US20220193250A1 - Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy - Google Patents

Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy Download PDF

Info

Publication number
US20220193250A1
US20220193250A1 US17/264,948 US201917264948A US2022193250A1 US 20220193250 A1 US20220193250 A1 US 20220193250A1 US 201917264948 A US201917264948 A US 201917264948A US 2022193250 A1 US2022193250 A1 US 2022193250A1
Authority
US
United States
Prior art keywords
antibody
transferrin receptor
cdr
muscle
oligonucleotide
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
US17/264,948
Other languages
English (en)
Inventor
Romesh R. Subramanian
Mohammed T. Qatanani
Timothy Weeden
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.)
Dyne Therapeutics Inc
Original Assignee
Dyne Therapeutics Inc
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 Dyne Therapeutics Inc filed Critical Dyne Therapeutics Inc
Priority to US17/264,948 priority Critical patent/US20220193250A1/en
Assigned to DYNE THERAPEUTICS, INC. reassignment DYNE THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SUBRAMANIAN, ROMESH R., QATANANI, Mohammed T., WEEDEN, TIMOTHY
Priority to US17/205,123 priority patent/US11111309B2/en
Publication of US20220193250A1 publication Critical patent/US20220193250A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C12N15/1137—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 against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02008—Hypoxanthine phosphoribosyltransferase (2.4.2.8)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position
    • 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
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • 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
    • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present application relates to targeting complexes for delivering molecular payloads (e.g., oligonucleotides) to cells and uses thereof, particularly uses relating to treatment of disease.
  • molecular payloads e.g., oligonucleotides
  • FSHD Facioscapulohumeral muscular dystrophy
  • DUX4 double homeobox 4
  • the DUX4 gene which encodes the DUX4 protein, is located in the D4Z4 repeat region on chromosome 4 and is typically expressed only in fetal development, after which it is repressed by hypermethylation of the D4Z4 repeats which surround and compact the DUX4 gene.
  • Two types of FSHD, Type 1 and Type 2 have been described.
  • Type 1 which accounts for about 95% of cases, is associated with deletions of D4Z4 repeats on chromosome 4.
  • FSHD1 FSHD1
  • the disclosure provides complexes that target muscle cells for purposes of delivering molecular payloads to those cells.
  • complexes provided herein are particularly useful for delivering molecular payloads that inhibit the expression or activity of DUX4, e.g., in a subject having or suspected of having Facioscapulohumeral muscular dystrophy (FSHD).
  • complexes provided herein comprise muscle-targeting agents (e.g., muscle targeting antibodies) that specifically bind to receptors on the surface of muscle cells for purposes of delivering molecular payloads to the muscle cells.
  • the complexes are taken up into the cells via a receptor mediated internalization, following which the molecular payload may be released to perform a function inside the cells.
  • complexes engineered to deliver oligonucleotides may release the oligonucleotides such that the oligonucleotides can inhibit DUX4 gene expression in the muscle cells.
  • the oligonucleotides are released by endosomal cleavage of covalent linkers connecting oligonucleotides and muscle-targeting agents of the complexes.
  • Some aspects of the disclosure comprise a complex comprising a muscle-targeting agent covalently linked to a molecular payload configured for inhibiting expression or activity of DUX4, wherein the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells.
  • the muscle-targeting agent is a muscle-targeting antibody.
  • a muscle-targeting antibody is an antibody that specifically binds to an extracellular epitope of a transferrin receptor (e.g., an epitope of the apical domain of the transferrin receptor).
  • a muscle-targeting antibody may specifically binds to an epitope of a sequence in the range of C89 to F760 of SEQ ID NO: 1-3.
  • the equilibrium dissociation constant (Kd) of binding of a muscle-targeting antibody to a transferrin receptor is in a range from 10 ⁇ 11 M to 10 ⁇ 6 M.
  • a muscle-targeting antibody of a complex competes for specific binding to an epitope of a transferrin receptor with an antibody listed in Table 1 (e.g., competes for specific binding to an epitope of a transferrin receptor with an Kd of less than or equal to 10 ⁇ 6 M, e.g., in a range of 10 ⁇ 11 M to 10 ⁇ 6 M).
  • a muscle-targeting antibody of a complex does not specifically bind to the transferrin binding site of a transferrin receptor and/or does not inhibit binding of transferrin to a transferrin receptor.
  • a muscle-targeting antibody of a complex is cross-reactive with extracellular epitopes of two or more of a human, non-human primate and rodent transferrin receptor.
  • a muscle-targeting antibody of a complex is configured to promote transferrin receptor mediated internalization of the molecular payload into a muscle cell.
  • a muscle-targeting antibody (e.g., muscle-targeting antibody is an antibody that specifically binds to an extracellular epitope of a transferrin receptor) is a chimeric antibody, wherein optionally the chimeric antibody is a humanized monoclonal antibody.
  • a muscle-targeting antibody may be in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.
  • a molecular payload of a complex is an oligonucleotide (e.g., an oligonucleotide that targets DUX4).
  • an oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NO: 45.
  • an oligonucleotide comprises SEQ ID NO: 45.
  • an oligonucleotide comprises a sequence that is complementary to at least 15 consecutive nucleotides of SEQ ID NO: 46.
  • an oligonucleotide comprises a region of complementarity to a DUX4 gene. In some embodiments, an oligonucleotide comprises an antisense strand that hybridizes, in a cell, with a wild-type DUX4 mRNA transcript encoded by the allele. In some embodiments, an oligonucleotide comprises an antisense strand that hybridizes, in a cell, with a mutant DUX4 mRNA transcript encoded by the allele. An oligonucleotide may comprise a strand complementary to the coding sequence of DUX4.
  • an oligonucleotide comprises a strand complementary to the non-coding sequence of DUX4. In some embodiments, an oligonucleotide may comprise a strand complementary to a 5′ or 3′ UTR sequence of DUX4. In some embodiments, an oligonucleotide mediates epigenetic silencing of DUX4.
  • An oligonucleotide of the disclosure may comprise at least one modified internucleotide linkage (e.g., a phosphorothioate linkage).
  • an oligonucleotide comprises phosphorothioate linkages in the Rp stereochemical conformation and in the Sp stereochemical conformation.
  • an oligonucleotide comprises phosphorothioate linkages that are all in the Rp stereochemical conformation.
  • an oligonucleotide comprises phosphorothioate linkages that are all in the Sp stereochemical conformation.
  • An oligonucleotide of the disclosure may comprise one or more modified nucleotides (e.g., 2′-modified nucleotides).
  • a modified nucleotide is a 2′-O-methyl, 2′-fluoro (2′-F), 2′-O-methoxyethyl (2′-MOE), or 2′, 4′-bridged nucleotide.
  • a modified nucleotides is a bridged nucleotide (e.g., selected from: 2′,4′-constrained 2′-O-ethyl (cEt) and locked nucleic acid (LNA) nucleotides).
  • an oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of the DUX4 mRNA transcript in a cell.
  • a gapmer oligonucleotide may comprise a central portion of 5 to 15 deoxyribonucleotides flanked by wings of 2 to 8 modified nucleotides (e.g., 2′-modified nucleotides).
  • an oligonucleotide is a mixmer oligonucleotide (e.g., a mixmer oligonucleotide inhibits translation of a DUX4 mRNA transcript).
  • a mixmer oligonucleotide may comprise two or more different 2′ modified nucleotides.
  • an oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of the DUX4 mRNA transcript.
  • An RNAi oligonucleotide may be a double-stranded oligonucleotide of 19 to 25 nucleotides in length.
  • an RNAi oligonucleotide comprises at least one 2′ modified nucleotide.
  • an oligonucleotide comprises a guide sequence for a genome editing nuclease.
  • an oligonucleotide is a phosphorodiamidite morpholino oligomer (PMO).
  • a molecular payload is a polypeptide (e.g., a polypeptide that inhibits DUX4 expression). In some embodiments, a molecular payload is a polypeptide that binds to a DUX4 enhancer sequence, thereby blocking recruitment of one or more activators of DUX4 expression.
  • a muscle-targeting agent is covalently linked to a molecular payload via a cleavable linker (e.g., a protease-sensitive linker, pH-sensitive linker, or glutathione-sensitive linker).
  • a protease-sensitive linker may comprise a sequence cleavable by a lysosomal protease and/or an endosomal protease.
  • a protease-sensitive linker comprises a valine-citrulline dipeptide sequence.
  • a pH-sensitive linker may be cleaved at a pH in a range of 4 to 6.
  • a muscle-targeting agent is covalently linked to a molecular payload via a non-cleavable linker (e.g., an alkane linker).
  • a non-cleavable linker e.g., an alkane linker
  • a muscle-targeting antibody comprises a non-natural amino acid to which an oligonucleotide can be covalently linked.
  • a muscle-targeting antibody is covalently linked to an oligonucleotide via conjugation to a lysine residue or a cysteine residue of the antibody.
  • an oligonucleotide is conjugated to a cysteine residue of the antibody via a maleimide-containing linker, optionally wherein the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group.
  • a muscle-targeting antibody is a glycosylated antibody that comprises at least one sugar moiety to which a oligonucleotide is covalently linked. In some embodiments, a glycosylated antibody that comprises at least one sugar moiety that is a branched mannose. In some embodiments, a muscle-targeting antibody is a glycosylated antibody that comprises one to four sugar moieties each of which is covalently linked to a separate oligonucleotide. In some embodiments, a muscle-targeting antibody is a fully-glycosylated antibody or a partially-glycosylated antibody. A partially-glycosylated antibody may be produced via chemical or enzymatic means. In some embodiments, a partially-glycosylated antibody is produced in a cell that is deficient for an enzyme in the N- or O-glycosylation pathway.
  • Some aspects of the disclosure comprise a method of delivering an molecular payload to a cell expressing transferrin receptor, the method comprising contacting the cell with a complex comprising a muscle-targeting agent covalently linked to a molecular payload configured for inhibiting expression or activity of DUX4.
  • Some aspects of the disclosure comprise a method of inhibiting expression or activity of DUX4 in a cell, the method comprising contacting the cell with a complex comprising a muscle-targeting agent covalently linked to a molecular payload configured for inhibiting expression or activity of DUX4 in an amount effective for promoting internalization of the molecular payload to the cell.
  • the cell is in vitro.
  • the cell is in a subject.
  • the subject is a human.
  • Some aspects of the disclosure comprise a method of treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy, the method comprising administering to the subject an effective amount of a complex comprising a muscle-targeting agent covalently linked to a molecular payload configured for inhibiting expression or activity of DUX4.
  • the subject has 10 or fewer D4Z4 repeats (e.g., the subject has 9, 8, 7, 6, 5, 4, 3, 2, or 1 D4Z4 repeats).
  • the subject has no D4Z4 repeats.
  • FIG. 1 depicts a non-limiting schematic showing the effect of transfecting cells with an siRNA.
  • FIG. 2 depicts a non-limiting schematic showing the activity of a muscle targeting complex comprising an siRNA.
  • FIGS. 4A-4E depict non-limiting schematics showing the tissue selectivity of a muscle targeting complex comprising an siRNA.
  • FIG. 5 depicts a non-limiting schematic showing the expression levels of DUX4 in three DUX4-expressing cell lines (A549, U-2 OS, and HepG2 cell lines) and immortalized skeletal muscle myoblasts (SkMC).
  • FIG. 6 depicts non-limiting schematics showing the ability of a phosphorodiamidate morpholino oligomer (PMO) version of an antisense oligonucleotide that targets DUX4 (FM10 PMO) to reduce expression levels of downstream DUX4 genes (ZSCAN1, MBDL3L2, TRIM43).
  • PMO phosphorodiamidate morpholino oligomer
  • aspects of the disclosure relate to a recognition that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects in muscle cells, it has proven challenging to effectively target such cells.
  • the present disclosure provides complexes comprising muscle-targeting agents covalently linked to molecular payloads in order to overcome such challenges.
  • the complexes are particularly useful for delivering molecular payloads that inhibit the expression or activity of target genes in muscle cells, e.g., in a subject having or suspected of having a rare muscle disease.
  • complexes are provided for targeting a DUX4 to treat subjects having FSHD.
  • complexes provided herein comprise oligonucleotides that inhibit expression of DUX4 in a subject that has one or more D4Z4 repeat deletions on chromosome 4.
  • complexes provided herein comprise molecular payloads such as guide molecules (e.g., guide RNAs) that are capable of targeting nucleic acid programmable nucleases (e.g., Cas9) to a DUX4 gene in order to inactivate the gene in muscle cells, for example, by removing a portion of the DUX4 gene, or by introducing an inactivating mutation or stop codon into the DUX4 gene.
  • nucleic programmable nucleases could be used to inactivate DUX4 that is aberrantly expressed in muscle cells.
  • Administering means to provide a complex to a subject in a manner that is physiologically and/or pharmacologically useful (e.g., to treat a condition in the subject).
  • an antibody refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen.
  • an antibody is a full-length antibody.
  • an antibody is a chimeric antibody.
  • an antibody is a humanized antibody.
  • an antibody is a Fab fragment, a F(ab′)2 fragment, a Fv fragment or a scFv fragment.
  • an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody.
  • an antibody is a diabody.
  • an antibody comprises a framework having a human germline sequence.
  • an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains.
  • an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or a light (L) chain variable region (abbreviated herein as VL).
  • an antibody comprises a constant domain, e.g., an Fc region.
  • an immunoglobulin constant domain refers to a heavy or light chain constant domain.
  • Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known.
  • the heavy chain of an antibody described herein can be an alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • the heavy chain of an antibody described herein can comprise a human alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • an antibody described herein comprises a human gamma 1 CH1, CH2, and/or CH3 domain.
  • the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region, such as any known in the art.
  • a human constant region sequence such as any known in the art.
  • human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.
  • the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation.
  • the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions.
  • linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
  • an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.
  • CDR refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • CDRs may be referred to as Kabat CDRs.
  • Sub-portions of CDRs may be designated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively.
  • These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
  • Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • Chimeric antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • complementary refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides.
  • complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleotides or two sets of nucleotides. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid (e.g., an mRNA), then the bases are considered to be complementary to each other at that position.
  • a target nucleic acid e.g., an mRNA
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil-type bases (U)
  • cytosine-type bases are complementary to guanosine-type bases (G)
  • universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • Covalently linked refers to a characteristic of two or more molecules being linked together via at least one covalent bond.
  • two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules.
  • two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds.
  • a linker may be a cleavable linker.
  • a linker may be a non-cleavable linker.
  • Cross-reactive As used herein and in the context of a targeting agent (e.g., antibody), the term “cross-reactive,” refers to a property of the agent being capable of specifically binding to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity.
  • an antibody that is cross-reactive against human and non-human primate antigens of a similar type or class e.g., a human transferrin receptor and non-human primate transferring receptor
  • an antibody is cross-reactive against a human antigen and a rodent antigen of a similar type or class. In some embodiments, an antibody is cross-reactive against a rodent antigen and a non-human primate antigen of a similar type or class. In some embodiments, an antibody is cross-reactive against a human antigen, a non-human primate antigen, and a rodent antigen of a similar type or class.
  • DUX4 refers to a gene that encodes double homeobox 4, a protein which is generally expressed during fetal development and in the testes of adult males.
  • DUX4 may be a human (Gene ID: 100288687), non-human primate (e.g., Gene ID: 750891, Gene ID: 100405864), or rodent gene (e.g., Gene ID: 306226).
  • expression of the DUX4 gene outside of fetal development and the testes is associated with facioscapulohumeral muscular dystrophy.
  • Facioscapulohumeral muscular dystrophy refers to a genetic disease caused by mutations in the DUX4 gene or SMCHD1 gene that is characterized by muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms. Two types of the disease, Type 1 and Type 2, have been described. Type I is associated with deletions in D4Z4 repeat regions on chromosome 4 which contain the DUX4 gene. Type 2 is associated with mutations in the SMCHD1 gene. Both Type 1 and Type 2 FSHD are characterized by aberrant production of the DUX4 protein after fetal development outside of the testes.
  • Facioscapulohumeral dystrophy the genetic basis for the disease, and related symptoms are described in the art (see, e.g. Campbell, A. E., et al., “Facioscapulohumeral dystrophy: Activatin an early embryonic transcriptional program in human skeletal muscle” Human Mol Genet. (2016); and Tawil, R. “Facioscapulohumeral muscular dystrophy” Handbook Clin. Neurol. (2018), 148: 541-548.)
  • FSHD Type 1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry #158900.
  • FSHD Type 2 is associated with OMIM Entry #158901.
  • framework refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
  • Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.
  • Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Humanized antibody refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences.
  • a non-human species e.g., a mouse
  • humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized anti-transferrin receptor antibodies and antigen binding portions are provided.
  • Such antibodies may be generated by obtaining murine anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.
  • an internalizing cell surface receptor refers to a cell surface receptor that is internalized by cells, e.g., upon external stimulation, e.g., ligand binding to the receptor.
  • an internalizing cell surface receptor is internalized by endocytosis.
  • an internalizing cell surface receptor is internalized by clathrin-mediated endocytosis.
  • an internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as, for example, phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake or constitutive clathrin-independent endocytosis.
  • the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and/or an extracellular domain, which may optionally further comprise a ligand-binding domain.
  • a cell surface receptor becomes internalized by a cell after ligand binding.
  • a ligand may be a muscle-targeting agent or a muscle-targeting antibody.
  • an internalizing cell surface receptor is a transferrin receptor.
  • Isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds transferrin receptor is substantially free of antibodies that specifically bind antigens other than transferrin receptor).
  • An isolated antibody that specifically binds transferrin receptor complex may, however, have cross-reactivity to other antigens, such as transferrin receptor molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • Kabat numbering The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.
  • the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • Molecular payload refers to a molecule or species that functions to modulate a biological outcome.
  • a molecular payload is linked to, or otherwise associated with a muscle-targeting agent.
  • the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide.
  • the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein.
  • the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a target gene.
  • Muscle-targeting agent refers to a molecule that specifically binds to an antigen expressed on muscle cells.
  • the antigen in or on muscle cells may be a membrane protein, for example an integral membrane protein or a peripheral membrane protein.
  • a muscle-targeting agent specifically binds to an antigen on muscle cells that facilitates internalization of the muscle-targeting agent (and any associated molecular payload) into the muscle cells.
  • a muscle-targeting agent specifically binds to an internalizing, cell surface receptor on muscles and is capable of being internalized into muscle cells through receptor mediated internalization.
  • the muscle-targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle-targeting agent is linked to a molecular payload.
  • Muscle-targeting antibody refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells.
  • a muscle-targeting antibody specifically binds to an antigen on muscle cells that facilitates internalization of the muscle-targeting antibody (and any associated molecular payment) into the muscle cells.
  • the muscle-targeting antibody specifically binds to an internalizing, cell surface receptor present on muscle cells.
  • the muscle-targeting antibody is an antibody that specifically binds to a transferrin receptor.
  • oligonucleotide refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length.
  • oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers, phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNAs), etc.
  • Oligonucleotides may be single-stranded or double-stranded.
  • an oligonucleotide may comprise one or more modified nucleotides (e.g. 2′-O-methyl sugar modifications, purine or pyrimidine modifications).
  • an oligonucleotide may comprise one or more modified internucleotide linkage.
  • an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.
  • Recombinant antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more details in this disclosure), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • One embodiment of the disclosure provides fully human antibodies capable of binding human transferrin receptor which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT publication No. WO 2005/007699 A2.
  • Region of complementarity refers to a nucleotide sequence, e.g., of a oligonucleotide, that is sufficiently complementary to a cognate nucleotide sequence, e.g., of a target nucleic acid, such that the two nucleotide sequences are capable of annealing to one another under physiological conditions (e.g., in a cell).
  • a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid.
  • a region of complementarity is partially complementary to a cognate nucleotide sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, or 4 mismatches compared with a cognate nucleotide sequence of a target nucleic acid.
  • the term “specifically binds” refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting to certain cells, e.g., muscle cells, through binding to the antigen, as described herein.
  • an antibody specifically binds to a target if the antibody has a K D for binding the target of at least about 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M, 10 ⁇ 12 M, 10 ⁇ 13 M, or less.
  • an antibody specifically binds to the transferrin receptor, e.g., an epitope of the apical domain of transferrin receptor.
  • a subject refers to a mammal.
  • a subject is non-human primate, or rodent.
  • a subject is a human.
  • a subject is a patient, e.g., a human patient that has or is suspected of having a disease.
  • the subject is a human patient who has or is suspected of having FSHD.
  • Transferrin receptor As used herein, the term, “transferrin receptor” (also known as TFRC, CD71, p90, or TFR1) refers to an internalizing cell surface receptor that binds transferrin to facilitate iron uptake by endocytosis.
