US20100184833A1 - Methods and means for treating dna repeat instability associated genetic disorders - Google Patents

Methods and means for treating dna repeat instability associated genetic disorders Download PDF

Info

Publication number
US20100184833A1
US20100184833A1 US12/377,160 US37716007A US2010184833A1 US 20100184833 A1 US20100184833 A1 US 20100184833A1 US 37716007 A US37716007 A US 37716007A US 2010184833 A1 US2010184833 A1 US 2010184833A1
Authority
US
United States
Prior art keywords
oligonucleotide
repeat
sequence
nucleotides
complementary
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
Application number
US12/377,160
Other languages
English (en)
Inventor
Josephus Johannes De Kimpe
Gerard Johannes Platenburg
Derick Gert Wansink
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PROSENTA Tech BV
Biomarin Technologies BV
Original Assignee
PROSENTA Tech BV
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by PROSENTA Tech BV filed Critical PROSENTA Tech BV
Assigned to PROSENSA TECHNOLOGIES B.V. reassignment PROSENSA TECHNOLOGIES B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DE KIMPE, JOSEPHUS JOHANNES, PLATENBURG, GERARD JOHANNES, WANSINK, DERICK GERT
Publication of US20100184833A1 publication Critical patent/US20100184833A1/en
Assigned to PROSENSA TECHNOLOGIES B.V. reassignment PROSENSA TECHNOLOGIES B.V. CHANGE OF ADDRESS Assignors: PROSENSA TECHNOLOGIES B.V.
Assigned to BIOMARIN TECHNOLOGIES B.V. reassignment BIOMARIN TECHNOLOGIES B.V. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: PROSENSA TECHNOLOGIES B.V.
Abandoned legal-status Critical Current

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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the current invention relates to the field of medicine, in particular to the treatment of genetic disorders associated with genes that have unstable repeats in their coding or non-coding sequences, most in particular unstable repeats in the human Huntington disease causing HD gene or the myotonic dystrophy type 1 causing DMPK gene.
  • SBMA X-linked spinal and bulbar muscular atrophy
  • DM1 myotonic dystrophy type 1
  • F fragile X syndrome
  • HD Huntington's disease
  • SCA gene family several spinocerebellar ataxias
  • Unstable repeat units are also found in untranslated regions, such as in myotonic dystrophy type 1 (DM1) in the 3′ UTR or in intronic sequences such as in myotonic dystrophy type 2 (DM2).
  • the normal number of repeats is around 5 to 37 for DMPK, but increases to premutation and full disease state two to ten fold or more, to 50, 100 and sometimes 1000 or more repeat units.
  • For DM2/ZNF9 increases to 10,000 or more repeats have been reported. (Cleary and Pearson, Cytogenet. Genome Res. 100: 25-55, 2003).
  • the causative gene for Huntington's disease, HD is located on chromosome 4. Huntington's disease is inherited in an autosomal dominant fashion. When the gene has more than 35 CAG trinucleotide repeats coding for a polyglutamine stretch, the number of repeats can expand in successive generations. Because of the progressive increase in length of the repeats, the disease tends to increase in severity and presents at an earlier age in successive generations, a process called anticipation.
  • the product of the, HD gene is the 348 kDa cytoplasmic protein huntingtin. Huntingtin has a characteristic sequence of fewer than 40 glutamine amino acid residues in the normal form; the mutated huntingtin causing the disease has more than 40 residues.
  • mutant huntingtin molecules in neuronal cells results in the formation of large protein deposits which eventually give rise to cell death, especially in the frontal lobes and the basal ganglia (mainly in the caudate nucleus).
  • the severity of the disease is generally proportional to the number of extra residues.
  • DM1 is the most common muscular dystrophy in adults and is an inherited, progressive, degenerative, multisystemic disorder of predominantly skeletal muscle, heart and brain. DM1 is caused by expansion of an unstable trinucleotide (CTG)n repeat in the 3′ untranslated region of the DMPK gene (myotonic dystrophy protein kinase) on human chromosome 19q (Brook et al, Cell, 1992). Type 2 myotonic dystrophy (DM2) is caused by a CCTG expansion in intron 1 of the ZNF9 gene, (Liguori et al, Science 2001).
  • CTG trinucleotide
  • DM2 is caused by a CCTG expansion in intron 1 of the ZNF9 gene, (Liguori et al, Science 2001).
  • DMPK transcripts bearing a long (CUG)n tract can form hairpin-like structures that bind proteins of the muscleblind family and subsequently aggregate in ribonuclear foci in the nucleus. These nuclear inclusions are thought to sequester muscleblind proteins, and potentially other factors, which then become limiting to the cell.
  • CUG long
  • ZNF9 RNA carrying the (CCUG)n expanded repeat form similar foci. Since muscleblind proteins are splicing factors, their depletion results in a dramatic rearrangement in splicing of other transcripts. Transcripts of many genes consequently become aberrantly spliced, for instance by inclusion of fetal exons, or exclusion of exons, resulting in non-functional proteins and impaired cell function.
  • a third approach by Furling et al. (Gene Therapy, Vol. 10, p 795-802, 2003) used a recombinant retrovirus expressing a 149-bp long antisense RNA to inhibit DMPK mRNA levels in human DM1 myoblasts.
  • a retrovirus was designed to provide DM1 cells with the 149 by long antisense RNA complementary to a 39 bp-long (CUG)13 repeat and a 110 by region following the repeat to increase specificity.
  • This method yielded a decrease in mutated (repeat expanded) DMPK transcript of 80%, compared to a 50% reduction in the wild type DMPK transcript and restoration of differentiation and functional characteristics in infected DM1 myoblasts.
  • this approach is not targeted selectively to the expanded repeat mRNA species, it depends on a very long antisense RNA and can only be used in combination with recombinant viral delivery techniques.
  • nucleid acid based methods that cause non-selective breakdown of both the affected repeat expanded allele and unaffected (normal) allele for genetic diseases that are associated with repeat instability and/or expansion.
  • the art employs sequences specific for the gene associated with the disease and does not provide a method that is applicable to several genetic disorders associated with repeat expansion.
  • the art only teaches methods that involve use of recombinant DNA vector delivery systems, which need to be adapted for each oligonucleotide and target cell and which still need to be further optimised.
  • the current invention provides a solution for these problems by using a short single stranded nucleic acid molecule that comprises or consists of a sequence, which is complementary to the expanded repeat region only, i.e. it does not rely on hybridisation to unique sequences in exons or introns of the repeat containing gene. Furthermore, it is not essential that the employed nucleic acid (oligonucleotide) reduces transcripts by the RNAse H mediated breakdown mechanism.
  • the current invention may cause a decrease in transcript levels by alterations in posttranscriptional processing and/or splicing of the premature RNA.
  • a decrease in transcript levels via alternative splicing and/or postranscriptional processing is thought to result in transcripts lacking the overly expanded or instable (tri)nucleotide repeat, but still possessing functional activities.
  • the reduction of aberrant transcripts by altered RNA processing and/or splicing may prevent accumulation and/or translation of aberrant, repeat expanded transcripts in cells.
  • RNA molecules and in particular RNA molecules comprising repetitive sequences are normally internally paired, forming a secondary structure comprising open loops and closed hairpin parts. Only the open parts are relatively accessible for complementary nucleic acids.
  • the short repeat stretches of a wild type transcript not associated with disease is often only 5 to about 20-40 repeats and due to the secondary structure relatively inaccessible for base pairing with a complementary nucleic acid.
  • the repeat units of the expanded repeat and disease associated allele is normally at least 2 fold expanded but usually even more, 3, 5, 10 fold, up to 100 or even more than 1000 fold expansion for some unstable repeat disorders.
  • This expansion increases the likelihood that part of the repeat is, at least temporarily, in an open loop structure and thereby more accessible to base pairing with a complementary nucleic acid molecule, relative to the wild type allele.
  • oligonucleotide is complementary to a repeat sequence present in both wildtype and repeat-expanded transcripts and could theoretically hybridise to both transcripts
  • the current invention teaches that oligonucleotides complementary to the repetitive tracts preferably hybridise to the disease-associated or disease-causing transcripts and leave the function of normal transcripts relatively unaffected. This selectivity is beneficial for treating diseases associated with repeat instability irrespective of the mechanism of reduction of the aberrant transcript.
  • the invention thus provides a method for the treatment of unstable cis-element DNA repeat associated genetic disorders, by providing nucleic acid molecules that are complementary to and/or capable of hybridising to the repetitive sequences only.
  • This method thereby preferentially targets the expanded repeat transcripts and leaves the transcripts of the normal, wild type allele relatively unaffected.
  • This is advantageous since the normal allele can thereby provide for the normal function of the gene, which is at least desirable and, depending on the particular gene with unstable DNA repeats, may in many cases be essential for the cell and/or individual to be treated.
  • this approach is not limited to a particular unstable DNA repeat associated genetic disorder, but may be applied to any of the known unstable DNA repeat diseases, such as, but not limited to: coding regions repeat diseases having a polyglutamine (CAG) repeat: Huntington's disease, Haw River syndrome, Kennedy's disease/spinobulbar muscular atrophy, spino-cerebellar ataxia, or diseases having polyalanine (GCG) repeats such as: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis/ptosis/epicanthus invensus syndrome, hand-foot-genital syndrome, synpolydactyly, oculopharyngeal muscular dystrophy, holoprosencephaly.
  • CAG polyglutamine
  • GCG polyalanine
  • Diseases with repeats in non-coding regions of genes to be treated according to the invention comprise the trinucleotide repeat disorders (mostly CTG and/or CAG and/or CCTG repeats): myotonic dystrophy type 1, myotonic dystrophy type 2. Friedreich's ataxia (mainly GAA), spino-cerebellar ataxia, autism. Furthermore, the method of the invention can be applied to fragile site associated repeat disorder comprising various fragile X-syndromes, Jacobsen syndrome and other unstable repetitive element disorders such as myoclonus epilepsy, facioscapulohumeral dystrophy and certain forms of diabetes mellitus type 2.
  • oligonucleotides specific for a repeat region may be administered directly to cells and it does not rely on vector-based delivery systems.
  • the use of plasmid or viral vectors is yet less desirable for therapeutic purposes because of current strict safety regulations for therapeutic recombinant DNA vectors, the production of sufficient recombinant vectors for broad clinical application and the limited control and reversibility of an exaggerated (or non-specific) response after application.
  • oligonucleotides that comprise or consist of a sequence that is complementary to repetitive sequences of expanded repeat transcripts, due to the expansion of their molecular target for hybridisation, have a much increased affinity and/or avidity for their target in comparison to oligonucleotides that are specific for unique sequences in a transcript.
  • oligonucleotide Because of this high affinity and avidity for the repeat expanded target transcript, lower amounts of oligonucleotide suffice to yield sufficient inhibition and/or reduction of the repeat expanded allele by RNase H degradation, RNA interference degradation or altered post-transcriptional processing (comprising but not limited to splicing or exon skipping) activities.
  • the oligonucleotides of the current invention which are complementary to repetitive sequences only, may be produced synthetically and are potent enough to be effective when delivered directly to cells using commonly applied techniques for direct delivery of oligonucleotides to cells and/or tissues. Recombinant vector delivery systems may, when desired, be circumvented by using the method and the oligonucleotide molecules of the current invention.
  • the current invention discloses and teaches the use of an oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans.
  • the invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders.
  • the invention also pertains to an oligonucleotide which can be used in the first aspect of the invention and/or applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.
  • An oligonucleotide of the invention may comprise a sequence that is complementary only to a repetitive sequence as defined below.
  • the repetitive sequence is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or more.
  • the oligonucleotide of the invention consists of a sequence that is complementary only to a repetitive sequence as defined below.
  • an oligonucleotide may comprise a sequence that is complementary only to a repetitive sequence as defined below and a targeting part, which is later on called a targeting ligand.
  • a repeat or repetitive element or repetitive sequence or repetitive stretch is herein defined as a repetition of at least 3, 4, 5, 10, 100, 1000 or more, of a repetitive unit or repetitive nucleotide unit or repeat nucleotide unit comprising a trinucleotide repetitive unit, or alternatively a 4, 5 or 6 nucleotide repetitive unit, in a transcribed gene sequence in the genome of a subject, including a human subject.
  • An oligonucleotide may be single stranded or double stranded. Double stranded means that the oligonucleotide is an heterodimer made of two complementary strands, such as in a siRNA. In a preferred embodiment, an oligonucleotide is single stranded.
  • a single stranded oligonucleotide has several advantages compared to a double stranded siRNA oligonucleotide: (i) its synthesis is expected to be easier than two complementary siRNA strands; (ii) there is a wider range of chemical modifications possible to optimise more effective uptake in cells, a better (physiological) stability and to decrease potential generic adverse effects; and (iii) siRNAs have a higher potential for non-specific effects and exaggerated pharmacology (e.g. less control possible of effectiveness and selectivity by treatment schedule or dose) and (iv) siRNAs are less likely to act in the nucleus and cannot be directed against introns.
  • the invention relates to the use of a single stranded oligonucleotide comprising or consisting of a sequence that is complementary only to a repetitive sequence in a human gene transcript for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans.
  • the oligonucleotide(s) preferably comprise at least 10 to about 50 consecutive nucleotides complementary to a repetitive element, more preferably 12 to 45 nucleotides, even more preferably 12 to 30, and most preferably 12 to 25 nucleotides complementary to a repetitive stretch, preferably having a trinucleotide repeat unit or a tetranucleotide repeat unit.
  • the oligonucleotide may be complementary to and/or capable of hybridizing to a repetitive stretch in a coding region of a transcript, preferably a polyglutamine (CAG) or a polyalanine (GCG) coding tract.
  • CAG polyglutamine
  • GCG polyalanine
  • the oligonucleotide may also be complementary to and/or capable of hybridizing to a non-coding region for instance 5′ or 3′ untranslated regions, or intronic sequences present in precursor RNA molecules.
  • the oligonucleotide to be used in the method of the invention comprises or consists of a sequence that is complementary to a repetitive element having as repetitive nucleotide unit a repetitive nucleotide unit selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n (GAA)n, (GCC)n and (CCUG)n. and said oligonucleotide being a single or double stranded oligonucleotide.
  • the oligonucleotide is double stranded.
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a polyglutamine (CAG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders Huntington's disease, several forms of spino-cerebellar ataxia or Haw River syndrome, X-linked spinal and bulbar muscular atrophy and/or dentatorubral-pallidoluysian atrophy caused by repeat expansions in the HD, HDL2/JPH3, SBMA/AR, SCA1/ATX1, SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCAT, SCA17, AR or DRPLA human genes.
  • CAG polyglutamine
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a polyalanine (GCG)n tract in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human disorders: infantile spasm syndrome, deidocranial dysplasia, blepharophimosis, hand-foot-genital disease, synpolydactyl), oculopharyngeal muscular dystrophy and/or holoprosencephaly, which are caused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13, OPDM/PABP2, TCFBR1 or ZIC2 human genes.
  • GCG polyalanine
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a (CUG)n repeat in a transcript and is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 1, spino-cerebrellar ataxia 8 and/or Huntington's disease-like 2 caused by repeat expansions in the DM1/DMPK, SCA8 or JPH3 genes respectively.
  • these genes are from human origin.
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCUG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder myotonic dystrophy type 2, caused by repeat expansions in the DM2/ZNF9 gene.
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a (CGG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of human fragile X syndromes, caused by repeat expansion in the FRAXA/FMR1, FRAXE/FMR2 and FRAXF/FAM11A genes.
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a (CCG)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Jacobsen syndrome, caused by repeat expansion in the FRA11B/CBL2 gene.
  • oligonucleotide that comprises or consists of a sequence that is complementary to a (GAA)n repeat in a transcript is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Friedreich's ataxia.
  • an oligonucleotide that comprises or consists of a sequence that is complementary to a (ATTCT)n repeat in an intron is particularly useful for the diagnosis, treatment and/or prevention of the human genetic disorder Spinocerebellar ataxia type 10 (SCA10).
  • SCA10 Spinocerebellar ataxia type 10
  • the repeat-complementary oligonucleotide to be used in the method of the invention may comprise or consist of RNA, DNA, Locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino phosphorodiamidates (PMO), ethylene bridged nucleic acid (ENA) or mixtures/hybrids thereof that comprise combinations of naturally occurring DNA and RNA nucleotides and synthetic, modified nucleotides.
  • LNA Locked nucleic acid
  • PNA peptide nucleic acid
  • PMO morpholino phosphorodiamidates
  • ENA ethylene bridged nucleic acid
  • mixtures/hybrids thereof that comprise combinations of naturally occurring DNA and RNA nucleotides and synthetic, modified nucleotides.
  • the phosphodiester backbone chemistry may further be replaced by other modifications, such as phosphorothioates or methylphosphonates.
  • Other oligonucleotide modifications exist and new ones are likely
  • the oligonucleotide comprises or consists of RNA nucleotides, DNA nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, morpholino phosphorodiamidates, ethylene-bridged nucleic acid (ENA) nucleotides or mixtures thereof with or without phosphorothioate containing backbones.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • ENA ethylene-bridged nucleic acid
  • Oligonucleotides containing at least in part naturally occurring DNA nucleotides are useful for inducing degradation of DNA-RNA hybrid molecules in the cell by RNase H activity (EC.3.1.26.4).
  • RNA ribonucleotides or RNA-like synthetic ribonucleotides comprising oligonucleotides may be applied in the method of the invention to form double stranded RNA-RNA hybrids that act as enzyme-dependent antisense through the RNA interference or silencing (RNAi/siRNA) pathways, involving target RNA recognition through sense-antisense strand pairing followed by target RNA degradation by the RNA-induced silencing complex (RISC).
  • RNAi/siRNA RNA interference or silencing
  • steric blocking antisense oligonucleotides interfere with gene expression or other precursor RNA or messenger RNA-dependent cellular processes, in particular but not limited to RNA splicing and exon skipping, by binding to a target sequence of RNA transcript and getting in the way of processes such as translation or blocking of splice donor or splice acceptor sites.
  • RNA splicing and exon skipping techniques using modified antisense oligonucleotides are well documented, known to the skilled artisan and may for instance be found in U.S. Pat. No. 6,210,892, WO9426887, WO04/083446 and WO02/24906.
  • steric hindrance may inhibit the binding of proteins, nuclear factors and others and thereby contribute to the decrease in nuclear accumulation or ribonuclear foci in diseases like DM1.
  • oligonucleotides of the invention which may comprise synthetic or modified nucleotides, complementary to (expanded) repetitive sequences are useful for the method of the invention for reducing or inactivating repeat containing transcripts via the siRNA/RNA interference or silencing pathway.
  • Single or double stranded oligonucleotides to be used in the method of the invention may comprise or consist of DNA nucleotides, RNA nucleotides, 2′-O substituted ribonucleotides, including alkyl and methoxy ethyl substitutions, peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino (PMO) antisense oligonucleotides and ethylene-bridged nucleotides (ENA) and combinations thereof, optionally chimeras with RNAse H dependent antisense. Integration of locked nucleic acids in the oligonucleotide changes the conformation of the helix after base pairing and increases the stability of the duplex.
  • PNAs Peptide nucleic acids
  • the backbone is a pseudopeptide rather than a sugar
  • PNAs are superior reagents in antisense and exon skipping applications of the invention.
  • the oligonucleotides to be used in the method of this invention comprise, at least in part or fully, 2′-O-methoxy ethyl phosphorothioate RNA nucleotides or 2′-O-methyl phosphorothioate RNA nucleotides.
  • Oligonucleotides comprising or consisting of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (CCG)n, (GAA)n, (GCC)n and (CCUG)n having a length of 10 to 50, more preferably 12 to 35, most preferably 12 to 25 nucleotides, and comprising 2′- ⁇ -methoxyethyl phosphorothioate RNA nucleotides, 2′-O-methyl phosphorothioate RNA nucleotides, LNA nucleotides or PMO nucleotides are most preferred for use in the invention for the diagnosis, treatment of prevention of cis-element repeat instability genetic disorders.
  • an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
  • an oligonucleotide of the invention and used in the invention comprises or consists of a sequence that is complementary to a repetitive sequence selected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and (CCUG)n, has a length of 10 to 50 nucleotides and is further characterized by:
  • the invention relates to a double stranded oligonucleotide with two complementary strands, the antisense strand, complementary only to a repetitive sequence in a human gene transcript
  • this double stranded oligonucleotide is preferably not the siRNA with antisense RNA strand (CUG) 7 and sense RNA strand (GCA) 7 applied to cultured monkey fibroblast (COS-7) or human neuroblastoma (SH-SY5Y) cell lines with or without transfection with a human Huntington gene exon 1 fused to GFP and as depicted in Wanzhao Liu et al (Wanzhao Liu et al, (2003), Proc. Japan Acad, 79: 293-298).
  • the invention does neither relate to the double stranded oligonucleotide siRNA (with antisense strand (CUG) 7 and sense strand (GCA) 7 ) nor to its use for the manufacture of a medicament for the treatment or prevention of Huntington disease, even more preferably for the treatment or prevention of Huntington disease gene exon 1 containing construct.
  • oligonucleotide may be sufficient for reducing the amount of repeat expanded transcripts, such as nuclear accumulated DMPK or ZNF9 transcripts or segments thereof or sufficient reduction of accumulation of repeat expanded HD protein, it is also within the scope of the invention to combine 2, 3, 4, 5 or more oligonucleotides.
  • the oligonucleotide comprising or consisting of a sequence that is complementary to a repetitive part of a transcript may be advantageously combined with oligonucleotides that comprise or consist of sequences that are complementary to and/or capable of hybridizing with unique sequences in a repeat containing transcript.
  • the method of the invention and the medicaments of the invention comprising repeat specific oligonucleotides may also be combined with any other treatment or medicament for cis-element repeat instability genetic disorders.
  • the oligonucleotide used in the method of the invention may be provided with a radioactive label or fluorescent label allowing detection of transcripts in samples, in cells in situ in vivo, ex vivo or in vitro.
  • labelled oligonucleotides may be used for diagnostic purposes, for visualisation of nuclear aggregates of DMPK or ZNF9 RNA transcript molecules with associated proteins.
  • Fluorescent labels may comprise Cy3, Cy5, FITC, TRITC, Rhodamine, GFP and the like.
  • Radioactive labels may comprise 3 H, 35 S, 32/33 P, 125 I.
  • Enzymes and/or immunogenic haptens such as digoxigenin, biotin and other molecular tags (HA, Myc, FLAG, VSV, lexA) may also be used. Accordingly, in a further aspect, the invention discloses an vitro or ex vivo detection and/or diagnostic method wherein a oligonucleotide as defined above is used.
  • the oligonucleotides for use according to the invention are suitable for direct administration to cells, tissues and/or organs in vivo of individuals affected by or at risk of developing a cis-element repeat instability disorder, and may be administered directly in vivo, ex vivo or in vitro.
  • the oligonucleotides may be provided by a nucleic acid vector capable of conferring expression of the oligonucleotide in human cells, in order to allow a sustainable source of the oligonucleotides.
  • Oligonucleotide molecules according to the invention may be provided to a cell, tissue, organ and/or subject to be treated in the form of an expression vector that is capable of conferring expression of the oligonucleotide in human cells.
  • the vector is preferably introduced in the cell by a gene delivery vehicle.
  • Preferred vehicles for delivery are viral vectors such as retroviral vectors, adeno-associated virus vectors (AAV), adenoviral vectors, Semliki Forest virus vectors (SFV), EBV vectors and the like.
  • viral vectors such as retroviral vectors, adeno-associated virus vectors (AAV), adenoviral vectors, Semliki Forest virus vectors (SFV), EBV vectors and the like.
  • plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of oligonucleotides.
  • Preferred for the current invention are those vectors wherein transcription is driven from polIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts.
  • a concentration of oligonucleotide which is ranged between about 0.1 nM and about 1 ⁇ M is used. More preferably, the concentration used is ranged between about 0.3 to about 400 nM, even more preferably between about 1 to about 200 nM. If several oligonucleotides are used, this concentration may refer to the total concentration of oligonucleotides or the concentration of each oligonucleotide added. The ranges of concentration of oligonucleotide(s) as given above are preferred concentrations for in vitro or ex vivo uses.
  • oligonueleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration of oligonucleotide(s) used may further vary and may need to be optimised any further.
  • the oligonucleotides to be used in the invention to prevent, treat or diagnose cis-element repeat instability disorders are synthetically produced and administered directly to cells, tissues, organs and/or patients in formulated form in pharmaceutically acceptable compositions.
  • the delivery of the pharmaceutical compositions to the subject is preferably carried out by one or more parenteral injections, e.g. intravenous and/or subcutaneous and/or intramuscular and/or intrathecal and/or intraventricular administrations, preferably injections, at one or at multiple sites in the human body.
  • An intrathecal or intraventricular administration is preferably realized by introducing a diffusion pump into the body of a subject. Several diffusion pumps are known to the skilled person.
  • compositions that are to be used to target the oligonucleotide molecules comprising or consisting of a sequence that is complementary to repetitive sequences may comprise various excipients such as diluents, fillers, preservatives, solubilisers and the like, which may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
  • excipients that will aid in delivery of the oligonucleotides to the cells and into the cells, in particular excipients capable of forming complexes, vesicles and/or liposomes that deliver substances and/or oligonucleotide(s) complexed or trapped in the vesicles or liposomes through a cell membrane.
  • excipients capable of forming complexes, vesicles and/or liposomes that deliver substances and/or oligonucleotide(s) complexed or trapped in the vesicles or liposomes through a cell membrane.
  • excipients capable of forming complexes, vesicles and/or liposomes that deliver substances and/or oligonucleotide(s) complexed or trapped in the vesicles or liposomes through a cell membrane.
  • Suitable substances comprise polyethylenimine (PEI), ExGen 500, synthetic amphiphils (SAINT-18
  • Lipofectin represents an example of liposomal transfection agents. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE).
  • DOTMA cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOPE neutral lipid dioleoylphosphatidylethanolamine
  • Another group of delivery systems are polymeric nanoparticles.
  • Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver oligonucleotides across cell membranes into cells.
  • PBCA butylcyanoacrylate
  • PHCA hexylcyanoacrylate
  • the cationic peptide protamine offers an alternative approach to formulate oligonucleotides as colloids.
  • This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of the oligonucleotides.
  • the skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver oligonucleotides for use in the current invention to deliver oligonucleotides for the treatment of cis-element repeat instability disorders in humans.
  • the oligonucleotide could be covalcntly or non-covalcntly linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
  • a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus.
  • ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.
  • an oligonucleotide in a medicament is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to cells and/or enhancing its intracellular delivery.
  • the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide of the invention and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of the oligonucleotide to the cell and/or enhancing its intracellular delivery.
  • the invention also pertains to a method for the reduction of repeat containing gene transcripts in a cell comprising the administration of a single strand or double stranded oligonucleotide molecule, preferably comprising 2′-O-substituted RNA phosphorothioate nucleotides such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothioate nucleotides or LNA nucleotides or PMO nucleotides, and having a length of 10 to 50 nucleotides that are complementary to the repetitive sequence only.
  • the nucleotides could be used in combination and/or with DNA phosphorothioate nucleotides.
  • FIG. 1 Northern blot of RNA isolated from myotubes transfected with different oligonucleotides or mock control.
  • the myotubes were derived from immorto mouse myoblast cell lines containing a transgenic human DMPK genes with (CTG)n repeat expansion length of approximately 500 next to its normal mouse DMPK gene without (CTG) repeat.
  • CCG human DMPK mRNA
  • mDMPK mouse DMPK
  • mDMPK mouse DMPK
  • FIG. 2 The human and mouse DMPK signals of FIG. 1 were quantified by phosphoimager analysis and normalized, to the GAPDH signal. The results are expressed relative to the mock treatment (set to 100).
  • FIG. 3 Northern blot of total RNA isolated from murine myotubes containing a mouse-human chimaeric DMPK gene in which the 3′ part of the mDMPK gene was replaced by the cognate segment of the human DMPK gene including a (CTG) 110 -repeat.
  • the blot was probed for DMPK mRNA (upper panel) and mouse GAPDH mRNA (bottom). Cells were transfected with antisense oligonucleotide PS58 or control.
  • FIG. 4 shows the response of DM500 myotubes treated with various concentrations of oligonucleotide PS58.
  • the expression of hDMPK was quantified via Northern blot analysis followed by phosphoimager analysis. The signal was normalised to the GAPDH signal and expressed relative to the response after mock treatment.
  • FIG. 5 shows the Northern blot of total RNA of DM500 myotubes transfected with 200 nM PS58 at different time points: 2 h, 4 h, 8 h and 48 h before harvesting. Mock treatment was performed 48 h before harvesting.
  • Northern blots show human and mouse DMPK and mouse GAPDH mRNA. These were quantified by phosphoimager and the normalized DMPK signal was expressed relative to mock treatment.
  • FIG. 6 shows the Northern blot of total RNA of DM500 myotubes harvested 2 d, 4 d, 6 d and 8 d after transfection with 200 nM PS58 or mock control. Northern blot analysis and quantification was performed as before.
  • FIG. 7 shows a Northern blot of total RNA from MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM) or mock control.
  • the myoblasts were derived from fibroblasts obtained from a congenital myotonic dystrophy type I patient bearing a hDMPK allele with a triplet repeat expansion length of approximately 1500 and a hDMPK allele with normal length of 11 repeats.
  • the Northern blot was hybridized with a human DMPK (hDMPK) probe and GAPDH mRNA probe.
  • the human DMPK signals were normalized to the GAPDH signal and expressed relative to mock control.
  • FIG. 8 shows the RT-PCR analysis of DM500 myotubes transfected with 20011M of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control.
  • RT-PCR analysis was performed with primers specific for hDMPK mRNA and three other gene transcripts with a naturally occurring (CUG) repeat in mice: Ptbp1 mRNA with a (CUG)6, Syndecan3 mRNA with a (CUG)6 and Taxilinbeta mRNA with a (CUG)9. The intensity of the signals were normalized to the actin signal and expressed relative to mock control.
  • FIG. 9 shows FISH analysis of DM500 myoblasts transfected with 200 nM PS58 (B) or mock control (A).
  • B nM PS58
  • A mock control
  • FIG. 10 shows the relative cell count for the presence of ribonuclear foci in the nucleus of DM500 myoblasts transfected with PS58 or mock control from the experiment depicted in FIG. 9 .
  • FIG. 11 shows the RT-PCR analysis of hDMPK mRNA in muscle of DM500 mice treated with PS58 or mock control.
  • PS58 (2 nmol) was injected in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h.
  • M. plantaris and M. gastrocnemius were isolated and RT-PCR was performed on total RNA for hDMPK and mouse actin.
  • the intensity of the hDMPK signal was normalized to the actin signal and the values expressed relative to mock control.
  • FIG. 12 shows a Northern blot analysis of DM500 myotubes treated with different oligonucleotides (200 nM) or mock control.
  • PS58, PS146 and PS147 carried a full 2′O-methyl phosphorothiate backbone, but differed in length, (CAG)7, (CUG)10 and (CUG)5, respectively.
  • PS142 has a complete phosphorothiate DNA backbone with a (CAG)7 sequence.
  • hDMPK and mDMPK signals were normalized to mouse GAPDH and expressed as percentage to mock control. Quantification is shown in the lower panel.
  • Immortomyoblast cell lines were derived from DM500 or CTG110 mice using standard techniques known to the skilled person. DM500 mice were derived from mice obtained from de Gourdon group in Paris. CTG110 mice are described below and present at the group of Wiering a and Wansink in Nijmegen. Immortomyoblast cell lines DM500 or CTG 110 with variable (CTG)n repeat length in the DMPK gene were grown subconfluent and maintained in a 5% CO 2 atmosphere at 33° C. on 0.1% gelatin coated dishes. Myoblast cells were grown subconfluent in DMEM supplemented with 20% FCS, 50 ⁇ g/ml gentamycin and 20 units of ⁇ -interferon/ml.
  • CTG variable
  • Myotube formation was induced by growing myoblast cells on Matrigel (BD Biosciences) coated dishes and placing a confluent myoblast culture at 37° C. and in DMEM supplemented with 5% horse serum and 50 ⁇ g/ml gentamycin. After five days on this low serum media contracting myotubes arose in culture and were transfected with the desired oligonucleotides. For transfection NaCl (500 mM, filter sterile), oligonucleotide and transfection reagens PEI (ExGen 500, Fermentas) were added in this specific order and directly mixed.
  • the oligonucleotide transfection solution contained a ratio of 5 ⁇ l ExGen 500 per ug oligonucleotide which is according to the instructions (ExGen 500, Fermentas). After 15 minutes of incubation at room temperature the oligonucleotide transfection solution was added to the low serum medium with the cultured myotubes and gently mixed. The final oligonucleotide concentration was 200 nM. Mock control treatment is carried out with transfection solution without an oligonucleotide. After four hours of incubation at 37° C., fresh medium was added to the culture (resulting in a dilution of approximately 2.3 ⁇ ) and incubation was extended overnight at 37° C.
  • the Northern blot was hybridized with a radioactive human DMPK (hDMPK) probe and a mouse GAPDH probe.
  • the probe used for DMPK is a human DMPK cDNA consisting of the DMPK open reading frame with full 3′ UTR and 11 CTGs.
  • the human and mouse DMPK signal were quantified by phosphoimager analysis and normalized to the GAPDH signal.
  • Primers that were used for the RT-PCR for hDMPK mRNA were situated in the 3′ untranslated part with the sequence 5 ′-GGGGGATCACAGACCATT-3′ and 5′-TCAATGCATCCAAAACGTGGA-3′ and for murine actin the primers were as followed: Actin sense 5′-GCTAYGAGCTGCCTGACGG-3′ and Actin antisense 5′-GAGGCCAGGATGGAGCC-3′.
  • PCR products were run on an agarose gel and the signal was quantified using Labworks 4.0 (UVP BioImaging systems, Cambridge, United Kingdom). The intensity of each band was normalized to the intensity of the corresponding actin band and expressed relative to mock control.
  • FIG. 1 shows the Northern blot of the isolated RNA from the oligonucleotide transfected myotubes visualized with the hDMPK probe and a GAPDH probe for loading control. Quantification of the human DMPK (with CTG repeat) and murine DMPK (without CTG repeat) signal on the Northerm blot is shown in FIG. 2 . The signal was normalized to murine GAPDH and expressed relative to mock control.
  • Table 2 indicates the level of hDMPK mRNA reduction that is caused by a specific oligonucleotide.
  • the minus ( ⁇ ) stands for no reduction and the number of positive signs (+) stands for the relative level of hDMPK mRNA break-down.
  • oligonucleotide PS58 specifically targeted to the repeat sequence, is much more potent in reducing or altering hDMPK transcripts than the other oligonucleotides complementary to unique sequences in the hDMPK transcripts.
  • FIG. 3 shows the effect of PS58 in murine immortomyotubes derived from CTG110 mice, a knock-in mouse containing a DMPK gene with the 3′ part of the human DMPK gene including a (CTG) repeat of approximately 110.
  • CTG a DMPK gene with the 3′ part of the human DMPK gene including a (CTG) repeat of approximately 110.
  • Northern blot analysis showed that the DMPK transcript containing the (CTG)110 repeat was reduced by the treatment with oligonucleotide PS58 but not after mock treatment.
  • the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1).
  • CTG approximate
  • the transfection was carried out with PS58 at different concentrations. Eighty four hours after start of treatment, the myotubes were harvested and Northern blot analysis was performed on isolated RNA as described above (see example 1).
  • FIG. 4 shows the quantification of the hDMPK mRNA signal preformed by phosphoimager analysis and normalized to the GAPDH signal at different concentrations. Under these conditions, a half maximal effect was observed at around 1 nM.
  • the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, prepared and transfected as described above (see example 1). However, in this example the transfection with 200 nM PS58 was carried out at different time points. Usually DM500 myotubes were harvested seven days after switching to low serum conditions to induce myotube formation. The standard procedure (as in example 1 and 2) was to start treatment (transfection) 48 h (two days) before harvesting. Now, treatment with PS58 was started 2 h-48 h ( FIG. 5 ) or 2 d-8 d ( FIG. 6 ) before harvesting. Northern blot analysis and quantification was performed as before.
  • FIG. 5 shows that expanded hDMPK mRNA in DM500 myotubes was decreased rapidly within 2 h of treatment with oligonucleotide PS58 compared to mock control treatment.
  • FIG. 6 shows a persistent decrease in expanded hDMPK mRNA in DM500 myotubes for at least 8 days.
  • cells were transfected in the myoblast stage (approximately 60% confluent, 33° C., high serum) and that they have received fresh medium on various occasions until harvesting (including a change to low serum at 37° C., two days after transfection).
  • Example 2 and 3 are indicative of a highly efficient inhibitory intervention by an oligonucleotide directed solely to the repeat expansion. The magnitude of this effect might be influenced by the relative low levels of hDMPK expression in these model cell systems, which normally is also seen in humans.
  • fibroblasts obtained from a human patient with congenital myotonic dystrophy type 1 were used. These patient cells carry one disease causing DMPK allele with a triplet repeat expansion length of 1500 and one normal DMPK allele with a repeat length of 11. The size of the (CTG)n expansion on both alleles was confirmed with PCR and Southern blotting.
  • Ad5Fib50MyoD, Crucell, Leiden MyoD-expressing adenovirus
  • the human DMPK signals were quantified by phosphoimager analysis and normalized to the GAPDH signal and expressed relative to mock control.
  • FIG. 7 shows the Northern blot analysis of the MyoD-transformed myoblasts treated with oligonucleotide PS58 (20 and 200 nM).
  • the results demonstrate an effective complete inhibition of the disease-causing hDMPK (CUG)1500 RNA transcript, while the smaller normal hDMPK (CUG)11 RNA transcript is only moderately affected at the two concentrations.
  • oligonucleotides directed to the repeat region exhibit selectivity towards the larger repeat size (or disease causing expansion).
  • the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured, transfected and analysed as described before in example 1.
  • the DM500 myotubes were treated 48 h before harvesting with 200 nM of oligonucleotide PS58, specific to the (CUG) repeat sequence only, oligonucleotide PS113, specific to a sequence in exon 1, or mock control.
  • RT-PCR analysis was performed on hDMPK mRNA expressed in this murine cell line (for primers see example 1) and on three other gene transcripts with a naturally occurring (CUG) repeat in mice, Ptbp1 with a (CUG)6, Syndecan3 with a (CUG)6 and Taxilinbeta with a (CUG)9.
  • the PCR primers used were for Ptbp1: 5′-TCTGTCCCTAATGTCCATGG-3′ and 5′-GCCATCTGCACAAGTGCGT-3′; for Syndecan3: 5′-GCTGTTGCTGCCACCGCT-3′ and 5′-GGCGCCTCGGGAGTGCTA-3′; and for Taxilinbeta: 5′-CTCAGCCCTGCTGCCTGT-3′ and 5′-CAGACCCATACGTGCTTATG-3′.
  • the PCR products were run on an agarose gel and signals were quantified using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, United Kingdom). The intensity of each signal was normalized to the corresponding actin signal and expressed relative to mock control.
  • FIG. 8 shows the RT-PCR results with a maximal inhibition of hDMPK mRNA expression by PS58.
  • the other gene transcripts carrying a naturally occurring small (CUG) repeat were not or only marginally affected by the oligonucleotide PS58, specific to the (CUG) repeat, compared to oligonucleotide PS113, which has no complementary sequence to these gene transcripts.
  • This example confirms the selectivity of an oligonucleotide, directed solely to the repeat region, towards the long repeat size (or disease causing expansion) compared to naturally occurring shorter repeat sizes.
  • the DM500 immortomyoblast cell line carrying a human DMPK gene with an approximate (CTG)500 repeat expansion was cultured and transfected with PS58 (200 nM).
  • CCG approximate
  • FISH analysis was carried out on the cells. Fourty eight hours after the start of the treatment, the cells were fixed with 4% formaldehyde, 5 mM MgCl 2 and 1 ⁇ PBS for 30 minutes. Hybridization with fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3 was performed overnight at 37° C. in a humid chamber. After hybridization the material was washed and mounted in mowiol and allowed to dry overnight.
  • Nuclear inclusions were visualized using a Bio-Rad MRC1024 confocal laser scanning microscope and LaserSharp2000 acquisition software. In total 50 cells were counted and scored for the presence of inclusions in the nuclei of these cells.
  • Literature indicates that DMPK mRNA containing a (CUG) expanded repeat accumulates and aggregates in the nucleus to form ribonuclear foci with regulatory nuclear proteins and transcription factors. Therefore, normal nuclear gene processing and cell function gets impaired.
  • FIG. 9 shows a mock treated cell containing ribonuclear inclusions in the nucleus, while these are no longer present in the cell nucleus after treatment with PS58.
  • FIG. 10 shows that the percentage of nuclei containing ribonuclear foci seen under control conditions in DM500 myotubes is strongly decreased by the treatment with PS58. This result demonstrates that inhibition of hDMPK mRNA expression also inhibits the disease related triplet repeat (CUG) rich inclusions.
  • CCG disease related triplet repeat
  • DM500 mice containing hDMPK with a (CTG)n expansion of approximately 500 triplets.
  • the DM500 mice were derived by somatic expansion from the DM300 mouse (e.g. see Gomes-Pereira M et al (2007) PLoS Genet. 2007 3(4): e52).
  • a (CTG) triplet repeat expansion of approximately 500 was confirmed by southern blot and PCR analysis.
  • PS58 was mixed with transfection agent ExGen 500 (Fermentas) according to the accompanying instructions for in vivo use.
  • PS58 (2 nmol, in the transfection solution with Exgen 500) was injected (40 ⁇ l) in the GPS complex of one-year-old DM500 mice and this procedure was repeated after 24 h.
  • DM500 mice were treated similarly with the transfection solution without PS58.
  • the mice were sacrificed, muscles were isolated and total RNA was isolated from the tissues (using Trizol, Invitrogen).
  • RT-PCR analysis was performed to detect hDMPK mRNA in the muscle similar as described above. The intensity of each band was performed using the Labworks 4.0 program (UVP BioImaging systems, Cambridge, United Kingdom) and normalized to the intensity of the corresponding actin band. Primer location is indicated in the figure.
  • FIG. 11 shows that in vivo treatment of DM500 mice with PS58 strongly reduced the presence of hDMPK mRNA containing a (CUG)n repeat expansion compared to mock treatment in the M. plantaris and M. gastrocnemius.
  • oligonucleotides in length and backbone chemistry
  • CTG CCGn repeat expansion
  • DM500 myotubes were cultured, transfected and analysed as described above in example 1.
  • Northern blots were quantified by phosphoimager analysis and DMPK signals were normalized to GAPDH.
  • the DM500 myotubes were treated with the following oligonucleotides (200 nM), all with a complete phosphorothioate backbone (see Table 3).
  • FIG. 12 shows that treatment of the DM500 myotubes results in a complete reduction of (CUG)n expanded hDMPK mRNA for all oligonucleotides tested.
  • the maximal effect obtainable is independent of oligonucleotide length, backbone modification or potential mechanism of inhibition by the employed single stranded oligonucleotides.
  • Fibroblasts (GM 00305) from a male patient with Huntington's Disease were obtained from Coriell Cell Repository (Camden, N.J., US) and cultured according to the accompanying instructions and standard techniques known to the skilled person in the art. Huntington patients carry one healthy and one disease-causing allele of the Huntington gene resulting in the expression of both mRNAs with respectively a normal number and an expanded number of (CAG) repeats, respectively.
  • the fibroblasts were transfected with a 21-mer 2′O-methyl phosphorothioate RNA antisense oligonucleotide PS57 with a (CUG)7 sequence, complementary to the (CAG) triplet repeat in Huntington mRNA. Transfection occurred at 100 or 200 nM in the presence of PEI as indicated by the manufacturer. Twenty four hours after transfection the cells were harvested and total RNA was isolated and analysed by RT-PCR. The Huntington transcript was determined using primers in downstream exon 64 (5′ GAAAG TCAGT CCGGG TAGAA CTTC 3 ′ and 5′ CAGAT ACCCG CTCCA TAGCA A 3′).
  • This method detects both types of Huntington mRNAs, the normal and mutant transcript with the additional (CAG) expansion.
  • GAPDH mRNA housekeeping gene
  • the signals were quantified and the total amount of Huntington mRNA was normalised to the amount of GAPDH mRNA in the same sample.
  • the results are expressed relative to a control treated (without oligonucleotide) sample from fibroblasts (which was to 100%).
  • PS57 an oligonucleotide directed only to the (CAG) repeat, induces a decrease in Huntington mRNA levels and these results are consistent with a selective inhibition of mutant over normal Huntington mRNA.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Neurology (AREA)
  • Analytical Chemistry (AREA)
  • Neurosurgery (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Psychology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US12/377,160 2006-08-11 2007-08-10 Methods and means for treating dna repeat instability associated genetic disorders Abandoned US20100184833A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP06118809.0 2006-08-11
EP06118809 2006-08-11
EP06119247.2 2006-08-21
EP06119247 2006-08-21
PCT/NL2007/050399 WO2008018795A1 (en) 2006-08-11 2007-08-10 Methods and means for treating dna repeat instability associated genetic disorders

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL2007/050399 A-371-Of-International WO2008018795A1 (en) 2006-08-11 2007-08-10 Methods and means for treating dna repeat instability associated genetic disorders

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/809,483 Division US9890379B2 (en) 2006-08-11 2015-07-27 Treatment of genetic disorders associated with DNA repeat instability

Publications (1)

Publication Number Publication Date
US20100184833A1 true US20100184833A1 (en) 2010-07-22

Family

ID=38668759

Family Applications (5)

Application Number Title Priority Date Filing Date
US12/377,160 Abandoned US20100184833A1 (en) 2006-08-11 2007-08-10 Methods and means for treating dna repeat instability associated genetic disorders
US14/809,483 Active US9890379B2 (en) 2006-08-11 2015-07-27 Treatment of genetic disorders associated with DNA repeat instability
US15/855,848 Active US10689646B2 (en) 2006-08-11 2017-12-27 Treatment of genetic disorders associated with DNA repeat instability
US16/660,387 Active US11274299B2 (en) 2006-08-11 2019-10-22 Methods and means for treating DNA repeat instability associated genetic disorders
US17/591,681 Pending US20220267765A1 (en) 2006-08-11 2022-02-03 Treatment of genetic disorders associated with dna repeat instability

Family Applications After (4)

Application Number Title Priority Date Filing Date
US14/809,483 Active US9890379B2 (en) 2006-08-11 2015-07-27 Treatment of genetic disorders associated with DNA repeat instability
US15/855,848 Active US10689646B2 (en) 2006-08-11 2017-12-27 Treatment of genetic disorders associated with DNA repeat instability
US16/660,387 Active US11274299B2 (en) 2006-08-11 2019-10-22 Methods and means for treating DNA repeat instability associated genetic disorders
US17/591,681 Pending US20220267765A1 (en) 2006-08-11 2022-02-03 Treatment of genetic disorders associated with dna repeat instability

Country Status (16)

Country Link
US (5) US20100184833A1 (pl)
EP (1) EP2049664B1 (pl)
JP (2) JP6047270B2 (pl)
AT (1) ATE524547T1 (pl)
AU (1) AU2007282224B2 (pl)
CA (1) CA2660523C (pl)
CY (1) CY1111975T1 (pl)
DK (1) DK2049664T3 (pl)
ES (1) ES2373246T3 (pl)
HK (1) HK1130510A1 (pl)
IL (1) IL196921A (pl)
NZ (1) NZ574807A (pl)
PL (1) PL2049664T3 (pl)
PT (1) PT2049664E (pl)
SI (1) SI2049664T1 (pl)
WO (1) WO2008018795A1 (pl)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090082547A1 (en) * 2003-04-29 2009-03-26 Iversen Patrick L Compositions for enhancing transport of molecules into cells
US20100016215A1 (en) * 2007-06-29 2010-01-21 Avi Biopharma, Inc. Compound and method for treating myotonic dystrophy
WO2013120003A1 (en) 2012-02-08 2013-08-15 Isis Pharmaceuticals, Inc. Modulation of rna by repeat targeting
US8835402B2 (en) 2009-06-26 2014-09-16 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
US8877725B2 (en) 2004-05-24 2014-11-04 Sarepta Therapeutics, Inc. Peptide conjugated, inosine-substituted antisense oligomer compound and method
US20150080452A1 (en) * 2006-09-21 2015-03-19 University Of Rochester Compositions and Methods Related to Protein Displacement Therapy for Myotonic Distrophy
US9161948B2 (en) 2011-05-05 2015-10-20 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
US9499583B2 (en) 2005-07-13 2016-11-22 Sarepta Therapeutics, Inc. Antibacterial antisense oligonucleotide and method
US9611471B2 (en) 2010-08-05 2017-04-04 Academisch Ziekenhuis Leiden Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
US20170260524A1 (en) * 2009-04-10 2017-09-14 Association Institut De Myologie Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease
US9890379B2 (en) 2006-08-11 2018-02-13 Biomarin Technologies B.V. Treatment of genetic disorders associated with DNA repeat instability
US20180066256A1 (en) * 2012-04-23 2018-03-08 Biomarin Technologies B.V. RNA Modulating Oligonucleotides with Improved Characteristics for the Treatment of Neuromuscular Disorders
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
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
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
US10888578B2 (en) 2016-12-19 2021-01-12 Sarepta Therapeutics, Inc. Exon skipping oligomer conjugates for muscular dystrophy
US11020417B2 (en) 2015-06-04 2021-06-01 Sarepta Therapeutics, Inc Methods and compounds for treatment of lymphocyte-related diseases and conditions
WO2021247020A1 (en) * 2020-06-03 2021-12-09 Triplet Therapeutics, Inc. Methods for the treatment of nucleotide repeat expansion disorders associated with msh3 activity
US11672786B2 (en) 2016-08-12 2023-06-13 The Hospital For Sick Children Methods of treating diseases associated with repeat instability

Families Citing this family (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7951934B2 (en) 2006-01-26 2011-05-31 Isis Pharmaceuticals, Inc. Compositions and their uses directed to huntingtin
CN101790385A (zh) 2007-07-12 2010-07-28 普罗森那技术公司 用于使化合物靶向多种选定器官、组织或肿瘤细胞的分子
JP2011510678A (ja) * 2008-02-08 2011-04-07 プロセンサ ホールディング ビーブイ Ｄｎａ反復不安定性関連遺伝性障害を治療するための方法及び手段
EP2317847B1 (en) * 2008-07-29 2019-04-17 The Board of Regents of The University of Texas System Selective inhibition of polyglutamine protein expression
EP2421971B1 (en) 2009-04-24 2016-07-06 BioMarin Technologies B.V. Oligonucleotide comprising an inosine for treating dmd
SI2475675T1 (sl) 2009-09-11 2017-03-31 Ionis Pharmaceuticals, Inc. Modulacija ekspresije huntingtina
WO2011097641A1 (en) * 2010-02-08 2011-08-11 Isis Pharmaceuticals, Inc. Methods and compositions useful in treatment of diseases or conditions related to repeat expansion
KR101981705B1 (ko) 2010-05-28 2019-05-24 사렙타 쎄러퓨틱스, 인코퍼레이티드 변형된 서브유니트간 결합 및/또는 말단 그룹을 갖는 올리고뉴클레오타이드 유사체
EP2595664B1 (en) * 2010-07-19 2018-10-17 Ionis Pharmaceuticals, Inc. Modulation of nuclear-retained rna
WO2012031243A2 (en) 2010-09-03 2012-03-08 Avi Biopharma, Inc. dsRNA MOLECULES COMPRISING OLIGONUCLEOTIDE ANALOGS HAVING MODIFIED INTERSUBUNIT LINKAGES AND/OR TERMINAL GROUPS
US9476043B2 (en) 2011-04-08 2016-10-25 Rula Zain-Luqman Diagnosis and treatment of friedreich's ataxia
AU2012246822B2 (en) * 2011-04-22 2017-05-25 Biomarin Technologies B.V. New compounds for treating, delaying and/or preventing a human genetic disorder such as myotonic dystrophy type 1 (DM1)
JP6478632B2 (ja) 2011-05-05 2019-03-06 サレプタ セラピューティクス， インコーポレイテッド ペプチドオリゴヌクレオチドコンジュゲート
EP2742135B2 (en) 2011-08-11 2020-06-10 Ionis Pharmaceuticals, Inc. Linkage modified gapped oligomeric compounds and uses thereof
EP2751269B1 (en) 2011-08-29 2016-03-23 Ionis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
EA202090338A1 (ru) 2011-11-18 2021-04-30 Сарепта Терапьютикс, Инк. Функционально-модифицированные олигонуклеотиды и их субъединицы
JP6496549B2 (ja) 2011-11-30 2019-04-03 サレプタ セラピューティクス， インコーポレイテッド 脊髄性筋萎縮症における誘発されたエクソン包含
ES2832531T3 (es) * 2011-11-30 2021-06-10 Sarepta Therapeutics Inc Oligonucleótidos para el tratamiento de enfermedades por expansión de repeticiones
WO2013142087A1 (en) 2012-03-20 2013-09-26 Sarepta Therapeutics, Inc. Boronic acid conjugates of oligonucleotide analogues
EP3027617A4 (en) 2013-07-31 2017-04-12 Ionis Pharmaceuticals, Inc. Methods and compounds useful in conditions related to repeat expansion
TW201536329A (zh) * 2013-08-09 2015-10-01 Isis Pharmaceuticals Inc 用於調節失養性肌強直蛋白質激酶(dmpk)表現之化合物及方法
US20160201064A1 (en) * 2013-08-16 2016-07-14 Rana Therapeutics, Inc. Compositions and methods for modulating expression of frataxin
JP2016531570A (ja) 2013-08-16 2016-10-13 ラナ セラピューティクス インコーポレイテッド ユークロマチン領域を標的とするオリゴヌクレオチド
MX2016004452A (es) * 2013-10-07 2017-04-27 Academisch Ziekenhuis Leiden Oligonucleotido antisentido dirigido a la eliminacion de sitios de escisión proteolitica, la mutacion de hchwa-d y de expansiones de repeticiones de trinucleotidos.
IL247600B (en) 2014-03-19 2022-06-01 Ionis Pharmaceuticals Inc Preparations for the modulation of ataxin 2 expression
US10006027B2 (en) * 2014-03-19 2018-06-26 Ionis Pharmaceuticals, Inc. Methods for modulating Ataxin 2 expression
WO2016088797A1 (ja) * 2014-12-02 2016-06-09 国立大学法人東京医科歯科大学 Ａｌｓの原因タンパク毒性を軽減する核酸
GB201504124D0 (en) 2015-03-11 2015-04-22 Proqr Therapeutics B V Oligonucleotides
JP6987041B2 (ja) 2015-08-28 2021-12-22 サレプタ セラピューティクス，インコーポレイテッド 脊髄性筋萎縮症におけるエクソン包含のための修飾アンチセンスオリゴマー
US10760076B2 (en) * 2015-10-05 2020-09-01 Proqr Therapeutics Ii B.V. Use of single-stranded antisense oligonucleotide in prevention or treatment of genetic diseases involving a trinucleotide repeat expansion
MA45328A (fr) 2016-04-01 2019-02-06 Avidity Biosciences Llc Compositions acide nucléique-polypeptide et utilisations de celles-ci
MX2019008199A (es) 2017-01-06 2019-11-25 Avidity Biosciences Llc Composiciones de acido nucleico polipeptido y metodos de induccion de la omision de exon.
US11613754B2 (en) 2017-02-20 2023-03-28 Northwestern University Toxic RNAi active seed sequences for killing cancer cells
JP2020508685A (ja) 2017-03-03 2020-03-26 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア サプレッサーｔＲＮＡ及びデアミナーゼによる変異のＲＮＡターゲティング
GB201711809D0 (en) 2017-07-21 2017-09-06 Governors Of The Univ Of Alberta Antisense oligonucleotide
MA51103A (fr) 2017-12-06 2020-10-14 Avidity Biosciences Inc Compositions et procédés de traitement de l'atrophie musculaire et de la dystrophie myotonique
WO2019200161A1 (en) * 2018-04-11 2019-10-17 Design Therapeutics Inc. Methods and compounds for the treatment of genetic disease
AU2019310097A1 (en) 2018-07-25 2021-02-04 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing ATXN2 expression
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
MX2021001284A (es) 2018-08-02 2021-07-15 Dyne Therapeutics Inc Complejos dirigidos al musculo y sus usos para el tratamiento de distrofia muscular facioescapulohumeral.
AU2019406199A1 (en) 2018-12-21 2021-07-29 Avidity Biosciences, Inc. Anti-transferrin receptor antibodies and uses thereof
AU2020321466A1 (en) 2019-07-26 2022-02-17 Proqr Therapeutics Ii B.V. Ophthalmic compositions comprising viscosifying polymers and nucleic acids
CN115038789A (zh) 2019-12-02 2022-09-09 塑造治疗公司 治疗性编辑
CN115666589A (zh) 2020-03-19 2023-01-31 艾维迪提生物科学公司 治疗面肩肱型肌营养不良的组合物和方法
TW202202173A (zh) 2020-03-27 2022-01-16 美商亞維代堤生物科學公司 治療肌肉萎縮症之組合物及方法
EP4127228A4 (en) 2020-03-30 2024-05-29 Neubase Therapeutics Inc MODIFIED PEPTIDE NUCLEIC ACID COMPOSITIONS
CN115667513A (zh) 2020-05-12 2023-01-31 田边三菱制药株式会社 用于调节Ataxin 3表达的化合物、方法和药物组合物
US11633498B2 (en) 2021-07-09 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
WO2023034870A2 (en) 2021-09-01 2023-03-09 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing dmpk expression
IL311452A (en) 2021-09-16 2024-05-01 Avidity Biosciences Inc Compositions and methods for the treatment of FSHD muscular dystrophy
US11931421B2 (en) 2022-04-15 2024-03-19 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy

Citations (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
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
US6172208B1 (en) * 1992-07-06 2001-01-09 Genzyme Corporation Oligonucleotides modified with conjugate groups
US6210892B1 (en) * 1998-10-07 2001-04-03 Isis Pharmaceuticals, Inc. Alteration of cellular behavior by antisense modulation of mRNA processing
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
US6369038B1 (en) * 1991-04-25 2002-04-09 Genset Closed antisense and sense oligonucleotides and their applications
US20020049173A1 (en) * 1999-03-26 2002-04-25 Bennett C. Frank Alteration of cellular behavior by antisense modulation of mRNA processing
US20020055481A1 (en) * 2000-08-25 2002-05-09 Jcr Pharmaceuticals Co., Ltd. Pharmaceutical composition for treatment of Duchenne muscular dystrophy
US6399575B1 (en) * 1998-11-10 2002-06-04 Auburn University Methods and compositions for targeting compounds to the central nervous system
US20020115824A1 (en) * 2000-11-30 2002-08-22 Engler Jeffrey A. Receptor-mediated uptake of peptides that bind the human transferrin receptor
US20030109476A1 (en) * 2001-08-07 2003-06-12 Kmiec Eric B. Compositions and methods for the prevention and treatment of Huntington's disease
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
US6670461B1 (en) * 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US20040132684A1 (en) * 2000-05-09 2004-07-08 Sampath Umashanker Polymeric nucleoside prodrugs
US20040226056A1 (en) * 1998-12-22 2004-11-11 Myriad Genetics, Incorporated Compositions and methods for treating neurological disorders and diseases
US20050246794A1 (en) * 2002-11-14 2005-11-03 Dharmacon Inc. Functional and hyperfunctional siRNA
US20050277133A1 (en) * 2001-05-18 2005-12-15 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US7001994B2 (en) * 2001-01-18 2006-02-21 Genzyme Corporation Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins
US20060074034A1 (en) * 2001-09-17 2006-04-06 Collins Douglas A Cobalamin mediated delivery of nucleic acids, analogs and derivatives thereof
US20060148740A1 (en) * 2005-01-05 2006-07-06 Prosensa B.V. Mannose-6-phosphate receptor mediated gene transfer into muscle cells
US7118893B2 (en) * 1997-08-19 2006-10-10 Regents Of The University Of Minnesota SCA7 gene and methods of use
US7189530B2 (en) * 1998-09-01 2007-03-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7202210B2 (en) * 2001-02-08 2007-04-10 Wyeth Modified and stabilized GDF propeptides and uses thereof
US20070082861A1 (en) * 2002-11-25 2007-04-12 Masafumi Matsuo Ena nucleic acid drugs modifying splicing in mrna precursor
US20070141628A1 (en) * 2005-12-15 2007-06-21 Cunningham Scott D Polyethylene binding peptides and methods of use
US7250404B2 (en) * 1989-03-21 2007-07-31 Vical Incorporated Lipid-mediated polynucleotide administration to deliver a biologically active peptide and to induce a cellular immune response
US20070275914A1 (en) * 2003-03-07 2007-11-29 Muthiah Manoharan Therapeutic Compositions
US20070292408A1 (en) * 2004-12-03 2007-12-20 University Of Massachusetts Spinal Muscular Atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US20080039418A1 (en) * 2006-01-26 2008-02-14 Freier Susan M Compositions and their uses directed to huntingtin
US7355018B2 (en) * 2003-09-30 2008-04-08 Regeneron Pharmaceuticals, Inc. Modified IGF1 polypeptides with increased stability and potency
US7405193B2 (en) * 2000-05-31 2008-07-29 Serono Genetics Institute S.A. Use of Acrp30 globular head to promote increases in muscle mass and muscle differentiation
US20080207538A1 (en) * 2004-03-11 2008-08-28 Lawrence David S Enhanced Production of Functional Proteins From Defective Genes
US20080249294A1 (en) * 2001-05-18 2008-10-09 Sirna Therapeutics, Inc. RNA Interference Mediated Inhibition of Gene Expression Using Short Interfering Nucleic Acid (siNA)
US7442782B2 (en) * 2001-05-11 2008-10-28 Regents Of The University Of Minnesota Intron associated with myotonic dystrophy type 2 and methods of use
US7514551B2 (en) * 2003-04-03 2009-04-07 Enzo Life Sciences, Inc. Multisignal labeling reagents, and processes and uses therefor
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
US7589189B2 (en) * 2003-05-14 2009-09-15 Japan Science And Technology Agency Inhibition of the expression of huntingtin gene
US7655785B1 (en) * 2002-11-14 2010-02-02 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof
US20100099750A1 (en) * 2002-02-20 2010-04-22 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MUSCARINIC COLINERGIC RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7771727B2 (en) * 2002-03-01 2010-08-10 The Administrators Of The Tulane Educational Fund Conjugates of therapeutic or cytotoxic agents and biologically active peptides
US7807816B2 (en) * 2004-06-28 2010-10-05 University Of Western Australia Antisense oligonucleotides for inducing exon skipping and methods of use thereof

Family Cites Families (105)

* Cited by examiner, † Cited by third party
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
US5766847A (en) 1988-10-11 1998-06-16 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Process for analyzing length polymorphisms in DNA regions
DE3834636A1 (de) 1988-10-11 1990-04-19 Max Planck Gesellschaft Verfahren zur analyse von laengenpolymorphismen in dna-bereichen
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
JPH07501204A (ja) 1991-06-28 1995-02-09 マサチューセッツ インスティテュート オブ テクノロジー 局所的オリゴヌクレオチド療法
US6200747B1 (en) 1992-01-28 2001-03-13 North Shore University Hospital Research Corp. Method and kits for detection of fragile X specific, GC-rich DNA sequences
US5869252A (en) 1992-03-31 1999-02-09 Abbott Laboratories Method of multiplex ligase chain reaction
US5418139A (en) 1993-02-10 1995-05-23 University Of Iowa Research Foundation Method for screening for cardiomyopathy
CA2116280A1 (en) 1993-03-05 1994-09-06 Marcy E. Macdonald Huntingtin dna, protein and uses thereof
US5695933A (en) * 1993-05-28 1997-12-09 Massachusetts Institute Of Technology Direct detection of expanded nucleotide repeats in the human genome
US5741645A (en) 1993-06-29 1998-04-21 Regents Of The University Of Minnesota Gene sequence for spinocerebellar ataxia type 1 and method for diagnosis
US5627263A (en) 1993-11-24 1997-05-06 La Jolla Cancer Research Foundation Integrin-binding peptides
DE4342605A1 (de) 1993-12-14 1995-06-22 Buna Gmbh Funktionalisierte Olefinhomo- und -copolymere
US5962332A (en) 1994-03-17 1999-10-05 University Of Massachusetts Detection of trinucleotide repeats by in situ hybridization
WO1995030774A1 (en) 1994-05-05 1995-11-16 Beckman Instruments, Inc. Oligonucleotide repeat arrays
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
BR9707529A (pt) 1996-02-14 2000-01-04 Isis Pharmaceuticals Inc Oligunucleotìdeo especificamente hibridizável com dna ou rna.
US6251589B1 (en) 1996-07-18 2001-06-26 Srl, Inc. Method for diagnosing spinocerebellar ataxia type 2 and primers therefor
WO1998018920A1 (fr) 1996-10-30 1998-05-07 Srl, Inc. Fragments d'adnc du gene responsable de l'ataxie spino-cerebelleuse de type 2
US5853995A (en) 1997-01-07 1998-12-29 Research Development Foundation Large scale genotyping of diseases and a diagnostic test for spinocerebellar ataxia type 6
US20020137890A1 (en) 1997-03-31 2002-09-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
WO1998049345A1 (en) 1997-04-29 1998-11-05 Trustees Of Boston University Methods and compositions for targeted dna differential display
US6280938B1 (en) 1997-08-19 2001-08-28 Regents Of The University Of Minnesota SCA7 gene and method of use
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 (ja) 1998-01-26 2000-02-28 新潟大学長 Ｃａｇリピート病の治療薬
KR100280219B1 (ko) 1998-02-26 2001-04-02 이수빈 삼핵산 반복 서열을 이용한 신경정신 질환의 진단 방법 및 진단 시약
US6322978B1 (en) 1998-04-20 2001-11-27 Joslin Diabetes Center, Inc. Repeat polymorphism in the frataxin gene and uses therefore
US5945290A (en) 1998-09-18 1999-08-31 Isis Pharmaceuticals, Inc. Antisense modulation of RhoA expression
CN1319133A (zh) 1998-09-25 2001-10-24 儿童医疗中心有限公司 选择性地调节蛋白激酶活性的短肽
US6133031A (en) 1999-08-19 2000-10-17 Isis Pharmaceuticals Inc. Antisense inhibition of focal adhesion kinase expression
US6379698B1 (en) 1999-04-06 2002-04-30 Isis Pharmaceuticals, Inc. Fusogenic lipids and vesicles
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
WO2001079283A1 (en) 2000-04-13 2001-10-25 University Of British Columbia Modulating cell survival by modulating huntingtin function
AU2001261063A1 (en) 2000-04-28 2001-11-12 Xiao Xiao Dna sequences encoding dystrophin minigenes and methods of use thereof
JP2003531915A (ja) 2000-05-01 2003-10-28 ハイブリドン・インコーポレイテッド ヌクレオシドの位置的修飾によるオリゴヌクレオチドＣｐＧ誘導性免疫刺激の調節
CN1326990A (zh) 2000-06-07 2001-12-19 上海博德基因开发有限公司 一种新的多肽——人类dna cgg重复结合蛋白16.17和编码这种多肽的多核苷酸
US20030124523A1 (en) 2000-06-22 2003-07-03 Asselbergs Fredericus Alphonsus Maria Organic compounds
US6794192B2 (en) 2000-06-29 2004-09-21 Pfizer Inc. Target
RU2165149C1 (ru) 2000-07-03 2001-04-20 Шапошников Валерий Геннадьевич Система формования и упаковки изделий из сахарной ваты
EP1366160B1 (en) 2000-10-06 2008-07-09 The Regents Of The University Of Michigan Mini-dystrophin nucleic acid and peptide sequences
EP1356278A4 (en) * 2000-10-27 2006-12-27 Baylor College Medicine METHODS AND COMPOSITIONS FOR IDENTIFYING AND TREATING NEURODEGENERATIVE DISORDERS
US6623927B1 (en) 2000-11-08 2003-09-23 Council Of Scientific And Industrial Research Method of detection of allelic variants of SCA2 gene
CA2526831C (en) 2001-05-18 2012-07-31 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (sina)
IL143379A (en) 2001-05-24 2013-11-28 Yissum Res Dev Co Oligonucleotide against human ache isoform r and its uses
ATE386548T1 (de) 2001-07-06 2008-03-15 Topigen Pharmaceuticals Inc Verfahren zur erhöhung der in vivo wirksamkeit von oligonukleotiden und zur entzündungshemmung in säugetieren
JP2005507244A (ja) 2001-08-10 2005-03-17 ノバルティス アクチエンゲゼルシャフト アテローム硬化性病変に結合するペプチド
KR20030035047A (ko) 2001-10-29 2003-05-09 (주)바이오코돈 Bmp-4 유전자 발현을 이용한 편평태선 질환의 치료 및진단방법
AU2002363253A1 (en) 2001-11-01 2003-05-12 Gpc Biotech Inc. Endothelial-cell binding peptides for diagnosis and therapy
US20030134790A1 (en) 2002-01-11 2003-07-17 University Of Medicine And Dentistry Of New Jersey Bone Morphogenetic Protein-2 And Bone Morphogenetic Protein-4 In The Treatment And Diagnosis Of Cancer
WO2003069330A1 (en) 2002-02-11 2003-08-21 The Trustees Of Columbia University In The City Of New York System and method for identifying proteins involved in force-initiated signal transduction
US20050096284A1 (en) 2002-02-20 2005-05-05 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20040101852A1 (en) 2002-11-21 2004-05-27 Isis Pharmaceuticals Inc. Modulation of CGG triplet repeat binding protein 1 expression
ITRM20020253A1 (it) 2002-05-08 2003-11-10 Univ Roma Molecole chimeriche di snrna con sequenze antisenso per le giunzioni di splicing del gene della distrofina e applicazioni terapeutiche.
EP1380644A1 (en) 2002-07-08 2004-01-14 Kylix B.V. The use of specified TCF target genes to identify drugs for the treatment of cancer, in particular colorectal cancer, in which TCF/beta-catenin/WNT signalling plays a central role
WO2004011060A2 (en) 2002-07-26 2004-02-05 Mirus Corporation Delivery of molecules and complexes to mammalian cells in vivo
US20050255086A1 (en) 2002-08-05 2005-11-17 Davidson Beverly L Nucleic acid silencing of Huntington's Disease gene
EP1529105B1 (en) 2002-08-12 2012-10-03 Société de commercialisation des produits de la recherche appliquée SOCPRA Sciences Santé et Humaines s.e.c. Methods to reprogram splice site selection in pre-messenger rnas
GB0219143D0 (en) 2002-08-16 2002-09-25 Univ Leicester Modified tailed oligonucleotides
ATE358136T1 (de) 2002-10-23 2007-04-15 Ct For Res And Technology Hell Prionen-bindende peptidsequenzen
US7892793B2 (en) 2002-11-04 2011-02-22 University Of Massachusetts Allele-specific RNA interference
GB0228079D0 (en) 2002-12-02 2003-01-08 Laxdale Ltd Huntington's Disease
CA2526669A1 (en) 2003-06-02 2004-12-16 Wyeth Use of myostatin (gdf8) inhibitors in conjunction with corticosteroids for treating neuromuscular disorders
ES2302898T3 (es) 2003-07-11 2008-08-01 Lbr Medbiotech B.V. Transferencia de genes a celulas musculares mediada por el receptor de manosa-6-fosfato.
US20050054752A1 (en) 2003-09-08 2005-03-10 O'brien John P. Peptide-based diblock and triblock dispersants and diblock polymers
EP1680439A2 (en) 2003-10-14 2006-07-19 Kernel Biopharma Inc. Dual phase-pna conjugates for the delivery of pna through the blood brain barrier
US20050191636A1 (en) 2004-03-01 2005-09-01 Biocept, Inc. Detection of STRP, such as fragile X syndrome
EP1735443A2 (en) 2004-04-14 2006-12-27 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED TREATMENT OF POLYGLUTAMINE (POLYQ) REPEAT EXPANSION DISEASES USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP1752536A4 (en) 2004-05-11 2008-04-16 Alphagen Co Ltd POLYNUCLEOTIDE CAUSING RNA INTERFERENCE AND METHOD OF REGULATING GENE EXPRESSION WITH THE USE OF THE SAME
CA2567810A1 (en) 2004-05-27 2005-12-08 Acceleron Pharma Inc. Cerberus/coco derivatives and uses thereof
EP1618881A1 (en) 2004-07-20 2006-01-25 Santhera Pharmaceuticals (Schweiz) GmbH Use of non-glucocorticoid steroids for the treatment of muscular dystrophy
WO2006017522A2 (en) 2004-08-03 2006-02-16 University Of Utah Research Foundation Use of antisense oligonucleotides to effect translation modulation
ITRM20040568A1 (it) 2004-11-18 2005-02-18 Uni Degli Studi Di Roma Tor Vergata Uso della tecnica "phage display" per l'identificazione di peptidi con capacita' di legame a cellule staminali/progenitore, peptidi cosi' ottenuti e loro usi.
US20120122801A1 (en) 2005-01-05 2012-05-17 Prosensa B.V. Mannose-6-phosphate receptor mediated gene transfer into muscle cells
CA2596588C (en) 2005-01-31 2017-06-27 University Of Iowa Research Foundation Nucleic acid silencing of huntington's disease gene
CN101184841A (zh) 2005-04-22 2008-05-21 莱顿教学医院 通过干扰SR蛋白的结合以及干扰RNA二级结构调节前mRNA中的外显子识别
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
CN101501193B (zh) 2006-08-11 2013-07-03 普罗森那技术公司 用于治疗与dna重复不稳定性相关的遗传病的方法和手段
CA2660523C (en) 2006-08-11 2019-03-19 Prosensa Technologies B.V. Methods and means for treating dna repeat instability associated genetic disorders
AU2008273096B2 (en) 2007-07-12 2013-05-02 Academisch Ziekenhuis Leiden Molecules for targeting compounds to various selected organs or tissues
CN101790385A (zh) 2007-07-12 2010-07-28 普罗森那技术公司 用于使化合物靶向多种选定器官、组织或肿瘤细胞的分子
EP2614827B1 (en) 2007-10-26 2017-06-28 Academisch Ziekenhuis Leiden Means and methods for counteracting muscle disorders
JP2011510678A (ja) 2008-02-08 2011-04-07 プロセンサ ホールディング ビーブイ Ｄｎａ反復不安定性関連遺伝性障害を治療するための方法及び手段
EP2119783A1 (en) 2008-05-14 2009-11-18 Prosensa Technologies B.V. Method for efficient exon (44) skipping in Duchenne Muscular Dystrophy and associated means
WO2010006237A2 (en) 2008-07-11 2010-01-14 Alnylam Pharmaceuticals, Inc. Phosphorothioate oligonucleotides non-nucleosidic phosphorothiotes as delivery agents for irna agents
EP2317847B1 (en) 2008-07-29 2019-04-17 The Board of Regents of The University of Texas System Selective inhibition of polyglutamine protein expression
US20110294870A1 (en) 2008-12-04 2011-12-01 Opko Curna, Llc Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene
EP2421971B1 (en) 2009-04-24 2016-07-06 BioMarin Technologies B.V. Oligonucleotide comprising an inosine for treating dmd
UA105390C2 (ru) 2009-06-08 2014-05-12 Міраджен Терапьютікс МОТИВЫ ХИМИЧЕСКИХ МОДИФИКАЦИЙ ДЛЯ ИНГИБИТОРОВ И МИМЕТИКОВ мкРНК
ES2616561T3 (es) 2009-12-24 2017-06-13 Biomarin Technologies B.V. Molécula para tratar un trastorno inflamatorio
WO2011097614A1 (en) 2010-02-08 2011-08-11 Isis Pharmaceuticals, Inc. Mehods and compositions useful in diseases or conditions related to repeat expansion
WO2011097641A1 (en) 2010-02-08 2011-08-11 Isis Pharmaceuticals, Inc. Methods and compositions useful in treatment of diseases or conditions related to repeat expansion
EP2595664B1 (en) 2010-07-19 2018-10-17 Ionis Pharmaceuticals, Inc. Modulation of nuclear-retained rna
CA2806616C (en) 2010-08-20 2015-08-11 Replicor Inc. Oligonucleotide chelate complexes
US10017764B2 (en) 2011-02-08 2018-07-10 Ionis Pharmaceuticals, Inc. Oligomeric compounds comprising bicyclic nucleotides and uses thereof
AU2012246822B2 (en) 2011-04-22 2017-05-25 Biomarin Technologies B.V. New compounds for treating, delaying and/or preventing a human genetic disorder such as myotonic dystrophy type 1 (DM1)
JP6478632B2 (ja) 2011-05-05 2019-03-06 サレプタ セラピューティクス， インコーポレイテッド ペプチドオリゴヌクレオチドコンジュゲート
ES2832531T3 (es) 2011-11-30 2021-06-10 Sarepta Therapeutics Inc Oligonucleótidos para el tratamiento de enfermedades por expansión de repeticiones
EP4043039A1 (en) 2012-01-27 2022-08-17 BioMarin Technologies B.V. Rna modulating oligonucleotides with improved characteristics for the treatment of duchenne and becker muscular dystrophy
US20140378533A1 (en) 2012-02-08 2014-12-25 Isis Pharmaceuticals, Inc. Modulation of rna by repeat targeting
CN110025628B (zh) 2012-04-23 2023-03-31 维科医疗有限公司 治疗神经肌肉障碍的具有改善特性的rna调节寡核苷酸
EP2870246B1 (en) 2012-07-03 2019-09-11 BioMarin Technologies B.V. Oligonucleotide for the treatment of muscular dystrophy patients

Patent Citations (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5541308A (en) * 1986-11-24 1996-07-30 Gen-Probe Incorporated Nucleic acid probes for detection and/or quantitation of non-viral organisms
US7250404B2 (en) * 1989-03-21 2007-07-31 Vical Incorporated Lipid-mediated polynucleotide administration to deliver a biologically active peptide and to induce a cellular immune response
US6369038B1 (en) * 1991-04-25 2002-04-09 Genset Closed antisense and sense oligonucleotides and their applications
US5593974A (en) * 1991-06-28 1997-01-14 Massachusetts Institute Of Technology Localized oligonucleotide therapy
US6172208B1 (en) * 1992-07-06 2001-01-09 Genzyme Corporation Oligonucleotides modified with conjugate groups
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
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
US5968909A (en) * 1995-08-04 1999-10-19 Hybridon, Inc. Method of modulating gene expression with reduced immunostimulatory response
US6329501B1 (en) * 1997-05-29 2001-12-11 Auburn University Methods and compositions for targeting compounds to muscle
US7118893B2 (en) * 1997-08-19 2006-10-10 Regents Of The University Of Minnesota SCA7 gene and methods of use
US6670461B1 (en) * 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US7189530B2 (en) * 1998-09-01 2007-03-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US6172216B1 (en) * 1998-10-07 2001-01-09 Isis Pharmaceuticals Inc. Antisense modulation of BCL-X expression
US6210892B1 (en) * 1998-10-07 2001-04-03 Isis Pharmaceuticals, Inc. Alteration of cellular behavior by antisense modulation of mRNA processing
US6399575B1 (en) * 1998-11-10 2002-06-04 Auburn University Methods and compositions for targeting compounds to the central nervous system
US20040226056A1 (en) * 1998-12-22 2004-11-11 Myriad Genetics, Incorporated Compositions and methods for treating neurological disorders and diseases
US20020049173A1 (en) * 1999-03-26 2002-04-25 Bennett C. Frank Alteration of cellular behavior by antisense modulation of mRNA processing
US6653466B2 (en) * 1999-05-21 2003-11-25 Jcr Pharmaceuticals Co., Ltd. Pharmaceutical composition for treatment of duchenne muscular dystrophy
US20010056077A1 (en) * 1999-05-21 2001-12-27 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
US20100081627A1 (en) * 2000-05-09 2010-04-01 Reliable Biopharmaceutical, Inc. Polymeric nucleoside prodrugs
US20040132684A1 (en) * 2000-05-09 2004-07-08 Sampath Umashanker Polymeric nucleoside prodrugs
US7405193B2 (en) * 2000-05-31 2008-07-29 Serono Genetics Institute S.A. Use of Acrp30 globular head to promote increases in muscle mass and muscle differentiation
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
US20020115824A1 (en) * 2000-11-30 2002-08-22 Engler Jeffrey A. Receptor-mediated uptake of peptides that bind the human transferrin receptor
US7001994B2 (en) * 2001-01-18 2006-02-21 Genzyme Corporation Methods for introducing mannose 6-phosphate and other oligosaccharides onto glycoproteins
US7202210B2 (en) * 2001-02-08 2007-04-10 Wyeth Modified and stabilized GDF propeptides and uses thereof
US7442782B2 (en) * 2001-05-11 2008-10-28 Regents Of The University Of Minnesota Intron associated with myotonic dystrophy type 2 and methods of use
US20080249294A1 (en) * 2001-05-18 2008-10-09 Sirna Therapeutics, Inc. RNA Interference Mediated Inhibition of Gene Expression Using Short Interfering Nucleic Acid (siNA)
US20050277133A1 (en) * 2001-05-18 2005-12-15 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
US20030109476A1 (en) * 2001-08-07 2003-06-12 Kmiec Eric B. Compositions and methods for the prevention and treatment of Huntington's disease
US20060074034A1 (en) * 2001-09-17 2006-04-06 Collins Douglas A Cobalamin mediated delivery of nucleic acids, analogs and derivatives thereof
US20100099750A1 (en) * 2002-02-20 2010-04-22 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MUSCARINIC COLINERGIC RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7771727B2 (en) * 2002-03-01 2010-08-10 The Administrators Of The Tulane Educational Fund Conjugates of therapeutic or cytotoxic agents and biologically active peptides
US7655785B1 (en) * 2002-11-14 2010-02-02 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof
US20050246794A1 (en) * 2002-11-14 2005-11-03 Dharmacon Inc. Functional and hyperfunctional siRNA
US7902160B2 (en) * 2002-11-25 2011-03-08 Masafumi Matsuo ENA nucleic acid drugs modifying splicing in mRNA precursor
US20070082861A1 (en) * 2002-11-25 2007-04-12 Masafumi Matsuo Ena nucleic acid drugs modifying splicing in mrna precursor
US20070275914A1 (en) * 2003-03-07 2007-11-29 Muthiah Manoharan Therapeutic Compositions
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
US7514551B2 (en) * 2003-04-03 2009-04-07 Enzo Life Sciences, Inc. Multisignal labeling reagents, and processes and uses therefor
US7589189B2 (en) * 2003-05-14 2009-09-15 Japan Science And Technology Agency Inhibition of the expression of huntingtin gene
US7355018B2 (en) * 2003-09-30 2008-04-08 Regeneron Pharmaceuticals, Inc. Modified IGF1 polypeptides with increased stability and potency
US20080207538A1 (en) * 2004-03-11 2008-08-28 Lawrence David S Enhanced Production of Functional Proteins From Defective Genes
US20120029058A1 (en) * 2004-06-28 2012-02-02 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
US20110015258A1 (en) * 2004-06-28 2011-01-20 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
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
US20070292408A1 (en) * 2004-12-03 2007-12-20 University Of Massachusetts Spinal Muscular Atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US20060148740A1 (en) * 2005-01-05 2006-07-06 Prosensa B.V. Mannose-6-phosphate receptor mediated gene transfer into muscle cells
US20070141628A1 (en) * 2005-12-15 2007-06-21 Cunningham Scott D Polyethylene binding peptides and methods of use
US20080039418A1 (en) * 2006-01-26 2008-02-14 Freier Susan M Compositions and their uses directed to huntingtin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Devore et al. (Integrated DNA Technologies, 2005 pages 1-11; see page 7). *
Tanaje (Biotechniques (1998) vol. 24:472-476). *
Thomsen et al. (RNA (2005) vol 11:1745-1748). *

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10905782B2 (en) 2003-04-29 2021-02-02 Sarepta Therapeutics, Inc. Compositions for enhancing transport of molecules into cells
US10300149B2 (en) 2003-04-29 2019-05-28 Sarepta Therapeutics, Inc. Compositions for enhancing transport of molecules into cells
US9572899B2 (en) 2003-04-29 2017-02-21 Avi Biopharma, Inc. Compositions for enhancing transport of molecules into cells
US20090082547A1 (en) * 2003-04-29 2009-03-26 Iversen Patrick L Compositions for enhancing transport of molecules into cells
US8877725B2 (en) 2004-05-24 2014-11-04 Sarepta Therapeutics, Inc. Peptide conjugated, inosine-substituted antisense oligomer compound and method
US9499583B2 (en) 2005-07-13 2016-11-22 Sarepta Therapeutics, Inc. Antibacterial antisense oligonucleotide and method
US10144762B2 (en) 2005-07-13 2018-12-04 Sarepta Therapeutics, Inc. Antibacterial antisense oligonucleotide and method
US9890379B2 (en) 2006-08-11 2018-02-13 Biomarin Technologies B.V. Treatment of genetic disorders associated with DNA repeat instability
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
US20150080452A1 (en) * 2006-09-21 2015-03-19 University Of Rochester Compositions and Methods Related to Protein Displacement Therapy for Myotonic Distrophy
US9371527B2 (en) * 2006-09-21 2016-06-21 University Of Rochester Compositions and methods related to protein displacement therapy for myotonic distrophy
US20120058946A1 (en) * 2007-06-29 2012-03-08 Avi Biopharma, Inc. Compound and method for treating myotonic dystrophy
US11236329B2 (en) 2007-06-29 2022-02-01 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
US8741863B2 (en) * 2007-06-29 2014-06-03 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
US20100016215A1 (en) * 2007-06-29 2010-01-21 Avi Biopharma, Inc. Compound and method for treating myotonic dystrophy
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
US20170260524A1 (en) * 2009-04-10 2017-09-14 Association Institut De Myologie Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease
US10465191B2 (en) * 2009-04-10 2019-11-05 Association Institut De Myologie Tricyclo-DNA antisense oligonucleotides, compositions, and methods for the treatment of disease
US10927378B2 (en) 2009-06-26 2021-02-23 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
US10106796B2 (en) 2009-06-26 2018-10-23 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
US8835402B2 (en) 2009-06-26 2014-09-16 Sarepta Therapeutics, Inc. Compound and method for treating myotonic dystrophy
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
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
US9611471B2 (en) 2010-08-05 2017-04-04 Academisch Ziekenhuis Leiden Antisense oligonucleotide directed removal of proteolytic cleavage sites from proteins
US10487326B2 (en) 2011-05-05 2019-11-26 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
US11225662B2 (en) 2011-05-05 2022-01-18 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
US11732259B2 (en) 2011-05-05 2023-08-22 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
US9161948B2 (en) 2011-05-05 2015-10-20 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
US9862946B2 (en) 2011-05-05 2018-01-09 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
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
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
WO2013120003A1 (en) 2012-02-08 2013-08-15 Isis Pharmaceuticals, Inc. Modulation of rna by repeat targeting
US11345915B2 (en) 2012-04-23 2022-05-31 Vico Therapeutics B.V. RNA modulating oligonucleotides with improved characteristics for the treatment of neuromuscular disorders
US20180066256A1 (en) * 2012-04-23 2018-03-08 Biomarin Technologies B.V. RNA Modulating Oligonucleotides with Improved Characteristics for the Treatment of Neuromuscular Disorders
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
US11020417B2 (en) 2015-06-04 2021-06-01 Sarepta Therapeutics, Inc Methods and compounds for treatment of lymphocyte-related diseases and conditions
US11672786B2 (en) 2016-08-12 2023-06-13 The Hospital For Sick Children Methods of treating diseases associated with repeat instability
US11642364B2 (en) 2016-12-19 2023-05-09 Sarepta Therapeutics, Inc. Exon skipping oligomer conjugates for muscular dystrophy
US10888578B2 (en) 2016-12-19 2021-01-12 Sarepta Therapeutics, Inc. Exon skipping oligomer conjugates for muscular dystrophy
WO2021247020A1 (en) * 2020-06-03 2021-12-09 Triplet Therapeutics, Inc. Methods for the treatment of nucleotide repeat expansion disorders associated with msh3 activity

Also Published As

Publication number Publication date
EP2049664B1 (en) 2011-09-14
JP5934408B2 (ja) 2016-06-15
US20220267765A1 (en) 2022-08-25
EP2049664A1 (en) 2009-04-22
IL196921A0 (en) 2011-08-01
JP2015157813A (ja) 2015-09-03
IL196921A (en) 2013-11-28
US11274299B2 (en) 2022-03-15
AU2007282224B2 (en) 2013-08-29
AU2007282224A1 (en) 2008-02-14
US9890379B2 (en) 2018-02-13
DK2049664T3 (da) 2012-01-02
PL2049664T3 (pl) 2012-02-29
CA2660523C (en) 2019-03-19
US20180112214A1 (en) 2018-04-26
US10689646B2 (en) 2020-06-23
ATE524547T1 (de) 2011-09-15
JP2010500023A (ja) 2010-01-07
PT2049664E (pt) 2012-01-03
CA2660523A1 (en) 2008-02-14
NZ574807A (en) 2011-01-28
ES2373246T3 (es) 2012-02-01
US20160053254A1 (en) 2016-02-25
JP6047270B2 (ja) 2016-12-21
US20200140857A1 (en) 2020-05-07
CY1111975T1 (el) 2015-11-04
WO2008018795A1 (en) 2008-02-14
HK1130510A1 (en) 2009-12-31
SI2049664T1 (sl) 2012-04-30

Similar Documents

Publication Publication Date Title
US11274299B2 (en) Methods and means for treating DNA repeat instability associated genetic disorders
US8263760B2 (en) Methods and means for treating DNA repeat instability associated genetic disorders
US11427820B2 (en) Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA
AU2009310557B2 (en) Methods and means for efficient skipping of exon 45 in Duchenne Muscular Dystrophy pre-mRNA
CN101501193B (zh) 用于治疗与dna重复不稳定性相关的遗传病的方法和手段
JP2023543494A (ja) 視神経萎縮症の治療

Legal Events

Date Code Title Description
AS Assignment

Owner name: PROSENSA TECHNOLOGIES B.V., NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DE KIMPE, JOSEPHUS JOHANNES;PLATENBURG, GERARD JOHANNES;WANSINK, DERICK GERT;REEL/FRAME:023984/0753

Effective date: 20090323

AS Assignment

Owner name: PROSENSA TECHNOLOGIES B.V., NETHERLANDS

Free format text: CHANGE OF ADDRESS;ASSIGNOR:PROSENSA TECHNOLOGIES B.V.;REEL/FRAME:034913/0884

Effective date: 20110704

AS Assignment

Owner name: BIOMARIN TECHNOLOGIES B.V., NETHERLANDS

Free format text: CHANGE OF NAME;ASSIGNOR:PROSENSA TECHNOLOGIES B.V.;REEL/FRAME:036732/0042

Effective date: 20150908

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION