29 , wherein the mRNA encodes a functional dystrophin protein.
31. The method according to claim 30 , wherein the functional dystrophin protein comprises at least two domains, wherein at least one of the domains is encoded by the mRNA as a result of skipping of at least part of an exon in the dystrophin pre-mRNA.
32. The method according to claim 25 , wherein contacting results in activation of a cryptic splice site in a contacted exon.
33. The method according to claim 25 , wherein the exon inclusion signal is present in an exon comprising a strong splice donor/acceptor pair.
34. The method according to claim 29 , wherein the translation results in a mutant dystrophin protein or a normal dystrophin protein.
35. The method according to claim 34 , wherein the mutant dystrophin protein is equivalent to a dystrophin protein of a Becker patient.
36. The method according to claim 25 , wherein the antisense-oligonucleotide is capable of specifically inhibiting an exon inclusion signal of exon number 51.
37. The method according to claim 25 , wherein the antisense-oligonucleotide comprises a nucleic acid.
38. The method according to claim 37 , wherein the nucleic acid comprises a 2′-O-methyl-oligoribonucleotide or a 2′-O-methyl-phosphorothioate.
39. The method according to claim 25 , wherein the antisense-oligonucleotide contains between 15-25 nucleotides.
40. The method according to claim 25 , further comprising:
providing the cell with another antisense-oligonucleotide capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.
41. A method for at least in part decreasing the production of an aberrant dystrophin protein in a cell, the cell comprising dystrophin pre-mRNA comprising exons coding for the aberrant dystrophin protein, the method comprising:
providing the cell with an antisense-oligonucleotide having between 14-40 nucleotides, capable of specifically inhibiting an exon inclusion signal of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53, and
allowing translation of mRNA produced from splicing of the dystrophin pre-mRNA.
42. The method according to claim 41 , wherein the antisense-oligonucleotide exhibiting the specific inhibition of an exon inclusion signal is obtainable by a method comprising:
providing a second cell with the antisense-oligonucleotide, the cell having pre-mRNA containing the exon,
culturing the second cell to form mRNA in the second cell from the pre-mRNA in the second cell, and
determining whether the exon is absent from the thus formed mRNA in the second cell.
43. The method according to claim 41 , wherein the antisense-oligonucleotide is capable of specifically inhibiting an exon inclusion signal of exon number 51.
44. The method according to claim 41 , wherein the dystrophin pre-mRNA comprises a Duchenne Muscular Dystrophy (DMD) deletion of one or more exons selected from the groups consisting of exons 3-7, 4-7, 5-7, 6-7, 18-44, 35-43, 44, 44-47, 45, 45-54, 45-52, 50, 50-52, 45-50, 46-47, 46-48, 46-49, 46-51, 46-53, 48-52, 48-50, 49-50, 49-52, 52, 52-63, 51, 51 55, 53, and 53-55.
45. The method according to claim 41 , wherein the antisense-oligonucleotide comprises a nucleic acid.
46. The method according to claim 21, wherein the nucleic acid comprises a 2′-O-methyl-oligoribonucleotide or a 2′-O-methyl-phosphorothioate oligoribonucleotide.
47. The method according to claim 45 , wherein the antisense-oligonucleotide contains between 15-25 nucleotides.
48. The method according to claim 41 , further comprising:
providing the cell with another antisense-oligonucleotide capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.
49. A method for determining whether a compound is able specifically inhibit an exon inclusion signal of an exon of a dystrophin pre-mRNA, the exon being selected from the group consisting of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53 and combinations thereof of the dystrophin pre-mRNA, wherein the compound has complementarity to a part of the selected exon, the method comprising:
providing a cell having a dystrophin pre-mRNA containing the exon with the compound,
culturing the cell to allow the formation of an mRNA from the dystrophin pre-mRNA, and
determining whether the exon is absent from the mRNA.
50. The method according to claim 49 further comprising determining in vitro the relative binding affinity of the compound to an RNA molecule comprising the exon.
51. A compound identified by the method according to claim 49 .
52. A nucleic acid delivery vehicle comprising the compound of claim 51 , or the complement thereof.
53. A nucleic acid delivery vehicle comprising the compound of claim 51 .
54. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising:
administering to the subject the compound of claim 51 .
55. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising administering to the subject the nucleic acid delivery vehicle of claim 52 .
56. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising:
administering to the subject an antisense-oligonucleotide, having between 14-40 nucleotides, and having complementarity to an element located within an exon in the pre-mRNA, the exon being selected from the group consisting of exon number 2, 8, 29, 43, 44, 45, 46, 50, 51, 52, or 53 and combinations thereof of a dystrophin gene encoding an aberrant protein,
wherein the antisense-oligonucleotide specifically inhibits and interferes with an exon inclusion signal of the exon in the pre-mRNA, thus forming a final mRNA excluding the exon, but encoding a mutant dystrophin protein having Becker mutant's functionality.
57. The method according to claim 56 , wherein the antisense-oligonucleotide that specifically inhibits and interferes with an exon inclusion signal of the exon in the pre-mRNA is obtainable by a method comprising:
providing a cell with the antisense-oligonucleotide, the cell having a pre-mRNA containing the exon, culturing the cell to form mRNA in the cell from the pre-mRNA in the cell, and determining whether the exon is absent from the thus formed mRNA in the cell.
58. The method according to claim 56 , wherein the antisense-oligonucleotide contains between 15-25 nucleotides.
59. A non-human animal provided with the compound of claim 51 .
60. The non-human animal of claim 59 , further comprising a nucleic acid encoding a human protein.
61. The non-human animal of claim 60 , further comprising a silencing mutation in a gene encoding an animal homologue of the human protein.
62. A composition comprising:
a nucleic acid or a functional equivalent thereof capable of inhibiting an exon inclusion signal in exon 2, 8, 29, 43, 44, 45, 46, 50, 51 or 53 of a dystrophin pre-mRNA, and having between 14-40 nucleotides.
63. The composition of claim 62 , further comprising:
a further nucleic acid or a functional equivalent thereof capable of inhibiting an exon inclusion signal present in another exon of the dystrophin pre-mRNA.
64. The composition of claim 62 , wherein the nucleic acid is an antisense-oligonucleotide containing between 15-25 nucleotides.
65. A nucleic acid delivery vehicle comprising:
an antisense-oligonucleotide capable of inhibiting an exon-inclusion signal in at least one of exons 2, 8, 29, 43, 44, 45, 50, 51, 52 or 53 of a dystrophin pre-mRNA, wherein the antisense-oligonucleotide contains between 14-40 nucleotides, or
the complement of said antisense-oligonucleotide.
66. The nucleic acid delivery vehicle of claim 65 , wherein the antisense-oligonucleotide contains between 15-25 nucleotides.
67. An antisense-oligonucleotide comprising one a nucleic acid sequence selected from the group consisting of
a functional part of any thereof, derivative of any thereof, and analogue of any thereof having the same exon skipping activity in kind, but not necessarily in amount.
68. The antisense-oligonucleotide of claim 67 , containing between 14-40 nucleotides.
69. A nucleic acid delivery vehicle capable of expressing the antisense-oligonucleotide of claim 67 .
70. The nucleic acid delivery vehicle of claim 69 , wherein the nucleic acid delivery vehicle is a single stranded virus.