  • a transferrin receptor may be of human (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.
  • multiple human transcript variants have been characterized that encoded different isoforms of the receptor (e.g., as annotated under GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).
  • a complex that comprise a targeting agent, e.g. an antibody, covalently linked to a molecular payload.
  • a complex comprises a muscle-targeting antibody covalently linked to a oligonucleotide.
  • a complex may comprise an antibody that specifically binds a single antigenic site or that binds to at least two antigenic sites that may exist on the same or different antigens.
  • a complex may be used to modulate the activity or function of at least one gene, protein, and/or nucleic acid.
  • the molecular payload present with a complex is responsible for the modulation of a gene, protein, and/or nucleic acids.
  • a molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and/or nucleic acid in a cell.
  • a molecular payload is an oligonucleotide that targets a DUX4 in muscle cells.
  • a complex comprises a muscle-targeting agent, e.g. an anti-transferrin receptor antibody, covalently linked to a molecular payload, e.g. an antisense oligonucleotide that targets a DUX4.
  • a muscle-targeting agent e.g. an anti-transferrin receptor antibody
  • a molecular payload e.g. an antisense oligonucleotide that targets a DUX4.
  • muscle-targeting agents e.g., for delivering a molecular payload to a muscle cell.
  • such muscle-targeting agents are capable of binding to a muscle cell, e.g., via specifically binding to an antigen on the muscle cell, and delivering an associated molecular payload to the muscle cell.
  • the molecular payload is bound (e.g., covalently bound) to the muscle targeting agent and is internalized into the muscle cell upon binding of the muscle targeting agent to an antigen on the muscle cell, e.g., via endocytosis. It should be appreciated that various types of muscle-targeting agents may be used in accordance with the disclosure.
  • the muscle-targeting agent may comprise, or consist of, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide).
  • a nucleic acid e.g., DNA or RNA
  • a peptide e.g., an antibody
  • a lipid e.g., a microvesicle
  • a sugar moiety e.g., a polysaccharide
  • muscle-targeting agents that specifically bind to an antigen on muscle, such as skeletal muscle, smooth muscle, or cardiac muscle.
  • any of the muscle-targeting agents provided herein bind to (e.g., specifically bind to) an antigen on a skeletal muscle cell, a smooth muscle cell, and/or a cardiac muscle cell.
  • muscle-specific cell surface recognition elements e.g., cell membrane proteins
  • muscle-specific cell surface recognition elements e.g., cell membrane proteins
  • molecules that are substrates for muscle uptake transporters are useful for delivering a molecular payload into muscle tissue. Binding to muscle surface recognition elements followed by endocytosis can allow even large molecules such as antibodies to enter muscle cells.
  • molecular payloads conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells via binding to transferrin receptor, which may then be endocytosed, e.g., via clathrin-mediated endocytosis.
  • muscle-targeting agents may be useful for concentrating a molecular payload (e.g., oligonucleotide) in muscle while reducing toxicity associated with effects in other tissues.
  • the muscle-targeting agent concentrates a bound molecular payload in muscle cells as compared to another cell type within a subject.
  • the muscle-targeting agent concentrates a bound molecular payload in muscle cells (e.g., skeletal, smooth, or cardiac muscle cells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than an amount in non-muscle cells (e.g., liver, neuronal, blood, or fat cells).
  • a toxicity of the molecular payload in a subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered to the subject when bound to the muscle-targeting agent.
  • a muscle recognition element e.g., a muscle cell antigen
  • a muscle-targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter.
  • a muscle-targeting agent may be an antibody that enters a muscle cell via transporter-mediated endocytosis.
  • a muscle targeting agent may be a ligand that binds to cell surface receptor on a muscle cell. It should be appreciated that while transporter-based approaches provide a direct path for cellular entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.
  • the muscle-targeting agent is an antibody.
  • the high specificity of antibodies for their target antigen provides the potential for selectively targeting muscle cells (e.g., skeletal, smooth, and/or cardiac muscle cells). This specificity may also limit off-target toxicity.
  • Examples of antibodies that are capable of targeting a surface antigen of muscle cells have been reported and are within the scope of the disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al. “Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide” Nature 1988; 333: 861-3; Song K. S., et al. “Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells.
  • Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins” J Biol Chem 1996; 271: 15160-5; and Weisbart R. H. et al., “Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb” Mol Immunol. 2003 March, 39(13):78309; the entire contents of each of which are incorporated herein by reference.
  • Transferrin receptors are internalizing cell surface receptors that transport transferrin across the cellular membrane and participate in the regulation and homeostasis of intracellular iron levels.
  • transferrin receptor binding proteins which are capable of binding to transferrin receptor.
  • binding proteins e.g., antibodies
  • binding proteins that bind to transferrin receptor are internalized, along with any bound molecular payload, into a muscle cell.
  • an antibody that binds to a transferrin receptor may be referred to as an anti-transferrin receptor antibody.
  • Antibodies that bind, e.g. specifically bind, to a transferrin receptor may be internalized into the cell, e.g. through receptor-mediated endocytosis, upon binding to a transferrin receptor.
  • anti-transferrin receptor antibodies may be produced, synthesized, and/or derivatized using several known methodologies, e.g. library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (D ⁇ ez, P. et al. “High-throughput phage-display screening in array format”, Enzyme and microbial technology, 2015, 79, 34-41; Christoph M. H. and Stanley, J. R. “Antibody Phage Display: Technique and Applications” J Invest Dermatol. 2014, 134:2; Engleman, Edgar (Ed.) “Human Hybridomas and Monoclonal Antibodies.” 1985, Springer.).
  • an anti-transferrin antibody has been previously characterized or disclosed.
  • Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g. U.S. Pat. No. 4,364,934, filed Dec. 4, 1979, “Monoclonal antibody to a human early thymocyte antigen and methods for preparing same”; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, “Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells”; U.S. Pat. No. 9,708,406, filed May 20, 2014, “Anti-transferrin receptor antibodies and methods of use”; U.S. Pat. No. 9,611,323, filed Dec.
  • anti-transferrin receptor antibodies may be used in the complexes disclosed herein.
  • anti-transferrin receptor antibodies including associated references and binding epitopes, are listed in Table 1.
  • the anti-transferrin receptor antibody comprises the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies provided herein, e.g., anti-transferrin receptor antibodies listed in Table 1.
  • the muscle-targeting agent is an anti-transferrin receptor antibody.
  • an anti-transferrin receptor antibody specifically binds to a transferrin protein having an amino acid sequence as disclosed herein.
  • an anti-transferrin receptor antibody may specifically bind to any extracellular epitope of a transferrin receptor or an epitope that becomes exposed to an antibody, including the apical domain, the transferrin binding domain, and the protease-like domain.
  • an anti-transferrin receptor antibody binds to an amino acid segment of a human or non-human primate transferrin receptor, as provided in SEQ ID Nos. 1-3 in the range of amino acids C89 to F760.
  • an anti-transferrin receptor antibody specifically binds with binding affinity of at least about 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M, 10 ⁇ 12 M, 10 ⁇ 13 M, or less.
  • Anti-transferrin receptor antibodies used herein may be capable of competing for binding with other anti-transferrin receptor antibodies, e.g. OKT9, 8D3, that bind to transferrin receptor with 10 ⁇ 3 M, 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, or less.
  • transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens ) is as follows:
  • Non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) is as follows:
  • non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis ) is as follows:
  • mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1 , Mus musculus ) is as follows:
  • an anti-transferrin receptor antibody binds to an amino acid segment of the receptor as follows: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFE DLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLG TGDPYTPGFPSFNHTQFPPSRSS GLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCR MVTSESKNVKLTVSNVLKE (SEQ ID NO: 5) and does not inhibit the binding interactions between transferrin receptors and transferrin and/or human hemochromatosis protein (also known as HFE).
  • HFE human hemochromatosis protein
  • an antibody may also be produced through the generation of hybridomas (see, e.g., Kohler, G and Milstein, C. “Continuous cultures of fused cells secreting antibody of predefined specificity” Nature, 1975, 256: 495-497).
  • the antigen-of-interest may be used as the immunogen in any form or entity, e.g., recombinant or a naturally occurring form or entity.
  • Hybridomas are screened using standard methods, e.g.
  • Antibodies may also be produced through screening of protein expression libraries that express antibodies, e.g., phage display libraries. Phage display library design may also be used, in some embodiments, (see, e.g. U.S. Pat. No. 5,223,409, filed Mar. 1, 1991, “Directed evolution of novel binding proteins”; WO 1992/18619, filed Apr.
  • an antigen-of-interest may be used to immunize a non-human animal, e.g., a rodent or a goat.
  • an antibody is then obtained from the non-human animal, and may be optionally modified using a number of methodologies, e.g., using recombinant DNA techniques. Additional examples of antibody production and methodologies are known in the art (see, e.g. Harlow et al. “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988.).
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation.
  • the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • a glycosylated antibody is fully or partially glycosylated.
  • an antibody is glycosylated by chemical reactions or by enzymatic means.
  • an antibody is glycosylated in vitro or inside a cell, which may optionally be deficient in an enzyme in the N- or O-glycosylation pathway, e.g. a glycosyltransferase.
  • an antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “Modified antibody, antibody-conjugate and process for the preparation thereof”.
  • transferrin receptor antibodies provided herein bind specifically to transferrin receptor (e.g., human transferrin receptor). Transferrin receptors are internalizing cell surface receptors that transport transferrin across the cellular membrane and participate in the regulation and homeostasis of intracellular iron levels. In some embodiments, transferrin receptor antibodies provided herein bind specifically to transferrin receptor from human, non-human primates, mouse, rat, etc. In some embodiments, transferrin receptor antibodies provided herein bind to human transferrin receptor. In some embodiments, transferrin receptor antibodies provided herein specifically bind to human transferrin receptor. In some embodiments, transferrin receptor antibodies provided herein specifically bind to transferrin receptor. In some embodiments, transferrin receptor antibodies provided herein bind to an apical domain of human transferrin receptor. In some embodiments, transferrin receptor antibodies provided herein specifically bind to an apical domain of human transferrin receptor.
  • transferrin receptor antibodies of the present disclosure include one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 1.
  • transferrin receptor antibodies include the CDR-H1, CDR-H2, and CDR-H3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1.
  • anti-transferrin receptor antibodies include the CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1.
  • anti-transferrin antibodies include the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the disclosure also includes any nucleic acid sequence that encodes a molecule comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1.
  • antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity/affinity of an antibody for an antigen.
  • anti-transferrin receptor antibodies of the disclosure may include at least the heavy and/or light chain CDR3s of any one of the anti-transferrin receptor antibodies selected from Table 1.
  • any of the anti-transferrin receptor antibodies of the disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 1.
  • CDR e.g., CDR-H or CDR-L sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 1.
  • the position of one or more CDRs along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) region of an antibody described herein can vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the position defining a CDR of any antibody described herein can vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the CDR position of any one of the antibodies described herein, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the length of one or more CDRs along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) region of an antibody described herein can vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the amino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the amino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or CDR-H3 described herein can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described herein (e.g., CDRS from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). Any method can be used to ascertain whether immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained, for example, using binding assays and conditions described in the art.
  • transferrin receptor e.g., human transferrin receptor
  • any of the anti-transferrin receptor antibodies of the disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the antibodies may include one or more CDR sequence(s) from any of the anti-transferrin receptor antibodies selected from Table 1 containing up to 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of the CDRs provided herein (e.g., CDRs from any of the anti-transferrin receptor antibodies selected from Table 1) so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived).
  • transferrin receptor e.g., human transferrin receptor
  • any of the amino acid variations in any of the CDRs provided herein may be conservative variations.
  • Conservative variations can be introduced into the CDRs at positions where the residues are not likely to be involved in interacting with a transferrin receptor protein (e.g., a human transferrin receptor protein), for example, as determined based on a crystal structure.
  • a transferrin receptor protein e.g., a human transferrin receptor protein
  • Some aspects of the disclosure provide transferrin receptor antibodies that comprise one or more of the heavy chain variable (VH) and/or light chain variable (VL) domains provided herein.
  • any of the VH domains provided herein include one or more of the CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein, for example, any of the CDR-H sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 1.
  • any of the VL domains provided herein include one or more of the CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein, for example, any of the CDR-L sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 1.
  • anti-transferrin receptor antibodies of the disclosure include any antibody that includes a heavy chain variable domain and/or a light chain variable domain of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • anti-transferrin receptor antibodies of the disclosure include any antibody that includes the heavy chain variable and light chain variable pairs of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • anti-transferrin receptor antibodies having a heavy chain variable (VH) and/or a light chain variable (VL) domain amino acid sequence homologous to any of those described herein.
  • the anti-transferrin receptor antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence and/or any light chain variable sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the homologous heavy chain variable and/or a light chain variable amino acid sequences do not vary within any of the CDR sequences provided herein.
  • the degree of sequence variation e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%
  • any of the anti-transferrin receptor antibodies provided herein comprise a heavy chain variable sequence and a light chain variable sequence that comprises a framework sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • an anti-transferrin receptor antibody which specifically binds to transferrin receptor (e.g., human transferrin receptor), comprises a light chain variable VL domain comprising any of the CDR-L domains (CDR-L1, CDR-L2, and CDR-L3), or CDR-L domain variants provided herein, of any of the anti-transferrin receptor antibodies selected from Table 1.
  • transferrin receptor e.g., human transferrin receptor
  • an anti-transferrin receptor antibody which specifically binds to transferrin receptor (e.g., human transferrin receptor), comprises a light chain variable VL domain comprising the CDR-L1, the CDR-L2, and the CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence comprising one, two, three or four of the framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the anti-transferrin receptor antibody comprises one, two, three or four of the framework regions of a light chain variable region sequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three or four of the framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the light chain variable framework region that is derived from said amino acid sequence consists of said amino acid sequence but for the presence of up to 10 amino acid substitutions, deletions, and/or insertions, preferably up to 10 amino acid substitutions.
  • the light chain variable framework region that is derived from said amino acid sequence consists of said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues being substituted for an amino acid found in an analogous position in a corresponding non-human, primate, or human light chain variable framework region.
  • an anti-transferrin receptor antibody that specifically binds to transferrin receptor comprises the CDR-L1, the CDR-L2, and the CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the antibody further comprises one, two, three or all four VL framework regions derived from the VL of a human or primate antibody.
  • the primate or human light chain framework region of the antibody selected for use with the light chain CDR sequences described herein can have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with a light chain framework region of a non-human parent antibody.
  • the primate or human antibody selected can have the same or substantially the same number of amino acids in its light chain complementarity determining regions to that of the light chain complementarity determining regions of any of the antibodies provided herein, e.g., any of the anti-transferrin receptor antibodies selected from Table 1.
  • the primate or human light chain framework region amino acid residues are from a natural primate or human antibody light chain framework region having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity with the light chain framework regions of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • an anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable kappa subfamily.
  • an anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable lambda subfamily.
  • any of the anti-transferrin receptor antibodies provided herein comprise a light chain variable domain that further comprises a light chain constant region.
  • the light chain constant region is a kappa, or a lambda light chain constant region.
  • the kappa or lambda light chain constant region is from a mammal, e.g., from a human, monkey, rat, or mouse.
  • the light chain constant region is a human kappa light chain constant region.
  • the light chain constant region is a human lambda light chain constant region. It should be appreciated that any of the light chain constant regions provided herein may be variants of any of the light chain constant regions provided herein.
  • the light chain constant region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any of the light chain constant regions of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.
  • an anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule.
  • an anti-transferrin receptor antibody comprises any of the VL domains, or VL domain variants, and any of the VH domains, or VH domain variants, wherein the VL and VH domains, or variants thereof, are from the same antibody clone, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
  • the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1
  • an antibody of the disclosure can bind to a target antigen (e.g., transferrin receptor) with relatively high affinity, e.g., with a K D less than 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or lower.
  • a target antigen e.g., transferrin receptor
  • anti-transferrin receptor antibodies can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM.
  • the disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and that have an affinity of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower).
  • a transferrin receptor protein e.g., human transferrin receptor
  • 50 nM or lower e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower.
  • the affinity and binding kinetics of the anti-transferrin receptor antibody can be tested using any suitable method including but not limited to biosensor technology (e.g., OCTET or BIACORE).
  • an antibody of the disclosure can bind to a target antigen (e.g., transferrin receptor) with relatively high affinity, e.g., with a K D less than 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or lower.
  • a target antigen e.g., transferrin receptor
  • anti-transferrin receptor antibodies can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM.
  • the disclosure also includes antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and that have an affinity of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower).
  • a transferrin receptor protein e.g., human transferrin receptor
  • 50 nM or lower e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower.
  • the affinity and binding kinetics of the anti-transferrin receptor antibody can be tested using any suitable method including but not limited to biosensor technology (e.g., OCTET or BIACORE).
  • the muscle-targeting agent is a transferrin receptor antibody (e.g., the antibody and variants thereof as described in International Application Publication WO 2016/081643, incorporated herein by reference).
  • the heavy chain and light chain CDRs of the antibody according to different definition systems are provided in Table 1.1.
  • the different definition systems e.g., the Kabat definition, the Chothia definition, and/or the contact definition have been described. See, e.g., (e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).
  • VH heavy chain variable domain
  • VH light chain variable domain sequences
  • VH (SEQ ID NO: 33) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMH WVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHY WGQGTSVTVSS VL (SEQ ID NO: 34) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAW YQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQ YSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLE LK
  • the transferrin receptor antibody of the present disclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) as compared with the CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1. “Collectively” means that the total number of amino acid variations in all of the three heavy chain CDRs is within the defined range.
  • the transferrin receptor antibody of the present disclosure may comprise a CDR-L1, a CDR-L2, and a CDR-L3, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variation) as compared with the CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, at least one of which contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the counterpart heavy chain CDR as shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure may comprise CDR-L1, a CDR-L2, and a CDR-L3, at least one of which contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the counterpart light chain CDR as shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises a CDR-L3, which contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) as compared with the CDR-L3 as shown in Table 1.1. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 containing one amino acid variation as compared with the CDR-L3 as shown in Table 1.1. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31 according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32 according to the Contact definition system).
  • QHFAGTPLT SEQ ID NO: 31 according to the Kabat and Chothia definition system
  • QHFAGTPL SEQ ID NO: 32 according to the Contact definition system
  • the transferrin receptor antibody of the present disclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1 and a CDR-L2 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31 according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32 according to the Contact definition system).
  • the transferrin receptor antibody of the present disclosure comprises heavy chain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs as shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises light chain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs as shown in Table 1.1.
  • the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 34.
  • the transferrin receptor antibody of the present disclosure comprises a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure comprises a VL containing no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL as set forth in SEQ ID NO: 34.
  • the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth in SEQ ID NO: 34.
  • the transferrin receptor antibody of the present disclosure is a humanized antibody (e.g., a humanized variant of an antibody).
  • the transferrin receptor antibody of the present disclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1, and comprises a humanized heavy chain variable region and/or a humanized light chain variable region.
  • Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat, or rabbit
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
  • humanization is achieved by grafting the CDRs (e.g., as shown in Table 1.1) into the IGKV1-NL1*01 and IGHV1-3*01 human variable domains.
  • the transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 as compared with the VL as set forth in SEQ ID NO: 34, and/or one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 as compared with the VL as set forth in SEQ ID NO: 34, and/or amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure is a humanized antibody and contains the residues at positions 43 and 48 of the VL as set forth in SEQ ID NO: 34.
  • the transferrin receptor antibody of the present disclosure is a humanized antibody and contains the residues at positions 48, 67, 69, 71, and 73 of the VH as set forth in SEQ ID NO: 33.
  • VH and VL amino acid sequences of an example humanized antibody that may be used in accordance with the present disclosure are provided:
  • Humanized VH (SEQ ID NO: 35) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMH WVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATL TVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHY WGQGTMVTVSS Humanized VL (SEQ ID NO: 36) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAW YQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTD YSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLE LK
  • the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 35.
  • the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 36.
  • the transferrin receptor antibody of the present disclosure comprises a VH containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VH as set forth in SEQ ID NO: 35.
  • the transferrin receptor antibody of the present disclosure comprises a VL containing no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the VL as set forth in SEQ ID NO: 36.
  • the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as set forth in SEQ ID NO: 35.
  • the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth in SEQ ID NO: 36.
  • the transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at one or more of positions 43 and 48 as compared with the VL as set forth in SEQ ID NO: 34, and/or amino acid substitutions at one or more of positions 48, 67, 69, 71, and 73 as compared with the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure is a humanized variant comprising a S43A and/or a V48L mutation as compared with the VL as set forth in SEQ ID NO: 34, and/or one or more of A67V, L691, V71R, and K73T mutations as compared with the VH as set forth in SEQ ID NO: 33
  • the transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 as compared with the VL as set forth in SEQ ID NO: 34, and/or amino acid substitutions at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 as compared with the VH as set forth in SEQ ID NO: 33.
  • the transferrin receptor antibody of the present disclosure is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the transferrin receptor antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody.
  • Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species.
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human.
  • amino acid modifications can be made in the variable region and/or the constant region.
  • the heavy chain of any of the transferrin receptor antibodies as described herein may comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof).
  • the heavy chain constant region can of any suitable origin, e.g., human, mouse, rat, or rabbit.
  • the heavy chain constant region is from a human IgG (a gamma heavy chain), e.g., IgG1, IgG2, or IgG4.
  • An exemplary human IgG1 constant region is given below:
  • the light chain of any of the transferrin receptor antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art.
  • CL is a kappa light chain.
  • the CL is a lambda light chain.
  • the CL is a kappa light chain, the sequence of which is provided below:
  • the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 39.
  • the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 40.
  • the transferrin receptor antibody described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39.
  • the transferrin receptor antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40.
  • the transferrin receptor antibody of the present disclosure comprises a heavy chain containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the heavy chain as set forth in SEQ ID NO: 39.
  • the transferrin receptor antibody of the present disclosure comprises a light chain containing no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the light chain as set forth in SEQ ID NO: 40.
  • the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 41.
  • the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 42.
  • the transferrin receptor antibody described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41.
  • the transferrin receptor antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 42.
  • the transferrin receptor antibody of the present disclosure comprises a heavy chain containing no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the heavy chain of humanized antibody as set forth in SEQ ID NO: 39.
  • the transferrin receptor antibody of the present disclosure comprises a light chain containing no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the light chain of humanized antibody as set forth in SEQ ID NO: 40.
  • the transferrin receptor antibody is an antigen binding fragment (FAB) of an intact antibody (full-length antibody).
  • FAB antigen binding fragment
  • Antigen binding fragment of an intact antibody (full-length antibody) can be prepared via routine methods.
  • F(ab′)2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments.
  • Exemplary FABs amino acid sequences of the transferrin receptor antibodies described herein are provided below:
  • Heavy Chain FAB (VH + a portion of human IgG1 constant region) (SEQ ID NO: 43) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMH WVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCP Heavy Chain FAB (humanized VH + a portion of human IgG1 constant region) (SEQ ID NO: 44) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMH WVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATL TVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHY
  • the transferrin receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain antibodies, bi-specific antibodies, or nanobodies.
  • the transferrin receptor antibody described herein is a scFv.
  • the transferrin receptor antibody described herein is a scFv-Fab (e.g., scFv fused to a portion of a constant region).
  • the transferrin receptor antibody described herein is a scFv fused to a constant region (e.g., human IgG1 constant region as set forth in SEQ ID NO: 39).
  • the muscle-targeting antibody is an antibody that specifically binds hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63.
  • the muscle-targeting antibody is an antibody that specifically binds a myogenic precursor protein.
  • myogenic precursor proteins include, without limitation, ABCG2, M-Cadherin/Cadherin-15, Caveolin-1, CD34, FoxK1, Integrin alpha 7, Integrin alpha 7 beta 1, MYF-5, MyoD, Myogenin, NCAM-1/CD56, Pax3, Pax7, and Pax9.
  • the muscle-targeting antibody is an antibody that specifically binds a skeletal muscle protein.
  • Exemplary skeletal muscle proteins include, without limitation, alpha-Sarcoglycan, beta-Sarcoglycan, Calpain Inhibitors, Creatine Kinase MM/CKMM, eIF5A, Enolase 2/Neuron-specific Enolase, epsilon-Sarcoglycan, FABP3/H-FABP, GDF-8/Myostatin, GDF-11/GDF-8, Integrin alpha 7, Integrin alpha 7 beta 1, Integrin beta 1/CD29, MCAM/CD146, MyoD, Myogenin, Myosin Light Chain Kinase Inhibitors, NCAM-1/CD56, and Troponin I.
  • the muscle-targeting antibody is an antibody that specifically binds a smooth muscle protein.
  • smooth muscle proteins include, without limitation, alpha-Smooth Muscle Actin, VE-Cadherin, Caldesmon/CALD1, Calponin 1, Desmin, Histamine H2 R, Motilin R/GPR38, Transgelin/TAGLN, and Vimentin.
  • antibodies to additional targets are within the scope of this disclosure and the exemplary lists of targets provided herein are not meant to be limiting.
  • conservative mutations can be introduced into antibody sequences (e.g., CDRs or framework sequences) at positions where the residues are not likely to be involved in interacting with a target antigen (e.g., transferrin receptor), for example, as determined based on a crystal structure.
  • a target antigen e.g., transferrin receptor
  • one, two or more mutations are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity.
  • one, two or more mutations are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Pat. No. 5,677,425.
  • the number of cysteine residues in the hinge region of the CH1 domain can be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.
  • one, two or more mutations are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell.
  • an Fc receptor e.g., an activated Fc receptor
  • Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for an Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporated herein by reference.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half-life of the antibody in vivo.
  • an IgG constant domain, or FcRn-binding fragment thereof preferably an Fc or hinge-Fc domain fragment
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo.
  • the antibodies can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and/or the third constant (CH3) domain (residues 341-447 of human IgG1), with numbering according to the EU index in Kabat (Kabat E A et al., (1991) supra).
  • the constant region of the IgG1 of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU index as in Kabat. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference.
  • an antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.
  • one, two or more amino acid substitutions are introduced into an IgG constant domain Fc region to alter the effector function(s) of the anti-transferrin receptor antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating antibody thereby increasing tumor localization. See, e.g., U.S. Pat. Nos.
  • one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on Fc region, which may reduce Fc receptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276: 6591-604).
  • one or more amino in the constant region of a muscle-targeting antibody described herein can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in International Publication No. WO 94/29351.
  • the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor.
  • ADCC antibody dependent cellular cytotoxicity
  • the heavy and/or light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies or antigen-binding fragments, as described elsewhere herein.
  • any variant, CDR-grafted, chimeric, humanized, or composite antibodies derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will maintain the ability to specifically bind transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor relative to the original antibody from which it is derived.
  • the antibodies provided herein comprise mutations that confer desirable properties to the antibodies.
  • the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgG1-like hinge sequence.
  • any of the antibodies may include a stabilizing ‘Adair’ mutation.
  • antibodies of this disclosure may optionally comprise constant regions or parts thereof.
  • a VL domain may be attached at its C-terminal end to a light chain constant domain like C ⁇ or C ⁇ .
  • a VH domain or portion thereof may be attached to all or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass.
  • Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of this may disclosure include VH and VL domains, or an antigen binding portion thereof, combined with any suitable constant regions.
  • Some aspects of the disclosure provide muscle-targeting peptides as muscle-targeting agents.
  • Short peptide sequences e.g., peptide sequences of 5-20 amino acids in length
  • cell-targeting peptides have been described in Vines e., et al., A. “Cell-penetrating and cell-targeting peptides in drug delivery” Biochim Biophys Acta 2008, 1786: 126-38; Jarver P., et al., “In vivo biodistribution and efficacy of peptide mediated delivery” Trends Pharmacol Sci 2010; 31: 528-35; Samoylova T.
  • the muscle-targeting agent is a muscle-targeting peptide that is from 4 to 50 amino acids in length.
  • the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
  • Muscle-targeting peptides can be generated using any of several methods, such as phage display.
  • a muscle-targeting peptide may bind to an internalizing cell surface receptor that is overexpressed or relatively highly expressed in muscle cells, e.g. a transferrin receptor, compared with certain other cells.
  • a muscle-targeting peptide may target, e.g., bind to, a transferrin receptor.
  • a peptide that targets a transferrin receptor may comprise a segment of a naturally occurring ligand, e.g., transferrin.
  • a peptide that targets a transferrin receptor is as described in U.S. Pat. No. 6,743,893, filed Nov.
  • a peptide that targets a transferrin receptor is as described in Kawamoto, M. et al, “A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.” BMC Cancer. 2011 Aug. 18; 11:359.
  • a peptide that targets a transferrin receptor is as described in U.S. Pat. No. 8,399,653, filed May 20, 2011, “TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY”.
  • muscle-specific peptides were identified using phage display library presenting surface heptapeptides.
  • the muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 6).
  • This peptide displayed improved specificity for binding to heart and skeletal muscle tissue after intravenous injection in mice with reduced binding to liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display.
  • a 12 amino acid peptide was identified by phage display library for muscle targeting in the context of treatment for DMD. See, Yoshida D., et al., “Targeting of salicylate to skin and muscle following topical injections in rats.” Int J Pharm 2002; 231: 177-84; the entire contents of which are hereby incorporated by reference.
  • a 12 amino acid peptide having the sequence SKTFNTHPQSTP SEQ ID NO: 7
  • this muscle-targeting peptide showed improved binding to C2C12 cells relative to the ASSLNIA (SEQ ID NO: 6) peptide.
  • an additional method for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types includes in vitro selection, which has been described in Ghosh D., et al., “Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting” J Virol 2005; 79: 13667-72; the entire contents of which are incorporated herein by reference. By pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types, non-specific cell binders were selected out. Following rounds of selection the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) appeared most frequently. Accordingly, in some embodiments, the muscle-targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).
  • a muscle-targeting agent may an amino acid-containing molecule or peptide.
  • a muscle-targeting peptide may correspond to a sequence of a protein that preferentially binds to a protein receptor found in muscle cells.
  • a muscle-targeting peptide contains a high propensity of hydrophobic amino acids, e.g. valine, such that the peptide preferentially targets muscle cells.
  • a muscle-targeting peptide has not been previously characterized or disclosed. These peptides may be conceived of, produced, synthesized, and/or derivatized using any of several methodologies, e.g.
  • phage displayed peptide libraries binding peptide libraries
  • one-bead one-compound peptide libraries or positional scanning synthetic peptide combinatorial libraries.
  • Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, B. P. and Brown, K. C. “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2, 1020-1081; Samoylova, T. I. and Smith, B. F. “Elucidation of muscle-binding peptides by phage display screening.” Muscle Nerve, 1999, 22:4. 460-6.).
  • a muscle-targeting peptide has been previously disclosed (see, e.g. Writer M. J.
  • Exemplary muscle-targeting peptides comprise an amino acid sequence of the following group: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSLNIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13), and DDTRHWG (SEQ ID NO: 14).
  • a muscle-targeting peptide may comprise about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids.
  • Muscle-targeting peptides may comprise naturally-occurring amino acids, e.g.
  • a muscle-targeting peptide may be linear; in other embodiments, a muscle-targeting peptide may be cyclic, e.g. bicyclic (see, e.g. Silvana, M. G. et al. Mol. Therapy, 2018, 26:1, 132-147.).
  • a muscle-targeting agent may be a ligand, e.g. a ligand that binds to a receptor protein.
  • a muscle-targeting ligand may be a protein, e.g. transferrin, which binds to an internalizing cell surface receptor expressed by a muscle cell. Accordingly, in some embodiments, the muscle-targeting agent is transferrin, or a derivative thereof that binds to a transferrin receptor.
  • a muscle-targeting ligand may alternatively be a small molecule, e.g. a lipophilic small molecule that preferentially targets muscle cells relative to other cell types.
  • Exemplary lipophilic small molecules that may target muscle cells include compounds comprising cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerine, alkyl chains, trityl groups, and alkoxy acids.
  • a muscle-targeting agent may be an aptamer, e.g. an RNA aptamer, which preferentially targets muscle cells relative to other cell types.
  • a muscle-targeting aptamer has not been previously characterized or disclosed.
  • These aptamers may be conceived of, produced, synthesized, and/or derivatized using any of several methodologies, e.g. Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies have been characterized in the art and are incorporated by reference (Yan, A. C. and Levy, M. “Aptamers and aptamer targeted delivery” RNA biology, 2009, 6:3, 316-20; Germer, K. et al.
  • RNA aptamers and their therapeutic and diagnostic applications Int. J. Biochem. Mol. Biol. 2013; 4: 27-40.
  • a muscle-targeting aptamer has been previously disclosed (see, e.g. Phillippou, S. et al. “Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers.” Mol Ther Nucleic Acids. 2018, 10:199-214; Thiel, W. H. et al. “Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.” Mol Ther. 2016, 24:4, 779-87.).
  • Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14.
  • an aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer or a peptide aptamer.
  • an aptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 1-3 kDa, or smaller.
  • One strategy for targeting a muscle cell is to use a substrate of a muscle transporter protein, such as a transporter protein expressed on the sarcolemma.
  • the muscle-targeting agent is a substrate of an influx transporter that is specific to muscle tissue.
  • the influx transporter is specific to skeletal muscle tissue.
  • Two main classes of transporters are expressed on the skeletal muscle sarcolemma, (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which facilitate efflux from skeletal muscle tissue and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle.
  • ATP adenosine triphosphate
  • ABS solute carrier
  • the muscle-targeting agent is a substrate that binds to an ABC superfamily or an SLC superfamily of transporters.
  • the substrate that binds to the ABC or SLC superfamily of transporters is a naturally-occurring substrate.
  • the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, for example, a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.
  • the muscle-targeting agent is a substrate of an SLC superfamily of transporters.
  • SLC transporters are either equilibrative or use proton or sodium ion gradients created across the membrane to drive transport of substrates.
  • Exemplary SLC transporters that have high skeletal muscle expression include, without limitation, the SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (S
  • the muscle-targeting agent is a substrate of an equilibrative nucleoside transporter 2 (ENT2) transporter.
  • ENT2 equilibrative nucleoside transporter 2
  • ENT2 has one of the highest mRNA expressions in skeletal muscle.
  • human ENT2 hENT2
  • Human ENT2 facilitates the uptake of its substrates depending on their concentration gradient.
  • ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases.
  • the muscle-targeting agent is an ENT2 substrate.
  • Exemplary ENT2 substrates include, without limitation, inosine, 2′,3′-dideoxyinosine, and calofarabine.
  • any of the muscle-targeting agents provided herein are associated with a molecular payload (e.g., oligonucleotide payload).
  • the muscle-targeting agent is covalently linked to the molecular payload.
  • the muscle-targeting agent is non-covalently linked to the molecular payload.
  • the muscle-targeting agent is a substrate of an organic cation/carnitine transporter (OCTN2), which is a sodium ion-dependent, high affinity carnitine transporter.
  • OCTN2 organic cation/carnitine transporter
  • the muscle-targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2.
  • the carnitine, mildronate, acetylcarnitine, or derivative thereof is covalently linked to the molecular payload (e.g., oligonucleotide payload).
  • a muscle-targeting agent may be a protein that is protein that exists in at least one soluble form that targets muscle cells.
  • a muscle-targeting protein may be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis.
  • hemojuvelin may be full length or a fragment, or a mutant with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a functional hemojuvelin protein.
  • a hemojuvelin mutant may be a soluble fragment, may lack a N-terminal signaling, and/or lack a C-terminal anchoring domain.
  • hemojuvelin may be annotated under GenBank RefSeq Accession Numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be appreciated that a hemojuvelin may be of human, non-human primate, or rodent origin.
  • a molecular payload for modulating a biological outcome, e.g., the transcription of a DNA sequence, the expression of a protein, or the activity of a protein.
  • a molecular payload is linked to, or otherwise associated with a muscle-targeting agent.
  • such molecular payloads are capable of targeting to a muscle cell, e.g., via specifically binding to a nucleic acid or protein in the muscle cell following delivery to the muscle cell by an associated muscle-targeting agent. It should be appreciated that various types of muscle-targeting agents may be used in accordance with the disclosure.
  • the molecular payload may comprise, or consist of, an oligonucleotide (e.g., antisense oligonucleotide), a peptide (e.g., a peptide that binds a nucleic acid or protein associated with disease in a muscle cell), a protein (e.g., a protein that binds a nucleic acid or protein associated with disease in a muscle cell), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein associated with disease in a muscle cell).
  • an oligonucleotide e.g., antisense oligonucleotide
  • a peptide e.g., a peptide that binds a nucleic acid or protein associated with disease in a muscle cell
  • a protein e.g., a protein that binds a nucleic acid or protein associated with disease in a muscle cell
  • the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a DUX4.
  • exemplary molecular payloads are described in further detail herein, however, it should be appreciated that the exemplary molecular payloads provided herein are not meant to be limiting.
  • any suitable oligonucleotide may be used as a molecular payload, as described herein.
  • the oligonucleotide may be designed to cause degradation of an mRNA (e.g., the oligonucleotide may be a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation).
  • the oligonucleotide may be designed to block translation of an mRNA (e.g., the oligonucleotide may be a mixmer, an siRNA or an aptamer that blocks translation).
  • an oligonucleotide may be designed to cause degradation and block translation of an mRNA.
  • an oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for directing activity of an enzyme (e.g., a gene editing enzyme).
  • an enzyme e.g., a gene editing enzyme
  • Other examples of oligonucleotides are provided herein. It should be appreciated that, in some embodiments, oligonucleotides in one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one format to the other format.
  • oligonucleotide may be used as a molecular payload, as described herein.
  • oligonucleotides useful for targeting DUX4 are provided in U.S. Pat. No. 9,988,628, published on Feb. 2, 2017, entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Pat. No. 9,469,851, published Oct. 30, 2014, entitled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; US Patent Application Publication 20120225034, published on Sep.
  • the oligonucleotide is an antisense oligonucleotide, a morpholino, a siRNA, a shRNA, or another nucleotide which hybridizes with the target DUX4 gene or mRNA.
  • oligonucleotides may have a region of complementarity to a sequence as set forth as: Human DUX4, corresponding to NCBI sequence NM_001293798.1 (SEQ ID NO: 15) as below and/or Mouse DUX4, corresponding to NCBI sequence NM_001081954.1 (SEQ ID NO: 16), as below.
  • the oligonucleotide may have a region of complementarity to a hypomethylated, contracted D4Z4 repeat, as in Daxinger, et al., “Genetic and Epigenetic Contributors to FSHD,” published in Curr Opin Genet Dev in 2015, Lim J-W, et al., DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep. 1; 24(17): 4817-4828, the contents of each of which are incorporated in their entireties.
  • oligonucleotides may have a region of complementarity to a sequence set forth as follows, which is an example human DUX4 gene sequence (NM_001293798.1) (SEQ ID NO: 15):
  • oligonucleotides may have a region of complementarity to a sequence set forth as follows, which is an example mouse DUX4 gene sequence (SEQ ID NO: 16) (NM_001081954.1):
  • an oligonucleotide may have a region of complementarity to DUX4 gene sequences of multiple species, e.g., selected from human, mouse and non-human species.
  • an oligonucleotide that targets DUX4 is a FM10 sequence. In some embodiments, an oligonucleotide that targets DUX4 is a phosphorodiamidate morpholino version of a FM10 sequence. In some embodiments, an oligonucleotide that targets DUX4 comprises the sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 45). In some embodiments, an oligonucleotide that targets DUX4 comprises a sequence that is complementary to at least 15 consecutive nucleotides of
  • muscle specific E3 ubiquitin ligases are overexpressed in FSHD and function in muscle atrophy (see, e.g., Vanderplanck, C. et al. “The FSHD Atrophic Myotube Phenotype Is Caused by DUX4 Expression” PLoS One 6,10:e26820, 2011).
  • downregulation of these ligases presents a viable therapeutic strategy.
  • an oligonucleotide may target, e.g., inhibit the expression of, a muscle specific E3 ubiquitin ligase implicated in FSHD, such as MuRF1 (also known as TRIM63) and MAFbx (also known as Fbx032).
  • an oligonucleotide may have a region of complementarity to at least one MuRF1 gene sequence, e.g. human MuRF1 (NCBI Gene ID 84676). In some embodiments, an oligonucleotide may have a region of complementarity to at least one MAFbx gene sequence, e.g. human MAFbx (NCBI Gene ID 114907).
  • Oligonucleotides may be of a variety of different lengths, e.g., depending on the format.
  • an oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • the oligonucleotide is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 21 to 23 nucleotides in lengths, etc.
  • a complementary nucleic acid sequence of an oligonucleotide for purposes of the present disclosure is specifically hybridizable or specific for the target nucleic acid when binding of the sequence to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) to cause a loss of activity (e.g., inhibiting translation) or expression (e.g., degrading a target mRNA) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the sequence to the target molecule e.g., mRNA
  • a loss of activity e.g., inhibiting translation
  • expression e.g., degrading a
  • an oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the consecutive nucleotides of an target nucleic acid.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target nucleic acid.
  • an oligonucleotide comprises region of complementarity to a target nucleic acid that is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length.
  • a region of complementarity of an oligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the region of complementarity is complementary with at least 8 consecutive nucleotides of a target nucleic acid.
  • an oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of target nucleic acid. In some embodiments the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit internally in a cell; minimizes TLR stimulation; or avoid pattern recognition receptors.
  • Any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same oligonucleotide.
  • nucleotide modifications may be used that make an oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact for a longer time than unmodified oligonucleotides.
  • modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Accordingly, oligonucleotides of the disclosure can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • an oligonucleotide may be of up to 50 or up to 100 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. Oligonucleotide modifications are described further herein.
  • an oligonucleotide include a 2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O—NMA).
  • a 2′-modified nucleotide e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O
  • an oligonucleotide can include at least one 2′-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2′-O-methyl modification.
  • an oligonucleotide comprises modified nucleotides in which the ribose ring comprises a bridge moiety connecting two atoms in the ring, e.g., connecting the 2′-O atom to the 4′-C atom.
  • the oligonucleotides are “locked,” e.g., comprise modified nucleotides in which the ribose ring is “locked” by a methylene bridge connecting the 2′-O atom and the 4′-C atom.
  • LNAs are described in International Patent Application Publication WO/2008/043753, published on Apr. 17, 2008, and entitled “RNA Antagonist Compounds For The Modulation Of PCSK9”, the contents of which are incorporated herein by reference in its entirety.
  • ENAs include ethylene-bridged nucleic acids (ENAs).
  • ENAs include, but are not limited to, 2′-0,4′-C-ethylene-bridged nucleic acids. Examples of ENAs are provided in International Patent Publication No. WO 2005/042777, published on May 12, 2005, and entitled “ APP/ENA Antisense ”; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005; the disclosures of which are incorporated herein by reference in their entireties.
  • the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • the oligonucleotide comprises a modified nucleotide disclosed in one of the following United States Patent or Patent Application Publications: U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Pat. No. 7,741,457, issued on Jun.
  • the oligonucleotide comprises at least one nucleotide modified at the 2′ position of the sugar, preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide.
  • RNA modifications include 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3′ end of the RNA.
  • the oligonucleotide may have at least one modified nucleotide that results in an increase in Tm of the oligonucleotide in a range of 1° C., 2° C., 3° C., 4° C., or 5° C. compared with an oligonucleotide that does not have the at least one modified nucleotide.
  • the oligonucleotide may have a plurality of modified nucleotides that result in a total increase in Tm of the oligonucleotide in a range of 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C. or more compared with an oligonucleotide that does not have the modified nucleotide.
  • the oligonucleotide may comprise alternating nucleotides of different kinds.
  • an oligonucleotide may comprise alternating deoxyribonucleotides or ribonucleotides and 2′-fluoro-deoxyribonucleotides.
  • An oligonucleotide may comprise alternating deoxyribonucleotides or ribonucleotides and 2′-O-methyl nucleotides.
  • An oligonucleotide may comprise alternating 2′-fluoro nucleotides and 2′-O-methyl nucleotides.
  • An oligonucleotide may comprise alternating bridged nucleotides and 2′-fluoro or 2′-O-methyl nucleotides.
  • oligonucleotide may contain a phosphorothioate or other modified internucleotide linkage. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, oligonucleotides comprise modified internucleotide linkages at the first, second, and/or third internucleoside linkage at the 5′ or 3′ end of the nucleotide sequence.
  • Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3′alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.
  • oligonucleotides may have heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).
  • heteroatom backbones such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and
  • internucleotidic phosphorus atoms of oligonucleotides are chiral, and the properties of the oligonucleotides are adjusted based on the configuration of the chiral phosphorus atoms.
  • appropriate methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev.
  • phosphorothioate containing oligonucleotides comprise nucleoside units that are joined together by either substantially all Sp or substantially all Rp phosphorothioate intersugar linkages.
  • such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. Pat. No. 5,587,261, issued on Dec. 12, 1996, the contents of which are incorporated herein by reference in their entirety.
  • chirally controlled oligonucleotides provide selective cleavage patterns of a target nucleic acid.
  • a chirally controlled oligonucleotide provides single site cleavage within a complementary sequence of a nucleic acid, as described, for example, in US Patent Application Publication 20170037399 A1, published on Feb. 2, 2017, entitled “CHIRAL DESIGN”, the contents of which are incorporated herein by reference in their entirety.
  • the oligonucleotide may be a morpholino-based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • PNAs Peptide Nucleic Acids
  • both a sugar and an internucleoside linkage (the backbone) of the nucleotide units of an oligonucleotide are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative publication that report the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
  • the oligonucleotide is a gapmer.
  • a gapmer oligonucleotide generally has the formula 5′-X-Y-Z-3′, with X and Z as flanking regions around a gap region Y.
  • the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, which are capable of recruiting an RNAse, such as RNAse H.
  • the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid.
  • the Y region is flanked both 5′ and 3′ by regions X and Z comprising high-affinity modified nucleotides, e.g., one to six modified nucleotides.
  • modified nucleotides include, but are not limited to, 2′ MOE or 2′OMe or Locked Nucleic Acid bases (LNA).
  • the flanking sequences X and Z may be of one to twenty nucleotides, one to eight nucleotides or one to five nucleotides in length, in some embodiments.
  • the flanking sequences X and Z may be of similar length or of dissimilar lengths.
  • the gap-segment Y may be a nucleotide sequence of five to twenty nucleotides, size to twelve nucleotides or six to ten nucleotides in length, in some embodiments.
  • the gap region of the gapmer oligonucleotides may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4′-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides.
  • the gap region comprises one or more unmodified internucleosides.
  • one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • the gap region and two flanking regions each independently comprise modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • modified internucleoside linkages e.g., phosphorothioate internucleoside linkages or other linkages
  • a gapmer may be produced using appropriate methods.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. patent publication Nos. US20090286969, US20100197762, and US20110112170; and PCT publication Nos. WO2008049085 and WO2009090182, each of which is herein incorporated by reference in its entirety.
  • an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern.
  • mixmers are oligonucleotides that comprise both naturally and non-naturally occurring nucleotides or comprise two different types of non-naturally occurring nucleotides typically in an alternating pattern.
  • Mixmers generally have higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule.
  • mixmers do not recruit an RNAse to the target molecule and thus do not promote cleavage of the target molecule.
  • Such oligonucleotides that are incapable of recruiting RNAse H have been described, for example, see WO2007/112754 or WO2007/112753.
  • the mixmer comprises or consists of a repeating pattern of nucleotide analogues and naturally occurring nucleotides, or one type of nucleotide analogue and a second type of nucleotide analogue.
  • a mixmer need not comprise a repeating pattern and may instead comprise any arrangement of modified nucleotides and naturally occurring nucleotides or any arrangement of one type of modified nucleotide and a second type of modified nucleotide.
  • the repeating pattern may, for instance be every second or every third nucleotide is a modified nucleotide, such as LNA, and the remaining nucleotides are naturally occurring nucleotides, such as DNA, or are a 2′ substituted nucleotide analogue such as 2′MOE or 2′ fluoro analogues, or any other modified nucleotide described herein. It is recognized that the repeating pattern of modified nucleotide, such as LNA units, may be combined with modified nucleotide at fixed positions—e.g. at the 5′ or 3′ termini.
  • a mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleotides, such as DNA nucleotides.
  • the mixmer comprises at least a region consisting of at least two consecutive modified nucleotide, such as at least two consecutive LNAs.
  • the mixmer comprises at least a region consisting of at least three consecutive modified nucleotide units, such as at least three consecutive LNAs.
  • the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogues, such as LNAs.
  • LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • Mixmers may be designed to comprise a mixture of affinity enhancing modified nucleotides, such as in non-limiting example LNA nucleotides and 2′-O-methyl nucleotides.
  • a mixmer comprises modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a mixmer may be produced using any suitable method.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Pat. No. 7,687,617.
  • a mixmer comprises one or more morpholino nucleotides.
  • a mixmer may comprise morpholino nucleotides mixed (e.g., in an alternating manner) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2′-O-Methyl nucleotides).
  • mixmers are useful for splice correcting or exon skipping, for example, as reported in Touznik A., et al., LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts Scientific Reports, volume 7, Article number: 3672 (2017), Chen S.
  • RNA Interference RNAi
  • oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA small interfering RNAs
  • mRNAs target nucleic acids
  • RNAi RNA interference pathway
  • Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA.
  • Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e. an antisense sequence, can be designed and prepared using appropriate methods (see, e.g., PCT Publication Number WO 2004/016735; and U.S. Patent Publication Nos. 2004/0077574 and 2008/0081791).
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single-stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense strands.
  • Double-stranded siRNA may comprise RNA strands that are the same length or different lengths. Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3′ end and/or the 5′ end of either or both strands).
  • shRNA Small hairpin RNA
  • a spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3′ end and/or the 5′ end of either or both strands).
  • a spacer sequence is may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA.
  • the overall length of the siRNA molecules can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule being designed. Generally between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.
  • siRNA molecule may comprise a 3′ overhang at one end of the molecule, The other end may be blunt-ended or have also an overhang (5′ or 3′).
  • the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different.
  • the siRNA molecule of the present disclosure comprises 3′ overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • microRNAs k. k. microRNA (miRNAs)
  • an oligonucleotide may be a microRNA (miRNA).
  • miRNAs are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript.
  • miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures.
  • pri-miRNAs large RNA precursors
  • pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
  • miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA.
  • the size range of the miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used.
  • oligonucleotides provided herein may be in the form of aptamers.
  • aptamer is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid in a cell.
  • the aptamer is a DNA aptamer or an RNA aptamer.
  • a nucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single-stranded nucleic acid aptamer may form helices and/or loop structures.
  • the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
  • Exemplary publications and patents describing aptamers and method of producing aptamers include, e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos.
  • oligonucleotides provided herein may be in the form of a ribozyme.
  • a ribozyme ribonucleic acid enzyme
  • Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, lncRNAs, and ribozymes, themselves.
  • Ribozymes may assume one of several physical structures, one of which is called a “hammerhead.”
  • a hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double-stranded stems I and III.
  • Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3′, 5′-phosphate diester to a 2′, 3′-cyclic phosphate diester.
  • this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
  • Modifications in ribozyme structure have also included the substitution or replacement of various non-core portions of the molecule with non-nucleotidic molecules.
  • Benseler et al. J. Am. Chem. Soc. (1993) 115:8483-8484) disclosed hammerhead-like molecules in which two of the base pairs of stem II, and all four of the nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol)bisphosphate, or bis(propanediol) phosphate.
  • Ma et al. Biochem.
  • Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92/07065; and U.S. Pat. Nos. 5,436,143 and 5,650,502) or can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in a cell.
  • the ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc.
  • the ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition).
  • the ribozyme RNA sequences maybe synthesized conventionally, for example, by using RNA polymerases such as T7 or SP6.
  • oligonucleotides are guide nucleic acid, e.g., guide RNA (gRNA) molecules.
  • a guide RNA is a short synthetic RNA composed of (1) a scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp), such as Cas9, and (2) a nucleotide spacer portion that defines the DNA target sequence (e.g., genomic DNA target) to which the gRNA binds in order to bring the nucleic acid programmable DNA binding protein in proximity to the DNA target sequence.
  • napDNAbp nucleic acid programmable DNA binding protein
  • the napDNAbp is a nucleic acid-programmable protein that forms a complex with (e.g., binds or associates with) one or more RNA(s) that targets the nucleic acid-programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence).
  • a nucleic acid-programmable nuclease when in a complex with an RNA, may be referred to as a nuclease:RNA complex.
  • Guide RNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.
  • gRNAs Guide RNAs
  • sgRNAs single-guide RNAs
  • gRNAs guide RNAs
  • gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (i.e., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein.
  • domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem-loop structure.
  • domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which is incorporated herein by reference.
  • a gRNA comprises two or more of domains (1) and (2), and may be referred to as an extended gRNA.
  • an extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein.
  • the gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex.
  • the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes .” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F.
  • Cas9 endonuclease for example, Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes .”
  • molecular payloads may comprise multimers (e.g., concatemers) of 2 or more oligonucleotides connected by a linker.
  • the oligonucleotide loading of a complex/conjugate can be increased beyond the available linking sites on a targeting agent (e.g., available thiol sites on an antibody) or otherwise tuned to achieve a particular payload loading content.
  • Oligonucleotides in a multimer can be the same or different (e.g., targeting different genes or different sites on the same gene or products thereof).
  • multimers comprise 2 or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, multimers comprise 2 or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, a multimer comprises 2 to 5, 2 to 10 or 4 to 20 oligonucleotides linked together.
  • a multimer comprises 2 or more oligonucleotides linked end-to-end (in a linear arrangement). In some embodiments, a multimer comprises 2 or more oligonucleotides linked end-to-end via a oligonucleotide based linker (e.g., poly-dT linker, an abasic linker). In some embodiments, a multimer comprises a 5′ end of one oligonucleotide linked to a 3′ end of another oligonucleotide. In some embodiments, a multimer comprises a 3′ end of one oligonucleotide linked to a 3′ end of another oligonucleotide.
  • a multimer comprises a 5′ end of one oligonucleotide linked to a 5′ end of another oligonucleotide. Still, in some embodiments, multimers can comprise a branched structure comprising multiple oligonucleotides linked together by a branching linker.
  • any suitable small molecule may be used as a molecular payload, as described herein.
  • the small molecule is as described in US Patent Application Publication 20170340606, published on Nov. 30, 2017, entitled “METHODS OF TREATING MUSCULAR DYSTROPHY” or as described in US Patent Application Publication 20180050043, published on Feb. 22, 2018, entitled “INHIBITION OF DUX4 EXPRESSION USING BROMODOMAIN AND EXTRA-TERMINAL DOMAIN PROTEIN INHIBITORS (BETi).
  • small molecule payloads are provided in Bosnakovski, D., et al., High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity, Skelet Muscle, February 2014, and Choi. S., et al., “Transcriptional Inhibitors Identified in a 160,000-Compound Small-Molecule DUX4 Viability Screen,” Journal of Biomolecular Screening, 2016.
  • the small molecule is a transcriptional inhibitor, such as SHC351, SHC540, SHC572.
  • the small molecule is STR00316 increases production or activity of another protein, such as integrin.
  • the small molecule is a bromodomain inhibitor (BETi), such as JQ1, PF1-1, I-BET-762, I-BET-151, RVX-208, or CPI-0610.
  • BETi bromodomain inhibitor
  • a protein is an enzyme. These peptides or proteins may be produced, synthesized, and/or derivatized using several methodologies, e.g. phage displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries.
  • the peptide or protein may bind a DME1 or DME2 enhancer to inhibit DUX4 expression, e.g., by blocking binding of an activator.
  • a gene expression construct may be a vector or a cDNA fragment.
  • a gene expression construct may be messenger RNA (mRNA).
  • mRNA messenger RNA
  • a mRNA used herein may be a modified mRNA, e.g., as described in U.S. Pat. No. 8,710,200, issued on Apr. 24, 2014, entitled “Engineered nucleic acids encoding a modified erythropoietin and their expression”.
  • a mRNA may comprise a 5′ methyl cap.
  • a mRNA may comprise a polyA tail, optionally of up to 160 nucleotides in length.
  • the gene expression construct may be expressed, e.g., overexpressed, within the nucleus of a muscle cell.
  • the gene expression construct encodes a oligonucleotide (e.g., an shRNA targeting DUX4) or a protein that downregulates the expression of DUX4 (e.g., a peptide or protein that binds to DME1 or DME2 enhancer to inhibit DUX4 expression, e.g., by blocking binding of an activator).
  • the gene expression construct encodes a oligonucleotide (e.g., an shRNA targeting MuRF1 or MAFbx) that downregulates the expression of MuRF1 or MAFbx, respectively.
  • the gene expression constructs encodes a protein that comprises at least one zinc finger.
  • the gene expression construct encodes a gene editing enzyme. Additional examples of nucleic acid constructs that may be used as molecular payloads are provided in International Patent Application Publication WO2017152149A1, published on Sep. 19, 2017, entitled, “CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER”; U.S. Pat. No. 8,853,377B2, issued on Oct.
  • Complexes described herein generally comprise a linker that connects a muscle-targeting agent to a molecular payload.
  • a linker comprises at least one covalent bond.
  • a linker may be a single bond, e.g., a disulfide bond or disulfide bridge, that connects a muscle-targeting agent to a molecular payload.
  • a linker may connect a muscle-targeting agent to a molecular payload through multiple covalent bonds.
  • a linker may be a cleavable linker.
  • a linker may be a non-cleavable linker.
  • a linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. Additionally, generally a linker does not negatively impact the functional properties of either the muscle-targeting agent or the molecular payload. Examples and methods of synthesis of linkers are known in the art (see, e.g. Kline, T. et al. “Methods to Make Homogenous Antibody Drug Conjugates.” Pharmaceutical Research, 2015, 32:11, 3480-3493; Jain, N. et al. “Current ADC Linker Chemistry” Pharm Res. 2015, 32:11, 3526-3540; McCombs, J. R. and Owen, S. C. “Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).
  • a precursor to a linker typically will contain two different reactive species that allow for attachment to both the muscle-targeting agent and a molecular payload.
  • the two different reactive species may be a nucleophile and/or an electrophile.
  • a linker is connected to a muscle-targeting agent via conjugation to a lysine residue or a cysteine residue of the muscle-targeting agent.
  • a linker is connected to a cysteine residue of a muscle-targeting agent via a maleimide-containing linker, wherein optionally the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group.
  • a linker is connected to a cysteine residue of a muscle-targeting agent or thiol functionalized molecular payload via a 3-arylpropionitrile functional group.
  • a linker is connected to a muscle-targeting agent and/or a molecular payload via an amide bond, a hydrazide, a triazole, a thioether or a disulfide bond.
  • a cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are generally cleavable only intracellularly and are preferably stable in extracellular environments, e.g. extracellular to a muscle cell.
  • Protease-sensitive linkers are cleavable by protease enzymatic activity. These linkers typically comprise peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length.
  • a peptide sequence may comprise naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids.
  • Non-naturally occurring amino acids include ⁇ -amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art.
  • a protease-sensitive linker comprises a valine-citrulline or alanine-citrulline dipeptide sequence.
  • a protease-sensitive linker can be cleaved by a lysosomal protease, e.g. cathepsin B, and/or an endosomal protease.
  • a pH-sensitive linker is a covalent linkage that readily degrades in high or low pH environments.
  • a pH-sensitive linker may be cleaved at a pH in a range of 4 to 6.
  • a pH-sensitive linker comprises a hydrazone or cyclic acetal.
  • a pH-sensitive linker is cleaved within an endosome or a lysosome.
  • a glutathione-sensitive linker comprises a disulfide moiety.
  • a glutathione-sensitive linker is cleaved by an disulfide exchange reaction with a glutathione species inside a cell.
  • the disulfide moiety further comprises at least one amino acid, e.g. a cysteine residue.
  • the linker is a Val-cit linker (e.g., as described in U.S. Pat. No. 6,214,345, incorporated herein by reference).
  • the val-cit linker before conjugation, has a structure of:
  • the val-cit linker after conjugation, has a structure of:
  • non-cleavable linkers may be used. Generally, a non-cleavable linker cannot be readily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitutions may include halogens, hydroxyl groups, oxygen species, and other common substitutions.
  • a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising a LPXT sequence, a thioether, a biotin, a biphenyl, repeating units of polyethylene glycol or equivalent compounds, acid esters, acid amides, sulfamides, and/or an alkoxy-amine linker.
  • sortase-mediated ligation will be utilized to covalently link a muscle-targeting agent comprising a LPXT sequence to a molecular payload comprising a (G). sequence (see, e.g. Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10.).
  • a linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), e.g. polyethylene oxide or polypropylene oxide.
  • a linker is connected to a muscle-targeting agent and/or molecular payload via a phosphate, thioether, ether, carbon-carbon, or amide bond.
  • a linker is connected to an oligonucleotide through a phosphate or phosphorothioate group, e.g. a terminal phosphate of an oligonucleotide backbone.
  • a linker is connected to an muscle-targeting agent, e.g. an antibody, through a lysine or cysteine residue present on the muscle-targeting agent
  • a linker is connected to a muscle-targeting agent and/or molecular payload by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the muscle-targeting agent, molecular payload, or the linker.
  • an alkyne may be a cyclic alkyne, e.g., a cyclooctyne.
  • an alkyne may be bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or substituted bicyclononyne.
  • a cyclooctane is as described in International Patent Application Publication WO2011136645, published on Nov. 3, 2011, entitled, “Fused Cyclooctyne Compounds And Their Use In Metal free Click Reactions”.
  • an azide may be a sugar or carbohydrate molecule that comprises an azide.
  • an azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine.
  • a sugar or carbohydrate molecule that comprises an azide is as described in International Patent Application Publication WO2016170186, published on Oct.
  • a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the muscle-targeting agent, molecular payload, or the linker is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “Modified antibody, antibody-conjugate and process for the preparation thereof”; or International Patent Application Publication WO2016170186, published on Oct.
  • a linker further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g., a HydraSpaceTM spacer.
  • a spacer is as described in Verkade, J. M. M. et al., “ A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody - Drug Conjugates ”, Antibodies, 2018, 7, 12.
  • a linker is connected to a muscle-targeting agent and/or molecular payload by the Diels-Alder reaction between a dienophile and a diene/hetero-diene, wherein the dienophile and the diene/hetero-diene may be located on the muscle-targeting agent, molecular payload, or the linker.
  • a linker is connected to a muscle-targeting agent and/or molecular payload by other pericyclic reactions, e.g. ene reaction.
  • a linker is connected to a muscle-targeting agent and/or molecular payload by an amide, thioamide, or sulfonamide bond reaction.
  • a linker is connected to a muscle-targeting agent and/or molecular payload by a condensation reaction to form an oxime, hydrazone, or semicarbazide group existing between the linker and the muscle-targeting agent and/or molecular payload.
  • a linker is connected to a muscle-targeting agent and/or molecular payload by a conjugate addition reactions between a nucleophile, e.g. an amine or a hydroxyl group, and an electrophile, e.g. a carboxylic acid or an aldehyde.
  • a nucleophile may exist on a linker and an electrophile may exist on a muscle-targeting agent or molecular payload prior to a reaction between a linker and a muscle-targeting agent or molecular payload.
  • an electrophile may exist on a linker and a nucleophile may exist on a muscle-targeting agent or molecular payload prior to a reaction between a linker and a muscle-targeting agent or molecular payload.
  • an electrophile may be an azide, a silicon centers, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or an activated sulfur center.
  • a nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilido group, or a thiol group.
  • complexes comprising any one the muscle targeting agent (e.g., a transferrin receptor antibodies) described herein covalently linked to any of the molecular payloads (e.g., an oligonucleotide) described herein.
  • the muscle targeting agent e.g., a transferrin receptor antibody
  • a linker Any of the linkers described herein may be used.
  • the linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide.
  • the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide, and wherein the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • antibodies can be linked to oligonucleotides with different stochiometries, a property that may be referred to as a drug to antibody ratios (DAR) with the “drug” being the oligonucleotide.
  • DAR drug to antibody ratios
  • a mixture of different complexes, each having a different DAR is provided.
  • an average DAR of complexes in such a mixture may be in a range of 1 to 3, 1 to 4, 1 to 5 or more.
  • DAR may be increased by conjugating oligonucleotides to different sites on an antibody and/or by conjugating multimers to one or more sites on antibody.
  • a DAR of 2 may be achieved by conjugating a single oligonucleotide to two different sites on an antibody or by conjugating a dimer oligonucleotide to a single site of an antibody.
  • the complex described herein comprises a transferrin receptor antibody (e.g., an antibody or any variant thereof as described herein) covalently linked to an oligonucleotide.
  • the complex described herein comprises a transferrin receptor antibody (e.g., an antibody or any variant thereof as described herein) covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker).
  • the linker e.g., a Val-cit linker
  • the linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide.
  • the linker e.g., a Val-cit linker
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.
  • a linker e.g., a Val-cit linker
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42.
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1, and wherein the complex comprises the structure of:
  • linker Val-cit linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody or any variant thereof as described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34, and wherein the complex comprises the structure of:
  • linker Val-cit linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody or any variant thereof as described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36, and wherein the complex comprises the structure of:
  • linker Val-cit linker is linked to the 5′ end, the 3′ end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody or any variant thereof as described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40, and wherein the complex comprises the structure of:
  • linker Val-cit linker is linked to the 5′ end, the 3′ end, or internally of an oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody or any variant thereof as described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42, and wherein the complex comprises the structure of:
  • linker Val-cit linker is linked to the 5′ end, the 3′ end, or internally of an oligonucleotide, and wherein the Val-cit linker is linked to the antibody (e.g., an antibody or any variant thereof as described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the antibody e.g., an antibody or any variant thereof as described herein
  • a thiol-reactive linkage e.g., via a cysteine in the antibody
  • complexes provided herein may be formulated in any suitable manner.
  • complexes provided herein are formulated in a manner suitable for pharmaceutical use.
  • complexes can be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the complexes in the formulation.
  • compositions comprising complexes and pharmaceutically acceptable carriers.
  • Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient amount of the complexes enter target muscle cells.
  • complexes are formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids.
  • compositions may include separately one or more components of complexes provided herein (e.g., muscle-targeting agents, linkers, molecular payloads, or precursor molecules of any one of them).
  • components of complexes provided herein e.g., muscle-targeting agents, linkers, molecular payloads, or precursor molecules of any one of them.
  • complexes are formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, complexes are formulated in basic buffered aqueous solutions (e.g., PBS).
  • formulations as disclosed herein comprise an excipient.
  • an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient.
  • an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).
  • a complex or component thereof e.g., oligonucleotide or antibody
  • a composition comprising a complex, or component thereof, described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).
  • a lyoprotectant e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone
  • a collapse temperature modifier e.g., dextran, ficoll, or gelatin
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, administration.
  • the route of administration is intravenous or subcutaneous.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • formulations include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Sterile injectable solutions can be prepared by incorporating the a complexes in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • a composition may contain at least about 0.1% of the a complex, or component thereof, or more, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • Complexes comprising a muscle-targeting agent covalently to a molecular payload as described herein are effective in treating FSHD.
  • complexes are effective in treating Type I FSHD.
  • complexes are effective in treating Type II FSHD.
  • FSHD is associated with deletions in D4Z4 repeat regions on chromosome 4 which contain the DUX4 gene.
  • FSHD is associated with mutations in the SMCHD1 gene.
  • a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject.
  • a subject may have myotonic dystrophy.
  • a subject has elevated expression of the DUX4 gene outside of fetal development and the testes.
  • the subject has facioscapulohumeral muscular dystrophy of Type I or Type II.
  • the subject having FSHD has mutations in the SMCHD1 gene.
  • the subject having FSHD has deletion mutations in D4Z4 repeat regions on chromosome 4.
  • An aspect of the disclosure includes a methods involving administering to a subject an effective amount of a complex as described herein.
  • an effective amount of a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload can be administered to a subject in need of treatment.
  • a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, e.g., as a bolus or by continuous infusion over a period of time.
  • intravenous administration may be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes.
  • a pharmaceutical composition may be in solid form, aqueous form, or a liquid form.
  • an aqueous or liquid form may be nebulized or lyophilized.
  • a nebulized or lyophilized form may be reconstituted with an aqueous or liquid solution.
  • compositions for intravenous administration may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
  • a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
  • a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload is administered via site-specific or local delivery techniques.
  • these techniques include implantable depot sources of the complex, local delivery catheters, site specific carriers, direct injection, or direct application.
  • a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload is administered at an effective concentration that confers therapeutic effect on a subject.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the severity of the disease, unique characteristics of the subject being treated, e.g. age, physical conditions, health, or weight, the duration of the treatment, the nature of any concurrent therapies, the route of administration and related factors. These related factors are known to those in the art and may be addressed with no more than routine experimentation.
  • an effective concentration is the maximum dose that is considered to be safe for the patient. In some embodiments, an effective concentration will be the lowest possible concentration that provides maximum efficacy.
  • Empirical considerations e.g. the half-life of the complex in a subject, generally will contribute to determination of the concentration of pharmaceutical composition that is used for treatment.
  • the frequency of administration may be empirically determined and adjusted to maximize the efficacy of the treatment.
  • an initial candidate dosage may be about 1 to 100 mg/kg, or more, depending on the factors described above, e.g. safety or efficacy.
  • a treatment will be administered once.
  • a treatment will be administered daily, biweekly, weekly, bimonthly, monthly, or at any time interval that provide maximum efficacy while minimizing safety risks to the subject.
  • the efficacy and the treatment and safety risks may be monitored throughout the course of treatment
  • the efficacy of treatment may be assessed using any suitable methods.
  • the efficacy of treatment may be assessed by evaluation of observation of symptoms associated with FSHD including muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms.
  • a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit activity or expression of a target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g. baseline level of gene expression prior to treatment.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1-5, 1-10, 5-15, 10-20, 15-30, 20-40, 25-50, or more days.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1, 2, 3, 4, 5, or 6 months.
  • a pharmaceutical composition may comprises more than one complex comprising a muscle-targeting agent covalently to a molecular payload.
  • a pharmaceutical composition may further comprise any other suitable therapeutic agent for treatment of a subject, e.g. a human subject having FSHD.
  • the other therapeutic agents may enhance or supplement the effectiveness of the complexes described herein.
  • the other therapeutic agents may function to treat a different symptom or disease than the complexes described herein.
  • HPRT hypoxanthine phosphoribosyltransferase
  • siHPRT sense 5′-UcCuAuGaCuGuAgAu 47 strand UuUaU-(CH 2 ) 6 NH 2 -3′
  • siCTRL sense 5′-UgUaAuAaCcAuAuCu 49 strand AcCuU-(CH 2 ) 6 NH 2 -3′
  • a muscle-targeting complex was generated comprising the HPRT siRNA used in Example 1 (siHPRT) covalently linked, via a non-cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker, to DTX-A-002, an anti-transferrin receptor antibody.
  • siHPRT HPRT siRNA used in Example 1
  • GMBS non-cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester
  • the GMBS linker was dissolved in dry DMSO and coupled to the 3′ end of the sense strand of siHPRT through amide bond formation under aqueous conditions. Completion of the reaction was verified by Kaiser test. Excess linker and organic solvents were removed by gel permeation chromatography. The purified, maleimide functionalized sense strand of siHPRT was then coupled to DTX-A-002 antibody using a Michael addition reaction.
  • antiTfR-siHPRT complexes comprising one or two siHPRT molecules covalently attached to DTX-A-002 antibody were purified. Densitometry confirmed that the purified sample of complexes had an average siHPRT to antibody ratio of 1.46. SDS-PAGE analysis demonstrated that >90% of the purified sample of complexes comprised DTX-A-002 linked to either one or two siHPRT molecules.
  • a control IgG2a-siHPRT complex was generated comprising the HPRT siRNA used in Example 1 (siHPRT) covalently linked via the GMBS linker to an IgG2a (Fab) antibody (DTX-A-003).
  • siHPRT HPRT siRNA used in Example 1
  • Fab IgG2a antibody
  • Densitometry confirmed that DTX-C-001 had an average siHPRT to antibody ratio of 1.46 and SDS-PAGE demonstrated that >90% of the purified sample of control complexes comprised DTX-A-003 linked to either one or two siHPRT molecules.
  • the antiTfR-siHPRT complex was then tested for cellular internalization and inhibition of HPRT in cellulo.
  • Hepa 1-6 cells which have relatively high expression levels of transferrin receptor, were incubated in the presence of vehicle (phosphate-buffered saline), IgG2a-siHPRT (100 nM), antiTfR-siCTRL (100 nM), or antiTfR-siHPRT (100 nM), for 72 hours. After the 72 hour incubation, the cells were isolated and assayed for expression levels of HPRT ( FIG. 2 ). Cells treated with the antiTfR-siHPRT demonstrated a reduction in HPRT expression by ⁇ 50% relative to the cells treated with the vehicle control.
  • mice C57BL/6 wild-type mice were intravenously injected with a single dose of a vehicle control (phosphate-buffered saline); siHPRT (2 mg/kg of RNA); IgG2a-siHPRT (2 mg/kg of RNA, corresponding to 9 mg/kg antibody complex); or antiTfR-siHPRT (2 mg/kg of RNA, corresponding to 9 mg/kg antibody complex.
  • a vehicle control phosphate-buffered saline
  • siHPRT 2 mg/kg of RNA
  • IgG2a-siHPRT 2 mg/kg of RNA, corresponding to 9 mg/kg antibody complex
  • antiTfR-siHPRT 2 mg/kg of RNA, corresponding to 9 mg/kg antibody complex.
  • Each experimental condition was replicated in four individual C57BL/6 wild-type mice. Following a three-day period after injection, the mice were euthanized and segmented into isolated tissue types. Individual tissue samples were subsequently assayed for expression levels of HPRT
  • mice treated with the antiTfR-siHPRT complex demonstrated a reduction in HPRT expression in gastrocnemius (31% reduction; p ⁇ 0.05) and heart (30% reduction; p ⁇ 0.05), relative to the mice treated with the siHPRT control ( FIGS. 3A-3B ). Meanwhile, mice treated with the IgG2a-siHPRT complex had HPRT expression levels comparable to the siHPRT control (little or no reduction in HPRT expression) for all assayed muscle tissue types.
  • mice treated with the antiTfR-siHPRT complex demonstrated no change in HPRT expression in non-muscle tissues such as brain, liver, lung, kidney, and spleen tissues ( FIGS. 4A-4E ).
  • DUX4-expressing cell lines A549, U-2 OS, and HepG2 cell lines
  • SkMC immortalized skeletal muscle myoblasts
  • FIG. 5 Three DUX4-expressing cell lines (A549, U-2 OS, and HepG2 cell lines) and immortalized skeletal muscle myoblasts (SkMC) were screened for expression of DUX4 mRNA ( FIG. 5 ).
  • Cells were seeded at a density of 10,000 cells/well and harvested for total RNA.
  • cDNA was synthesized from the total RNA extracts and qPCR was performed to determine concentration of DUX4 relative to a control gene (PPIB) in technical quadruplicate. These data were used to aid in the selection of the U-2 OS cell line for downstream development of DUX4-targeting oligonucleotides.
  • FM10 PMO phosphorodiamidate morpholino oligomer
  • FM10 PMO phosphorodiamidate morpholino oligomer
  • U-2 OS cells were seeded at a density of 10k cells/well before being allowed to recover overnight. Cells were then treated with either a control PMO (100 nM) or with the FM10 PMO (10 ⁇ M). Cells were incubated for 72 hours before being harvested for total RNA. cDNA was then synthesized from the total RNA extracts and qPCR was performed to determine expression of downstream DUX4 genes (ZSCAN1, MBDL3L2, TRIM43) in technical quadruplicate. All qPCR data were analyzed using a standard ⁇ CT method and were normalized to a plate-based negative control comprised of untreated cells (i.e., without any oligonucleotide). Results were then converted to fold change to evaluate efficacy.
  • DUX4 genes ZSCAN1, MBDL3L2, TRIM43
  • Example 5 Targeting DUX4 with a Muscle-Targeting Complex
  • a muscle-targeting complex comprising an antisense oligonucleotide that targets a mutant allele of DUX4 (DUX4 ASO) covalently linked, via a cathepsin cleavable linker, to DTX-A-002 (RI7 217 (Fab)), an anti-transferrin receptor antibody.
  • DUX4 ASO antisense oligonucleotide that targets a mutant allele of DUX4
  • DTX-A-002 RI7 217 (Fab)
  • Fab anti-transferrin receptor antibody
  • MC-Val-Cit-PABC-PNP maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate
  • MC-Val-Cit-PABC-PNP maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate
  • MC-Val-Cit-PABC-PNP p-nitrophenyl carbonate
  • the product of the antibody coupling reaction is then subjected to hydrophobic interaction chromatography (HIC-HPLC) to purify the muscle-targeting complex.
  • HIC-HPLC hydrophobic interaction chromatography
  • Densitometry and SDS-PAGE analysis of the purified complex allow for determination of the average ratio of ASO-to-antibody and total purity, respectively.
  • control complex comprising DUX4 ASO covalently linked via a Val-Cit linker to an IgG2a (Fab) antibody.
  • the purified muscle-targeting complex comprising DTX-A-002 covalently linked to DUX4 ASO is then tested for cellular internalization and inhibition of DUX4.
  • Disease-relevant muscle cells that have relatively high expression levels of transferrin receptor, are incubated in the presence of vehicle control (saline), muscle-targeting complex (100 nM), or control complex (100 nM) for 72 hours. After the 72 hour incubation, the cells are isolated and assayed for expression levels of DUX4.
  • sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid.
  • the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US17/264,948 2018-08-02 2019-08-02 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy Abandoned US20220193250A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/264,948 US20220193250A1 (en) 2018-08-02 2019-08-02 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US17/205,123 US11111309B2 (en) 2018-08-02 2021-03-18 Method of reducing expression of DUX4 in a muscle cell by administering an anti-transferrin receptor antibody linked to an oligonucleotide targeting DUX4

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201862713933P 2018-08-02 2018-08-02
US17/264,948 US20220193250A1 (en) 2018-08-02 2019-08-02 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
PCT/US2019/044990 WO2020028864A1 (fr) 2018-08-02 2019-08-02 Complexes de ciblage musculaire et leurs utilisations pour le traitement de la dystrophie musculaire facio-scapulo-humérale

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US17/264,905 Continuation-In-Part US20210308272A1 (en) 2018-08-02 2019-08-02 Muscle targeting complexes and uses thereof for treating myotonic dystrophy
PCT/US2019/044987 Continuation-In-Part WO2020028861A1 (fr) 2018-08-02 2019-08-02 Complexes de ciblage musculaire et utilisations associées pour le traitement de la dystrophie myotonique
PCT/US2019/044990 A-371-Of-International WO2020028864A1 (fr) 2018-08-02 2019-08-02 Complexes de ciblage musculaire et leurs utilisations pour le traitement de la dystrophie musculaire facio-scapulo-humérale

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/205,123 Continuation US11111309B2 (en) 2018-08-02 2021-03-18 Method of reducing expression of DUX4 in a muscle cell by administering an anti-transferrin receptor antibody linked to an oligonucleotide targeting DUX4

Publications (1)

Publication Number Publication Date
US20220193250A1 true US20220193250A1 (en) 2022-06-23

Family

ID=69232303

Family Applications (9)

Application Number Title Priority Date Filing Date
US17/264,948 Abandoned US20220193250A1 (en) 2018-08-02 2019-08-02 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US17/205,123 Active US11111309B2 (en) 2018-08-02 2021-03-18 Method of reducing expression of DUX4 in a muscle cell by administering an anti-transferrin receptor antibody linked to an oligonucleotide targeting DUX4
US17/400,295 Active US11286305B2 (en) 2018-08-02 2021-08-12 Complex comprising anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets DUX4 RNA
US17/671,707 Active US11390682B2 (en) 2018-08-02 2022-02-15 Methods of intravenouisly delivering anti-transferrin antibody/oligonucleotide complexes to subjects having muscular dystrophy
US17/846,738 Active US11518816B2 (en) 2018-08-02 2022-06-22 Methods of delivering an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
US17/936,483 Active 2039-09-20 US11787869B2 (en) 2018-08-02 2022-09-29 Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness
US18/184,905 Active US11795234B2 (en) 2018-08-02 2023-03-16 Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US18/184,741 Active US11795233B2 (en) 2018-08-02 2023-03-16 Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US18/467,851 Pending US20240067743A1 (en) 2018-08-02 2023-09-15 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy

Family Applications After (8)

Application Number Title Priority Date Filing Date
US17/205,123 Active US11111309B2 (en) 2018-08-02 2021-03-18 Method of reducing expression of DUX4 in a muscle cell by administering an anti-transferrin receptor antibody linked to an oligonucleotide targeting DUX4
US17/400,295 Active US11286305B2 (en) 2018-08-02 2021-08-12 Complex comprising anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets DUX4 RNA
US17/671,707 Active US11390682B2 (en) 2018-08-02 2022-02-15 Methods of intravenouisly delivering anti-transferrin antibody/oligonucleotide complexes to subjects having muscular dystrophy
US17/846,738 Active US11518816B2 (en) 2018-08-02 2022-06-22 Methods of delivering an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
US17/936,483 Active 2039-09-20 US11787869B2 (en) 2018-08-02 2022-09-29 Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness
US18/184,905 Active US11795234B2 (en) 2018-08-02 2023-03-16 Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US18/184,741 Active US11795233B2 (en) 2018-08-02 2023-03-16 Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US18/467,851 Pending US20240067743A1 (en) 2018-08-02 2023-09-15 Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy

Country Status (12)

Country Link
US (9) US20220193250A1 (fr)
EP (1) EP3830259A4 (fr)
JP (1) JP2021533200A (fr)
KR (1) KR20210081324A (fr)
CN (1) CN112912499A (fr)
AU (1) AU2019316103A1 (fr)
CA (1) CA3108289A1 (fr)
EA (1) EA202190416A1 (fr)
IL (1) IL280426A (fr)
MX (1) MX2021001284A (fr)
SG (1) SG11202100928QA (fr)
WO (1) WO2020028864A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11497815B2 (en) 2018-08-02 2022-11-15 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11518816B2 (en) 2018-08-02 2022-12-06 Dyne Therapeutics, Inc. Methods of delivering an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
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
US11648318B2 (en) 2021-07-09 2023-05-16 Dyne Therapeutics, Inc. Anti-transferrin receptor (TFR) antibody and uses thereof
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11931421B2 (en) 2022-04-15 2024-03-19 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11168141B2 (en) 2018-08-02 2021-11-09 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
KR20210129645A (ko) 2019-01-22 2021-10-28 코로 바이오, 인크. Rna-편집 올리고뉴클레오타이드 및 그의 용도
JP2022519184A (ja) * 2019-01-22 2022-03-22 コロ バイオ， インコーポレイテッド Ｒｎａ編集オリゴヌクレオチド及びその使用
AU2021237465A1 (en) * 2020-03-19 2022-10-13 Avidity Biosciences, Inc. Compositions and methods of treating Facioscapulohumeral muscular dystrophy
MX2022012265A (es) 2020-04-02 2023-01-11 Mirecule Inc Inhibicion dirigida utilizando oligonucleotidos ingenieria.
TW202227627A (zh) 2020-09-11 2022-07-16 美商愛羅海德製藥公司 用於抑制DUX4表現之RNAi藥劑、其組合物及使用方法
EP4271478A1 (fr) * 2020-12-31 2023-11-08 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour traiter la myopathie facio-scapulo-humérale
EP4359534A1 (fr) * 2021-06-21 2024-05-01 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de la maladie de pompe
WO2023283629A1 (fr) * 2021-07-09 2023-01-12 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs formulations pour traiter la dystrophie musculaire facio-scapulo-humérale
EP4124345A1 (fr) * 2021-07-30 2023-02-01 4basebio UK Ltd Nanoparticules pour l'administration musculaire
CA3226457A1 (fr) * 2021-07-30 2023-02-02 Amy WALKER Nanoparticules et peptides pour l'administration de charges a des cellules musculaires
PE20240895A1 (es) 2021-09-01 2024-04-24 Biogen Ma Inc Anticuerpos del receptor antitransferrina y usos de los mismos
AU2022345098A1 (en) * 2021-09-16 2024-04-04 Avidity Biosciences, Inc. Compositions and methods of treating facioscapulohumeral muscular dystrophy
WO2023086864A1 (fr) * 2021-11-12 2023-05-19 Dyne Therapeutics, Inc. Complexes de ciblage musculaire pour traiter la dystrophie musculaire facio-scapulo-humérale

Family Cites Families (231)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2631173A (en) 1949-01-04 1953-03-10 Phillips Petroleum Co Production of cyclooctadiene
IL54795A (en) 1978-05-26 1980-10-26 Yeda Res & Dev Analgesic compositions comprising substituted bicyclic and spiroaliphatic amines and certain novel compounds of these types
EP0425491B1 (fr) 1988-03-02 1994-07-20 The Uab Research Foundation Moteurs mecanochimiques reversibles composes de bio-elastomeres capables de moduler les transitions de temperature inverses, permettant l'interconversion de travail chimique et mecanique
WO1991004753A1 (fr) 1989-10-02 1991-04-18 Cetus Corporation Conjugues d'oligonucleotides non codants et leurs emplois therapeutiques
US7015315B1 (en) 1991-12-24 2006-03-21 Isis Pharmaceuticals, Inc. Gapped oligonucleotides
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US6255455B1 (en) 1996-10-11 2001-07-03 The Trustees Of The University Of Pennsylvania Rh(D)-binding proteins and magnetically activated cell sorting method for production thereof
CA2400659A1 (fr) 2000-02-22 2001-08-30 Banyu Pharmaceutical Co., Ltd. Composes a base d'imidazoline
US6784291B2 (en) 2000-05-04 2004-08-31 Avi Biopharma, Inc. Splice-region antisense composition and method
EP1191097A1 (fr) 2000-09-21 2002-03-27 Leids Universitair Medisch Centrum Induction du "exon-skipping" dans des cellules eukaryotes
US20040063654A1 (en) 2001-11-02 2004-04-01 Davis Mark E. Methods and compositions for therapeutic use of RNA interference
US7241444B2 (en) 2002-01-18 2007-07-10 Pierre Fabre Medicament Anti-IGF-IR antibodies and uses thereof
CA2478700C (fr) 2002-03-11 2012-10-16 Momenta Pharmaceuticals, Inc. Analyse de polysaccharides sulfates
PT2284266E (pt) 2002-11-14 2013-12-17 Thermo Fisher Scient Biosciences Inc Siarn contra tp53
DK3222722T3 (da) 2002-11-18 2019-06-17 Roche Innovation Ct Copenhagen As Antisense-design
EP1568769A4 (fr) 2002-11-25 2008-08-06 Masafumi Matsuo Medicaments d'acide nucleique ena modifiant l'epissage dans un precurseur mrna
WO2004058717A1 (fr) 2002-12-20 2004-07-15 X-Ceptor Therapeutics, Inc. Derives d'isoquinolinone et leur utilisation comme agents therapeutiques
PT1592793E (pt) 2003-02-10 2009-09-30 Santaris Pharma As Compostos oligoméricos para a modulação da expressão de survivina
WO2004083432A1 (fr) 2003-03-21 2004-09-30 Academisch Ziekenhuis Leiden Modulation de la reconnaissance d'exons dans le pre-arnm par interference avec la structure d'arn secondaire
CA2522700A1 (fr) 2003-04-18 2004-11-04 Cytovia, Inc. Procedes pour traiter des maladies entrainant l'induction d'apoptose et essais de criblage
US20050053981A1 (en) 2003-09-09 2005-03-10 Swayze Eric E. Gapped oligomeric compounds having linked bicyclic sugar moieties at the termini
US8404747B2 (en) 2004-03-05 2013-03-26 The General Hospital Corporation Compositions and methods for modulating interaction between polypeptides
US20080199960A1 (en) 2004-05-13 2008-08-21 Juliano Rudolph L Methods for the Delivery of Oligomeric Compounds
JP5697297B2 (ja) 2004-05-14 2015-04-08 ロゼッタ ジノミクス リミテッド マイクロｎａｓおよびその使用
EP2206781B1 (fr) 2004-06-28 2015-12-02 The University Of Western Australia Oligonucléotides antisens permettant d'induire un saut d'exon et leurs procédés d'utilisation
FR2874384B1 (fr) 2004-08-17 2010-07-30 Genethon Vecteur viral adeno-associe pour realiser du saut d'exons dans un gene codant une proteine a domaines dispensables
WO2006050262A2 (fr) 2004-11-01 2006-05-11 The Regents Of The University Of California Compositions et procedes de modification de biomolecules
US8431558B2 (en) 2004-11-01 2013-04-30 The Regents Of The University Of California Compositions and methods for modification of biomolecules
US20120122801A1 (en) 2005-01-05 2012-05-17 Prosensa B.V. Mannose-6-phosphate receptor mediated gene transfer into muscle cells
WO2006091796A2 (fr) 2005-02-22 2006-08-31 Acucela, Inc. Compositions et methodes destinees au diagnostic et au traitement de maladies retiniennes
US7807648B2 (en) 2005-10-06 2010-10-05 University Of Delaware G-rich polynucleotides for the treatment of Huntington's Disease
JP2007104971A (ja) 2005-10-14 2007-04-26 New Industry Research Organization ミオスタチンのスプライシング制御アンチセンスオリゴヌクレオチド
US20090214531A1 (en) 2006-01-30 2009-08-27 John Mekalanos Methods and Compositions for Treating and Preventing Bacterial Infections
EP2623610B1 (fr) 2006-02-10 2015-04-29 Life Technologies Corporation Marquage et détection de protéines modifiées post-traductionnellement
US8859752B2 (en) 2006-04-18 2014-10-14 The Trustees Of The University Of Pennsylvania SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)
EP2019692B1 (fr) 2006-05-05 2014-06-04 Isis Pharmaceuticals, Inc. Composés et procédés pour moduler l'expression de gccr
EP1857548A1 (fr) 2006-05-19 2007-11-21 Academisch Ziekenhuis Leiden Procédé et moyen permettant d'induire un saut d'exon
JP6047270B2 (ja) 2006-08-11 2016-12-21 バイオマリン テクノロジーズ ベー．フェー． Ｄｎａリピートの不安定性に関連した遺伝的障害を治療するための方法及び手段
ES2526295T5 (es) 2006-10-18 2021-05-04 Ionis Pharmaceuticals Inc Compuestos antisentido
CA2693178C (fr) 2006-11-29 2018-12-04 Nationwide Children's Hospital, Inc. Inhibition de la myostatine destinee a ameliorer le muscle et/ou a ameliorer la fonction musculaire
US8580756B2 (en) 2007-03-22 2013-11-12 Santaris Pharma A/S Short oligomer antagonist compounds for the modulation of target mRNA
EP2149605B1 (fr) 2007-03-22 2013-07-03 Santaris Pharma A/S Composés ARN antagonistes courts pour la modulation de l'ARNm cible
ES2639852T3 (es) 2007-10-26 2017-10-30 Academisch Ziekenhuis Leiden Medios y métodos para contrarrestar los trastornos musculares
USRE48468E1 (en) 2007-10-26 2021-03-16 Biomarin Technologies B.V. Means and methods for counteracting muscle disorders
EP2274423A2 (fr) 2008-04-04 2011-01-19 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléosides bicycliques et ayant une toxicité réduite
WO2009144481A2 (fr) 2008-05-30 2009-12-03 Isis Innovation Limited Conjugués pour la délivrance de composés biologiquement actifs
US8084601B2 (en) 2008-09-11 2011-12-27 Royal Holloway And Bedford New College Royal Holloway, University Of London Oligomers
BR122020021379B1 (pt) 2008-10-24 2021-05-11 Sarepta Therapeutics, Inc. oligômero morfolino fosforodiamidato, composição que compreende o mesmo e uso do dito oligômero para tratar distrofia muscular
SI2607484T1 (sl) 2008-10-27 2016-11-30 Biomarin Technologies B.V. Metode in sredstva za učinkovito preskakovanje eksona 45 v pre-mRNA Duchennove mišične distrofije
JP2012524540A (ja) 2009-04-24 2012-10-18 プロセンサ テクノロジーズ ビー．ブイ． Ｄｍｄを処置するためのイノシンを含むオリゴヌクレオチド
CA3185821A1 (fr) * 2009-05-08 2010-11-11 Curna, Inc. Traitement de maladies liees a la famille de la dystrophine par inhibition du produit antisens naturel de transcription vers la famille de la dmd
FR2947269B1 (fr) 2009-06-29 2013-01-18 Sanofi Aventis Nouveaux composes anticancereux
EP2480659A2 (fr) 2009-09-24 2012-08-01 Cellectis Réactifs à base de méganucléase et leurs utilisations pour traiter des maladies génétiques provoquées par des mutations de changement de phase/ non sens
KR101958491B1 (ko) 2009-11-12 2019-03-15 더 유니버시티 오브 웨스턴 오스트레일리아 안티센스 분자 및 이를 이용한 질환 치료방법
WO2011078797A2 (fr) 2009-12-22 2011-06-30 Singapore Health Services Pte. Ltd Oligonucléotides antisens et utilisations de ceux-ci
US8734799B2 (en) 2010-04-13 2014-05-27 The Regents Of The University Of California Unconjugated anti-TfR antibodies and compositions thereof for the treatment of cancer
EP2563753B9 (fr) 2010-04-27 2016-07-06 SynAffix B.V. Composés de cyclooctyne condensés et leur utilisation dans des click-réactions non métalliques
JP2013530154A (ja) 2010-05-28 2013-07-25 サレプタ セラピューティクス， インコーポレイテッド 修飾されたサブユニット間結合および／または末端基を有するオリゴヌクレオチドアナログ
IT1400425B1 (it) 2010-06-08 2013-05-31 Amsterdam Molecular Therapeutics Bv Modified snrnas for use in therapy.
AU2011277999A1 (en) 2010-07-12 2013-01-10 Covx Technologies Ireland Limited Multifunctional Antibody Conjugates
WO2012012443A2 (fr) 2010-07-19 2012-01-26 Bennett C Frank Modulation de l'expression de la protéine kinase de la dystrophie myotonique (dmpk)
EP2606152B1 (fr) 2010-08-18 2016-10-05 Fred Hutchinson Cancer Research Center Procédés de détermination de la présence ou du risque de développement de la dystrophie facio-scapulo-humérale (fshd)
TWI541024B (zh) 2010-09-01 2016-07-11 日本新藥股份有限公司 反義核酸
DK2426203T3 (en) 2010-09-02 2016-11-07 Université de Mons Agents that are useful in the treatment of muscular dystrophy, facioscapulohumeral
WO2012109395A1 (fr) 2011-02-08 2012-08-16 Isis Pharmaceuticals, Inc. Composés oligomères comprenant des nucléotides bicycliques et leurs utilisations
BR112013020273A2 (pt) 2011-02-08 2016-10-18 Charlotte Mecklenburg Hospital oligonucleotídeos antissenso
EP2699269A1 (fr) 2011-04-22 2014-02-26 Prosensa Technologies B.V. Nouveaux composés pour traiter, retarder et/ou prévenir un trouble génétique humain, tel que la dystrophie myotonique de type 1 (dm1)
CN107080847A (zh) 2011-06-24 2017-08-22 森彻斯有限公司 细胞外靶向药物缀合物
ES2648169T3 (es) 2011-07-25 2017-12-28 Nationwide Children's Hospital, Inc. Productos de virus recombinantes y métodos para inhibición de la expresión de DUX4
WO2013019623A2 (fr) 2011-07-29 2013-02-07 Fred Hutchinson Cancer Research Center Méthodes et compositions modulant la réponse immunitaire naturelle et/ou la myogenèse chez un mammifère
US20130066063A1 (en) 2011-09-09 2013-03-14 John Cooke Hodges Bicyclo[6.1.0]non-4-yne regents for chemical modification of oligonucleotides
EP2788026A4 (fr) 2011-12-05 2015-08-05 Univ Duke Immunogènes v1v2
US9725716B2 (en) 2011-12-06 2017-08-08 Ohio State Innovation Foundation and Research Institute at Nationwide Children's Hospital Non-ionic, low osmolar contrast agents for delivery of antisense oligonucleotides and treatment of disease
CN108486116A (zh) 2011-12-28 2018-09-04 日本新药株式会社 反义核酸
WO2013103800A1 (fr) 2012-01-06 2013-07-11 Bioalliance C.V. Anticorps de récepteur d'anti-transferrine et leurs procédés
RU2014138474A (ru) 2012-02-24 2016-04-10 СтемСентРкс, Инк. Новые модуляторы и способы применения
WO2013138662A1 (fr) 2012-03-16 2013-09-19 4S3 Bioscience, Inc. Conjugués antisens destinés à diminuer l'expression du dmpk
EP2841578B1 (fr) 2012-04-23 2017-06-07 BioMarin Technologies B.V. Oligonucléotides de modulation arn présentant des caractéristiques améliorées pour le traitement des troubles neuromusculaires
CN110257379B (zh) 2012-07-03 2023-08-11 马林生物科技有限公司 用于治疗肌肉萎缩症患者的寡核苷酸
EP2684892A1 (fr) 2012-07-13 2014-01-15 Association Française contre les Myopathies Compositions et procédés pour la thérapie génique de la dystrophie musculaire de Duchenne
WO2014059353A2 (fr) 2012-10-11 2014-04-17 Isis Pharmaceuticals, Inc. Composés oligomères comportant des nucléosides bicycliques et leurs utilisations
HUE062385T2 (hu) 2012-10-23 2023-10-28 Synaffix Bv Módosított antitest, antitest-konjugátum és eljárás ezek elõállítására
ES2701076T3 (es) 2012-11-24 2019-02-20 Hangzhou Dac Biotech Co Ltd Enlazadores hidrofílicos y sus usos para la conjugación de fármacos a las moléculas que se unen a las células
TR201903009T4 (tr) 2013-03-14 2019-03-21 Sarepta Therapeutics Inc Musküler distrofi tedavisine yönelik ekson atlama bileşimleri.
WO2014144978A2 (fr) 2013-03-15 2014-09-18 Sarepta Therapeutics, Inc. Compositions améliorées pour le traitement de la dystrophie musculaire
MA38632B1 (fr) 2013-05-20 2019-10-31 Genentech Inc Anticorps anti-récepteur de transferrine et procédés d'utilisation
WO2014189370A1 (fr) 2013-05-24 2014-11-27 Stichting Katholieke Universiteit Composés azadibenzocyclooctyne substitués et leur utilisation dans des réactions « click » sans métal
CA2914519A1 (fr) 2013-06-05 2014-12-11 Duke University Edition et regulation geniques a guidage arn
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
EP3038659A4 (fr) 2013-08-28 2017-07-26 AbbVie Stemcentrx LLC Conjugués anti-dll3 modifiés et procédés d'utilisation
WO2015057065A1 (fr) 2013-10-14 2015-04-23 Synaffix B.V. Anticorps glycomanipulé, conjugué d'anticorps et procédés pour leur préparation
US9987373B2 (en) 2013-10-14 2018-06-05 Synaffix B.V. Modified glycoprotein, protein-conjugate and process for the preparation thereof
EP2862928A1 (fr) 2013-10-18 2015-04-22 Université de Strasbourg Inhibiteur de dynamine 2 pour le traitement de myopathies centronucléaires
US10004814B2 (en) 2013-11-11 2018-06-26 Sirna Therapeutics, Inc. Systemic delivery of myostatin short interfering nucleic acids (siNA) conjugated to a lipophilic moiety
WO2015073848A1 (fr) 2013-11-14 2015-05-21 Shehadeh Lina A Méthodes et compositions utilisant un aptamère pour administrer des acides nucléiques dans des cellules promotrices et/ou souches endothéliales, cardiaques et de muscle lisse
US10266502B2 (en) 2014-01-24 2019-04-23 Synaffix B.V. Process for the cycloaddition of a halogenated 1,3-dipole compound with a (hetero)cycloalkyne
JP2017505770A (ja) 2014-01-24 2017-02-23 シンアフィックス ビー．ブイ． （ヘテロ）アリール１，３−双極子化合物と（ヘテロ）シクロアルキンとの環化付加のためのプロセス
LT3118311T (lt) 2014-03-12 2019-04-10 Nippon Shinyaku Co., Ltd. Priešprasminė nukleorūgštis
WO2015143062A1 (fr) 2014-03-18 2015-09-24 Fred Hutchinson Cancer Research Center Expression génique induite par dux4 en dystrophie musculaire facio-scapulo-humérale (fshd)
JP6750148B2 (ja) 2014-04-25 2020-09-02 公益財団法人野口研究所 糖鎖切断抗体の製造方法及び均一糖鎖抗体
TWI713450B (zh) 2014-05-23 2020-12-21 美商健臻公司 藉由使用反義寡核苷酸創造提前終止密碼子抑制或下調肝醣合成酶
SG10201912858VA (en) 2014-06-17 2020-02-27 Nippon Shinyaku Co Ltd Antisense nucleic acids
AR101449A1 (es) 2014-08-04 2016-12-21 Miragen Therapeutics Inc Inhibidores de myh7b y usos de los mismos
WO2016022027A1 (fr) 2014-08-04 2016-02-11 Synaffix B.V. Procédé pour la modification d'une glycoprotéine à l'aide d'une bêta-(1,4)-n-acétylgalactosaminyl-transférase ou d'un mutant correspondant
US20160075772A1 (en) 2014-09-12 2016-03-17 Regeneron Pharmaceuticals, Inc. Treatment of Fibrodysplasia Ossificans Progressiva
WO2016053107A1 (fr) 2014-10-03 2016-04-07 Synaffix B.V. Lieur de type sulfamide, conjugués de celui-ci et procédés de préparation
EP3218411B1 (fr) 2014-11-14 2022-01-12 Ossianix, Inc. Récepteurs d'antigène nouveaux variables (vnars) dirigé contre récepteur de transferrine et leur utilisation
EP3221362B1 (fr) 2014-11-19 2019-07-24 F.Hoffmann-La Roche Ag Anticorps anti-récepteur de transferrine et procédés d'utilisation
BR122024000903A2 (pt) 2014-11-21 2024-02-27 Merck Sharp & Dohme Llc Composto, composição, e, uso do composto
WO2016094374A1 (fr) 2014-12-09 2016-06-16 The Board Of Regents Of The University Of Texas System Compositions et méthodes de traitement de l'ataxie de friedreich
WO2016115490A1 (fr) 2015-01-16 2016-07-21 Ionis Pharmaceuticals, Inc. Composés et procédés de modulation de dux4
BR112017018383B1 (pt) 2015-02-27 2023-04-25 Murdoch University Compostos anti-sentido que induzem a inclusão de éxon2, composições farmacêuticas que compreendem ditos compostos e usos dos mesmos para tratar doença de armazenamento de glicogênio tipo ii
EP3134520B1 (fr) 2015-04-23 2017-12-20 Synaffix B.V. Procédé de modification d'une glycoprotéine utilisant une glycosyltransférase qui est ou est dérivée d'une beta- (1,4) -n-acetylgalactosaminyltransférase
HUE057720T2 (hu) 2015-05-04 2022-06-28 Cytomx Therapeutics Inc Aktiválható anti-CD71 antitestek és ezek alkalmazási eljárásai
US10675356B2 (en) 2015-05-19 2020-06-09 Sarepta Therapeutics, Inc. Peptide oligonucleotide conjugates
EP3325509B1 (fr) 2015-07-22 2020-12-16 Inatherys Anticorps anti-tfr et leur utilisation dans le traitement des maladies prolifératives et inflammatoires
LT3351633T (lt) 2015-09-15 2020-08-10 Nippon Shinyaku Co., Ltd. Priešprasmė nukleorūgštis
EP3353301A1 (fr) 2015-09-21 2018-08-01 Association Institut de Myologie Oligonucléotides antisens et leurs utilisations
WO2017049407A1 (fr) 2015-09-23 2017-03-30 UNIVERSITé LAVAL Modification du gène de la dystrophine et ses utilisations
CN108699555A (zh) 2015-10-09 2018-10-23 萨勒普塔医疗公司 用于治疗杜兴肌营养不良和相关病症的组合物和方法
TW201722439A (zh) 2015-10-09 2017-07-01 波濤生命科學有限公司 寡核苷酸組合物及其方法
PL3386534T3 (pl) 2015-12-08 2021-03-08 Regeneron Pharmaceuticals, Inc. Kompozycje i sposoby internalizacji enzymów
FR3044926B1 (fr) 2015-12-09 2020-01-31 Genethon Outils de therapie genique efficaces pour le saut de l'exon 53 de la dystrophine
US10874746B2 (en) 2016-02-08 2020-12-29 Synaffix B.V. Sulfamide linkers for use in bioconjugates
IL260983B (en) 2016-02-19 2022-07-01 Genisphere Llc Nucleic acid carriers and methods of medical use
MA45328A (fr) 2016-04-01 2019-02-06 Avidity Biosciences Llc Compositions acide nucléique-polypeptide et utilisations de celles-ci
FR3049951A1 (fr) 2016-04-12 2017-10-13 Univ Rabelais Francois Nouvelles constructions peptidiques et leur utilisation dans le traitement de la toxoplasmose
JP7033547B2 (ja) 2016-04-18 2022-03-10 サレプタ セラピューティクス， インコーポレイテッド 酸性アルファ－グルコシダーゼ遺伝子に関連する疾患を処置するためのアンチセンスオリゴマーおよびそれを用いる方法
MA45290A (fr) 2016-05-04 2019-03-13 Wave Life Sciences Ltd Procédés et compositions d'agents biologiquement actifs
WO2017205191A1 (fr) 2016-05-24 2017-11-30 Merck Sharp & Dohme Corp. Agonistes partiels du récepteur de l'insuline et analogues du glp-1
EP3463386A4 (fr) 2016-06-03 2020-03-04 Wave Life Sciences Ltd. Oligonucléotides, compositions et méthodes associées
US20190240346A1 (en) 2016-06-20 2019-08-08 Genahead Bio, Inc. Antibody-drug conjugate
US11427838B2 (en) 2016-06-29 2022-08-30 Vertex Pharmaceuticals Incorporated Materials and methods for treatment of myotonic dystrophy type 1 (DM1) and other related disorders
US20180028554A1 (en) 2016-07-05 2018-02-01 Biomarin Technologies B.V. Oligomers Having Bicyclic Scaffold Moeities
EP3485015A4 (fr) 2016-07-15 2020-07-29 Ionis Pharmaceuticals, Inc. Composés et méthodes de modulation de transcrit de la dystrophine
US20190151476A1 (en) 2016-07-19 2019-05-23 Duke University Therapeutic applications of cpf1-based genome editing
US10933081B2 (en) 2016-09-21 2021-03-02 Alnylam Pharmaceuticals, Inc. Myostatin iRNA compositions and methods of use thereof
CN110267970A (zh) 2016-11-16 2019-09-20 比奥马林技术公司 用于靶向各种选定器官或组织的物质
US10947540B2 (en) 2016-11-29 2021-03-16 Association Institut De Myologie Allele-specific silencing therapy for Dynamin 2-related diseases
MX2019006157A (es) 2016-11-28 2019-11-21 Univ Texas Prevención de la distrofia muscular mediante la edición de genes mediada por crispr/cpf1.
JP2020500541A (ja) 2016-12-08 2020-01-16 ザ ボード オブ リージェンツ オブ ザ ユニバーシティー オブ テキサス システム ヒト化デュシェンヌ型筋ジストロフィー変異を有するｄｍｄレポーターモデル
JOP20190166A1 (ar) 2017-01-05 2019-07-02 Univ Texas استراتيجية مثلى من أجل تعديلات تخطي إكسون باستخدام crispr/cas9 مع متواليات توجيه ثلاثي
CN110381980A (zh) 2017-01-06 2019-10-25 艾维迪提生物科学有限责任公司 核酸-多肽组合物以及诱导外显子跳读的方法
MA50546A (fr) 2017-06-07 2020-09-16 Regeneron Pharma Compositions et méthodes pour l'internalisation d'enzymes
GB201711809D0 (en) 2017-07-21 2017-09-06 Governors Of The Univ Of Alberta Antisense oligonucleotide
EP3684378A4 (fr) 2017-09-19 2021-06-16 Children's National Medical Center Gapmères et procédés d'utilisation de ces derniers pour le traitement de la dystrophie musculaire
EA201991450A1 (ru) 2017-09-22 2019-12-30 Сарепта Терапьютикс, Инк. Конъюгаты олигомеров для пропуска экзона при мышечной дистрофии
US20200282074A1 (en) 2017-09-22 2020-09-10 Avidity Biosciences, Inc. Nucleic acid-polypeptide compositions and methods of inducing exon skipping
WO2019067975A1 (fr) 2017-09-28 2019-04-04 Sarepta Therapeutics, Inc. Polythérapies pour traiter une dystrophie musculaire
WO2019070741A1 (fr) 2017-10-02 2019-04-11 Research Institute At Nationwide Children's Hospital Système de déciblage de miarn pour interférence spécifique d'un tissu
JP2021500862A (ja) 2017-10-04 2021-01-14 アビディティー バイオサイエンシーズ，インク． 核酸ポリペプチド組成物およびその使用
MA50578A (fr) 2017-11-09 2021-09-15 Vertex Pharma Systèmes crispr/cas pour le traitement de dmd
MX2020005860A (es) 2017-12-06 2020-09-09 Avidity Biosciences Inc Composiciones y metodos de tratamiento de atrofia muscular y distrofia miotonica.
EP3720504A1 (fr) 2017-12-06 2020-10-14 Synaffix B.V. Conjugués d'énédiyne
WO2019126641A2 (fr) 2017-12-21 2019-06-27 Ionis Pharmaceuticals, Inc. Modulation de l'expression de la frataxine
US20210095283A1 (en) 2018-01-04 2021-04-01 Avidity Biosciences, Inc. Heteroduplex nucleic acid molecules and uses thereof
WO2019136216A1 (fr) 2018-01-05 2019-07-11 The Board Of Regents Of The University Of Texas System Compositions crispr/cas9 thérapeutiques et méthodes d'utilisation
US20200370042A1 (en) 2018-01-31 2020-11-26 The Board Of Regents Of The University Of Texas System Compositions and methods for correcting dystrophin mutations in human cardiomyocytes
EP3746082A4 (fr) 2018-02-02 2021-12-08 Fred Hutchinson Cancer Research Center Méthodes permettant de traiter la dystrophie musculaire facio-scapulo-humérale
CA3090519A1 (fr) 2018-02-07 2019-08-15 Regeneron Pharmaceuticals, Inc. Procedes et compositions pour l'administration de proteines therapeutiques
AU2019252680A1 (en) 2018-04-12 2020-10-22 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
US20230132377A9 (en) 2018-05-11 2023-04-27 Alpha Anomeric Sas Oligonucleotides conjugates comprising 7'-5'-alpha-anomeric-bicyclic sugar nucleosides
TW202015726A (zh) 2018-05-30 2020-05-01 瑞士商諾華公司 Ｅｎｔｐｄ２抗體、組合療法、及使用該等抗體和組合療法之方法
WO2019241385A2 (fr) 2018-06-13 2019-12-19 Sarepta Therapeutics, Inc. Oligomères induisant un saut d'exon pour la dystrophie musculaire
EP3810775A1 (fr) 2018-06-21 2021-04-28 The Board Of Regents Of The University Of Texas System Correction des délétions de l'exon 43, de l'exon 45 ou de l'exon 52 de la dystrophine dans la dystrophie musculaire de duchenne
KR20210081324A (ko) 2018-08-02 2021-07-01 다인 세라퓨틱스, 인크. 근육 표적화 복합체 및 안면견갑상완 근육 이영양증을 치료하기 위한 그의 용도
KR20210081323A (ko) 2018-08-02 2021-07-01 다인 세라퓨틱스, 인크. 근육 표적화 복합체 및 근긴장성 이영양증을 치료하기 위한 그의 용도
CA3108328A1 (fr) 2018-08-02 2020-02-06 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de la fibrodysplasie ossifiante progressive
US20210324101A1 (en) 2018-08-02 2021-10-21 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating hypertrophic cardiomyopathy
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
EP3830130A4 (fr) 2018-08-02 2022-05-18 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de la myopathie centronucléaire
KR20210086601A (ko) 2018-08-02 2021-07-08 다인 세라퓨틱스, 인크. 근육 표적화 복합체 및 프리드라이히 운동실조를 치료하기 위한 그의 용도
CN112955153A (zh) 2018-08-02 2021-06-11 达因疗法公司 肌肉靶向复合物及其用于治疗肌养蛋白病的用途
JP2021533198A (ja) 2018-08-02 2021-12-02 ダイン セラピューティクス, インコーポレーテッドDyne Therapeutics, Inc. 筋標的化複合体およびそれらの使用
EP3829635A4 (fr) 2018-08-02 2022-05-11 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de l'atrophie musculaire
US11168141B2 (en) 2018-08-02 2021-11-09 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
KR20210054513A (ko) 2018-08-02 2021-05-13 다인 세라퓨틱스, 인크. 근육 표적화 복합체 및 폼페병을 치료하기 위한 그의 용도
IT201800009682A1 (it) 2018-10-22 2020-04-22 Ice Spa Coniugati di acidi biliari e loro derivati per la veicolazione di molecole attive
CN113260384A (zh) 2018-11-05 2021-08-13 西纳福克斯股份有限公司 用于靶向表达trop-2的肿瘤的抗体缀合物
IL297818B1 (en) 2018-12-21 2024-05-01 Avidity Biosciences Inc Anti-transferrin receptor antibodies and uses thereof
AU2020263487A1 (en) 2019-04-25 2021-12-16 Avidity Biosciences, Inc. Nucleic acid compositions and methods of multi-exon skipping
EP3980436A4 (fr) 2019-06-06 2023-12-20 Avidity Biosciences, Inc. Compositions d'acide nucléique-polypeptide et utilisations de celles-ci
AU2020287880A1 (en) 2019-06-06 2022-01-20 Avidity Biosciences, Inc. UNA amidites and uses thereof
WO2020247738A1 (fr) 2019-06-07 2020-12-10 Dyne Therapeutics, Inc. Procédés de préparation de complexes protéine-oligonucléotide
WO2021003573A1 (fr) 2019-07-09 2021-01-14 The Governors Of The University Of Alberta Saut des exons 45 à 55 à l'aide de cocktails adaptés aux mutations, de morpholinos antisens, dans le gène dmd
US20240148891A1 (en) 2019-10-18 2024-05-09 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
WO2021142217A1 (fr) 2020-01-10 2021-07-15 Dyne Thereapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour la modulation de milck1
CA3163295A1 (fr) 2020-01-10 2021-07-15 Romesh R. Subramanian Complexes de ciblage musculaire et utilisations associees pour le traitement de la dystrophie myotonique
WO2023283531A2 (fr) 2021-07-09 2023-01-12 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour la modulation de gènes associés à la santé musculaire
WO2021142331A1 (fr) 2020-01-10 2021-07-15 Dyne Therapeutics, Inc. Complexes de ciblage de muscle et leurs utilisations pour la modulation de gènes associés à une maladie du muscle cardiaque
US20230203180A1 (en) 2020-01-10 2023-06-29 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for modulation of genes associated with muscle health
WO2021142269A1 (fr) 2020-01-10 2021-07-15 Dyne Therapeutics, Inc. Complexes de ciblage de muscle et leurs utilisations pour la modulation de gènes associés à l'atrophie musculaire
WO2021142307A1 (fr) 2020-01-10 2021-07-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et utilisations de ces derniers pour le traitement de dystrophinopathies
WO2021142260A1 (fr) 2020-01-10 2021-07-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et utilisations associées pour la modulation d'acvr1
CN115335062A (zh) 2020-01-10 2022-11-11 达因疗法公司 肌肉靶向复合物及其用于调节与肌肉健康相关的基因的用途
US20230088865A1 (en) 2020-01-10 2023-03-23 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
EP4087876A4 (fr) 2020-01-10 2024-03-27 Dyne Therapeutics Inc Complexes de ciblage de muscle et leurs utilisation
US20230117883A1 (en) 2020-01-24 2023-04-20 Dyne Therapeutics, Inc. Muscle-targeting complexes and uses thereof in treating muscle atrophy
EP4096695A4 (fr) 2020-01-31 2024-02-28 Dyne Therapeutics Inc Anticorps anti-récepteur de la transferrine (tfr) et utilisations associées
WO2021154477A1 (fr) 2020-01-31 2021-08-05 Dyne Therapeutics, Inc. Anticorps anti-récepteur de la transferrine (tfr) et utilisations associées
US20230287108A1 (en) 2020-07-23 2023-09-14 Dyne Therapeutics, Inc. Muscle-targeting complexes and uses thereof
WO2022020109A1 (fr) 2020-07-23 2022-01-27 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations dans le traitement de l'atrophie musculaire
CA3186752A1 (fr) 2020-07-23 2022-01-27 Romesh R. Subramanian Complexes de ciblage musculaire et leurs utilisations dans le traitement de dystrophinopathies
US20230272065A1 (en) 2020-07-23 2023-08-31 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
CN116348138A (zh) 2020-07-23 2023-06-27 达因疗法公司 抗转铁蛋白受体（tfr）抗体及其用途
US20240016952A1 (en) 2020-07-23 2024-01-18 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
JP2023539341A (ja) 2020-09-01 2023-09-13 ウルトラジェニクス ファーマシューティカル インク． Ｄｕｘ４阻害剤およびその使用方法
KR20230079068A (ko) 2020-09-03 2023-06-05 다인 세라퓨틱스, 인크. 단백질-올리고뉴클레오티드 복합체의 제조 방법
US20240026356A1 (en) 2020-11-30 2024-01-25 Research Institute At Nationwide Children's Hospital Compositions and methods for treating facioscapulohumeral muscular dystrophy (fshd)
KR20230117182A (ko) 2020-12-04 2023-08-07 다인 세라퓨틱스, 인크. 항체-올리고뉴클레오티드 복합체 및 그의 용도
EP4271478A1 (fr) 2020-12-31 2023-11-08 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour traiter la myopathie facio-scapulo-humérale
IL304048A (en) 2020-12-31 2023-08-01 Dyne Therapeutics Inc Muscle targeting complexes and their uses for the treatment of myotonic dystrophy
WO2022159712A1 (fr) 2021-01-22 2022-07-28 Ionis Pharmaceuticals, Inc. Composés et méthodes pour réduire l'expression de dux4
EP4359534A1 (fr) 2021-06-21 2024-05-01 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de la maladie de pompe
CA3222816A1 (fr) 2021-06-21 2022-12-29 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leur utilisation pour traiter l'ataxie de friedreich
US11633498B2 (en) 2021-07-09 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
IL309936A (en) 2021-07-09 2024-03-01 Dyne Therapeutics Inc Muscle targeting complexes and formulations for the treatment of dystrophinopathy
EP4366741A2 (fr) 2021-07-09 2024-05-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations dans le traitement de dystrophinopathies
CA3226300A1 (fr) 2021-07-09 2023-01-12 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de dystrophinopathies
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
CA3226301A1 (fr) 2021-07-09 2023-01-12 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et utilisations associees pour le traitement de la dystrophie myotonique
EP4367247A2 (fr) 2021-07-09 2024-05-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations dans le traitement de dystrophinopathies
WO2023283629A1 (fr) 2021-07-09 2023-01-12 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs formulations pour traiter la dystrophie musculaire facio-scapulo-humérale
US11672872B2 (en) 2021-07-09 2023-06-13 Dyne Therapeutics, Inc. Anti-transferrin receptor antibody and uses thereof
EP4367147A1 (fr) 2021-07-09 2024-05-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de dystrophinopathies
EP4366783A1 (fr) 2021-07-09 2024-05-15 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et leurs utilisations pour le traitement de dystrophinopathies
KR20240058160A (ko) 2021-09-16 2024-05-03 다인 세라퓨틱스, 인크. 디스트로핀병증을 치료하기 위한 근육 표적화 복합체의 투여
WO2023077120A1 (fr) 2021-11-01 2023-05-04 Dyne Therapeutics, Inc. Complexes de ciblage musculaire pour traiter des dystrophinopathies
WO2023086864A1 (fr) 2021-11-12 2023-05-19 Dyne Therapeutics, Inc. Complexes de ciblage musculaire pour traiter la dystrophie musculaire facio-scapulo-humérale

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11787869B2 (en) 2018-08-02 2023-10-17 Dyne Therapeutics, Inc. Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness
US11518816B2 (en) 2018-08-02 2022-12-06 Dyne Therapeutics, Inc. Methods of delivering an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11633496B2 (en) 2018-08-02 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11833217B2 (en) 2018-08-02 2023-12-05 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11497815B2 (en) 2018-08-02 2022-11-15 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11795233B2 (en) 2018-08-02 2023-10-24 Dyne Therapeutics, Inc. Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11795234B2 (en) 2018-08-02 2023-10-24 Dyne Therapeutics, Inc. Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11648318B2 (en) 2021-07-09 2023-05-16 Dyne Therapeutics, Inc. Anti-transferrin receptor (TFR) antibody and uses thereof
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11759525B1 (en) 2021-07-09 2023-09-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US11679161B2 (en) 2021-07-09 2023-06-20 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US11672872B2 (en) 2021-07-09 2023-06-13 Dyne Therapeutics, Inc. Anti-transferrin receptor antibody and uses thereof
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
US11839660B2 (en) 2021-07-09 2023-12-12 Dyne Therapeutics, Inc. Anti-transferrin receptor antibody and uses thereof
US11844843B2 (en) 2021-07-09 2023-12-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US11633498B2 (en) 2021-07-09 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US11986537B2 (en) 2021-07-09 2024-05-21 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11931421B2 (en) 2022-04-15 2024-03-19 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy

Also Published As

Publication number Publication date
MX2021001284A (es) 2021-07-15
US11111309B2 (en) 2021-09-07
IL280426A (en) 2021-03-25
EP3830259A4 (fr) 2022-05-04
US11795233B2 (en) 2023-10-24
US11787869B2 (en) 2023-10-17
US20220324992A1 (en) 2022-10-13
WO2020028864A1 (fr) 2020-02-06
JP2021533200A (ja) 2021-12-02
KR20210081324A (ko) 2021-07-01
US20230049450A1 (en) 2023-02-16
US20210206868A1 (en) 2021-07-08
CA3108289A1 (fr) 2020-02-06
US20220169743A1 (en) 2022-06-02
WO2020028864A8 (fr) 2020-02-27
US20210380709A1 (en) 2021-12-09
US20230227569A1 (en) 2023-07-20
US11390682B2 (en) 2022-07-19
SG11202100928QA (en) 2021-02-25
AU2019316103A1 (en) 2021-03-11
EA202190416A1 (ru) 2021-06-23
EP3830259A1 (fr) 2021-06-09
US20230203181A1 (en) 2023-06-29
US20240067743A1 (en) 2024-02-29
US11795234B2 (en) 2023-10-24
US11518816B2 (en) 2022-12-06
CN112912499A (zh) 2021-06-04
US11286305B2 (en) 2022-03-29

Similar Documents

Publication Publication Date Title
US11795233B2 (en) Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11497815B2 (en) Muscle targeting complexes and uses thereof for treating dystrophinopathies
US20240148891A1 (en) Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11248056B1 (en) Muscle targeting complexes and uses thereof for treating dystrophinopathies
US20210322562A1 (en) Muscle targeting complexes and uses thereof for treating centronuclear myopathy
US20210317226A1 (en) Muscle targeting complexes and uses thereof for treating pompe disease
US20210308274A1 (en) Muscle targeting complexes and uses thereof for treating friedreich's ataxia
US20220378934A1 (en) Muscle-targeting complexes and uses thereof in treating muscle atrophy
US20210324101A1 (en) Muscle targeting complexes and uses thereof for treating hypertrophic cardiomyopathy
US20210322563A1 (en) Muscle targeting complexes and uses thereof for treating fibrodysplasia ossificans progressiva
US11911484B2 (en) Muscle targeting complexes and uses thereof for treating myotonic dystrophy
CA3108287A1 (fr) Complexes de ciblage musculaire et utilisations associees pour le traitement de la dystrophie myotonique
US20240067744A1 (en) Muscle targeting complexes and uses thereof for treating myotonic dystrophy

Legal Events

Date Code Title Description
AS Assignment

Owner name: DYNE THERAPEUTICS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SUBRAMANIAN, ROMESH R.;QATANANI, MOHAMMED T.;WEEDEN, TIMOTHY;SIGNING DATES FROM 20190827 TO 20190828;REEL/FRAME:055585/0658

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STCB Information on status: application discontinuation

Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION