WO2013120038A2 - Morpholino ciblant le dux4 pour le traitement de la myopathie facio-scapulo-humérale - Google Patents

Morpholino ciblant le dux4 pour le traitement de la myopathie facio-scapulo-humérale Download PDF

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WO2013120038A2
WO2013120038A2 PCT/US2013/025461 US2013025461W WO2013120038A2 WO 2013120038 A2 WO2013120038 A2 WO 2013120038A2 US 2013025461 W US2013025461 W US 2013025461W WO 2013120038 A2 WO2013120038 A2 WO 2013120038A2
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dux4
morpholino
antisense oligonucleotide
oligonucleotide
sequence
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PCT/US2013/025461
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WO2013120038A3 (fr
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Michael Kyba
Megan MULTHAUP
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Regents Of The University Of Minnesota
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2310/314—Phosphoramidates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring

Definitions

  • Facioscapulohumeral muscular dystrophy is a dominantly inherited degenerative myopathy affecting approximately one in 20,000 individuals.
  • FSHD affected patients can present with asymmetric weakness and wasting of facial muscles.
  • FSHD affected patients can also present with shoulder muscle wasting and/or upper arm muscle wasting.
  • FSHD disease pathology can progress to include distal anterior leg weakness, abdominal weakness, or hip-girdle muscle weakness (see, e.g., Tawil and Van Der Marrel, 2006. Muscle Nerve. 34(1): 1-15).
  • a genetic alteration that can be associated with FSHD is a contraction within the D4Z4 array, a 3.3 kb macrosatellite repeat located at the distal tip of chromosome 4 (4q35.2).
  • the D4Z4 array copy number can be polymorphic presumably due to intra-chromosomal conversion or crossover events. Individuals often carry chromosome 4 D4Z4 alleles bearing on the order of 100 repeats (see, e.g., Lemmers et al., 2007. Am. J. Hum. Genet. 81(5):884-94). FSHD patients presenting with the FSHD phenotype can carry 10 or fewer chromosome 4 D4Z4 repeats (see, e.g., Wijmenga et al., 1992. Nat. Genet. 2(l):26-30).
  • the present inventors have recognized, among other things, that a problem to be solved is the development of a therapy for FSHD.
  • the present subject matter can provide a solution to this problem, such as by identifying a causative gene.
  • the present subject matter can provide a solution to this problem, such as by identifying a therapeutic molecular target.
  • the present subject matter provides an antisense morpholino oligonucleotide, methods to prepare an antisense morpholino oligonucleotide, and uses of an antisense morpholino
  • oligonucleotide for example, to bind an RNA sequence located within the 5' untranslated region (UTR) upstream of the DUX4 translational start site.
  • UTR 5' untranslated region
  • either a contraction of the D4Z4 array or a second site mutation or epigenetic alteration allows for an increase in transcription at the 4q32.2 locus and this can result in FSHD.
  • the increase in transcription can lead to an overexpression of a gene product that is deleterious to skeletal muscle development and function.
  • the expression of a gene product can be lowered.
  • a target sequence within a gene can be identified.
  • a morpholino oligonucleotide can be designed to interact with the particular target sequence. The interaction can be sequence specific, so as to ultimately result in a stable
  • morpholino:RNA duplex Repression of gene product expression can be achieved by targeting a sequence within the UTR of an RNA message directly upstream of a translational start site.
  • an embodiment provides a morpholino oligonucleotide capable of inhibiting expression of a facioscapulohumeral muscular dystrophy candidate gene located within the polymorphic D4Z4 repeat region of chromosome 4q.
  • oligonucleotide can inhibit DUX4 expression.
  • Another embodiment provides a dendrimer delivery moiety.
  • a designed morpholino oligonucleotide can bind to the DUX4 gene product.
  • the morpholino oligonucleotide can bind within the DUX4 5' UTR of the RNA message.
  • the morpholino can disrupt translation of DUX4 by creating a stable, sequence specific morpholino:RNA duplex within DUX4.
  • the morpholino can disrupt ribosome binding such that DUX4 translation initiation is inhibited.
  • An embodiment provides an antisense oligonucleotide comprised of morpholino subunits and phosphorous-containing intersubunit linkages comprising a sequence of at least 12 contiguous base- pairing moieties complementary to SEQ ID NO: 3 (gcacagtccggc tgaggtgcac gggagcccgc cggcctctct ctgcccgcgt ccgtgaaattccggcc ggggctcacc gcgATGgccccccgacacc ctcggacagc (the ATG of DUX4 is capitalized; the sequence upstream of the ATG represents the UTR) wherein the antisense oligonucleotide is capable of binding to mRNA to form a heteroduplex structure.
  • the heteroduplex structure has a T m of dissociation of at least 45° C.
  • the morpholino subunits are joined by phosphorodiamidate backbone linkages.
  • the antisense oligonucleotide contains between 14-24 base-pairing moieties.
  • the antisense oligonucleotide comprises a targeting sequence of 25 contiguous base-pairing moieties complementary to SEQ ID NO:3.
  • the antisense oligonucleotide comprises SEQ ID NO: l.
  • One embodiment provides a composition comprising the antisense oligonucleotides described herein and a pharmaceutically acceptable carrier.
  • An embodiment provides a method of inhibiting expression of DUX4 comprising administering to a subject (e.g., a human) in need thereof an effective amount of the antisense oligonucleotides described herein or compositions containing them.
  • the antisense oligonucleotide is administered in a manner effective to reduce or eliminate a detectable symptom of FSHD in the subject.
  • Another embodiment provides a method to treat facioscapulohumeral muscular dystrophy comprising administering to a subject in need thereof an effective amount of the antisense oligonucleotides described herein or compositions containing them.
  • An embodiment provides a method of treating facioscapulohumeral muscular dystrophy comprising: administering a morpholino oligonucleotide to a subject in need thereof; wherein the morpholino oligonucleotide is administered in a dose effective to inhibit expression of a
  • the administration of a morpholino oligonucleotide is in a dose effective to inhibit expression of DUX4.
  • the administration of a morpholino oligonucleotide is capable of inhibiting DUX4 expression by binding to a sequence within the 5' UTR of DUX4.
  • the administration is by intraperitoneal or intravenous injection.
  • the administration comprises a dendrimer delivery moiety, such as an octa-guanidine dendrimer delivery moiety.
  • the translation of DUX4 is inhibited.
  • the nuclear processing of DUX4 RNA is inhibited.
  • One embodiment provides a method of treating facioscapulohumeral muscular dystrophy in a subject, comprising administering to a subject an effective amount of an antisense oligonucleotide of 12-40 morpholino subunits linked by phosphorous-containing intersubunit linkages which join a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit,; and comprising a sequence which forms a heteroduplex with a AUG start-site region of DUX4 or a region 5' thereof and/or 3' thereof; wherein the antisense oligonucleotide inhibits production of DUX4 mRNA or protein.
  • FIGURES 1 A-C illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.
  • FIGURES 1 A-C (A) provides a representation of chromosome 4 (not to scale), wherein the highly polymorphic D4Z4 repeat region, located on the q terminus at 4q35.2 is depicted. The red box indicates the region shown in Fig IB.
  • the representation further depicts a representation of one 3.3kb D4Z4 unit, wherein each of the 3.3kb D4Z4 units harbor two homeoboxes within a single open reading frame comprising the DUX4 gene.
  • C depicts the MK1 morpholino (SEQ ID NOs: l and 2).
  • the MK1 morpholino oligonucleotide was designed to inhibit DUX4 expression by binding to a sequence located within the 5'UTR of DUX4 directly upstream of the ATG (so as to prevent translation).
  • FIGURES 2A-B (A) provides the western blot analysis of DUX4 expression in 5'UTR
  • DUX4 mES cells that were induced with doxycycline and treated with various amounts of MK1 morpholino oligonucleotide.
  • B provides the DUX4 percent expression after protein levels were normalized to that of GAPDH.
  • a "morpholino oligonucleotide" is an oligonucleotide composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus- containing linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine.
  • the synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, all of which are incorporated herein by reference.
  • target refers a nucleotide region that surrounds the AUG start codon of a messenger RNA.
  • target sequence refers to a portion of the target RNA against which the oligonucleotide sequence is directed, that is, the sequence to which the oligonucleotide will hybridize by Watson-Crick base pairing of a complementary sequence.
  • the target sequence may be a contiguous region of the mRNA.
  • AUG start site region includes a 125 nucleotide region in both the 5' and 3' direction relative to the AUG start codon of mRNAs.
  • the region includes about 25 nucleotides downstream (i.e., in a 3' direction) and 100 nucleotides upstream (i.e., in a 5' direction).
  • Target and the oligonucleotide sequence analog are described as “complementary” to one another when hybridization occurs in an antiparallel configuration.
  • a oligonucleotide analog sequence may have “near” or “substantial” complementarity to the target sequence and still function for the purpose of the present invention, that is, still be “complementary.”
  • the oligonucleotide analog sequence compounds employed herein can have one mismatch with the target sequence out of 10 nucleotides, or one mismatch out of 20.
  • the antisense oligonucleotides employed have at least 90% sequence homology, such as at least 95% sequence homology, with the target as designated herein.
  • An oligonucleotide "specifically hybridizes" to a target if the oligomer hybridizes to the target under physiological conditions, with a T m greater than about 45°C, such as at least about 50°C, including about 60°C to about 80°C or higher. Such hybridization may also correspond to stringent hybridization conditions. At a given ionic strength and pH, the T m is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Such hybridization may occur with "near" or “substantial” complementary of the antisense oligomer to the target sequence, as well as with exact complementarity.
  • heteroduplex refers to a duplex between an oligonucleotide analog sequence and the complementary portion of a target RNA.
  • “Inhibit” generally means to reduce expression of RNA or protein or its activity as compared to an untreated subject or sample (such as by inhibiting about 10%, about 20%, about 30%, about 40%, about 50%, about 60% about 70% about 80%, about 90% or about 100%).
  • an “effective amount” generally means an amount that provides the desired local or systemic effect and or performance.
  • an “effective amount” of an antisense oligonucleotide, targeted against DUX4 is an amount effective to treat or reduce at least one symptom of FSHD or DUX4 production.
  • isolated refers to protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factor(s), cell or cells which are not associated with one or more protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factors, cells or one or more cellular components that are associated with the protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factor(s), cell or cells in vivo.
  • Subject means a vertebrate, such as a mammal, including a human.
  • Mammals include, but are not limited to, humans, farm animals, sport animals and companion animals. Included in the term "animal” is dog, cat, fish, gerbil, guinea pig, hamster, horse, rabbit, swine, mouse, monkey (e.g., ape, gorilla, chimpanzee, orangutan) rat, sheep, goat, cow and bird.
  • treat includes treating, ameliorating, or inhibiting a disease related condition and/ or a symptom of a disease related condition.
  • antisense oligonucleotides can knockdown gene expression.
  • An antisense oligonucleotide in accordance with the present subject matter can target, for example, the 5' UTR region of any number of FSHD candidate genes, particularly those located within the D4Z4 repeat region, such as, but not limited to FRG1, FRG2, CRYM, ANT1, ALP, PITX1, LRP2BP, TUBB4Q or DUX4.
  • Facioscapulohumeral muscular dystrophy is caused by a contraction of the D4Z4 macros atellite repeat array, located at the distal tip of chromosome 4(4q35.2). This modification alters chromatin and results in transcription from D4Z4.
  • the D4Z4 transcript encodes DUX4 (NM_001127386 mRNA; XP_001720134 protein,) a protein that causes pathological changes in myoblasts, including impaired differentiation, stress sensitivity and cell death.
  • Double homeobox, 4 also known as DUX4 is a protein which in humans is encoded by the DUX4 gene. This gene is located within a D4Z4 repeat array in the subtelomeric region of chromosome 4q. The D4Z4 repeat is polymorphic in length; a similar D4Z4 repeat array has been identified on chromosome 10. Each D4Z4 repeat unit has an open reading frame (named DUX4) that contains two homeoboxes; the repeat-array and ORF is conserved in other mammals.
  • the targets selected are sequences that span, are just downstream (within about 25 bases) or upstream (within 100 bases) of the AUG start codon of selected FSHD candidate genes.
  • the sequence for the DUX4 AUG start-site region is identified herein as SEQ ID NO:3.
  • an antisense oligonucleotide is complementary to at least 12 contiguous bases in SEQ IDNO:3.
  • An antisense morpholino oligonucleotide was designed to bind to an RNA sequence located within the 5'UTR upstream of the DUX4 translational start site.
  • MK1 DUX4 antisense morpholino oligonucleotide
  • MK1 has the following sequence: 5 ' - AATTTC ACGG ACGG ACGCGGGC AG A-3 ' (SEQ ID NO: 1).
  • Each morpholino oligonucleotide is comprised of a short chain of subunits, each subunit being comprised of a nucleic acid base, a morpholine ring, and a non-ionic
  • a morpholino oligonucleotide is conjugated to fluorescine reporter (MKl-fluor or MK1 -inverse- fluor) or as a Vivo-Morpholino covalently linked to an octa-guanidne dendrimer delivery moiety (Vivo- MK1, Vivo-MKl -inverse).
  • Antisense oligonucleotides are designed to hybridize to a region of the target sequence.
  • Selected targeting sequences can be made shorter, e.g., 12 bases, or longer, e.g., 40 bases, and include a number of mismatches, as long as the sequence is sufficiently complementary to allow hybridization with the target and forms a heteroduplex having a T m of 45°C or greater.
  • the degree of complementarity between the antisense oligonucleotide and the target is sufficient to form a stable duplex.
  • the region of complementarity of the antisense oligonucleotide with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g. 12-20 bases, or 12-25 bases or greater. Oligomers as long as 40 bases may be suitable, where at least a minimum number of bases, e.g., 12 bases, are complementary to the target sequence.
  • the antisense oligonucleotide may be 100% complementary to the nucleic acid target sequence, or it may include mismatches, e.g., to accommodate variants. Mismatches, if present, are less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligomer, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • an antisense oligonucleotide is not necessarily 100% complementary to the nucleic acid target sequence, it is effective to stably and specifically bind to the target sequence, such that a biological activity of the nucleic acid target, e.g., expression of DUX4 protein(s) and/ or mRNA, is modulated.
  • the stability of the duplex formed between the antisense oligonucleotide and the target sequence is a function of the binding T m and the susceptibility of the duplex to cellular enzymatic cleavage.
  • the T m of an antisense compound with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid
  • Each antisense oligomer should have a binding T m , with respect to a complementary-sequence RNA, of greater than body temperature and such as greater than 50°C, such as T m 's in the range 60-80°C or greater.
  • the T m of an oligomer, with respect to a complementary- based RNA hybrid can be increased by increasing the ratio of C:G paired bases in the duplex, and/or by increasing the length (in base pairs) of the heteroduplex.
  • an antisense oligonucleotide including a morpholino
  • the 3' modification can be, but is not limited to 3'- carboxyfluorescein, 3'-lissamine, 3'-dabcyl, 3'-primary amine, 3'-biotin, 3'-disulfide, 3' amine plus biotin, or any mixture thereof.
  • the 5'-end modification can be a 5'-primary amine, or 5'-dabcyl.
  • the antisense compounds can be prepared by methods available to the art.
  • the antisense oligonucleotide may be taken up by host cells by facilitated or active transport across the host cell membrane if administered in free (non-complexed) form, or by an endocytotic mechanism if administered in complexed form.
  • the antisense oligonucleotide is a substrate for a membrane transporter system (i.e. a membrane protein or proteins) capable of facilitating transport or actively transporting the oligomer across the cell membrane.
  • a membrane transporter system i.e. a membrane protein or proteins
  • the antisense oligonucleotide can be administered in complexed form, where the complexing agent is typically a polymer, e.g., a cationic lipid, polypeptide, or non-biological cationic polymer, having an opposite charge to any net charge on the antisense compound.
  • the complexing agent is typically a polymer, e.g., a cationic lipid, polypeptide, or non-biological cationic polymer, having an opposite charge to any net charge on the antisense compound.
  • Methods of forming complexes, including bilayer complexes, between anionic oligonucleotides and cationic lipid or other polymer components are well known.
  • the liposomal composition LipofectinTM Fraigner, Gadek et al.
  • DOTMA N-[l -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
  • DOPE neutral phospholipid
  • ethanolamine is widely used. After administration, the complex is taken up by cells through an endocytotic mechanism, typically involving particle encapsulation in endosomal bodies.
  • the antisense compound may also be administered in conjugated form with an arginine-rich peptide linked covalently to the 5' or 3' end of the antisense oligomer.
  • the peptide is typically 8-16 amino acids and consists of a mixture of arginine, and other amino acids including phenyalanine and cysteine.
  • the use of arginine-rich peptide conjugates can be used to enhance cellular uptake of the antisense oligomer (See, e.g. Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005).
  • liposomes may be employed to facilitate uptake of the antisense oligonucleotides into cells.
  • Hydrogels may also be used as vehicles for antisense oligonucleotide administration, for example, as described in WO 93/01286.
  • the oligonucleotides may be administered in microspheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432, 1987).
  • gas-filled microbubbles complexed with the antisense oligonucleotides can enhance delivery to target tissues, as described in U.S. Pat. No. 6,245,747.
  • An in vivo delivery strategy can employ peptides based on a tat or a penetratin peptide to achieve delivery of an antisense oligonucleotide, such as a morpholino oligonucleotide.
  • an antisense oligonucleotide such as a morpholino oligonucleotide
  • for in vivo use can be comprised of a morpholino oligonucleotide coupled with a dendrimer delivery moiety.
  • the antisense oligonucleotide, such as a morpholino oligonucleotide can be covalently linked to an octa-guanidine dendrimer delivery moiety.
  • a dendrimer can encapsulate a small molecule.
  • a surface of the dendrimer can be unconjugated (such as an amine terminated, cationic surface), or conjugated. Hydrogen bonding, van der Waals interaction, or electrostatic attraction between opposite charges on the dendrimer and cargo can be driving forces for the interaction leading to encapsulation.
  • a surface of the dendrimer can be conjugated with, for example, succinic acid, benzene disulphonic acid, or polyethylene glycol 200 (PEGylated dendrimer).
  • Cargo such as a morpholino oligonucleotide, can be conjugated to the dendrimer periphery. In an embodiment, cargo can be conjugated to a dendrimer focal point.
  • a dendrimer based drug delivery system can include a dendrimer scaffold such as, but not limited to, triazine, polypropyleneimine, polyester 'bow tie,' polyamidoamine (PAMAM), or poly-L- lysine.
  • a triazine core can be a key element in assembling a dendritic molecular transporter.
  • two sites of a tri-functional triazine can be used for branching side arms.
  • a third functional site can be used linking covalently with a morpholino
  • the dendritic moiety can be conjugated with a morpholino oligomer while the oligomer remains on the synthesis resin.
  • the protecting groups can be removed, and the oligomer can be detached from the synthesis resin.
  • a subsequent perguanidinylation process can result in a conjugate of morpholino with a transporter moiety of a triazine core scaffold presenting eight guanidine head groups.
  • oligomers with any given backbone can be determined by a simple in vivo test, in which a labeled compound is administered to an animal, and a body fluid sample, taken from the animal several hours after the oligomer is administered, is assayed for the presence of heteroduplex with target RNA.
  • one embodiment comprises contacting a cell with an antisense oligonucleotide effective to, for example, inhibit transcription, processing, and/or translation of a preselected mRNA.
  • a method provided herein can include a morpholino oligonucleotide capable of knocking down gene expression.
  • a morpholino oligonucleotide can block translation initiation in the cytosol by targeting the five prime untranslated region (5'UTR) of messenger RNA.
  • the morpholino oligonucleotide can interfere with translation of a target transcript by interfering with the progression of the ribosomal initiation complex.
  • a morpholino oligonucleotide can modify pre-mRNA processing.
  • a morpholino oligonucleotide can affect splicing in the nucleus by targeting splice junctions of splice regulatory sites.
  • a morpholino oligonucleotide can prevent binding of splice directing small nuclear ribonucleoprotein (such as snRNPs).
  • snRNPs small nuclear ribonucleoprotein
  • the prevention of snRNP binding can inhibit the formation of complexes that could otherwise bind to targets at the border of an intron of pri-mRNA and would otherwise form the splice lariat structure.
  • the morpholino oligonucleotide can modify splicing and can inhibit the formation of mature mRNA.
  • a morpholino oligonucleotide can inhibit miRNA maturation and activity by targeting mature miRNA or pri-miRNA. In an example, a morpholino oligonucleotide can inhibit a ribozyme. In an example, a morpholino oligonucleotide can result in a translational frameshift.
  • an effective therapeutic antisense oligonucleotide such as a morpholino oligonucleotide
  • a morpholino oligonucleotide can be designed to enter the cell of an adult animal in vivo.
  • systemic results can be achieved by intravenously injecting a morpholino.
  • Routes of antisense oligonucleotide delivery include, but are not limited to, various systemic routes, including oral and parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as inhalation, transdermal and topical delivery.
  • localized delivery can be achieved by injecting an antisense oligonucleotide, such as a morpholino oligonucleotide, directly into the area of interest.
  • an antisense oligonucleotide such as a morpholino oligonucleotide
  • the appropriate route may be determined by one of skill in the art, as appropriate to the condition of the subject under treatment.
  • the antisense oligonucleotide may be administered in any convenient vehicle which is physiologically acceptable.
  • a composition may include any of a variety of standard
  • saline phosphate buffered saline
  • PBS phosphate buffered saline
  • water aqueous ethanol
  • emulsions such as oil/water emulsions or triglyceride emulsions, tablets and capsules.
  • suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.
  • the antisense oligonucleotide is generally administered in an amount and manner effective to result in a peak blood concentration of at least 200-400 nM antisense oligonucleotide.
  • one or more doses of antisense oligonucleotide are administered, generally at regular intervals, for a period of about one to two weeks.
  • Doses for oral administration can be from about 5-1000 mg oligomer per 70 kg individual. In some cases, doses of greater than 500 mg oligomer/patient may be administered.
  • doses can include from about 100-1000 mg oligomer per 70 kg body weight.
  • the antisense oligonucleotide may be administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the oligomer is administered intermittently over a longer period of time.
  • the treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.
  • Generating a DUX4 expression construct with 5' UTR A 2.7 kb construct encoding the DUX4 ORF and 3' genomic sequences to the Eco RI site was extended by subcloning additional 5' UTR sequence. This was accomplished by synthesizing primers, Fwd (5'- ATTCTACTCGAGGC AC AGTCCGGCTGAGGTGC A-3 ' ; SEQ ID NO:4) and Rev (5'- C ACGAGGGAGC AGGGTGAC-3 ' ; SEQ ID NO:5), amplifying a 5' sequence using a genomic
  • Murine myoblast cell line C2C12 was purchased from ATCC. Derivative cell lines were made by inserting into C2C12 myoblasts and murine ES cells using an inducible cassette exchange system, where the gene of interest is targeted into a doxycycline regulated locus as described in Bosnakovshi et al., 2008. EMBO J. 27:2766-2779 and Iacovino et al., 2011. Stem Cells. 29(10): 1580- 1588. Several cell lines were generated in which expression of the DUX4-encoding D4Z4 transcript could be regulated by doxycycline. Each derivative cell line contained the endogenous DUX4 5'UTR in some respect. Expression constructs were designed to express DUX4 (GenBack accession no. AF117653) sequence or from a larger construct containing sequence downstream of the ORF.
  • the iC2C12 cell line was targeted with DUX4 (iC2C12-DUX4), the 5'UTR of DUX4 (iC2C12-5'UTR DUX4 ORF), or a 2.7 kb DNA sequence from the terminal D4Z4 repeat containing DUX4 (iC2C12-5'UTR DUX4(2.7)).
  • the iC2C12 lines were seeded onto 75 cm 2 flasks at a cell concentration between 1.5 x 10 5 and 1.0 x 10 6 .
  • the cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamax, and 1% antibiotic antimycotic at 37° C and 5% CO 2 . Medium was replaced or added every 2 to 3 days. Cultures were not permitted to become confluent.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • glutamax fetal bovine serum
  • antibiotic antimycotic 37° C and 5% CO 2
  • Inducible murine embryonic stem cells containing the 5'UTR DUX4 sequence (5'UTR DUX4 mES) were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
  • DMEM/F12 fetal calf serum
  • FBS fetal bovine serum
  • Glutamax 1% Glutamax
  • Non-essential amino acids 1% Non-essential amino acids
  • LIF Leukemia Inhibitory Factor
  • ⁇ -mercaptoethanol 1% penicillin/streptomycin at 37° C and 5% C0 2 .
  • Cells were passaged every 2 to 3 days, and medium was replaced or added every day. Differentiation of the ES cells was prevented.
  • the DUX4 antisense morpholino oligonucleotide was designed to bind to an RNA sequence located within the 5'UTR upstream of the DUX4 translational start site.
  • the DUX4 antisense morpholino oligonucleotide (MK1) has the following sequence: 5'- AATTTC ACGGACGGACGCGGGC AGA-3 ' (SEQ ID NO: 1).
  • a negative control was designed to contain an identical molecular composition, but with the inverse sequence of the DUX4 antisense morpholino oligonucleotide.
  • the negative control inverse morpholino oligonucleotide had the following sequence: 5'-AGACGGGCGCAGGCAGGCACTTTAA-3' (SEQ ID NO: 2).
  • Each morpholino oligonucleotide was comprised of a short chain of subunits, each subunit being comprised of a nucleic acid base, a morpholine ring, and a non-ionic phosphorodiamidate intersubunit linkage (GeneTools, Philomath, OR).
  • a morpholino oligonucleotide was conjugated to fluorescine reporter (MKl-fluor or MKl-inverse-fluor) or as a Vivo-Morpholino covalently linked to an octa-guanidne dendrimer delivery moiety (Vivo-MKl, Vivo-MKl-inverse).
  • Induced cells were then treated with morpholino oligonucleotide at varying concentrations in the presence of 6 ⁇ Endo-porter (GeneTools, Philomath, OR).
  • protein extracts were generated by lysing cells in IX lysis buffer/SDS loading dye (300mM Tris pH 6.8, 25% glycerol, 20% ⁇ -mercaptoethanol, 10% SDS, 0.02% bromophenol blue) at room temperature then boiled for 10 minutes at 99° C.
  • Equal volumes of protein extract were electrophoresed through a 10% Tris-HCl polyacrylamide-SDS separation gel having a 4 % polyacrylamide stacking gel for one hour at 150 volts.
  • the gel was transferred onto PVDF membrane using a Bio-Rad wet transfer system for one hour at 200 mAmps.
  • the membrane was washed three times in IX tris buffered saline (TBS; 1M Tris, pH7, 5M sodium chloride).
  • TBS IX tris buffered saline
  • the membrane was then washed three times in TBST (TBS plus 0.05% Tween 20).
  • the membrane was then blocked for one hour with Western Blocking Reagent (Roche Applied Science, Indianapolis, IN).
  • the membrane was then washed again three times with IX TBS, then three times with IX TBST.
  • the membrane was incubated overnight at 4° C in a suitable antibody.
  • the membrane was then washed three times with IX TBS and a further three times with IX TBST.
  • the membrane was next incubated for one hour at 4° C with a suitable secondary antibody.
  • the signal was visualized using the Chemiluminescent Substrate Detection Kit (Thermo Fisher Scientific Inc., Rockford, Illinois). Protein expression levels were quantified using ImageJ software and normalized to GAPDH expression levels.
  • Immunoreaction Enhancer Kit (Calbiochem EMD4Biosciences). Secondary antibodies were diluted in Solution 2 of the SignalBoostTM Immunoreaction Enhancer Kit. Suitable antibodies can be obtained from polyclonal sera, monospecific sera or from monoclonal antibody culture. Techniques for producing and processing monoclonal and polyclonal sera are abundantly known in the art (e.g. Reinherz et al. (1979) J. Immunol. 123, 1312 , Ritz et al. Nature (1980) 283, 583, and Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987). Animals suitable for raising the antibodies are e.g. cows, rabbits, mice, goats, donkeys, or chickens.
  • a rabbit monoclonal antibody recognizing DUX4 (E5-5, diluted 1 in 100, Biomol) was utilized and produced a 45 kDa band (+/- 5 kDa).
  • a murine monoclonal antibody recognizing DUX4 (9A12, diluted 1 in 100, Dixit et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18157-18162) produced a 50 kDa band (+/- 5 kDa).
  • Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) conjugated to peroxidase (G9295, diluted 1 in 1000, Sigma- Aldrich) produced a 37 kDa band (+/- 5 kDa).
  • Glyceraldehyde-3-Phosphate Dehydrogenase conjugated to peroxidase G9295, diluted 1 in 1000, Sigma- Aldrich
  • a peroxidase conjugated anti-rabbit IgG or a peroxidase conjugated anti-mouse IgG (whole molecule, SantaCruz, Gibbstwon, NJ) was also used.
  • Cell survival/ ATP Assays Cells were cultured as described above. DUX 4 expression was induced with doxycycline, prior to morpholino treatment, as described above. Cells were treated with morpholino. After 48 hours, cell viability was determined by quantifying the presence of ATP in each culture via CellTiter-Glo (Promega), as per the manufacturer's protocol.
  • the polymorphic D4Z4 repeat region is located on chromosome 4q ( Figure 1 A). As discussed previously, contraction and/or hypo-methylation of the D4Z4 repeat regions can be associated with the FSHD disease phenotype.
  • a morpholino-based oligonucleotide was designed to target a sequence within the DUX4 5'UTR of the RNA message directly upstream of the translational start site ( Figures 1 A-C).
  • RNA duplex is formed.
  • An example of an effective DUX4 targeting morpholino is a morpholino oligonucleotide that is targeted directly upstream of the DUX4 translational start site, thereby disrupting ribosome binding and reducing DUX4 translation initiation.
  • Cells were then transduced with the MK1 morpholino oligonucleotide or the inverse morpholino oligonucleotide at a concentration of 5 ⁇ or 10 ⁇ , or were left untreated. As previously described, the transduction was performed with the Endo-porter transfection system. Forty-eight hours following morpholino oligonucleotide treatment, cells were lysed and DUX4 expression levels were determined by western blot ( Figure 2). Expression levels were normalized to GAPDH and quantified as a percentage of total expression determined by mES 5'UTR DUX4 cells treated with 100 ng/ml doxycycline alone.
  • MK1 MK1 morpholino oligonucleotide
  • Embodiment 1 includes subject matter (such as a morpholino oligonucleotide) capable of inhibiting expression of a facioscapulohumeral muscular dystrophy candidate gene located within the highly polymorphic D4Z4 repeat region of chromosome 4q.
  • subject matter such as a morpholino oligonucleotide
  • Embodiment 2 the subject matter of Embodiment 1 can optionally include a morpholino oligonucleotide capable of inhibiting DUX4 expression.
  • Embodiment 3 the subject matter of one or any combination of Embodiments 1 - 2 can optionally include a dendrimer delivery moiety.
  • Embodiment 4 the subject matter of one or any combination of Embodiments 1 - 3 can optionally include an octa-guanidine dendrimer delivery moiety.
  • Embodiment 5 the subject matter of one or any combination of Embodiments 1 - 4 can optionally include a covalently linked morpholino oligonucleotide and octa-guanidine dendrimer delivery moiety.
  • Embodiment 6 the subject matter of one or any combination of Embodiments 1 - 5 can optionally include a morpholino oligonucleotide capable of binding to a sequence within the 5' UTR of DUX4.
  • Embodiment 7 the subject matter of one or any combination of Embodiments 1 - 6 can optionally include a morpholino oligonucleotide capable of interfering with pre-mRNA processing.
  • Embodiment 8 the subject matter of one or any combination of Embodiments 1 - 7 can optionally include a morpholino oligonucleotide that is capable of inducing a translational frameshift.
  • Embodiment 9 the subject matter of one or any combination of Embodiments 1 - 8 can optionally include a phosphorodiamidate backbone.
  • Embodiment 10 the subject matter of one or any combination of Embodiments 1 - 9 can optionally include a morpholino oligonucleotide that is effective in lowering the toxicity of the facioscapulohumeral muscular dystrophy candidate gene DUX4.
  • Embodiment 11 can include, or can optionally be combined with the subject matter of one or any combination of Embodiments 1-10 to include, subject matter (such as of treating
  • facioscapulohumeral muscular dystrophy comprising administering a morpholino oligonucleotide to a human in need thereof; wherein the morpholino oligonucleotide is administered in a dose effective to inhibit expression of a facioscapulohumeral muscular dystrophy candidate gene located within the highly polymorphic D4Z4 repeat region of chromosome 4q.
  • the method of Embodiment 11 can optionally further comprise the administration of a morpholino oligonucleotide in a dose effective to inhibit expression of DUX4.
  • the method of one or any combination of Embodiments 11 - 12 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting DUX4 expression by binding to a sequence within the 5' UTR of DUX4.
  • Embodiment 14 the method of one or any combination of Embodiments 11 - 13 can optionally further comprise the step of intraperitoneal injection.
  • the method of one or any combination of Embodiments 11 - 14 can optionally further comprise the step of intravenously injecting the morpholino oligonucleotide.
  • Embodiment 16 the method of one or any combination of Embodiments 11 - 15 can optionally further comprise the administration of a dendrimer delivery moiety.
  • Embodiment 17 the method of one or any combination of Embodiments 11 - 16 can optionally further comprise the administration of an octa-guanidine dendrimer delivery moiety.
  • Embodiment 18 the method of one or any combination of Embodiments 11 - 17 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting translation of DUX4.
  • Embodiment 19 the method of one or any combination of Embodiments 11 - 18 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting nuclear processing of DUX4 mRNA.
  • the method of one or any combination of Embodiments 11 - 19 can optionally further comprise the administration of a morpholino oligonucleotide effective in lowering the toxicity of the facioscapulohumeral muscular dystrophy candidate gene DUX4.

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Abstract

La présente invention concerne une composition et un procédé comprenant un oligonucléotide morpholino apte à inhiber l'expression du DUX4. Ledit oligonucléotide morpholino peut procurer un effet thérapeutique chez des individus atteints de dystrophie musculaire facio-scapulo-humérale. En outre, ladite composition et ledit procédé peuvent comprendre une fraction d'administration de dendrimère.
PCT/US2013/025461 2012-02-10 2013-02-09 Morpholino ciblant le dux4 pour le traitement de la myopathie facio-scapulo-humérale WO2013120038A2 (fr)

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WO2016195493A1 (fr) * 2015-06-02 2016-12-08 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Moyens et méthodes de traitement de la dystrophie musculaire facio-scapulo-humérale (fshd)
WO2017050836A1 (fr) * 2015-09-21 2017-03-30 Association Institut De Myologie Oligonucléotides antisens et leurs utilisations
US10258628B2 (en) 2016-10-26 2019-04-16 Genea Biocells USA (Holdings), Inc. Generation of muscle lineage cells and therapeutic uses thereof
EP3420109A4 (fr) * 2016-02-26 2019-11-06 Research Institute at Nationwide Children's Hospital Produits viraux recombinants et procédés d'induction d'un saut d'exon dux4
US10538763B2 (en) 2015-01-16 2020-01-21 Ionis Pharmaceuticals, Inc. Compounds and methods for modulation of DUX4
CN113710283A (zh) * 2019-03-29 2021-11-26 田边三菱制药株式会社 用于调节dux4的表达的化合物、方法和医药组合物
RU2768289C2 (ru) * 2016-04-02 2022-03-23 Рисерч Инститьют Эт Нэшнуайд Чилдрен'С Хоспитал Модифицированная промоторная система u6 для тканеспецифической экспрессии
US11795459B2 (en) 2020-04-02 2023-10-24 Mirecule, Inc. Targeted inhibition using engineered oligonucleotides
EP4087924A4 (fr) * 2020-01-10 2024-01-17 Dyne Therapeutics Inc Complexes de ciblage de muscle et leurs utilisations pour traiter la dystrophie musculaire facio-scapulo-humérale
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy

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US20050054012A1 (en) * 2002-10-11 2005-03-10 Tupler Rossella G. Methods of detecting and treating facioscapulohumeral muscular dystrophy
FR2919616A1 (fr) * 2007-08-02 2009-02-06 Ecole Norm Superieure Lyon Polynucleotides insulateurs derives de l'element d4z4 et leurs utilisations en transgenese
US20090280517A1 (en) * 2008-03-18 2009-11-12 University Of Maryland Methods of diagnosis and prognosis for a muscular dystrophy
EP2175037B1 (fr) * 2008-09-26 2017-10-11 Genomic Vision Procédé pour analyser les réseaux de répétitions en tandem du D4Z4 d'un acide nucléique et kit correspondant
US8802438B2 (en) * 2010-04-16 2014-08-12 Children's Medical Center Corporation Compositions, kits, and methods for making induced pluripotent stem cells using synthetic modified RNAs
WO2012024535A2 (fr) * 2010-08-18 2012-02-23 Fred Hutchinson Cancer Research Center Procédés de détermination de la présence ou du risque de développement de la dystrophie facio-scapulo-humérale (fshd)
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US10538763B2 (en) 2015-01-16 2020-01-21 Ionis Pharmaceuticals, Inc. Compounds and methods for modulation of DUX4
WO2016195493A1 (fr) * 2015-06-02 2016-12-08 Academisch Ziekenhuis Leiden H.O.D.N. Lumc Moyens et méthodes de traitement de la dystrophie musculaire facio-scapulo-humérale (fshd)
WO2017050836A1 (fr) * 2015-09-21 2017-03-30 Association Institut De Myologie Oligonucléotides antisens et leurs utilisations
EP3420109A4 (fr) * 2016-02-26 2019-11-06 Research Institute at Nationwide Children's Hospital Produits viraux recombinants et procédés d'induction d'un saut d'exon dux4
US11180755B2 (en) 2016-02-26 2021-11-23 Research Institute At Nationwide Children's Hospital Recombinant virus products and methods for inducing DUX4 exon skipping
US11939579B2 (en) 2016-04-02 2024-03-26 Research Institute At Nationwide Children's Hospital Modified U6 promoter system for tissue specific expression
RU2768289C2 (ru) * 2016-04-02 2022-03-23 Рисерч Инститьют Эт Нэшнуайд Чилдрен'С Хоспитал Модифицированная промоторная система u6 для тканеспецифической экспрессии
US11345913B2 (en) 2016-04-02 2022-05-31 Research Institute At Nationwide Children's Hospital Modified U6 promoter system for tissue specific expression
US10258628B2 (en) 2016-10-26 2019-04-16 Genea Biocells USA (Holdings), Inc. Generation of muscle lineage cells and therapeutic uses thereof
CN113710283A (zh) * 2019-03-29 2021-11-26 田边三菱制药株式会社 用于调节dux4的表达的化合物、方法和医药组合物
EP4087924A4 (fr) * 2020-01-10 2024-01-17 Dyne Therapeutics Inc Complexes de ciblage de muscle et leurs utilisations pour traiter la dystrophie musculaire facio-scapulo-humérale
US11795459B2 (en) 2020-04-02 2023-10-24 Mirecule, Inc. Targeted inhibition using engineered oligonucleotides
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy

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