71. The nucleic acid delivery vehicle of claim 70 , wherein said single stranded virus comprises an adeno-associated virus.
72. A method for directing splicing of a dystrophin pre-mRNA in a subject, the method comprising administering to the subject the nucleic acid delivery vehicle of claim 53 .
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/383,897 US20090228998A1 (en) | 2000-09-21 | 2009-03-30 | Induction of exon skipping in eukaryotic cells |
US14/331,934 US20140350076A1 (en) | 2000-09-21 | 2014-07-15 | Induction of exon skipping in eukaryotic cells |
US14/712,753 US20150322434A1 (en) | 2000-09-21 | 2015-05-14 | Induction of exon skipping in eukaryotic cells |
US14/839,200 US20150361424A1 (en) | 2000-09-21 | 2015-08-28 | Induction of exon skipping in eukaryotic cells |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00203283.7 | 2000-09-21 | ||
EP00203283A EP1191097A1 (en) | 2000-09-21 | 2000-09-21 | Induction of exon skipping in eukaryotic cells |
PCT/NL2001/000697 WO2002024906A1 (en) | 2000-09-21 | 2001-09-21 | Induction of exon skipping in eukaryotic cells |
US10/395,031 US7973015B2 (en) | 2000-09-21 | 2003-03-21 | Induction of exon skipping in eukaryotic cells |
US12/383,897 US20090228998A1 (en) | 2000-09-21 | 2009-03-30 | Induction of exon skipping in eukaryotic cells |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/395,031 Continuation US7973015B2 (en) | 2000-09-21 | 2003-03-21 | Induction of exon skipping in eukaryotic cells |
US11/982,285 Continuation US20080209581A1 (en) | 2000-09-21 | 2007-10-31 | Induction of exon skipping in eukaryotic cells |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/331,934 Continuation US20140350076A1 (en) | 2000-09-21 | 2014-07-15 | Induction of exon skipping in eukaryotic cells |
Publications (1)
Publication Number | Publication Date |
---|---|
US20090228998A1 true US20090228998A1 (en) | 2009-09-10 |
Family
ID=8172043
Family Applications (6)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/395,031 Expired - Fee Related US7973015B2 (en) | 2000-09-21 | 2003-03-21 | Induction of exon skipping in eukaryotic cells |
US11/982,285 Abandoned US20080209581A1 (en) | 2000-09-21 | 2007-10-31 | Induction of exon skipping in eukaryotic cells |
US12/383,897 Abandoned US20090228998A1 (en) | 2000-09-21 | 2009-03-30 | Induction of exon skipping in eukaryotic cells |
US14/331,934 Abandoned US20140350076A1 (en) | 2000-09-21 | 2014-07-15 | Induction of exon skipping in eukaryotic cells |
US14/712,753 Abandoned US20150322434A1 (en) | 2000-09-21 | 2015-05-14 | Induction of exon skipping in eukaryotic cells |
US14/839,200 Abandoned US20150361424A1 (en) | 2000-09-21 | 2015-08-28 | Induction of exon skipping in eukaryotic cells |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/395,031 Expired - Fee Related US7973015B2 (en) | 2000-09-21 | 2003-03-21 | Induction of exon skipping in eukaryotic cells |
US11/982,285 Abandoned US20080209581A1 (en) | 2000-09-21 | 2007-10-31 | Induction of exon skipping in eukaryotic cells |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/331,934 Abandoned US20140350076A1 (en) | 2000-09-21 | 2014-07-15 | Induction of exon skipping in eukaryotic cells |
US14/712,753 Abandoned US20150322434A1 (en) | 2000-09-21 | 2015-05-14 | Induction of exon skipping in eukaryotic cells |
US14/839,200 Abandoned US20150361424A1 (en) | 2000-09-21 | 2015-08-28 | Induction of exon skipping in eukaryotic cells |
Country Status (17)
Country | Link |
---|---|
US (6) | US7973015B2 (en) |
EP (14) | EP1191097A1 (en) |
JP (7) | JP4846965B2 (en) |
AT (1) | ATE409224T2 (en) |
AU (4) | AU2002211062C1 (en) |
CA (1) | CA2423044C (en) |
CY (10) | CY1109601T1 (en) |
DE (1) | DE60135936D1 (en) |
DK (10) | DK2594640T3 (en) |
ES (11) | ES2561292T3 (en) |
HK (8) | HK1184821A1 (en) |
LT (3) | LT2940139T (en) |
NZ (1) | NZ524853A (en) |
PT (7) | PT2801618T (en) |
SI (1) | SI2801618T1 (en) |
TR (1) | TR201810606T4 (en) |
WO (1) | WO2002024906A1 (en) |
Cited By (27)
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 |
US20110046360A1 (en) * | 2002-11-25 | 2011-02-24 | Masafumi Matsuo | ENA NUCLEIC ACID DRUGS MODIFYING SPLICING IN mRNA PRECURSOR |
US8637483B2 (en) | 2009-11-12 | 2014-01-28 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
EP2796425A1 (en) | 2013-04-24 | 2014-10-29 | Corning Incorporated | Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients |
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 |
US9605019B2 (en) | 2011-07-19 | 2017-03-28 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
US9611471B2 (en) | 2010-08-05 | 2017-04-04 | Academisch Ziekenhuis Leiden | Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins |
US9617547B2 (en) | 2012-07-13 | 2017-04-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant |
US9695211B2 (en) | 2008-12-02 | 2017-07-04 | Wave Life Sciences Japan, Inc. | Method for the synthesis of phosphorus atom modified nucleic acids |
US9744183B2 (en) | 2009-07-06 | 2017-08-29 | Wave Life Sciences Ltd. | Nucleic acid prodrugs and methods of use thereof |
US9890379B2 (en) | 2006-08-11 | 2018-02-13 | Biomarin Technologies B.V. | Treatment of genetic disorders associated with DNA repeat instability |
US9926544B2 (en) | 2014-01-24 | 2018-03-27 | Am-Pharma B.V. | Chimeric alkaline phosphatase-like proteins |
US9982257B2 (en) | 2012-07-13 | 2018-05-29 | Wave Life Sciences Ltd. | Chiral control |
US10144933B2 (en) | 2014-01-15 | 2018-12-04 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator |
US10149905B2 (en) | 2014-01-15 | 2018-12-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having antitumor effect and antitumor agent |
US10160969B2 (en) | 2014-01-16 | 2018-12-25 | Wave Life Sciences Ltd. | Chiral design |
US10167309B2 (en) | 2012-07-13 | 2019-01-01 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
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 |
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 |
US10322173B2 (en) | 2014-01-15 | 2019-06-18 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent |
US10428019B2 (en) | 2010-09-24 | 2019-10-01 | Wave Life Sciences Ltd. | Chiral auxiliaries |
US10450568B2 (en) | 2015-10-09 | 2019-10-22 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US10570380B2 (en) | 2014-01-24 | 2020-02-25 | Am-Pharma B.V. | Downstream processing of an alkaline phosphatase |
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 |
USRE48960E1 (en) | 2004-06-28 | 2022-03-08 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
Families Citing this family (98)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1282699B1 (en) | 2000-05-04 | 2012-11-21 | Sarepta Therapeutics, Inc. | Splice-region antisense composition and method |
EP1191097A1 (en) | 2000-09-21 | 2002-03-27 | Leids Universitair Medisch Centrum | Induction of exon skipping in eukaryotic cells |
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. |
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 |
ES2500921T3 (en) | 2003-04-29 | 2014-10-01 | Sarepta Therapeutics, Inc. | Compositions to enhance the transport and antisense efficacy of nucleic acid analogs in cells |
US20050288246A1 (en) * | 2004-05-24 | 2005-12-29 | Iversen Patrick L | Peptide conjugated, inosine-substituted antisense oligomer compound and method |
FR2874384B1 (en) * | 2004-08-17 | 2010-07-30 | Genethon | ADENO-ASSOCIATED VIRAL VECTOR FOR PRODUCING EXON JUMP IN A GENE ENCODING A PROTEIN WITH DISPENSABLE DOMAINS |
EP1855694B1 (en) | 2005-02-09 | 2020-12-02 | Sarepta Therapeutics, Inc. | Antisense composition for treating muscle atrophy |
EP1877555A2 (en) * | 2005-04-22 | 2008-01-16 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mrna by interfering with the binding of sr proteins and by interfering with secondary rna structure. |
US8067571B2 (en) | 2005-07-13 | 2011-11-29 | Avi Biopharma, Inc. | Antibacterial antisense oligonucleotide and method |
US7785834B2 (en) * | 2005-11-10 | 2010-08-31 | Ercole Biotech, Inc. | Soluble TNF receptors and their use in treatment of disease |
WO2007058894A2 (en) * | 2005-11-10 | 2007-05-24 | The University Of North Carolina At Chapel Hill | Splice switching oligomers for tnf superfamily receptors and their use in treatment of disease |
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 |
US20090264353A1 (en) * | 2007-10-19 | 2009-10-22 | Santaris Pharma A/S | Splice Switching Oligomers for TNF Superfamily Receptors and their Use in Treatment of Disease |
JP5864100B2 (en) * | 2007-06-29 | 2016-02-17 | サレプタ セラピューティクス インコーポレイテッド | Tissue-specific peptide conjugates and methods |
US20100016215A1 (en) * | 2007-06-29 | 2010-01-21 | Avi Biopharma, Inc. | Compound and method for treating myotonic dystrophy |
CN101815535B (en) | 2007-07-12 | 2013-04-24 | 普罗森那技术公司 | Molecules for targeting compounds to various selected organs or tissues |
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 |
USRE48468E1 (en) | 2007-10-26 | 2021-03-16 | Biomarin Technologies B.V. | Means and methods for counteracting muscle disorders |
WO2009099326A1 (en) | 2008-02-08 | 2009-08-13 | Prosensa Holding Bv | Methods and means for treating dna repeat instability associated genetic disorders |
US8084601B2 (en) | 2008-09-11 | 2011-12-27 | Royal Holloway And Bedford New College Royal Holloway, University Of London | Oligomers |
JP2012524540A (en) | 2009-04-24 | 2012-10-18 | プロセンサ テクノロジーズ ビー.ブイ. | Oligonucleotides containing inosine for treating DMD |
US20110269665A1 (en) | 2009-06-26 | 2011-11-03 | Avi Biopharma, Inc. | Compound and method for treating myotonic dystrophy |
US20120270930A1 (en) | 2009-10-29 | 2012-10-25 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Methods and compositions for dysferlin exon-skipping |
CA2785451C (en) | 2009-12-24 | 2019-01-22 | Prosensa Technologies B.V. | Molecule for treating an inflammatory disorder |
TWI541024B (en) | 2010-09-01 | 2016-07-11 | 日本新藥股份有限公司 | Antisense nucleic acid |
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 |
US9161948B2 (en) | 2011-05-05 | 2015-10-20 | Sarepta Therapeutics, Inc. | Peptide oligonucleotide conjugates |
WO2013033407A2 (en) | 2011-08-30 | 2013-03-07 | The Regents Of The University Of California | Identification of small molecules that enhance therapeutic exon skipping |
AU2012305053B2 (en) | 2011-09-05 | 2017-12-21 | Stichting Radboud Universitair Medisch Centrum | Antisense oligonucleotides for the treatment of Leber congenital amaurosis |
US20130085139A1 (en) | 2011-10-04 | 2013-04-04 | Royal Holloway And Bedford New College | Oligomers |
CA2861247C (en) * | 2011-12-28 | 2021-11-16 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
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 |
JP6460983B2 (en) * | 2012-07-03 | 2019-01-30 | バイオマリン テクノロジーズ ベー.フェー. | Oligonucleotides for the treatment of patients with muscular dystrophy |
RS60318B1 (en) | 2012-08-01 | 2020-07-31 | Ikaika Therapeutics Llc | Mitigating tissue damage and fibrosis via anti-ltbp4 antibody |
AU2015227733B2 (en) | 2014-03-12 | 2019-10-31 | National Center Of Neurology And Psychiatry | Antisense nucleic acids |
WO2015171918A2 (en) | 2014-05-07 | 2015-11-12 | Louisiana State University And Agricultural And Mechanical College | Compositions and uses for treatment thereof |
AU2015286663B2 (en) | 2014-07-10 | 2021-09-23 | Stichting Radboud Universitair Medisch Centrum | Antisense oligonucleotides for the treatment of usher syndrome type 2 |
JP6837429B2 (en) * | 2014-08-11 | 2021-03-03 | ザ ボード オブ リージェンツ オブ ザ ユニバーシティー オブ テキサス システム | Prevention of muscular dystrophy by editing CRISPR / CAS9-mediated genes |
CN107073091A (en) | 2014-09-07 | 2017-08-18 | 西莱克塔生物科技公司 | Method and composition for weakening the antiviral transfer vector immune response of exon skipping |
US20170266320A1 (en) | 2014-12-01 | 2017-09-21 | President And Fellows Of Harvard College | RNA-Guided Systems for In Vivo Gene Editing |
GB201504124D0 (en) | 2015-03-11 | 2015-04-22 | Proqr Therapeutics B V | Oligonucleotides |
MA41795A (en) | 2015-03-18 | 2018-01-23 | Sarepta Therapeutics Inc | EXCLUSION OF AN EXON INDUCED BY ANTISENSE COMPOUNDS IN MYOSTATIN |
EP3302489A4 (en) | 2015-06-04 | 2019-02-06 | Sarepta Therapeutics, Inc. | Methods and compounds for treatment of lymphocyte-related diseases and conditions |
US10144931B2 (en) | 2015-09-15 | 2018-12-04 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acids |
EP3858993A1 (en) | 2015-10-09 | 2021-08-04 | Sarepta Therapeutics, Inc. | Compositions and methods for treating duchenne muscular dystrophy and related disorders |
WO2017136435A1 (en) | 2016-02-01 | 2017-08-10 | The Usa, As Represented By The Secretary, Department Of Health And Human Services Office Of Technology Transfer National Institute Of Health | Compounds for modulating fc-epsilon-ri-beta expression and uses thereof |
AU2017257292A1 (en) | 2016-04-25 | 2018-12-06 | Proqr Therapeutics Ii B.V. | Oligonucleotides to treat eye disease |
AU2017290231A1 (en) | 2016-06-30 | 2019-02-07 | Sarepta Therapeutics, Inc. | Exon skipping oligomers for muscular dystrophy |
SG10201607303YA (en) * | 2016-09-01 | 2018-04-27 | Agency Science Tech & Res | Antisense oligonucleotides to induce exon skipping |
GB201616202D0 (en) | 2016-09-23 | 2016-11-09 | Proqr Therapeutics Ii Bv | Antisense oligonucleotides for the treatment of eye deisease |
WO2018109011A1 (en) | 2016-12-13 | 2018-06-21 | Stichting Katholieke Universiteit | Antisense oligonucleotides for the treatment of stargardt disease |
MX2019006989A (en) | 2016-12-19 | 2019-08-16 | Sarepta Therapeutics Inc | Exon skipping oligomer conjugates for muscular dystrophy. |
BR112019012664A2 (en) | 2016-12-19 | 2020-01-21 | Sarepta Therapeutics Inc | exon jump oligomer conjugates for muscular dystrophy |
MD3554553T2 (en) | 2016-12-19 | 2022-10-31 | Sarepta Therapeutics Inc | Exon skipping oligomer conjugates for muscular dystrophy |
US10961537B2 (en) | 2017-07-18 | 2021-03-30 | Csl Behring Gene Therapy, Inc. | Compositions and methods for treating beta-hemoglobinopathies |
GB201711809D0 (en) | 2017-07-21 | 2017-09-06 | Governors Of The Univ Of Alberta | Antisense oligonucleotide |
SG10202002990XA (en) | 2017-08-04 | 2020-05-28 | Skyhawk Therapeutics Inc | Methods and compositions for modulating splicing |
EP3665304A4 (en) * | 2017-08-11 | 2021-04-28 | Agency for Science, Technology and Research | Method for screening splicing variants or events |
EA201991450A1 (en) | 2017-09-22 | 2019-12-30 | Сарепта Терапьютикс, Инк. | OLIGOMER CONJUGATES FOR EXONISM SKIP IN 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 |
US20190142974A1 (en) | 2017-10-13 | 2019-05-16 | Selecta Biosciences, Inc. | Methods and compositions for attenuating anti-viral transfer vector igm responses |
GB201803010D0 (en) | 2018-02-26 | 2018-04-11 | Royal Holloway & Bedford New College | Neurodegenerative disorders |
US10765760B2 (en) | 2018-05-29 | 2020-09-08 | Sarepta Therapeutics, Inc. | Exon skipping oligomer conjugates for muscular dystrophy |
EP3824086A1 (en) | 2018-07-19 | 2021-05-26 | Stichting Katholieke Universiteit | Antisense oligonucleotides rescue aberrant splicing of abca4 |
KR20210081324A (en) | 2018-08-02 | 2021-07-01 | 다인 세라퓨틱스, 인크. | Muscle targeting complexes and their use for treating facioscapulohumeral muscular dystrophy |
CA3108282A1 (en) | 2018-08-02 | 2020-02-06 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
US11168141B2 (en) | 2018-08-02 | 2021-11-09 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
JP2022510673A (en) | 2018-12-04 | 2022-01-27 | スティッチング カソリーケ ウニベルシテイト | Antisense oligonucleotide rescues abnormal splicing of ABCA4 |
EP3897745A1 (en) | 2018-12-23 | 2021-10-27 | CSL Behring LLC | Haematopoietic stem cell-gene therapy for wiskott-aldrich syndrome |
CA3123045A1 (en) | 2018-12-23 | 2020-07-02 | Csl Behring L.L.C. | Donor t-cells with kill switch |
WO2020148400A1 (en) | 2019-01-16 | 2020-07-23 | Stichting Katholieke Universiteit | Antisense oligonucleotides for use in the treatment of crpc |
WO2020157014A1 (en) | 2019-01-28 | 2020-08-06 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of leber's congenital amaurosis |
JP2022521467A (en) | 2019-02-05 | 2022-04-08 | スカイホーク・セラピューティクス・インコーポレーテッド | Methods and compositions for regulating splicing |
WO2020163544A1 (en) | 2019-02-06 | 2020-08-13 | Skyhawk Therapeutics, Inc. | Methods and compositions for modulating splicing |
US20220177894A1 (en) | 2019-04-02 | 2022-06-09 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for immunotherapy |
AU2020259856A1 (en) | 2019-04-18 | 2021-11-18 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of usher syndrome |
CN114206396A (en) | 2019-05-28 | 2022-03-18 | 西莱克塔生物科技公司 | Methods and compositions for attenuating an immune response against an antiviral transfer vector |
WO2020254249A1 (en) | 2019-06-21 | 2020-12-24 | Proqr Therapeutics Ii B.V. | Delivery of nucleic acids for the treatment of auditory disorders |
JP2022543474A (en) | 2019-08-08 | 2022-10-12 | ユーシーエル ビジネス リミテッド | Antisense oligonucleotides rescue ABCA4 aberrant splicing |
WO2021084021A1 (en) | 2019-10-31 | 2021-05-06 | Stichting Katholieke Universiteit | Allele-specific silencing therapy for dfna9 using antisense oligonucleotides |
EP4112083A1 (en) | 2020-02-28 | 2023-01-04 | Nippon Shinyaku Co., Ltd. | Antisense nucleic acid inducing skipping of exon 51 |
EP4114945A1 (en) | 2020-03-04 | 2023-01-11 | ProQR Therapeutics II B.V. | Antisense oligonucleotides for use in the treatment of usher syndrome |
AU2021294317A1 (en) | 2020-06-26 | 2023-02-23 | Csl Behring Llc | Donor T-cells with kill switch |
WO2022090256A1 (en) | 2020-10-26 | 2022-05-05 | Proqr Therapeutics Ii B.V. | Antisense oligonucleotides for the treatment of stargardt disease |
CN112430645A (en) * | 2020-12-09 | 2021-03-02 | 北京华瑞康源生物科技发展有限公司 | Relative quantitative method and kit for detecting human DMD gene copy number by multiple real-time fluorescence PCR method |
IL308353A (en) | 2021-05-10 | 2024-01-01 | Entrada Therapeutics Inc | Compositions and methods for modulating tissue distribution of intracellular therapeutics |
AU2022298774A1 (en) | 2021-06-23 | 2023-12-14 | Entrada Therapeutics, Inc. | Antisense compounds and methods for targeting cug repeats |
EP4359526A1 (en) | 2021-06-25 | 2024-05-01 | Stichting Radboud universitair medisch centrum | Allele-specific silencing therapy for dfna21 using antisense oligonucleotides |
US11638761B2 (en) | 2021-07-09 | 2023-05-02 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy |
US11771776B2 (en) | 2021-07-09 | 2023-10-03 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating dystrophinopathies |
WO2023064367A1 (en) | 2021-10-12 | 2023-04-20 | Selecta Biosciences, Inc. | Methods and compositions for attenuating anti-viral transfer vector igm responses |
EP4215614A1 (en) | 2022-01-24 | 2023-07-26 | Dynacure | Combination therapy for dystrophin-related diseases |
WO2023172624A1 (en) | 2022-03-09 | 2023-09-14 | Selecta Biosciences, Inc. | Immunosuppressants in combination with anti-igm agents and related dosing |
DE102022124232A1 (en) | 2022-09-21 | 2024-03-21 | Carl von Ossietzky Universität Oldenburg, Körperschaft des öffentlichen Rechts | Antisense oligonucleotides for the treatment of Joubert syndrome |
WO2024074668A1 (en) | 2022-10-06 | 2024-04-11 | Stichting Radboud Universitair Medisch Centrum | Antisense oligonucleotides for treatment of usher 2a. exons 30-31 |
WO2024074670A1 (en) | 2022-10-06 | 2024-04-11 | Stichting Radboud Universitair Medisch Centrum | Antisense oligonucleotides for treatment of usher 2a. exon 68 |
Citations (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5034506A (en) * | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US5418139A (en) * | 1993-02-10 | 1995-05-23 | University Of Iowa Research Foundation | Method for screening for cardiomyopathy |
US5541308A (en) * | 1986-11-24 | 1996-07-30 | Gen-Probe Incorporated | Nucleic acid probes for detection and/or quantitation of non-viral organisms |
US5593974A (en) * | 1991-06-28 | 1997-01-14 | Massachusetts Institute Of Technology | Localized oligonucleotide therapy |
US5608046A (en) * | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
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 |
US5658764A (en) * | 1992-01-28 | 1997-08-19 | North Shore University Hospital Research Corp. | Method and kits for detection of fragile X specific, GC-rich DNA sequences |
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 |
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 |
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 |
US5869252A (en) * | 1992-03-31 | 1999-02-09 | Abbott Laboratories | Method of multiplex ligase chain reaction |
US5916808A (en) * | 1993-05-11 | 1999-06-29 | The University Of North Carolina At Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
US5962332A (en) * | 1994-03-17 | 1999-10-05 | University Of Massachusetts | Detection of trinucleotide repeats by in situ hybridization |
US5968909A (en) * | 1995-08-04 | 1999-10-19 | Hybridon, Inc. | Method of modulating gene expression with reduced immunostimulatory response |
US6172216B1 (en) * | 1998-10-07 | 2001-01-09 | Isis Pharmaceuticals Inc. | Antisense modulation of BCL-X expression |
US6329501B1 (en) * | 1997-05-29 | 2001-12-11 | Auburn University | Methods and compositions for targeting compounds to muscle |
US20010056077A1 (en) * | 1999-05-21 | 2001-12-27 | Jcr Pharmaceuticals Co., Ltd | Pharmaceutical composition for treatment of duchenne muscular dystrophy |
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 |
US20020055481A1 (en) * | 2000-08-25 | 2002-05-09 | Jcr Pharmaceuticals Co., Ltd. | Pharmaceutical composition for treatment of Duchenne muscular dystrophy |
US6653467B1 (en) * | 2000-04-26 | 2003-11-25 | Jcr Pharmaceutical Co., Ltd. | Medicament for treatment of Duchenne muscular dystrophy |
US20030235845A1 (en) * | 2000-09-21 | 2003-12-25 | Van Ommen Garrit-Jan Boudewijn | Induction of exon skipping in eukaryotic cells |
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) |
US20080200409A1 (en) * | 2004-06-28 | 2008-08-21 | Stephen Donald Wilson | Antisense Oligonucleotides For Inducing Exon Skipping and Methods of Use Thereof |
US7534879B2 (en) * | 2003-03-21 | 2009-05-19 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure |
US8084601B2 (en) * | 2008-09-11 | 2011-12-27 | Royal Holloway And Bedford New College Royal Holloway, University Of London | Oligomers |
Family Cites Families (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6867195B1 (en) * | 1989-03-21 | 2005-03-15 | Vical Incorporated | Lipid-mediated polynucleotide administration to reduce likelihood of subject's becoming infected |
FR2675803B1 (en) * | 1991-04-25 | 1996-09-06 | Genset Sa | CLOSED, ANTISENSE AND SENSE OLIGONUCLEOTIDES AND THEIR APPLICATIONS. |
AU659482B2 (en) | 1991-06-28 | 1995-05-18 | Massachusetts Institute Of Technology | Localized oligonucleotide therapy |
US6172208B1 (en) * | 1992-07-06 | 2001-01-09 | Genzyme Corporation | Oligonucleotides modified with conjugate groups |
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 |
US6300060B1 (en) * | 1995-11-09 | 2001-10-09 | Dana-Farber Cancer Institute, Inc. | Method for predicting the risk of prostate cancer morbidity and mortality |
JP4293636B2 (en) | 1996-02-14 | 2009-07-08 | アイシス・ファーマシューティカルス・インコーポレーテッド | Oligonucleotide with sugar-modified gap |
JP3950480B2 (en) * | 1996-07-18 | 2007-08-01 | 株式会社エスアールエル | Method for detecting causal gene of spinocerebellar degeneration type 2 and primer therefor |
US20020137890A1 (en) * | 1997-03-31 | 2002-09-26 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US6280938B1 (en) * | 1997-08-19 | 2001-08-28 | Regents Of The University Of Minnesota | SCA7 gene and method of use |
US6514755B1 (en) * | 1998-08-18 | 2003-02-04 | Regents Of The University Of Minnesota | SCA7 gene and methods of use |
US6794499B2 (en) * | 1997-09-12 | 2004-09-21 | Exiqon A/S | Oligonucleotide analogues |
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 |
ES2226414T3 (en) * | 1998-06-10 | 2005-03-16 | Biognostik Gesellschaft Fur Biomolekulare Diagnostik Mbh | STIMULATION OF THE IMMUNE SYSTEM |
US6924355B2 (en) * | 1998-09-01 | 2005-08-02 | Genentech, Inc. | PRO1343 polypeptides |
IL142828A0 (en) * | 1998-09-25 | 2002-03-10 | Childrens Medical Center | 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 |
JP2000125448A (en) † | 1998-10-14 | 2000-04-28 | Yazaki Corp | Electrical junction box |
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 |
JP2000256547A (en) † | 1999-03-10 | 2000-09-19 | Sumitomo Dow Ltd | Resin composition for heat-resistant card |
US20030236214A1 (en) * | 1999-06-09 | 2003-12-25 | Wolff Jon A. | Charge reversal of polyion complexes and treatment of peripheral occlusive disease |
AU5625500A (en) * | 1999-06-18 | 2001-01-09 | Emory University | Huntington disease cellular model: stably transfected pc12 cells expressing mutant huntingtin |
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 |
WO2001085751A1 (en) * | 2000-05-09 | 2001-11-15 | Reliable Biopharmaceutical, Inc. | Polymeric compounds useful as prodrugs |
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 |
JP4836366B2 (en) * | 2000-08-25 | 2011-12-14 | 雅文 松尾 | Duchenne muscular dystrophy treatment |
JP3995996B2 (en) * | 2002-06-21 | 2007-10-24 | エスアイアイ・プリンテック株式会社 | Ink jet head and ink jet recording apparatus |
WO2009054725A2 (en) * | 2007-10-26 | 2009-04-30 | Academisch Ziekenhuis Leiden | Means and methods for counteracting muscle disorders |
CA2785451C (en) * | 2009-12-24 | 2019-01-22 | Prosensa Technologies B.V. | Molecule for treating an inflammatory disorder |
ES2907250T3 (en) * | 2012-01-27 | 2022-04-22 | Biomarin Tech Bv | RNA-modulating oligonucleotides with improved characteristics for the treatment of Duchenne and Becker muscular dystrophy |
JP5794194B2 (en) † | 2012-04-19 | 2015-10-14 | 東京エレクトロン株式会社 | Substrate processing equipment |
-
2000
- 2000-09-21 EP EP00203283A patent/EP1191097A1/en not_active Withdrawn
-
2001
- 2001-09-21 EP EP13170251.6A patent/EP2636742B1/en not_active Expired - Lifetime
- 2001-09-21 EP EP13170239.1A patent/EP2636740B1/en not_active Expired - Lifetime
- 2001-09-21 EP EP18173900.4A patent/EP3382021A1/en not_active Withdrawn
- 2001-09-21 PT PT141763003T patent/PT2801618T/en unknown
- 2001-09-21 DK DK12198465.2T patent/DK2594640T3/en active
- 2001-09-21 ES ES12198465.2T patent/ES2561292T3/en not_active Expired - Lifetime
- 2001-09-21 EP EP14176300.3A patent/EP2801618B1/en not_active Expired - Lifetime
- 2001-09-21 ES ES13170251.6T patent/ES2628349T3/en not_active Expired - Lifetime
- 2001-09-21 EP EP12198485.0A patent/EP2594641B1/en not_active Expired - Lifetime
- 2001-09-21 AU AU2002211062A patent/AU2002211062C1/en not_active Expired
- 2001-09-21 EP EP10177969.2A patent/EP2284264B1/en not_active Expired - Lifetime
- 2001-09-21 PT PT101779692T patent/PT2284264T/en unknown
- 2001-09-21 EP EP05076770.6A patent/EP1619249B2/en not_active Expired - Lifetime
- 2001-09-21 DK DK15166264.0T patent/DK2940139T3/en active
- 2001-09-21 TR TR2018/10606T patent/TR201810606T4/en unknown
- 2001-09-21 DK DK10177969.2T patent/DK2284264T3/en active
- 2001-09-21 EP EP01979073A patent/EP1320597A2/en not_active Ceased
- 2001-09-21 DK DK05076770.6T patent/DK1619249T4/en active
- 2001-09-21 EP EP12198465.2A patent/EP2594640B1/en not_active Expired - Lifetime
- 2001-09-21 WO PCT/NL2001/000697 patent/WO2002024906A1/en active IP Right Grant
- 2001-09-21 DK DK14176300.3T patent/DK2801618T3/en active
- 2001-09-21 CA CA2423044A patent/CA2423044C/en not_active Expired - Lifetime
- 2001-09-21 JP JP2002529499A patent/JP4846965B2/en not_active Expired - Lifetime
- 2001-09-21 DK DK13170245.8T patent/DK2636741T3/en active
- 2001-09-21 ES ES12198497.5T patent/ES2561294T3/en not_active Expired - Lifetime
- 2001-09-21 AU AU1106202A patent/AU1106202A/en active Pending
- 2001-09-21 DK DK12198517.0T patent/DK2602322T3/en active
- 2001-09-21 PT PT15166264T patent/PT2940139T/en unknown
- 2001-09-21 ES ES05076770T patent/ES2315788T5/en not_active Expired - Lifetime
- 2001-09-21 ES ES15166264.0T patent/ES2690049T3/en not_active Expired - Lifetime
- 2001-09-21 DK DK13170251.6T patent/DK2636742T3/en active
- 2001-09-21 DK DK12198497.5T patent/DK2594642T3/en active
- 2001-09-21 ES ES14176300.3T patent/ES2629747T3/en not_active Expired - Lifetime
- 2001-09-21 EP EP13170245.8A patent/EP2636741B1/en not_active Revoked
- 2001-09-21 SI SI200131062A patent/SI2801618T1/en unknown
- 2001-09-21 DE DE60135936T patent/DE60135936D1/en not_active Expired - Lifetime
- 2001-09-21 ES ES13170239.1T patent/ES2610568T3/en not_active Expired - Lifetime
- 2001-09-21 NZ NZ524853A patent/NZ524853A/en not_active IP Right Cessation
- 2001-09-21 ES ES12198517.0T patent/ES2567417T3/en not_active Expired - Lifetime
- 2001-09-21 ES ES12198485.0T patent/ES2561293T3/en not_active Expired - Lifetime
- 2001-09-21 LT LTEP15166264.0T patent/LT2940139T/en unknown
- 2001-09-21 PT PT05076770T patent/PT1619249E/en unknown
- 2001-09-21 EP EP15166264.0A patent/EP2940139B1/en not_active Expired - Lifetime
- 2001-09-21 LT LTEP14176300.3T patent/LT2801618T/en unknown
- 2001-09-21 DK DK12198485.0T patent/DK2594641T3/en active
- 2001-09-21 PT PT121984652T patent/PT2594640E/en unknown
- 2001-09-21 PT PT131702516T patent/PT2636742T/en unknown
- 2001-09-21 AT AT05076770T patent/ATE409224T2/en active
- 2001-09-21 PT PT131702458T patent/PT2636741E/en unknown
- 2001-09-21 ES ES13170245.8T patent/ES2581285T3/en not_active Expired - Lifetime
- 2001-09-21 LT LTEP10177969.2T patent/LT2284264T/en unknown
- 2001-09-21 EP EP12198517.0A patent/EP2602322B1/en not_active Expired - Lifetime
- 2001-09-21 ES ES10177969.2T patent/ES2609421T3/en not_active Expired - Lifetime
- 2001-09-21 EP EP12198497.5A patent/EP2594642B1/en not_active Expired - Lifetime
-
2003
- 2003-03-21 US US10/395,031 patent/US7973015B2/en not_active Expired - Fee Related
-
2007
- 2007-10-31 US US11/982,285 patent/US20080209581A1/en not_active Abandoned
- 2007-11-13 AU AU2007234488A patent/AU2007234488B2/en not_active Expired
-
2008
- 2008-12-23 CY CY20081101494T patent/CY1109601T1/en unknown
-
2009
- 2009-03-30 US US12/383,897 patent/US20090228998A1/en not_active Abandoned
-
2011
- 2011-03-23 AU AU2011201325A patent/AU2011201325B2/en not_active Expired - Fee Related
- 2011-04-27 JP JP2011098952A patent/JP2011200235A/en active Pending
-
2013
- 2013-10-29 HK HK13112164.0A patent/HK1184821A1/en not_active IP Right Cessation
- 2013-10-29 HK HK13112162.2A patent/HK1184819A1/en not_active IP Right Cessation
- 2013-10-29 HK HK13112163.1A patent/HK1184820A1/en not_active IP Right Cessation
- 2013-10-29 HK HK13112161.3A patent/HK1184818A1/en not_active IP Right Cessation
- 2013-12-18 JP JP2013260728A patent/JP6126983B2/en not_active Expired - Lifetime
-
2014
- 2014-01-24 HK HK14100770.0A patent/HK1188249A1/en not_active IP Right Cessation
- 2014-01-24 HK HK14100771.9A patent/HK1188250A1/en not_active IP Right Cessation
- 2014-07-15 US US14/331,934 patent/US20140350076A1/en not_active Abandoned
-
2015
- 2015-04-27 HK HK15104035.2A patent/HK1203554A1/en not_active IP Right Cessation
- 2015-05-14 US US14/712,753 patent/US20150322434A1/en not_active Abandoned
- 2015-08-28 US US14/839,200 patent/US20150361424A1/en not_active Abandoned
-
2016
- 2016-01-29 JP JP2016014974A patent/JP6250078B2/en not_active Expired - Lifetime
- 2016-03-10 CY CY20161100207T patent/CY1117521T1/en unknown
- 2016-03-11 CY CY20161100216T patent/CY1117318T1/en unknown
- 2016-03-11 CY CY20161100215T patent/CY1117522T1/en unknown
- 2016-04-28 HK HK16104896.9A patent/HK1216906A1/en not_active IP Right Cessation
- 2016-05-23 CY CY20161100443T patent/CY1117887T1/en unknown
- 2016-07-22 CY CY20161100724T patent/CY1117852T1/en unknown
-
2017
- 2017-03-09 CY CY20171100302T patent/CY1118972T1/en unknown
- 2017-06-29 CY CY20171100691T patent/CY1119026T1/en unknown
- 2017-07-14 CY CY20171100750T patent/CY1119088T1/en unknown
- 2017-08-07 JP JP2017152082A patent/JP6425775B2/en not_active Expired - Lifetime
- 2017-12-26 JP JP2017248568A patent/JP6511124B2/en not_active Expired - Lifetime
-
2018
- 2018-09-19 CY CY20181100969T patent/CY1121139T1/en unknown
-
2019
- 2019-02-18 JP JP2019026486A patent/JP2019073555A/en active Pending
Patent Citations (56)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
US5608046A (en) * | 1990-07-27 | 1997-03-04 | Isis Pharmaceuticals, Inc. | Conjugated 4'-desmethyl nucleoside analog compounds |
US5593974A (en) * | 1991-06-28 | 1997-01-14 | Massachusetts Institute Of Technology | Localized oligonucleotide therapy |
US5658764A (en) * | 1992-01-28 | 1997-08-19 | 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 |
US5418139A (en) * | 1993-02-10 | 1995-05-23 | University Of Iowa Research Foundation | Method for screening for cardiomyopathy |
US5976879A (en) * | 1993-05-11 | 1999-11-02 | The University Of North Carolina At Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
US5916808A (en) * | 1993-05-11 | 1999-06-29 | The University Of North Carolina At Chapel Hill | Antisense oligonucleotides which combat aberrant splicing and methods of using the same |
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 |
US5962332A (en) * | 1994-03-17 | 1999-10-05 | University Of Massachusetts | Detection of trinucleotide repeats by in situ hybridization |
US5968909A (en) * | 1995-08-04 | 1999-10-19 | Hybridon, Inc. | Method of modulating gene expression with reduced immunostimulatory response |
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) |
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 |
US6329501B1 (en) * | 1997-05-29 | 2001-12-11 | Auburn University | Methods and compositions for targeting compounds to muscle |
US6172216B1 (en) * | 1998-10-07 | 2001-01-09 | Isis Pharmaceuticals Inc. | Antisense modulation of BCL-X expression |
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 |
US20010056077A1 (en) * | 1999-05-21 | 2001-12-27 | Jcr Pharmaceuticals Co., Ltd | Pharmaceutical composition for treatment of duchenne muscular dystrophy |
US6653466B2 (en) * | 1999-05-21 | 2003-11-25 | Jcr Pharmaceuticals Co., Ltd. | Pharmaceutical composition for treatment of duchenne muscular dystrophy |
US6653467B1 (en) * | 2000-04-26 | 2003-11-25 | Jcr Pharmaceutical Co., Ltd. | Medicament for treatment of Duchenne muscular dystrophy |
US20020055481A1 (en) * | 2000-08-25 | 2002-05-09 | Jcr Pharmaceuticals Co., Ltd. | Pharmaceutical composition for treatment of Duchenne muscular dystrophy |
US6727355B2 (en) * | 2000-08-25 | 2004-04-27 | Jcr Pharmaceuticals Co., Ltd. | Pharmaceutical composition for treatment of Duchenne muscular dystrophy |
US20030235845A1 (en) * | 2000-09-21 | 2003-12-25 | Van Ommen Garrit-Jan Boudewijn | Induction of exon skipping in eukaryotic cells |
US7534879B2 (en) * | 2003-03-21 | 2009-05-19 | Academisch Ziekenhuis Leiden | Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure |
US20080200409A1 (en) * | 2004-06-28 | 2008-08-21 | Stephen Donald Wilson | 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 |
US20110015258A1 (en) * | 2004-06-28 | 2011-01-20 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20110015253A1 (en) * | 2004-06-28 | 2011-01-20 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20110046203A1 (en) * | 2004-06-28 | 2011-02-24 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US7960541B2 (en) * | 2004-06-28 | 2011-06-14 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20110263686A1 (en) * | 2004-06-28 | 2011-10-27 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20130274313A1 (en) * | 2004-06-28 | 2013-10-17 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20120022144A1 (en) * | 2004-06-28 | 2012-01-26 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120022145A1 (en) * | 2004-06-28 | 2012-01-26 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120029058A1 (en) * | 2004-06-28 | 2012-02-02 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120029059A1 (en) * | 2004-06-28 | 2012-02-02 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120029060A1 (en) * | 2004-06-28 | 2012-02-02 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120029057A1 (en) * | 2004-06-28 | 2012-02-02 | The University Of Western Australia | Antisense Oligonucleotides for Inducing Exon Skipping and Methods of Use Thereof |
US20120041050A1 (en) * | 2004-06-28 | 2012-02-16 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8232384B2 (en) * | 2004-06-28 | 2012-07-31 | University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20130116310A1 (en) * | 2004-06-28 | 2013-05-09 | 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 |
US8455635B2 (en) * | 2004-06-28 | 2013-06-04 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US7807816B2 (en) * | 2004-06-28 | 2010-10-05 | 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 |
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 |
US20130217755A1 (en) * | 2004-06-28 | 2013-08-22 | 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 |
US20130253180A1 (en) * | 2004-06-28 | 2013-09-26 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US20130253033A1 (en) * | 2004-06-28 | 2013-09-26 | The University Of Western Australia | Antisense oligonucleotides for inducing exon skipping and methods of use thereof |
US8084601B2 (en) * | 2008-09-11 | 2011-12-27 | Royal Holloway And Bedford New College Royal Holloway, University Of London | Oligomers |
Non-Patent Citations (4)
Title |
---|
Habara et al (J Med Genet 2009;46:542-547) * |
Jou et al (HUMAN MUTATION 5:86-93 (1995)) * |
Sertic et al (Coll. Atropol. 21: 151-156, 1997) * |
Yu et al (Proc. Nat. Acad. Sci. USA 90: 6340-6344, 1993) * |
Cited By (85)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8624019B2 (en) | 2002-11-25 | 2014-01-07 | Masafumi Matsuo | ENA nucleic acid drugs modifying splicing in mRNA precursor |
US9657049B2 (en) | 2002-11-25 | 2017-05-23 | Masafumi Matsuo | ENA nucleic acid pharmaceuticals capable of modifying splicing of mRNA precursors |
US20110046360A1 (en) * | 2002-11-25 | 2011-02-24 | Masafumi Matsuo | ENA NUCLEIC ACID DRUGS MODIFYING SPLICING IN mRNA PRECURSOR |
US9657050B2 (en) | 2002-11-25 | 2017-05-23 | Masafumi Matsuo | ENA nucleic acid pharmaceuticals capable of modifying splicing of mRNA precursors |
US9243026B2 (en) | 2002-11-25 | 2016-01-26 | Daiichi Sankyo Company, Limited | ENA nucleic acid pharmaceuticals capable of modifying splicing of mRNA precursors |
US10781451B2 (en) | 2004-06-28 | 2020-09-22 | 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 |
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 |
US8524880B2 (en) | 2004-06-28 | 2013-09-03 | 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 |
US10421966B2 (en) | 2004-06-28 | 2019-09-24 | 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 |
USRE47691E1 (en) | 2004-06-28 | 2019-11-05 | 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 |
US9018368B2 (en) | 2004-06-28 | 2015-04-28 | 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 |
US9035040B2 (en) | 2004-06-28 | 2015-05-19 | 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 |
US9605262B2 (en) | 2004-06-28 | 2017-03-28 | 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 |
US20110015258A1 (en) * | 2004-06-28 | 2011-01-20 | 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 |
US9249416B2 (en) | 2004-06-28 | 2016-02-02 | 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 |
US10227590B2 (en) | 2004-06-28 | 2019-03-12 | 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 |
US10995337B2 (en) | 2004-06-28 | 2021-05-04 | 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 |
USRE47769E1 (en) | 2004-06-28 | 2019-12-17 | 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 |
US8455635B2 (en) | 2004-06-28 | 2013-06-04 | 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 |
US10689646B2 (en) | 2006-08-11 | 2020-06-23 | 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 |
US9890379B2 (en) | 2006-08-11 | 2018-02-13 | Biomarin Technologies B.V. | Treatment of genetic disorders associated with DNA repeat instability |
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 |
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 |
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 |
US9447416B2 (en) | 2008-10-24 | 2016-09-20 | Sarepta Therapeutics, Inc. | Multiple exon skipping compositions for DMD |
US9447417B2 (en) | 2008-10-24 | 2016-09-20 | 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 |
US8871918B2 (en) | 2008-10-24 | 2014-10-28 | 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 |
US9453225B2 (en) | 2008-10-24 | 2016-09-27 | 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 |
US9695211B2 (en) | 2008-12-02 | 2017-07-04 | Wave Life Sciences Japan, Inc. | Method for the synthesis of phosphorus atom modified nucleic acids |
US10329318B2 (en) | 2008-12-02 | 2019-06-25 | Wave Life Sciences Ltd. | Method for the synthesis of phosphorus atom modified nucleic acids |
US10307434B2 (en) | 2009-07-06 | 2019-06-04 | Wave Life Sciences Ltd. | Nucleic acid prodrugs and methods of use thereof |
US9744183B2 (en) | 2009-07-06 | 2017-08-29 | Wave Life Sciences Ltd. | Nucleic acid prodrugs and methods of use thereof |
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 |
US10287586B2 (en) | 2009-11-12 | 2019-05-14 | 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 |
US10781450B2 (en) | 2009-11-12 | 2020-09-22 | Sarepta Therapeutics, Inc. | Antisense molecules and methods for treating pathologies |
US8637483B2 (en) | 2009-11-12 | 2014-01-28 | The University Of Western Australia | Antisense molecules and methods for treating pathologies |
US10590421B2 (en) | 2010-08-05 | 2020-03-17 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Antisense oligonucleotide directed removal of proteolytic cleavage sites, the HCHWA-D mutation, and trinucleotide repeat expansions |
US10364432B2 (en) | 2010-08-05 | 2019-07-30 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins |
US9611471B2 (en) | 2010-08-05 | 2017-04-04 | Academisch Ziekenhuis Leiden | Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins |
US10428019B2 (en) | 2010-09-24 | 2019-10-01 | Wave Life Sciences Ltd. | Chiral auxiliaries |
US10280192B2 (en) | 2011-07-19 | 2019-05-07 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
US9605019B2 (en) | 2011-07-19 | 2017-03-28 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
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 |
US10167309B2 (en) | 2012-07-13 | 2019-01-01 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
US9982257B2 (en) | 2012-07-13 | 2018-05-29 | Wave Life Sciences Ltd. | Chiral control |
US9617547B2 (en) | 2012-07-13 | 2017-04-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant |
US10590413B2 (en) | 2012-07-13 | 2020-03-17 | Wave Life Sciences Ltd. | Chiral control |
US9217148B2 (en) | 2013-03-14 | 2015-12-22 | Sarepta Therapeutics, Inc. | Exon skipping compositions for treating muscular dystrophy |
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 |
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 |
EP2796425A1 (en) | 2013-04-24 | 2014-10-29 | Corning Incorporated | Delamination resistant pharmaceutical glass containers containing active pharmaceutical ingredients |
US10144933B2 (en) | 2014-01-15 | 2018-12-04 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator |
US10149905B2 (en) | 2014-01-15 | 2018-12-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having antitumor effect and antitumor agent |
US10322173B2 (en) | 2014-01-15 | 2019-06-18 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent |
US10160969B2 (en) | 2014-01-16 | 2018-12-25 | Wave Life Sciences Ltd. | Chiral design |
US10570380B2 (en) | 2014-01-24 | 2020-02-25 | Am-Pharma B.V. | Downstream processing of an alkaline phosphatase |
US9926544B2 (en) | 2014-01-24 | 2018-03-27 | Am-Pharma B.V. | Chimeric alkaline phosphatase-like proteins |
US10822597B2 (en) | 2014-01-24 | 2020-11-03 | Am-Pharma B.V. | Chimeric alkaline phosphatase-like proteins |
US11746340B2 (en) | 2014-01-24 | 2023-09-05 | Am-Pharma B.V. | Chimeric alkaline phosphatase-like proteins |
US10450568B2 (en) | 2015-10-09 | 2019-10-22 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7973015B2 (en) | Induction of exon skipping in eukaryotic cells | |
AU2002211062A1 (en) | Induction of exon skipping in eukaryotic cells | |
AU2012200761B2 (en) | Induction of exon skipping in eukaryotic cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ACADEMISCH ZIEKENHUIS LEIDEN, NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VAN OMMEN, GARRIT-JAN B.;VAN DEUTEKOM, JUDITH C. T.;DEN DUNNEN, JOHANNES T.;REEL/FRAME:022533/0844 Effective date: 20030414 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |