[go: up one dir, main page]

WO2025174995A1 - Methods of using activin a and myostatin signaling inhibitors - Google Patents

Methods of using activin a and myostatin signaling inhibitors

Info

Publication number
WO2025174995A1
WO2025174995A1 PCT/US2025/015776 US2025015776W WO2025174995A1 WO 2025174995 A1 WO2025174995 A1 WO 2025174995A1 US 2025015776 W US2025015776 W US 2025015776W WO 2025174995 A1 WO2025174995 A1 WO 2025174995A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
exon
activin
myostatin
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.)
Pending
Application number
PCT/US2025/015776
Other languages
French (fr)
Inventor
Jasbir S. Seehra
Jennifer Lachey
Ffolliott Martin FISHER
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.)
Keros Therapeutics Inc
Original Assignee
Keros 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 Keros Therapeutics Inc filed Critical Keros Therapeutics Inc
Publication of WO2025174995A1 publication Critical patent/WO2025174995A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Glucocorticoids have been the standard of care in DMD and help preserve muscle strength and function, leading to extension of independent ambulation for several years. While glucocorticoids help to maintain muscle function in DMD patients, long-term treatment with them can have significant negative side effects, including fluid retention, hyperglycemia, severe weight gain with fat deposits in the abdomen, face and neck, bone fragility, cataracts, high blood pressure and mood effects, leading many patients to forego long-term treatment. Additionally, glucocorticoid treatment is associated with lean mass loss, an effect mediated by myostatin.
  • the present invention features methods of treating Duchenne muscular dystrophy (DMD) by administering a combination of an activin A and myostatin signaling inhibitor described herein (e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap) and a dystrophin exon skipping therapy (e.g., an exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy including a sequence of Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy including a sequence of Table 22), an exon
  • E1 A method of treating a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
  • a method of increasing lean mass in a subject having DMD comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
  • a method of increasing dystrophin expression in a subject having DMD comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
  • E6 The method of E5, wherein the dystrophin exon skipping therapy is an exon 44 skipping therapy.
  • E7 The method of E6, wherein the exon 44 skipping therapy comprises a sequence having at least
  • E9 The method of any one of E6-E8, wherein the exon 44 skipping therapy is NS-089/NCNP-02, AOC 1044, or ENTR-601 -44.
  • E28 The method of E27, wherein the exon 53 skipping therapy comprises the sequence of any one of SEQ ID NOs: 1029-1031 , 1746-1793, and 1806-1809.
  • E30 The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 2 skipping.
  • E31 The method of E30, wherein the dystrophin exon skipping therapy is an exon 2 skipping therapy.
  • E38 The method of E37, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) sequence in Table 1 and a light chain variable region (LCVR) sequence in Table 1 , such as an HCVR sequence and an LCVR sequence from the same row of Table 1 .
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • E39 The method of any one of E35, E37, and E38, wherein the activin A antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 2 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 2).
  • E45 The method of E44, wherein the myostatin antibody or an antigen binding fragment thereof has a HCVR sequence in Table 4 and a LCVR sequence in Table 4, such as an HCVR sequence and an LCVR sequence from the same row of Table 4.
  • E47 The method of any one of E42 and E44-E46, wherein the myostatin antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 8 (e.g., a heavy chain and light chain sequence from the same row of Table 8).
  • a heavy chain and light chain sequence provided in Table 8 (e.g., a heavy chain and light chain sequence from the same row of Table 8).
  • E50 The method of E49, wherein the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 ), and a myostatin HCVR and LCVR from Table 4 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, and a LCVR sequence having at least
  • E51 The method of E50, wherein the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 and a myostatin HCVR and LCVR from Table 4.
  • E52 The method of any one of E49-E51 , wherein the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 2 (e.g., an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 2) and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 5 (e.g., a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 5).
  • Table 2 e.g., an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 2
  • E53 The method of E35, wherein the anti-myostatin protein is an anti-myostatin adnectin recombinant protein.
  • E59 The method of E57, wherein the ActRII antibody or an antigen binding fragment thereof has a
  • HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 10 and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 10 (e.g., a HCVR sequence in Table 10 and a LCVR sequence in Table 10, such as an HCVR sequence and an LCVR sequence from the same row of Table 10).
  • E61 The method of any one of E57, E59, and E60, wherein the ActRII antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 11 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 11 ).
  • E62 The method of any one of E57 and E59-E61 , wherein the ActRII antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 12 (e.g., a heavy chain and light chain sequence from the same row of Table 12).
  • a heavy chain and light chain sequence provided in Table 12 (e.g., a heavy chain and light chain sequence from the same row of Table 12).
  • E65 The method of E64, wherein the ActRII ligand trap is an ActRIIA ligand trap.
  • E66 The method of E65, wherein the ActRIIA ligand trap is a composition of Table 28 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 28).
  • E67 The method of E65, wherein the ActRIIA ligand trap comprises an extracellular portion of wildtype ActRIIA (e.g., SEQ ID NO: 2 or SEQ ID NO: 783).
  • E69 The method of E64, wherein the ActRII ligand trap is an ActRIIB ligand trap.
  • E70 The method of E69, wherein the ActRIIB ligand trap comprises an extracellular portion of wildtype ActRIIB (e.g., SEQ ID NO: 4 or a portion thereof).
  • E73 The method of E69, wherein the ActRIIB ligand trap comprises the sequence of any one of SEQ ID NOs: 871 -876 (e.g., the sequence of any one of SEQ ID NOs: 871 -876 fused to an Fc domain monomer, by way of a linker).
  • E74 The method of E64, wherein the ActRII ligand trap is an ActRII chimera ligand trap.
  • E75 The method of E74, wherein the ActRII chimera ligand trap is a composition of Table 30 or Table
  • E77 The method of E76, wherein the chimera has the sequence of SEQ ID NO: 895.
  • E78 The method of E76, wherein the chimera has the sequence of SEQ ID NO: 888.
  • E81 The method of E80, wherein is E or D, Xs is N or S, and X
  • E83 The method of E76, wherein the chimera has the sequence of any one of SEQ ID NOs: 898-919.
  • E84 The method of E83, wherein the chimera has the sequence of SEQ ID NO: 917.
  • E85 The method of E83, wherein the chimera has the sequence of SEQ ID NO: 901 .
  • E86 The method of E83, wherein the chimera has the sequence of SEQ ID NO: 916.
  • E87 The method of any one of E76-E86, wherein the polypeptide further includes an Fc domain monomer fused to the C-terminus of the polypeptide (e.g., the C-terminus of the chimera) by way of a linker.
  • E90 The method of E88, wherein the polypeptide has the sequence of SEQ ID NO: 1162.
  • E92 The method of any one of E87-E91 , wherein the polypeptide is in the form of a dimer (e.g., a homodimer).
  • a dimer e.g., a homodimer
  • E93 The method of any one of E1 -E92, wherein the method increases lean mass.
  • E94 The method of any one of E1 -E93, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in lean mass compared to administration of the dystrophin exon skipping therapy alone.
  • E97 The method of any one of E1 -E96, wherein the method increases muscle strength.
  • E99 The method of any one of E1 -E98, wherein the method increases dystrophin expression.
  • E100 The method of any one of E1 -E99, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase of dystrophin expression compared to administration of either agent alone.
  • E101 The method of any one of E1 -E100, wherein the method slows or inhibits the progression of DMD, reduces or inhibits muscle atrophy or wasting, preserves ambulation or slows the loss of ambulation, or reduces or inhibits respiratory and/or cardiac complications (e.g., compared to administration of the dystrophin exon skipping therapy alone).
  • E102 The method of any one of E1 -E101 , wherein the subject is a human.
  • any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds.
  • “administration” refers to providing or giving a subject a therapeutic agent (e.g., an activin A and myostatin signaling inhibitor or a dystrophin exon skipping therapy described herein), by any effective route. Exemplary routes of administration are described herein below.
  • anti-myostatin protein refers to any protein that binds specifically to myostatin and reduces myostatin signaling.
  • exemplary anti-myostatin proteins include myostatin antibodies or antigen binding fragments thereof and anti-myostatin adnectin recombinant proteins.
  • antibody is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • Antibody fragments include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • extracellular activin receptor type HA (ActRIIA) variant and “ActRIIA variant” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIA, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 1 shown below).
  • the sequence of the wild-type, human ActRIIA precursor protein is shown below (SEQ ID NO: 1 ), in which the signal peptide is italicized and the extracellular portion is bold.
  • an extracellular ActRIIA variant has a sequence of any one of SEQ ID NOs: 789- 855 (Table 14).
  • an extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 2).
  • extracellular activin receptor type IIB (ActRIIB) variant and “ActRIIB variant” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIB, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 3 shown below).
  • the sequence of the wild-type, human ActRIIB is shown below (SEQ ID NO: 3), in which the signal peptide is italicized and the extracellular portion is bold.
  • the ActRII chimeras result from the substitution of one or more amino acid sequence corresponding a p-sheet from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA) and/or from the substitution of one or more intervening sequence (e.g., a sequence between the p- sheets) from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA).
  • one or more intervening sequence e.g., a sequence between the p- sheets
  • the C-terminal extension can be one or more amino acids, such as 1 -6 amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids).
  • the C-terminal extension may include amino acids from the corresponding position of wild-type ActRIIA (for an ActRIIA variant) or from the corresponding position of wild-type ActRIIA or ActRIIB (for an ActRII chimera).
  • Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 1090) (a six amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used.
  • percent (%) identity refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.
  • the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence is calculated as follows:
  • serum half-life refers to, in the context of administering a therapeutic protein to a subject, the time required for plasma concentration of the protein in the subject to be reduced by half.
  • the protein can be redistributed or cleared from the bloodstream, or degraded, e.g., by proteolysis. Serum half-life comparisons can be made by comparing the serum half-life of Fc fusion proteins.
  • binding affinity refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum total of non-covalent interactions between a molecule and its binding partner. Unless indicated otherwise, binding affinity refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair.
  • the binding affinity between two molecules is commonly described by the dissociation constant (KD) or the affinity constant (KA). TWO molecules that have low binding affinity for each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. TWO molecules that have high affinity for each other generally bind readily, tend to remain bound longer, and exhibit a small KD.
  • KD dissociation constant
  • KA affinity constant
  • TWO molecules that have high affinity for each other generally bind readily, tend to remain bound longer, and exhibit a small KD.
  • the KD of two interacting molecules may be determined using methods and techniques well known in the art, e.g., surface plasmon resonance. KD is calculated as the ratio of
  • polypeptide describes a single polymer in which the monomers are amino acid residues which are covalently conjugated together through amide bonds.
  • a polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.
  • the term “host cell” refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express proteins from their corresponding nucleic acids.
  • the nucleic acids are typically included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.).
  • a host cell may be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or a HEK293 cell).
  • the terms “effective amount” and “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a subject having DMD, it is an amount of the composition, polypeptide, nucleic acid, or vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, polypeptide, nucleic acid, or vector.
  • treatment and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results.
  • beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable.
  • RNA refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after a portion of a pre-processed (or precursor) RNA has been removed by splicing.
  • the mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • DBA2J mice treated with TBS intraperitoneal injection served as controls (WT).
  • the lean mass was determined by nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec) on days -1 , 25, and 55 of the study. Data are shown as a percentage of lean mass change from baseline measurements. From left to right at each timepoint: WT: Vehicle, D2MDX: Vehicle, D2MDX: Chimera 1/2b-mFc, D2MDX: PMO-1 , D2MDX: PMO-1 + Chimera 1/2b-mFc. Data are shown as mean ⁇ SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test. * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer).
  • Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ⁇ SEM. *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer).
  • Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ⁇ SEM. *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally.
  • the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • Mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer).
  • GX2 pCT 5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer.
  • Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ⁇ SEM. *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • WT intraperitoneal injection
  • Data are shown as mean ⁇ SEM.
  • D2MDX Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****p ⁇ 0.0001 ;
  • WT intraperitoneal injection
  • Data are shown as mean ⁇ SEM.
  • WT controls
  • Data in FIG. 11 A are shown as a percentage of exon skipping, as mean ⁇ SEM.
  • FIG. 11 B shows a representative image of a gel showing exon skipping.
  • WT TBS intraperitoneal injection
  • Data are shown as mean ⁇ SEM.
  • TBS vehicle
  • PMO-1 PMO-1 in combination with Chimera 1/2b-mFc
  • WT controls
  • FIG. 13A is a graph showing percentage of dystrophin-positive fibers as mean ⁇ SEM.
  • FIG. 13B is a series of representative images showing dystrophin protein localization.
  • FIG. 14A is a graph showing % of CD31 - Sca.1 - cells.
  • the invention features methods of co-administering a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
  • the activin A and myostatin signaling inhibitor can be an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap.
  • Activin type II receptors are single transmembrane domain receptors that modulate signals for ligands in the transforming growth factor p (TGF-p) superfamily.
  • Ligands in the TGF-p superfamily are involved in a host of physiological processes, such as muscle growth, vascular growth, cell differentiation, homeostasis, and osteogenesis.
  • Examples of ligands in the TGF-p superfamily include, e.g., activin, inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9).
  • GDFs growth differentiation factors
  • BMPs bone morphogenetic proteins
  • Myostatin and activins are known to play a role in the regulation of skeletal muscle growth. For example, mice without myostatin show a large increase in skeletal muscle mass. Methods that reduce or inhibit activin A and myostatin signaling could, therefore, be used in
  • the present invention is based, in part, on the discovery by the present inventors that the combination of an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy further increased muscle strength and dystrophin expression in a mouse model of DMD compared to administration of the activin A and myostatin signaling inhibitor or the dystrophin exon skipping therapy alone and further increased lean mass compared to administration of the dystrophin exon skipping therapy alone.
  • administration of the activin A and myostatin signaling inhibitor may lead to increased numbers of satellite cells, which may allow for increased cellular uptake of the dystrophin exon skipping therapy.
  • the activin A and myostatin signaling inhibitor is an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein).
  • an anti-myostatin protein e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein.
  • the activin A antibody is garetosmab (also known as REGN-2477). Additional activin A antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2015017576, WO2013074557, W02008031061 , and WO2023147107; US Patent Application Publication No. US20150359850; and US Patent Nos. 9,718,881 , 10,526,403, 8,309,082, 8,753,627, and 10,100,109, each of which is incorporated herein by reference.
  • the activin A antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , such as any one of SEQ ID NOs: 5, 7, 9, 10, 11 , 13, 15, 17, 18, 19, 21 , 23, 25, 27, 29, 31 , 32, 34, 36, 37, 39, 41 , 43, 45, 47, 48, 50, 52, and 54) and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 , such as any one of SEQ ID NOs: 6, 8, 12, 14, 16, 20, 22, 24, 26, 28, 30, 33, 35, 38, 40, 42, 44, 46, 49, 51 , and 53.
  • the activin A antibody or an antigen binding fragment thereof apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 1 .
  • the activin A antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 1 .
  • the activin A antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 1 .
  • the activin A antibody or an antigen-binding fragment thereof has the CDR sequences described in Table 2 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3).
  • the activin A antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 2, such as any one of SEQ ID NOs: 58, 64, 70, 76, 82, 88, 100, 119, 130, 141 , and 147; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 59, 65, 71 , 77, 83, 92, 101 , 110, 114, 120, 125, 131 , 142, and 148; a light chain variable CDR3 sequence having at least 90% (e
  • a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 56, 62, 68, 74, 80, 86, 91 , 97, 99, 104, 106, 108, 1 13, 1 17, 123, 128, 133, 135, 139, 145, and 151 ; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 2, such as any one of SEQ ID NOs: 57, 63, 69, 75, 81 ,
  • the activin A antibody or an antigen-binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 3.
  • the activin A antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence from the same row of Table 3.
  • the heavy chain and light chain have the sequence of SEQ ID NOs: 1094 and 1095; 1096 and 1097; 1098 and 1099; 1100 and 1101 ; 1102 and 1095; 1103 and 1104; 1105 and 1106; 1107 and 1106; 1108 and 1109; 1110 and 1111 ; 1112 and 1113; 1114 and 1115; 1116 and 1117; 1118 and 1117; 1119 and 1120; 1121 and 1122; 1123 and 1124; or 1125 and 1126 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the sequence of the sequence
  • the anti-myostatin protein is a myostatin antibody or an antigen binding fragment thereof.
  • the myostatin antibody is domagrozumab (also known as PF- 06252616), landogrozumab (also known as LY2495655), trevogrumab (also known as REGN-1033), or apitegromab (SRK-015). Additional myostatin antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos.
  • the myostatin antibody or antigen binding fragment thereof includes an HCVR sequence of any one of SEQ ID NOs: 278, 280-284, 286, 287, 289-291 , 293, 295, 297, 299, 301 , 303, and 305-316 and an LCVR sequence of any one of SEQ ID NOs: 279, 285, 288, 292, 294, 296, 298, 300, 302, and 304.
  • the myostatin antibody or an antigen-binding fragment thereof has the CDR sequences described in Table 5, 6, or 7 (i.e. , a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3).
  • the myostatin antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 358, 363, 368, 374, 377, 389, 395, 400, 405, 413, 419, 425, 431 , 436, 442, 445, 449, 454, 458, 466, 469, 473, 476, 478, 516, and 517-519; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 36
  • the myostatin antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 516; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1260; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 510; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)
  • the ActRII antibody or an antigen-binding fragment thereof has the CDR sequences described in Table 1 1 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3).
  • an ActRIIA ligand trap including an extracellular ActRIIA variant e.g., any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)
  • an ActRIIA ligand trap including an extracellular ActRIIA variant e.g., any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) with the sequence TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26 can have a substitution of the amino acid E for the amino acid K at position X24.
  • ActRIIA variants having the sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26 have reduced or weak binding to BMP9 (e.g., reduced binding to BMP9 compared to BMP9 binding of wild-type ActRIIA).
  • an ActRIIA ligand trap including an extracellular ActRIIA variant may further include a C-terminal extension (e.g., one more additional amino acids at the C-terminus of the ActRIIA variant).
  • the C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA.
  • the C- terminal extension is amino acid sequence NP.
  • a sequence including the C-terminal extension NP is SEQ ID NO: 854 (e.g., SEQ ID NO: 852 with a C-terminal extension of NP).
  • the C-terminal extension is amino acid sequence NPVTPK (SEQ ID NO: 1090).
  • a sequence including the C-terminal extension NPVTPK (SEQ ID NO: 1090) is SEQ ID NO: 855 (e.g., SEQ ID NO: 852 with a C-terminal extension of NPVTPK (SEQ ID NO: 1090)).
  • the C-terminal extension can add one or more amino acids at the C-terminus of the ActRIIA variant (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids).
  • an ActRIIA ligand trap including an extracellular ActRIIA variant may further include an Fc domain monomer, which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIA variant by way of a linker or other covalent bonds.
  • a polypeptide including an extracellular ActRIIA variant fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.
  • an ActRIIA ligand trap described herein has a serum half-life of at least 7 days in humans.
  • the ActRIIA ligand trap may bind to activin A with a KD of 10 pM or higher.
  • the ActRIIA ligand trap does not bind to BMP9 or activin A.
  • the ActRIIA ligand trap binds to activin A, activin B, and/or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA).
  • the ActRIIA ligand trap that has reduced or weak binding to BMP9 has the sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26. In some embodiments, the ActRIIA ligand trap does not substantially bind to human BMP9.
  • the ActRIIA ligand trap may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM).
  • a KD of about 800 pM or less e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM.
  • the ActRIIA ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM).
  • the ActRIIA ligand trap is sotatercept (also known as ACE-011 ). Additional ActRIIA ligand traps that may be used in the methods described herein include those described in International Patent Application Publication No. W02007062188 and US Patent Nos.
  • the extracellular ActRIIB variant has an N-terminal truncation of 1 -7 amino acids (e.g., 1 , 2, 3, 4, 5, 6, or 7 amino acids).
  • An N-terminal truncation can be produced by removing 1 -7 amino acids from the N-terminus of an of an ActRIIB variant shown in Tables 15 and 16.
  • the N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE) are retained in the N-terminally truncated ActRIIB variants). Additional ActRIIB variants having an N-terminal truncation are provided below:
  • the ActRIIB ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM)
  • the ActRIIB ligand trap may also bind to growth and differentiation factor 11 (GDF-11 ) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or
  • the ActRII ligand trap is an ActRII chimera ligand trap.
  • the ActRII chimera ligand traps contain portions of extracellular ActRIIA (e.g., human ActRIIA) and extracellular ActRIIB (e.g., human ActRIIB).
  • the ActRII chimera ligand traps described herein contain an N- terminal portion of extracellular ActRIIB (SEQ ID NO: 4 shown above) joined to a C-terminal portion of extracellular ActRIIA (SEQ ID NO: 2 shown above) such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA).
  • the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the six amino acids found at the N-terminus of extracellular ActRIIA joined to the fifth amino acid of extracellular ActRIIB.
  • the N- terminus of the ActRII chimera included in the ActRII chimera ligand trap begins with the first amino acid located at the N-terminus of extracellular ActRIIB. Accordingly, in some embodiments, the N-terminal portion of ActRIIB begins with the amino acid in the fifth position of SEQ ID NO: 4 (A), while in other embodiments (e.g., in embodiments in which the six amino acids found at the N-terminus of extracellular ActRIIA are not included in the chimera), the N-terminal portion of ActRIIB begins with the amino acid in the first position of SEQ ID NO: 4 (G).
  • the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the first ten amino acids found at the N-terminus of extracellular ActRIIA joined to the ninth amino acid of extracellular ActRIIB, in which case the N-terminal portion of ActRIIB begins with the amino acid in the ninth position of SEQ ID NO: 4.
  • Amino acid substitutions can alter the activity and/or binding affinity of the extracellular ActRII chimeras.
  • the extracellular ActRII chimeras bind to activin A, activin B, myostatin, and/or GDF11 with sufficient affinity to compete with endogenous activin receptors for binding to one or more of these ligands.
  • the extracellular ActRII chimeras have reduced, weak, or no substantial binding to BMP9 (e.g., compared to wild-type ActRIIB).
  • BMP9 binding may be reduced in extracellular ActRII chimeras containing the amino acid sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X3, X4, X5, and Xe.
  • BMP9 binding is reduced in extracellular ActRII chimeras containing the amino acid sequence KKDS (SEQ ID NO: 1245) or TKDS (SEQ ID NO: 1246) at positions X3, X4, X5, and Xe.
  • a polypeptide including an extracellular ActRII chimera e.g., any one of SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919)
  • TEEN SEQ ID NO: 1092
  • X3, X4, X5, and Xe can have a substitution of the amino acid K for the amino acid E at position X4.
  • a polypeptide including an extracellular ActRII chimera e.g., any one of SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919)
  • sequence TKEN SEQ ID NO: 1093
  • X3, X4, X5, and Xe can have a substitution of the amino acid E for the amino acid K at position X4.
  • TEEN SEQ ID NO: 1092
  • TKEN SEQ ID NO: 1093
  • extracellular ActRII chimeras e.g., the chimeras in Tables 17 and 18, e.g., SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919).
  • the ActRII chimera has the sequence of an ActRII chimera listed in Table , below. Table 20. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 1128-1158
  • a polypeptide containing an extracellular ActRII chimera of Table 20 may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 300 pM and about 1 pM).
  • the polypeptide containing an extracellular ActRII chimera of Table 20 may bind to GDF-8 with a KD of approximately 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM).
  • a KD of approximately 800 pM or less e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM.
  • the polypeptide containing an extracellular ActRII chimera of Table 20 may also bind to human BMP10 with a KD of about 1 pM or higher (e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM or higher).
  • a KD of about 1 pM or higher e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM or higher.
  • the extracellular ActRII chimeras described herein have an N-terminal truncation of 1 -9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids).
  • the N-terminal truncation can involve the removal of 1 -9 amino acids from the N-terminus of any of the chimeras shown in Tables 17- 20.
  • the N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimera ligand traps).
  • a polypeptide containing an extracellular ActRII chimera described herein has a serum half-life of at least 7 days in humans.
  • the polypeptide containing an ActRII chimera described herein may bind to activin A with a KD of 1 pM or higher (e.g., 10 pM or higher).
  • the polypeptide containing an ActRII chimera described herein binds to activin A, activin B, and/or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., compared to wild-type extracellular ActRIIB).
  • the polypeptide containing an ActRII chimera described herein does not substantially bind to human BMP9.
  • Dystrophin exon 44 skipping therapies in clinical trials include NS-089/NCNP-02 (phase 2), AOC 1044 (phase 1/2), and ENTR-601 -44 (phase 1 ).
  • NS-089/NCNP-02 (also known as brogidirsen) is a PMO having the sequence of SEQ ID NO: 949 and is described in International Patent Application Publication No. WO2015194520 and Watanabe et al. (Mol. Ther. Nucleic Acids 34, 2023), each of which is incorporated herein by reference.
  • Additional dystrophin exon 44 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2015194520, WO2023196400, and WO2023034817, each of which is incorporated herein by reference.
  • the exon 44 skipping therapy includes 18-30 consecutive nucleotides of SEQ ID NO: 985, in which the first nucleotide starts at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • any one or more of the thymine bases (T) may optionally be a uracil base (U).
  • an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 45 skipping in combination with a dystrophin exon 45 skipping therapy.
  • Casimersen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 45 skipping (under brand name AMONDYS 45TM). It can be administered as an intravenous infusion over 35 to 60 minutes via an in-line 0.2 micron filter at a dose of 30 milligrams per kilogram of body weight once weekly.
  • Casimersen is a PMO having the sequence of SEQ ID NO: 991 .
  • Eteplirsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 51 skipping (under brand name EXONDYS 51 TM). It can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly. Eteplirsen is a PMO having the sequence of SEQ ID NO: 1000.
  • Drisapersen is a 2'-O-methyl phosphorothioate modified oligonucleotide having the sequence of SEQ ID NO: 1002 and is described in International Patent Application Publication No. WQ2009054725A2.
  • PGN-EDO51 is a PMO conjugated to a peptide and is described in International Patent Application Publication No. WO2022192749, which is incorporated herein by reference.
  • Suvodirsen also known as WVE-210201
  • Sp 2’-fluoro-2'-deoxy-modified phosphorothioate
  • P phosphodiester modified oligonucleotide having the sequence of SEQ ID NO: 1028 and is described in International Patent Application Publication No.
  • the exon 51 skipping therapy includes a sequence listed in Table 24.
  • the exon 51 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 24, such as any one of SEQ ID NOs: 1000-1028, 1247-1256, 1581 -1745, 1805, or 1897.
  • the exon 51 skipping therapy has a sequence provided in Table 24.
  • any one or more of the thymine bases (T) may optionally be a uracil base (U).
  • any one of SEQ ID NOs: 1002-1028 or 1247-1256 contains U in place of T.
  • an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 53 skipping in combination with a dystrophin exon 53 skipping therapy.
  • Golodirsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping (under brand name VYONDYS 53TM). It can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly.
  • Golodirsen is a PMO having the sequence of SEQ ID NO: 1029.
  • Viltolarsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping (under brand name VILTEPSOTM). It can be administered as an intravenous infusion over 60 minutes at a dose of 80 mg/kg once weekly. Viltolarsen is a PMO having the sequence of SEQ ID NO: 1030.
  • WVE-N531 (phase 1 b/2a).
  • WVE-N531 is a 2’- F and 2’-OMe modified oligonucleotide having the sequence of SEQ ID NO: 1031 with 17 stereodefined internucleotide linkages, 14 of which are Sp phosphorothioate linkages, and three as Rp N-(1 ,3- dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages, and is described in WO2023168014A2, which is incorporated herein by reference.
  • Additional dystrophin exon 53 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2018118662A1 , W02020004675A1 , and WO2023168014A2, each of which is incorporated herein by reference.
  • any one or more of the thymine bases (T) may optionally be a uracil base (U).
  • SEQ ID NO: 1031 contains U in place of T.
  • an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 2 skipping in combination with a dystrophin exon 2 skipping therapy.
  • Exon 2 skipping therapies in clinical trials include scAAV9.U7.ACCA (phase 1/2).
  • scAAV9.U7.ACCA (SEQ ID NO: 1032) contains four copies of the U7snRNA in a self-complementary genome, with two copies targeting the splice acceptor site and two targeting the splice donor, encapsulated in AAV9 (scAAV9.U7.ACCA), as described in International Patent Application Publication No. WO2022067257A1 , which is incorporated herein by reference.
  • the exon 2 skipping therapy includes a sequence listed in Table 26. In some embodiments, the exon 2 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table
  • dystrophin exon 2 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication No. WO2022067257A1 , which is incorporated herein by reference.
  • the exon 2 skipping therapy has the sequence in Table 26. Table 26. Exemplary exon 2 skipping therapies
  • any one or more of the thymine bases (T) may optionally be a uracil base (U).
  • the dystrophin exon-skipping therapies disclosed herein may contain naturally occurring (e.g., DNA or RNA) and/or modified nucleotides (e.g., naturally occurring nucleotides with one or more modifications in order to increase the stability and/or therapeutic efficiency in vivo).
  • modified nucleotides e.g., naturally occurring nucleotides with one or more modifications in order to increase the stability and/or therapeutic efficiency in vivo.
  • Modifications that will improve the efficacy of an exon-skipping therapy of the disclosure such as a stabilizing modification and/or a modification that reduces RNase H activation in order to avoid degradation of the targeted transcript are known in the art (see, e.g., Bennett and Swayze, Annu. Rev. Pharmacol. Toxicol. 50:259- 293, 2010; and Juliano, Nucleic Acids Res. 19;44(14):6518-48, 2016).
  • the dystrophin exon-skipping therapies may include one or more modifications, each of which is independently a backbone modification, nucleobase modification, sugar modification, or conjugation.
  • the modification is a backbone modification.
  • oligonucleotides with backbone modifications include morpholinos, phosphorodiamidate morpholino oligomers (PMO), peptide nucleic acid (PNA), phosphorothioate (PS) oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, stereochemically pure phosphorothioate (PS) oligonucleotides, phosphoramidite oligonucleotides, P-ethoxy oligonucleotides, boranephosphate oligonucleotides; thiophosphoramidate oligonucleotides, and methylphosphonate oligonucleotides.
  • the modification is a nucleobase modification.
  • modified nucleobases include bicycle modified oligonucleotides, Bicyclic Nucleic Acid (BNA), tricycle modified oligonucleotides, tricyclo-DNA-antisense oligonucleotides (ASOs), 5-methyl substitution on pyrimidine nucleobases (e.g., 5-methylcytosine), 5-substituted pyrimidine analogues, 2-Thio-thymine modified oligonucleotides, and purine modified oligonucleotides.
  • BNA Bicyclic Nucleic Acid
  • ASOs tricyclo-DNA-antisense oligonucleotides
  • 5-methyl substitution on pyrimidine nucleobases e.g., 5-methylcytosine
  • 2-Thio-thymine modified oligonucleotides 2-Thio-thymine modified oligonucleotides
  • the modification is a sugar modification.
  • oligonucleotides with sugar modifications include Locked Nucleic Acid (LNA) oligonucleotides, 2’,4’-Methyleneoxy Bridged Nucleic Acid (BNA), ethylene-bridged nucleic acid (ENA) oligonucleotides, constrained ethyl (cEt) oligonucleotides, oligonucleotides with modifications at the 2' position of the sugar (e.g., 2’-0-Me RNA (2’- OMe), 2’-O-Methoxyethyl (MOE), and 2’-Fluoro), and oligonucleotides with modifications at the 4' position of the sugar (e.g., 4’-Thio).
  • LNA Locked Nucleic Acid
  • BNA BNA
  • EAA ethylene-bridged nucleic acid
  • cEt constrained ethyl
  • MOE 2’-O-Met
  • the modification is a conjugation.
  • the oligonucleotides may be conjugated to another molecule, such as tri-ethylene glycol (TEG) (e.g., a TEG group attached at the 5’ terminus of the oligonucleotide via a phosphate group), N-acetyl galactosamine (GalNAc) oligonucleotide conjugates such as 5’-GalNAc and 3’-GalNAc oligonucleotides conjugates, lipid oligonucleotide conjugates, cell penetrating peptide (CPP) oligonucleotide conjugates, targeted oligonucleotide conjugates, antibody-oligonucleotide conjugates, and polymer-oligonucleotide conjugate such as with PEGylation or targeting ligands.
  • TEG tri-ethylene glycol
  • GalNAc N-acetyl galactosamine
  • CPP cell penetrating peptid
  • the exon-skipping therapy of any one of SEQ ID NOs: 949-991 , 1000, 1001 , 1003-1027, 1029, 1030, and 1247-1256 may be a phosphorodiamidate morpholino oligomer (PMO), as described in International Patent Application Publication Nos.
  • PMO phosphorodiamidate morpholino oligomer
  • the exon-skipping therapy of SEQ ID NO: 1002 may be a 2'-O-methyl phosphorothioate oligomer, as described in International Patent Application Publication No. W02009054725A2, which is incorporated herein by reference.
  • the exon-skipping therapy of SEQ ID NO: 1031 may be a 2’-F and 2’-OMe modified oligonucleotide with 17 stereodefined internucleotide linkages, 14 of which are identified as Sp phosphorothioate linkages, and three as Rp N-(1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages.
  • internucleotide linkages can be illustrated as: 5’-SSRSSRSSOSSSOSSSRSS-3’, where ‘S’, ‘R’, and ‘O’ represent Sp phosphorothioate linkage, Rp N-(1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage, and phosphate linkages, respectively, as described in International Patent Application Publication No. WO2023168014A2, which is incorporated herein by reference.
  • the exon-skipping therapy of SEQ ID NO: 1028 may be a 2'-fluoro-2'- deoxy-modified phosphorothioate (Sp)/phosphodiester modified oligonucleotide, as described in International Patent Application Publication No. WO2019217784A1 , which is incorporated herein by reference.
  • the exon-skipping therapy of SEQ ID NO: 992 may be 2'-O,4'-C-ethylene- bridged nucleic acid phosphorothioate modified oligonucleotide, as described in International Patent Application Publication No. W02022020107A1 , which is incorporated herein by reference.
  • the exon-skipping therapy of any one of SEQ ID NO: 984-990, 1001 , or 1247-1256 may be a PMO conjugated to a peptide (e.g., a cell-penetrating peptide, a muscle-targeting peptide, a cyclic peptide, or an endosomal escape vehicle), as described in International Patent Application Publication Nos. WO2022232478A1 , WQ2022192749, and WQ2023034817A1 , each of which is incorporated herein by reference.
  • a peptide e.g., a cell-penetrating peptide, a muscle-targeting peptide, a cyclic peptide, or an endosomal escape vehicle
  • the exon-skipping therapy of any one of SEQ ID NOs: 950-983 or 1003- 1027 may be a PMO conjugated to an antibody or antigen-binding fragment thereof (e.g., a transferrin antibody or antigen-binding fragment thereof), as described in International Patent Application Publication No. WQ2023196400A2 and US Patent Application Publication No. US20230045002A1 , each of which is incorporated herein by reference.
  • an antibody or antigen-binding fragment thereof e.g., a transferrin antibody or antigen-binding fragment thereof
  • the exon-skipping therapy of SEQ ID NO: 1897 may include the following modifications: cytosine nucleotides are 5-methylcytosine, guanine and cytosine nucleotides are locked nucleic acids (LNA), a tri-ethylene glycol (TEG) group is attached to the 5' terminus via a phosphate group, the internucleoside linkages are phosphorothioate linkages, and the non-LNA nucleotides are 2'- OMe nucleotides, as described in U.S. Patent Application Publication No. US20230416741 A1 , which is incorporated herein by reference.
  • LNA locked nucleic acids
  • TEG tri-ethylene glycol
  • ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer of an immunoglobulin or a fragment of an Fc domain to increase the serum half-life of the polypeptide.
  • An ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which form an Fc domain in the dimer.
  • an Fc domain is the protein structure that is found at the C-terminus of an immunoglobulin.
  • An Fc domain includes two Fc domain monomers that are dimerized by the interaction between the CH3 antibody constant domains.
  • a wild-type Fc domain forms the minimum structure that binds to an Fc receptor, e.g., FcyRI, FcyRlla, FcyRllb, FcyRllla, FcyRlllb, FcyRIV.
  • an Fc domain may be mutated to lack effector functions, typical of a “dead” Fc domain.
  • an Fc domain may include specific amino acid substitutions that are known to minimize the interaction between the Fc domain and an Fey receptor.
  • an Fc domain does not induce any immune system- related response.
  • the Fc domain in a dimer of an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and an Fey receptor.
  • the sequence of an Fc domain monomer that may be included in an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below (SEQ ID NO: 1033):
  • the Fc domain monomer fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof lacks a terminal lysine.
  • An exemplary sequence for a wild-type Fc domain monomer lacking the terminal lysine is provided below (SEQ ID NO: 1035):
  • an Fc domain is from an IgG 1 antibody and includes amino acid substitutions L12A, L13A, and G15A, relative to the sequence of SEQ ID NO: 1033. In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions D43A, K100A, and N212A, relative to the sequence of SEQ ID NO: 1033. In some embodiments, the terminal lysine is absent from the Fc domain monomer having the sequence of SEQ ID NO: 1033 or SEQ ID NO: 1034.
  • an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein may be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 1033, SEQ ID NO: 1034, or SEQ ID NO: 1035) through conventional genetic or chemical means, e.g., chemical conjugation.
  • a linker e.g., a spacer
  • the Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof.
  • the Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD.
  • the Fc domain monomer can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4).
  • the Fc domain monomer is an IgG 1 Fc domain monomer (e.g., a human lgG1 Fc domain monomer).
  • an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain.
  • the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization.
  • the Fc domain contains a hinge domain.
  • the Fc domain can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4).
  • the Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.
  • one or more amino acids with large sidechains may be introduced to the CH3-CH3 dimer interface to hinder dimer formation due to steric clash.
  • one or more amino acids with small sidechains e.g., alanine, valine, or threonine
  • Methods of introducing amino acids with large or small sidechains in the CH3 domain are described in, e.g., Ying et al. ⁇ J Biol Chem. 287:19399-19408, 2012), U.S.
  • one or more amino acid residues in the CH3 domain that make up the CH3-CH3 interface between two Fc domains are replaced with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable depending on the specific charged amino acids introduced.
  • positively charged amino acid residues e.g., lysine, arginine, or histidine
  • negatively charged amino acid residues e.g., aspartic acid or glutamic acid
  • an Fc domain includes one or more of the following amino acid substitutions: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351 H, L351 N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K
  • a spacer can contain motifs of GGGGA (SEQ ID NO: 1042) or GGGGS (SEQ ID NO: 1043), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 1069) and GGGGSGGGGSGGGGS (SEQ ID NO: 1070).
  • an amino acid spacer between an Fc domain monomer and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein may be GGG, GGGA (SEQ ID NO: 1039), GGGG (SEQ ID NO: 1041 ), GGGAG (SEQ ID NO: 1071 ), GGGAGG (SEQ ID NO: 1072), or GGGAGGG (SEQ ID NO:
  • polypeptides of SEQ ID NOs: 1159-1243 may be expressed including a C- terminal lysine, which then may be proteolytically cleaved upon expression of the polypeptide (e.g., the polypeptides of SEQ ID NOs: 1159-1243 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue).
  • the polypeptides of SEQ ID NOs: 1159-1243 may also be expressed without including the C-terminal lysine residue.
  • Table 27 Polypeptides containing an ActRII variant fused to an Fc domain monomer by way of a linker
  • the activin A and myostatin signaling inhibitors described herein can be produced from a host cell.
  • a host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids.
  • the nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like).
  • transformation, transfection e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like.
  • the choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.
  • a nucleic acid sequence encoding the amino acid sequence of a polypeptide described herein may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, ligation, and overlap extension PCR.
  • a nucleic acid molecule encoding a polypeptide described herein may be obtained using standard techniques, e.g., gene synthesis.
  • a nucleic acid molecule encoding a wild-type extracellular ActRIIA or ActRIIB may be mutated to include specific amino acid substitutions using standard techniques in the art, e.g., QuikChangeTM mutagenesis.
  • Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.
  • a nucleic acid sequence encoding a polypeptide described herein may be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells.
  • Many vectors are available in the art and can be used for the purpose of the invention.
  • Each vector may include various components that may be adjusted and optimized for compatibility with the particular host cell.
  • the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding protein of interest, and a transcription termination sequence.
  • mammalian cells may be used as host cells for the invention.
  • mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells.
  • HEK human embryonic kidney
  • CHO Chinese hamster ovary
  • HeLa HeLa
  • COS Chinese hamster ovary
  • PC3, Vero Chinese hamster ovary
  • CHO Chinese hamster ovary
  • HeLa HeLa
  • COS Chinese hamster ovary
  • PC3, Vero Chinese hamster ovary
  • CHO Chinese hamster ovary
  • co// strains include, but are not limited to, E. coli 294 (ATCC® 31 ,446), E. coli h 1776 (ATCC®31 ,537, E. coli BL21 (DE3) (ATCC® BAA- 1025), and E. coli RV308 (ATCC® 31 ,608).
  • E. coli 294 ATCC® 31 ,446
  • E. coli h 1776 ATCC®31 ,537
  • E. coli BL21 (DE3) ATCC® BAA- 1025
  • E. coli RV308 ATCC® 31 ,608.
  • Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the polypeptide expressed.
  • the above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection.
  • host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012.
  • Host cells used to produce the polypeptides described herein may be grown in media known in the art and suitable for culturing of the selected host cells.
  • suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293TM Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640.
  • suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin.
  • Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10%.
  • the pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
  • the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture media. Protein recovery may involve filtering the cell culture media to remove cell debris.
  • the proteins may be further purified.
  • a polypeptide described herein may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • marker amino acid sequence is a hexa-histidine peptide (His- tag), which binds to nickel-functionalized agarose affinity column with micromolar affinity.
  • His- tag hexa-histidine peptide
  • Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).
  • the polypeptides described herein can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, and alphaviral vector)) containing a nucleic acid molecule encoding the polypeptide.
  • a vector such as a viral vector (e.g., a retroviral vector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, and alphaviral vector)
  • a vector such as a viral vector (e.g., a retroviral vector, adenoviral vector
  • the vector once inside a cell of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.) will promote expression of the polypeptide, which is then secreted from the cell. If treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood may be collected from the subject and the protein purified from the blood by methods known in the art.
  • a pharmaceutical composition of the invention includes an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer as the therapeutic protein.
  • an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA
  • nucleic acid molecules encoding a polypeptide described herein or vectors containing such nucleic acid molecules may be administered using a hydrodynamic injection platform.
  • a nucleic acid molecule encoding a polypeptide described herein is put under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid).
  • the plasmid is often delivered rapidly in a large fluid volume intravenously.
  • Hydrodynamic injection uses controlled hydrodynamic pressure in veins to enhance cell permeability such that the elevated pressure from the rapid injection of the large fluid volume results in fluid and plasmid extravasation from the vein.
  • the expression of the nucleic acid molecule is driven primarily by the liver. In mice, hydrodynamic injection is often performed by injection of the plasmid into the tail vein.
  • mRNA molecules encoding a polypeptide described herein may be administered using hydrodynamic injection.
  • the pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms.
  • the pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules).
  • dosage forms e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules).
  • therapeutic proteins are dosed at 0.1 -100 mg/kg, e.g., 0.5-50 mg/kg.
  • compositions that include a polypeptide described herein may be administered to a subject in need thereof, for example, one or more times (e.g., 1 -10 times or more) daily, weekly, biweekly, every four weeks, monthly, bimonthly, quarterly, biannually, annually, or as medically necessary.
  • pharmaceutical compositions that include a polypeptide described herein may be administered to a subject in need thereof weekly, biweekly, every four weeks, monthly, bimonthly, or quarterly. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may increase as the medical condition improves or decrease as the health of the patient declines.
  • the activin A and myostatin signaling inhibitors described herein can reduce or inhibit the activity of negative regulators of skeletal muscle (e.g., activin A and myostatin). Therefore, the activin A and myostatin signaling inhibitors can be used to treat diseases in which muscle cells have increased susceptibility to damage and death, such as DMD.
  • an activin A and myostatin signaling inhibitor described herein e.g., a polypeptide including an ActRII chimera
  • a dystrophin exon skipping therapy further increased muscle strength and dystrophin expression compared to administration of the activin A and myostatin signaling inhibitor or the dystrophin exon skipping therapy alone and further increased lean mass compared to administration of the dystrophin exon skipping therapy alone
  • the activin A and myostatin signaling inhibitors described herein can be used to treat a subject having DMD in combination with a dystrophin exon skipping therapy (e.g., to increase lean mass (e.g., muscle mass), muscle strength, and/or dystrophin expression, or to ameliorate (e.g., reduce) muscle loss).
  • An activin A and myostatin signaling inhibitor described herein e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap
  • an activin A and myostatin signaling inhibitor described herein e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap
  • an activin A and myostatin signaling inhibitor described herein e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein
  • treatment with the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy is started concurrently (e.g., the subject begins treatment with both agents at approximately the same time, e.g., begins treatment with both agents during the same day, week, or month).
  • the subject has been undergoing treatment with the dystrophin exon skipping therapy prior to co-administration of an activin A and myostatin signaling inhibitor described herein (e.g., the subject has been undergoing treatment with the dystrophin exon skipping therapy for at least two weeks (e.g., 2 weeks or longer, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) prior to co-administration of an activin A and myostatin signaling inhibitor described herein).
  • 2 weeks or longer such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months
  • the method can further include evaluating body weight, lean mass, muscle mass, muscle strength, and/or dystrophin expression after administration of an activin A and myostatin signaling inhibitor described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an activin A and myostatin signaling inhibitor described herein).
  • an activin A and myostatin signaling inhibitor described herein e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an activin A and myostatin signaling inhibitor described herein.
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 44 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 - 44, or a dystrophin exon 44 skipping therapy provided in Table 21 ).
  • a dystrophin exon 44 skipping therapy e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 - 44, or a dystrophin exon 44 skipping therapy provided in Table 21 .
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 45 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22).
  • a dystrophin exon 45 skipping therapy e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22.
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 50 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23).
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 51 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24).
  • a dystrophin exon 51 skipping therapy e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24.
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 53 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy provided in Table 25).
  • a dystrophin exon 53 skipping therapy e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy provided in Table 25.
  • the subject has a confirmed mutation of the DMD gene that is amenable to exon 2 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26).
  • a dystrophin exon 2 skipping therapy e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26.
  • an activin A and myostatin signaling inhibitor described herein can maintain or increase lean mass (e.g., muscle mass, such as skeletal muscle mass) in a subject undergoing treatment with a dystrophin exon skipping therapy.
  • the activin A and myostatin signaling inhibitor may increase lean mass as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone.
  • co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in lean mass compared to administration of either agent alone.
  • administration of an activin A and myostatin signaling inhibitor described herein increases muscle strength.
  • the activin A and myostatin signaling inhibitor may increase muscle strength as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone.
  • administration of the activin A and myostatin signaling inhibitor leads to an amelioration of muscle loss as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone.
  • co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater amelioration of muscle loss compared to administration of either agent alone. In some embodiments, co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle strength compared to administration of either agent alone.
  • administration of the activin A and myostatin signaling inhibitor leads to an increase in dystrophin expression (e.g., increase in dystrophin mRNA levels and/or increase in dystrophin protein levels) as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone.
  • coadministration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in dystrophin expression compared to administration of either agent alone.
  • the methods described herein e.g., co-administration of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor
  • increase lean mass e.g., increase muscle mass (e.g., skeletal muscle mass)
  • ameliorate muscle loss increase muscle strength, and/or increase dystrophin expression.
  • the methods described herein may increase lean mass, increase muscle mass, ameliorate muscle loss, increase muscle strength, or increase dystrophin expression (e.g., increase dystrophin mRNA levels and/or increase dystrophin protein levels) compared to measurements obtained prior to treatment (e.g., treatment with the combination of the dystrophin exon skipping therapy and the activin A and myostatin signaling inhibitor) or compared to measurements obtained from subjects treated with a dystrophin exon skipping therapy or an activin A and myostatin signaling inhibitor alone.
  • dystrophin expression e.g., increase dystrophin mRNA levels and/or increase dystrophin protein levels
  • the methods described herein slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications (e.g., beathing difficulties, shortness of breath, cardiomyopathy, or respiratory and/or heart failure) (e.g., compared to outcomes in subjects treated with a dystrophin exon skipping therapy or an activin A and myostatin signaling inhibitor alone).
  • the method can further include evaluating body weight, lean mass, muscle mass, muscle loss, muscle strength, dystrophin expression (e.g., dystrophin mRNA levels and/or dystrophin protein levels), or other DMD symptoms (e.g., ambulation, respiratory function, or cardiac function) after administration of an activin A and myostatin signaling inhibitor described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an activin A and myostatin signaling inhibitor described herein in combination with a dystrophin exon skipping therapy).
  • dystrophin expression e.g., dystrophin mRNA levels and/or dystrophin protein levels
  • other DMD symptoms e.g., ambulation, respiratory function, or cardiac function
  • an activin A and myostatin signaling inhibitor described herein e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,
  • the activin A and myostatin signaling inhibitor may be an activin A antibody or an antigen-binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen-binding fragment thereof or an anti-myostatin adnectin recombinant protein) (i.e., a combination of an activin A antibody and an anti-myostatin protein).
  • the activin A and myostatin signaling inhibitor may be an ActRII antibody or an antigen-binding fragment thereof.
  • the activin A and myostatin signaling inhibitor may be an ActRII ligand trap, such as an ActRII ligand trap including an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877- 919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)).
  • an extracellular ActRII variant e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)
  • an extracellular ActRIIB variant having the sequence of any one
  • the activin A and myostatin signaling inhibitor is administered at a dosage ranging from 0.01 to 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 0.3 to about 30 mg/kg.
  • an ActRII ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-854 (e.g., SEQ ID NOs: 789-854) or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., 898-919)) that further includes a C-terminal extension of one or more amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids) may be used as the therapeutic protein.
  • an extracellular ActRIIA variant e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-854 (e.g., SEQ ID NOs: 789-854) or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., 898-919)
  • a dimer formed by the interaction of two Fc domain monomers that are each fused to a polypeptide including an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784- 855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) may be used as the therapeutic protein.
  • an extracellular ActRII variant e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784- 855 (e.g., SEQ ID NOs: 789-855)
  • an extracellular ActRIIB variant having the sequence of
  • the dystrophin exon skipping therapy is an exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy provided in Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22), an exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23), an exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24), an exon 53 skipping therapy (e.g., golodir
  • the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy are administered at the same or at similar frequencies.
  • both the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy can be administered once a week, once every two weeks, once every four weeks, or once a month.
  • Example 1 Effect of an ActRII chimera alone or in combination with a dystrophin exon skipping therapy in a mouse model of DMD
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally and the dose of Chimera 1/2b-mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • the lean mass was determined by nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec) on days -1 , 25, and 55 of the study. Lean mass results are shown in FIG. 2. Data are shown as a percentage of lean mass change from baseline measurements. Data are shown as mean ⁇ SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test, * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • mice were sacrificed on day 58 and total RNA was extracted from the quadriceps of the mice.
  • the dystrophin mRNA levels were determined by qRT-PCR (QuantStudio 7 Pro, ThermoFisher Scientific). Dystrophin expression results are shown in FIG. 4. Data are shown as relative expression to housekeeping genes. Data are shown as mean ⁇ SEM. Statistics are shown using 1 -way ANOVA with a Tukey’s multiple comparison test. * P ⁇ 0.05, ** P ⁇ 0.01 , *“ P ⁇ 0.001 , *“* P ⁇ 0.0001 .
  • D2MDX mice exhibited lower trabecular bone volume fraction and minimum moment of inertia in Z axis and increased trabecular pattern factor.
  • Chimera 1/2b-mFc and PMO-1 combination treatment significantly improved bone volume and bone structure associated with greater bone strength.
  • Chimera 1/2b-mFc ameliorated muscle loss in a DMD mouse model.
  • the observation that Chimera 1/2b-mFc treatment augmented PMO-1 induced dystrophin expression highlighted the potential synergistic benefit of using an activin A and myostatin signaling inhibitor and dystrophin exon skipping therapy combination therapy for DMD.
  • vehicle Tris-buffered saline (TBS)
  • PMO-1 a phosphorodiamidate morpholino oligomer having the sequence of SEQ ID NO: 1244 (ggccaaacctcggcttacctgaaat)
  • PMO-1 a phosphorodiamidate morpholino oligomer having the sequence of SEQ ID NO: 12
  • TBS and Chimera 1/2b-mFc were administered intraperitoneally and the dose of Chimera 1 /2b- mFc was 10 mg/kg.
  • PMO-1 was administered intravenously at a dose of 25 mg/kg.
  • the treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively.
  • the tricep myofiber cross-sectional area was determined. Triceps were snap-frozen, cross-sectioned on a cryostat (8 pm), and slide-mounted. The sections were stained with a Laminin primary antibody (ABCAM®, ab11576, Rat, lgG1 , 5 pg/mL) and an Alexa Fluor 555 secondary antibody (INVITROGEN®, A21434, Goat-anti-Rat, IgG, 4 pg/mL). Images were captured at 20x magnification using a ZEISS® Axioscan7. The files were exported, converted to .sis format, and analyzed using ARMS® Vision4D software (2023, Version 4.1 .2, Carl Zeiss AG).
  • a cell-pose model was used for myofiber segmentation to determine cross-sectional area of individual fibers to determine mean CSA per sample. Creation parameters were as follows: cell pose-based segmenter using CPx model at 60 pm with a minimum diameter of 15 pm detection, segment morphology objects erosion by 5 pixels to disclude basal lamina and an object feature filter to only include objects greater than or equal to 50 pm 2 for artifact removal. Data export Surface Area (Voxel) to .xls before compiling area of each individual fiber into a mean CSA per sample. Tricep myofiber cross-sectional area results are shown in FIG. 10. Data are shown as average CSA in pm 2 . Data are shown as mean ⁇ SEM.
  • a representative image of a gel showing exon skipping is provided in FIG. 1 1 B.
  • WT Vehicle group
  • D2MDX Vehicle group, no exon skipping was observed, consistent with the expected mutation profile without treatment.
  • PMO-1 -treated group partial exon skipping was observed, indicated by the presence of a band that migrated further on the gel compared to the full-length dystrophin band.
  • D2MDX PMO-1 + Chimera 1/2b-mFc, P ⁇ 0.0001 ;
  • D2MDX Vehicle vs.
  • the combination of PMO-1 and Chimera 1 /2b-mFc resulted in significantly higher dystrophin protein expression relative to Vehicle or PMO-1 alone groups.
  • the distribution of dystrophin protein localization within the muscle was determined by immunofluorescence imaging. Initially, the tricep tissues were embedded in optimal cutting temperature (OCT) compound and snap frozen in 2-methyl-butane cooled with liquid nitrogen. The embedded tissues were cryosectioned into 8 pm slices and mounted onto slides. On Day 1 , a hydrophobic barrier was created around each slide with a PAP pen and the slides were rehydrated in 1 X PBS. Antigen retrieval was performed using DAKO® Target Retrieval Solution in a water bath at 90°C for 20 minutes, followed by cooling to room temperature for 20 minutes. The slides were washed with PBS and permeabilized using 0.3% TritonX-100 for 20 minutes.
  • OCT optimal cutting temperature
  • the slides were blocked with a solution containing 0.1 % TritonX and 10% BSA in PBS for one hour at room temperature.
  • the primary antibodies anti-dystrophin (ABCAM® ab15277) rabbit IgG
  • the slides were incubated overnight at 4°C.
  • the slides were washed to remove unbound primary antibodies and the secondary antibodies (Alexa FluorTM 488 (A1 1034) goat antirabbit IgG) were applied in a similar blocking solution for one hour at room temperature in the dark.
  • the slides were then washed again to remove unbound secondary antibodies, and nuclear staining was performed using a DAPI working solution.
  • the slides were mounted using PROLONGTM Glass Mountant, dried overnight at 2-8°C, sealed with nail polish, and cleaned thoroughly before imaging using a ZEISS® Axioscan7 slide scanner with a 20x objective with automated tile stitching.
  • a series of representative images showing dystrophin protein localization is provided in FIG. 13B and highlights the increased expression and improved localization of dystrophin in the combined PMO-1 and Chimera 1/2b-mFc treatment group.
  • the combination of PMO-1 and Chimera 1/2b-mFc treatment significantly increased dystrophin localization and percentage of dystrophin-positive fibers with an increase in the distribution of dystrophin across the muscle section.
  • markers of satellite cells CD31 , Sca.1 , CD34, a7 integrin, and CD106
  • Satellite cell population results are shown in FIG. 14A.
  • Data are shown as percent of CD31 - Sca.1 - cells.
  • Satellite cell differentiation marker results are shown in FIG. 14B.
  • Data are shown as relative expression of paired box 7 (Pax7), myogenic factor 5 (Myf5), and myoblast determination protein 1 (MyoD).
  • Data are shown as mean ⁇ SEM.
  • Treatment with Chimera 1/2b-mFc increased the pool of satellite cells in wild type mice.
  • Molecular markers demonstrated commitment/differentiation of satellite cells to muscle.
  • Example 4 Treatment of a subject having DMD by administration of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor
  • a physician of skill in the art can treat a subject, such as a human patient, having DMD so as to increase lean mass (e.g., muscle mass), increase muscle strength, increase dystrophin expression, slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications.
  • lean mass e.g., muscle mass
  • an activin A and myostatin signaling inhibitor e.g., an activin A antibody or antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein
  • an ActRII antibody e.g., an ActRII ligand trap
  • an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898
  • composition containing the activin A and myostatin signaling inhibitor may be administered to the subject, for example, by parenteral injection (e.g., subcutaneous or intravenous injection) in combination with the dystrophin exon skipping therapy, which may be administered by intravenous injection or infusion (e.g., for casimersen, eteplirsen, golodirsen, viltolarsen, NS-089/NCNP-02, AOC 1044, NS-050/NCNP-03, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , WVE-N531 , ENTR-601 -44, or scAAV9.U7.ACCA) or by subcutaneous injection (e.g., for renadirsen or drisapersen).
  • parenteral injection e.g., subcutaneous or intravenous injection
  • the activin A and myostatin signaling inhibitor is administered in a therapeutically effective amount, such as from 0.01 to 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg).
  • 0.01 to 500 mg/kg e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10,
  • the activin A and myostatin signaling inhibitor e.g., a polypeptide including an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919), such as an extracellular ActRIIA variant, ActRIIB variant, or ActRII chimera fused to an Fc domain or Fc domain monomer) is administered bimonthly, once a month, once every four weeks, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more) and the dys
  • the activin A and myostatin signaling inhibitor is administered in an amount sufficient to increase lean mass (e.g., skeletal muscle mass), increase muscle strength, increase dystrophin expression, slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications.
  • lean mass e.g., skeletal muscle mass
  • a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient’s lean mass, muscle strength, or dystrophin expression using standard clinical tests. A finding that the patient exhibits increased lean mass, muscle strength, or dystrophin expression following administration of the composition compared to test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Psychology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention features methods of treating a subject having Duchenne muscular dystrophy by administering an activin A and myostatin signaling inhibitor in combination with a dystrophin exon skipping therapy. The activin A and myostatin signaling inhibitor may be an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an extracellular ActRII ligand trap.

Description

METHODS OF USING ACTIVIN A AND MYOSTATIN SIGNALING INHIBITORS
Sequence Listing
The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 7, 2025, is named 51184-056WO4_Sequence_Listing_2_7_25.xml and is 1 ,777,373 bytes in size.
Background of the Invention
Duchenne muscular dystrophy (DMD) is a genetic disorder and the most common form of muscular dystrophy. The National Organization for Rare Disorders estimates that 1 in every 3500 males globally is affected by DMD, in which patients lack functional dystrophin, a protein that is an important structural component of muscle cells that helps to promote myofiber stability. Lack of dystrophin results in contraction-induced muscle damage. This damage initiates a process of asynchronous regeneration leading to a loss of muscle mass with increased adipose infiltration and fibrosis. The replacement of muscle fibers with fatty and fibrotic tissue leads to progressive loss of muscle strength and function leading to immobility and respiratory and cardiac complications. The lack of functional dystrophin results in weakening of cardiac muscles, eventually leading to cardiomyopathy and limiting the life span of patients.
Glucocorticoids have been the standard of care in DMD and help preserve muscle strength and function, leading to extension of independent ambulation for several years. While glucocorticoids help to maintain muscle function in DMD patients, long-term treatment with them can have significant negative side effects, including fluid retention, hyperglycemia, severe weight gain with fat deposits in the abdomen, face and neck, bone fragility, cataracts, high blood pressure and mood effects, leading many patients to forego long-term treatment. Additionally, glucocorticoid treatment is associated with lean mass loss, an effect mediated by myostatin.
Exon skipping therapies have been explored to compensate for dystrophin deficiency by inducing the production of truncated but functional dystrophin. However, poor target tissue delivery, low efficacy, and high dose-induced off-target toxicity hinder the development of exon skipping therapies as an effective therapy.
Accordingly, there exists a need for effective treatment options for DMD.
Summary of the Invention
The present invention features methods of treating Duchenne muscular dystrophy (DMD) by administering a combination of an activin A and myostatin signaling inhibitor described herein (e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap) and a dystrophin exon skipping therapy (e.g., an exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy including a sequence of Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy including a sequence of Table 22), an exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy including a sequence of Table 23), an exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy including a sequence of Table 24), an exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy including a sequence of Table 25), or an exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy including a sequence of Table 26). The methods described herein may increase lean mass (e.g., muscle mass), increase muscle strength, increase dystrophin expression, and/or ameliorate muscle loss.
Exemplary embodiments of the invention are described in the enumerated paragraphs below. E1 . A method of treating a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
E2. A method of increasing lean mass in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
E3. A method of increasing muscle strength in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
E4. A method of increasing dystrophin expression in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
E5. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene (the gene encoding dystrophin) that is amenable to exon 44 skipping.
E6. The method of E5, wherein the dystrophin exon skipping therapy is an exon 44 skipping therapy.
E7. The method of E6, wherein the exon 44 skipping therapy comprises a sequence having at least
90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 949-990, 1264-1431 , and 1794- 1796.
E8. The method of E7, wherein the exon 44 skipping therapy comprises the sequence of any one of SEQ ID NOs: 949-990, 1264-1431 , and 1794-1796.
E9. The method of any one of E6-E8, wherein the exon 44 skipping therapy is NS-089/NCNP-02, AOC 1044, or ENTR-601 -44.
E10. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 45 skipping.
E11 . The method of E10, wherein the dystrophin exon skipping therapy is an exon 45 skipping therapy.
E12. The method of E11 , wherein the exon 45 skipping therapy comprises a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 991 , 992, 1432-1545, and 1797- E13. The method of E12, wherein the exon 45 skipping therapy comprises the sequence of any one of SEQ ID NOs: 991 , 992, 1432-1545, and 1797-1801 .
E14. The method of any one of E11 -E13, wherein the exon 45 skipping therapy is casimersen or renadirsen.
E15. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 50 skipping.
E16. The method of E15, wherein the dystrophin exon skipping therapy is an exon 50 skipping therapy.
E17. The method of E16, wherein the exon 50 skipping therapy comprises a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 993-999, 1546-1580, and 1802- 1804.
E18. The method of E17, wherein the exon 50 skipping therapy comprises the sequence of any one of SEQ ID NOs: 993-999, 1546-1580, and 1802-1804.
E19. The method of any one of E16-E18, wherein the exon 50 skipping therapy is NS-050/NCNP-03.
E20. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 51 skipping.
E21 . The method of E20, wherein the dystrophin exon skipping therapy is an exon 51 skipping therapy.
E22. The method of E21 , wherein the exon 51 skipping therapy comprises a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1000-1028, 1247-1256, 1581 - 1745, 1805, and 1897.
E23. The method of E22, wherein the exon 51 skipping therapy comprises the sequence of any one of SEQ ID NOs: 1000-1028, 1247-1256, 1581 -1745, 1805, and 1897.
E24. The method of any one of E21 -E23, wherein the exon 51 skipping therapy is eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, or BMN-351.
E25. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 53 skipping.
E26. The method of E25, wherein the dystrophin exon skipping therapy is an exon 53 skipping therapy.
E27. The method of E26, wherein the exon 53 skipping therapy comprises a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of any one of SEQ ID NOs: 1029-1031 , 1746-1793, and 1806-1809.
E28. The method of E27, wherein the exon 53 skipping therapy comprises the sequence of any one of SEQ ID NOs: 1029-1031 , 1746-1793, and 1806-1809.
E29. The method of any one of E26-E28, wherein the exon 53 skipping therapy is golodirsen, viltolarsen, or WVE-N531 .
E30. The method of any one of E1 -E4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 2 skipping. E31 . The method of E30, wherein the dystrophin exon skipping therapy is an exon 2 skipping therapy.
E32. The method of E31 , wherein the exon 2 skipping therapy comprises a sequence having at least
90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of SEQ ID NO: 1032.
E33. The method of E32, wherein the exon 2 skipping therapy comprises the sequence of SEQ ID NO: 1032.
E34. The method of any one of E31 -E33, wherein the exon 2 skipping therapy is scAAV9.U7.ACCA.
E35. The method of any one of E1 -E34, wherein the activin A and myostatin signaling inhibitor is an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein) (i.e., the subject is administered both an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein)).
E36. The method of E35, wherein the activin A antibody is garetosmab.
E37. The method of E35, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 and a light chain variable region (LCVR) sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 (e.g., an HCVR sequence in Table 1 and an LCVR sequence in Table 1 , such as an HCVR sequence and an LCVR sequence from the same row of Table 1 ).
E38. The method of E37, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) sequence in Table 1 and a light chain variable region (LCVR) sequence in Table 1 , such as an HCVR sequence and an LCVR sequence from the same row of Table 1 .
E39. The method of any one of E35, E37, and E38, wherein the activin A antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 2 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 2).
E40. The method of any one of E35 and E37-E39, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 3 (e.g., a heavy chain and light chain sequence from the same row of Table 3).
E41 . The method of E40, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence provided in Table 3 (e.g., a heavy chain and light chain sequence from the same row of Table 3).
E42. The method of E35, wherein the anti-myostatin protein is a myostatin antibody or an antigen binding fragment thereof.
E43. The method of E42, wherein the myostatin antibody is domagrozumab, landogrozumab, trevogrumab, or apitegromab (SRK-015). E44. The method of E42, wherein the myostatin antibody or an antigen binding fragment thereof has a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4 and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4 (e.g., a HCVR sequence in Table 4 and a LCVR sequence in Table 4, such as an HCVR sequence and an LCVR sequence from the same row of Table 4).
E45. The method of E44, wherein the myostatin antibody or an antigen binding fragment thereof has a HCVR sequence in Table 4 and a LCVR sequence in Table 4, such as an HCVR sequence and an LCVR sequence from the same row of Table 4.
E46. The method of any one of E42, E44, and E45, wherein the myostatin antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 5, Table 6, or Table 7 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 5).
E47. The method of any one of E42 and E44-E46, wherein the myostatin antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 8 (e.g., a heavy chain and light chain sequence from the same row of Table 8).
E48. The method of E47, wherein the myostatin antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence provided in Table 8 (e.g., a heavy chain and light chain sequence from the same row of Table 8).
E49. The method of E42, wherein the myostatin antibody or an antigen binding fragment thereof is a bi-specific antibody or an antigen binding fragment thereof that also binds to activin A.
E50. The method of E49, wherein the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 ), and a myostatin HCVR and LCVR from Table 4 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, and a LCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4).
E51 . The method of E50, wherein the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 and a myostatin HCVR and LCVR from Table 4.
E52. The method of any one of E49-E51 , wherein the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 2 (e.g., an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 2) and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 5 (e.g., a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 5).
E53. The method of E35, wherein the anti-myostatin protein is an anti-myostatin adnectin recombinant protein.
E54. The method of E53, wherein the anti-myostatin adnectin recombinant protein is taldefgrobep alfa.
E55. The method of E53, wherein the anti-myostatin adnectin recombinant protein comprises a sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a sequence provided in Table 9.
E56. The method of E55, wherein the anti-myostatin adnectin recombinant protein comprises a sequence provided in Table 9.
E57. The method of any one of E1 -E34, wherein the activin A and myostatin signaling inhibitor is an ActRII antibody or an antigen binding fragment thereof.
E58. The method of E57, wherein the ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861 .
E59. The method of E57, wherein the ActRII antibody or an antigen binding fragment thereof has a
HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 10 and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 10 (e.g., a HCVR sequence in Table 10 and a LCVR sequence in Table 10, such as an HCVR sequence and an LCVR sequence from the same row of Table 10).
E60. The method of E59, wherein the ActRII antibody or an antigen binding fragment thereof has a HCVR sequence in Table 10 and a LCVR sequence in Table 10, such as an HCVR sequence and an LCVR sequence from the same row of Table 10.
E61 . The method of any one of E57, E59, and E60, wherein the ActRII antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 11 (e.g., a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 11 ).
E62. The method of any one of E57 and E59-E61 , wherein the ActRII antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 12 (e.g., a heavy chain and light chain sequence from the same row of Table 12).
E63. The method of E62, wherein the ActRII antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence provided in Table 12 (e.g., a heavy chain and light chain sequence from the same row of Table 12).
E64. The method of any one of E1 -E34, wherein the activin A and myostatin signaling inhibitor is an ActRII ligand trap.
E65. The method of E64, wherein the ActRII ligand trap is an ActRIIA ligand trap.
E66. The method of E65, wherein the ActRIIA ligand trap is a composition of Table 28 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 28). E67. The method of E65, wherein the ActRIIA ligand trap comprises an extracellular portion of wildtype ActRIIA (e.g., SEQ ID NO: 2 or SEQ ID NO: 783).
E68. The method of E65, wherein the ActRIIA ligand trap is sotatercept.
E69. The method of E64, wherein the ActRII ligand trap is an ActRIIB ligand trap.
E70. The method of E69, wherein the ActRIIB ligand trap comprises an extracellular portion of wildtype ActRIIB (e.g., SEQ ID NO: 4 or a portion thereof).
E71 . The method of E69, wherein the ActRIIB ligand trap is BIIB1 10, ALG-802, luspatercept, ramatercept, or ACE-2494.
E72. The method of E69, wherein the ActRIIB ligand trap is a composition of Table 29 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 29).
E73. The method of E69, wherein the ActRIIB ligand trap comprises the sequence of any one of SEQ ID NOs: 871 -876 (e.g., the sequence of any one of SEQ ID NOs: 871 -876 fused to an Fc domain monomer, by way of a linker).
E74. The method of E64, wherein the ActRII ligand trap is an ActRII chimera ligand trap.
E75. The method of E74, wherein the ActRII chimera ligand trap is a composition of Table 30 or Table
31 (e.g., a polypeptide, nucleic acid molecule, vector, or pharmaceutical composition of Table 30 or Table 31 ).
E76. The method of E75, wherein the composition of Table 30 is a polypeptide comprising an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 877-897, wherein Xi is D or R, X2 is I, F, E, D, Y, S, N, Q, or T, X3 is N or T, X4 is A or E, X5 is T or K, X6 is E or K, X7 is E or D, Xs is N or S, and Xg is Q, E, K, R, D, or N, optionally wherein the chimera is truncated from the N-terminus by deletion of 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, wherein the chimera retains the two amino acids before the first cysteine.
E77. The method of E76, wherein the chimera has the sequence of SEQ ID NO: 895.
E78. The method of E76, wherein the chimera has the sequence of SEQ ID NO: 888.
E79. The method of E76, wherein the chimera has the sequence of SEQ ID NO: 881 .
E80. The method of any one of E76-E79, wherein Xi is D, X2 is I, F, or E, X3 is N or T, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xs is N or S, and X9 is E or Q.
E81 . The method of E80, wherein is E or D, Xs is N or S, and X
E82. The method of E81 , wherein Xi is D, X2 is F, X3 is N, X4 is E, X5 is K, Xs is K, X7 is D, Xs is S, and
X9 is Q.
E83. The method of E76, wherein the chimera has the sequence of any one of SEQ ID NOs: 898-919.
E84. The method of E83, wherein the chimera has the sequence of SEQ ID NO: 917.
E85. The method of E83, wherein the chimera has the sequence of SEQ ID NO: 901 .
E86. The method of E83, wherein the chimera has the sequence of SEQ ID NO: 916.
E87. The method of any one of E76-E86, wherein the polypeptide further includes an Fc domain monomer fused to the C-terminus of the polypeptide (e.g., the C-terminus of the chimera) by way of a linker.
E88. The method of E87, wherein the polypeptide has the sequence of any one of SEQ ID NOs: 1 159- 1242. E89. The method of E88, wherein the polypeptide has the sequence of SEQ ID NO: 1195.
E90. The method of E88, wherein the polypeptide has the sequence of SEQ ID NO: 1162.
E91 . The method of E88, wherein the polypeptide has the sequence of SEQ ID NO: 1194.
E92. The method of any one of E87-E91 , wherein the polypeptide is in the form of a dimer (e.g., a homodimer).
E93. The method of any one of E1 -E92, wherein the method increases lean mass.
E94. The method of any one of E1 -E93, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in lean mass compared to administration of the dystrophin exon skipping therapy alone.
E95. The method of any one of E1 -E94, wherein the method increases muscle mass (e.g., skeletal muscle mass).
E96. The method of any one of E1 -E95, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle mass compared to administration of the dystrophin exon skipping therapy alone.
E97. The method of any one of E1 -E96, wherein the method increases muscle strength.
E98. The method of any one of E1 -E97, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle strength compared to administration of either agent alone.
E99. The method of any one of E1 -E98, wherein the method increases dystrophin expression.
E100. The method of any one of E1 -E99, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase of dystrophin expression compared to administration of either agent alone.
E101 . The method of any one of E1 -E100, wherein the method slows or inhibits the progression of DMD, reduces or inhibits muscle atrophy or wasting, preserves ambulation or slows the loss of ambulation, or reduces or inhibits respiratory and/or cardiac complications (e.g., compared to administration of the dystrophin exon skipping therapy alone).
E102. The method of any one of E1 -E101 , wherein the subject is a human.
Definitions
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the invention. Terms such as "a", "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
As used herein, the term “about” refers to a value that is within 10% above or below the value being described.
As used herein, any values provided in a range of values include both the upper and lower bounds, and any values contained within the upper and lower bounds. As used herein, “administration” refers to providing or giving a subject a therapeutic agent (e.g., an activin A and myostatin signaling inhibitor or a dystrophin exon skipping therapy described herein), by any effective route. Exemplary routes of administration are described herein below.
As used herein, “anti-myostatin protein” refers to any protein that binds specifically to myostatin and reduces myostatin signaling. Exemplary anti-myostatin proteins include myostatin antibodies or antigen binding fragments thereof and anti-myostatin adnectin recombinant proteins.
The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
“Antibody fragments” include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
As used herein, the terms “extracellular activin receptor type HA (ActRIIA) variant” and “ActRIIA variant” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIA, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 1 shown below). The sequence of the wild-type, human ActRIIA precursor protein is shown below (SEQ ID NO: 1 ), in which the signal peptide is italicized and the extracellular portion is bold.
Wild-type, human ActRIIA precursor protein (SEQ ID NO: 1 ):
/WGZW/WAF/ FL/SCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFAT WKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPT SNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPL QLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRG TSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHR DIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRID MYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKH AGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL An extracellular ActRIIA variant may have a sequence of any one of SEQ ID NOs: 784-855. In particular embodiments, an extracellular ActRIIA variant has a sequence of any one of SEQ ID NOs: 789- 855 (Table 14). In some embodiments, an extracellular ActRIIA variant may have at least 85% (e.g., at least 85%, 87%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 2).
As used herein, the terms “extracellular activin receptor type IIB (ActRIIB) variant” and “ActRIIB variant” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor, ActRIIB, that has at least one amino acid substitution relative to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 3 shown below). The sequence of the wild-type, human ActRIIB is shown below (SEQ ID NO: 3), in which the signal peptide is italicized and the extracellular portion is bold.
Wild-type human ActRIIB (SEQ ID NO: 3):
MTAPWI/A /./.WGS/.CAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCY ASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAG
GPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPP PSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMK HENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSY LHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVG TRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQ HPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSL IRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI
An extracellular ActRIIB variant may have a sequence of any one of SEQ ID NOs: 856-876. In particular embodiments, an extracellular ActRIIB variant has a sequence of any one of SEQ ID NOs: 857- 876 (Table 16). In some embodiments, an extracellular ActRIIB variant may have at least 85% (e.g., at least 85%, 87%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIB (SEQ ID NO: 4). The extracellular ActRIIB variant may also have an N-terminal truncation of 1 -7 amino acids relative to the extracellular portion of ActRIIB.
As used herein, the terms “extracellular activin receptor type II (ActRII) chimera,” “extracellular ActRII chimera,” and “ActRII chimera” refer to a peptide including a soluble, extracellular portion of the single transmembrane receptor ActRIIB and a soluble, extracellular portion of the single transmembrane receptor ActRIIA. In some embodiments, the ActRII chimeras described herein result from joining an N- terminal portion of extracellular ActRIIB to a C-terminal portion of extracellular ActRIIA such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). The extracellular ActRII chimera may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to a wild-type extracellular ActRIIB (e.g., bold portion of the sequence of SEQ ID NO: 3 shown above), and one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to a wild-type extracellular ActRIIA (e.g., bold portion of the sequence of SEQ ID NO: 1 shown above). In other embodiments, the ActRII chimeras result from the substitution of one or more amino acid sequence corresponding a p-sheet from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA) and/or from the substitution of one or more intervening sequence (e.g., a sequence between the p- sheets) from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA). For example, an ActRII chimera may be produced by replacing one or more amino acid sequence corresponding to a p-sheet in ActRIIB with an amino acid sequence corresponding to the p- sheet from ActRIIA. The extracellular ActRII chimera may also have an N-terminal truncation of 1 -9 amino acids relative to the extracellular portion of ActRIIB or ActRIIA. The sequences of wild-type, human ActRIIB (SEQ ID NO: 3) and wild-type, human ActRIIA (SEQ ID NO: 1 ) are shown in the definitions above, in which the signal peptide is italicized and the extracellular portion is bold. An extracellular ActRII chimera may have the sequence of any one of SEQ ID NOs: 877-919 or 1 128-1 158. In particular embodiments, an extracellular ActRII chimera has the sequence of any one of SEQ ID NOs: 898-919 (Table 18). As used herein, the term “extracellular activin receptor type II (ActRII) variant” refers to an extracellular ActRIIA variant, an extracellular ActRIIB variant, or an extracellular ActRII chimera described herein.
As used herein, the term “N-terminal truncation” refers to a deletion of 1 -7 amino acids (e.g., 1 , 2, 3, 4, 5, 6, or 7 amino acids) from the N-terminus of an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876)) or a deletion of 1 -9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) from the N-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)). The N-terminal truncation can remove amino acids up to two amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimeras).
As used herein, the term “linker” refers to a linkage between two elements, e.g., peptides or protein domains. An ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer. The Fc domain monomer increases stability or improves pharmacokinetic properties of the polypeptide and can be fused to the polypeptide by way of a linker. A linker can be a covalent bond or a spacer. The term “bond” refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. The term “spacer” refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1 -200 amino acid sequence) occurring between two elements, e.g., peptides or protein domains, to provide space and/or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to the spaced peptides via the polypeptide backbone). The formation of disulfide bonds, e.g., between two hinge regions that form an Fc domain, is not considered a linker.
As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers. An Fc domain has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fc domain that includes at least a CH2 domain and a CH3 domain. An Fc domain monomer includes second and third antibody constant domains (CH2 and CH3). In some embodiments, the Fc domain monomer also includes a hinge domain. An Fc domain does not include any portion of an immunoglobulin that is capable of acting as an antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR). In the wild-type Fc domain, the two Fc domain monomers dimerize by the interaction between the two CH3 antibody constant domains, as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead Fc domain.” In certain embodiments, each of the Fc domain monomers in an Fc domain includes amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and an Fey receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. An Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Additionally, an Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.
As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell, e.g., a human muscle cell).
As used herein, the term “fused” is used to describe the combination or attachment of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides in tandem series can be fused to form one contiguous protein structure, e.g., a polypeptide, through chemical conjugation, a chemical bond, a peptide linker, or any other means of covalent linkage. In some embodiments of a polypeptide described herein, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof (e.g., an extracellular ActRII variant, e.g., an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)), an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876)), or an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) may be fused in tandem series to the N- or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 1033, SEQ ID NO: 1034, or SEQ ID NO: 1035) by way of a linker. For example, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is fused to an Fc domain monomer by way of a peptide linker, in which the N-terminus of the peptide linker is fused to the C-terminus of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof through a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the Fc domain monomer through a chemical bond, e.g., a peptide bond.
As used herein, the terms “increasing” and “decreasing” refer to modulating resulting in, respectively, greater or lesser amounts, of function, expression, or activity of a metric relative to a reference. For example, subsequent to administration of an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy in a method described herein, the amount of a marker of a metric (e.g., body weight, lean mass, muscle mass, muscle strength, or dystrophin expression) as described herein may be increased or decreased in a subject relative to the amount of the marker prior to administration. Generally, the metric is measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least one week, one month, 3 months, or 6 months, after a treatment regimen has begun.
As used herein, the term “C-terminal extension” refers to the addition of one or more amino acids to the C-terminus of an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) or to the C-terminus of an extracellular ActRII chimera (e.g., an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)). The C-terminal extension can be one or more amino acids, such as 1 -6 amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids). The C-terminal extension may include amino acids from the corresponding position of wild-type ActRIIA (for an ActRIIA variant) or from the corresponding position of wild-type ActRIIA or ActRIIB (for an ActRII chimera). Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 1090) (a six amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used.
As used herein, the term “percent (%) identity” refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows:
100 x (fraction of A/B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.
In particular embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits from 50% to 100% identity across the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.
As used herein, the term “serum half-life” refers to, in the context of administering a therapeutic protein to a subject, the time required for plasma concentration of the protein in the subject to be reduced by half. The protein can be redistributed or cleared from the bloodstream, or degraded, e.g., by proteolysis. Serum half-life comparisons can be made by comparing the serum half-life of Fc fusion proteins.
As used herein, the term “lean mass” refers to a component of body composition which includes, e.g., lean mass, body fat, and body fluid. Normally lean mass is calculated by subtracting the weights of body fat and body fluid from total body weight. Typically, a subject’s lean mass is between 60% and 90% of total body weight. In the present invention, administration of an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy to a subject having DMD maintains or increases the subject’s lean mass.
As used herein, the term “affinity” or “binding affinity” refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum total of non-covalent interactions between a molecule and its binding partner. Unless indicated otherwise, binding affinity refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair. The binding affinity between two molecules is commonly described by the dissociation constant (KD) or the affinity constant (KA). TWO molecules that have low binding affinity for each other generally bind slowly, tend to dissociate easily, and exhibit a large KD. TWO molecules that have high affinity for each other generally bind readily, tend to remain bound longer, and exhibit a small KD. The KD of two interacting molecules may be determined using methods and techniques well known in the art, e.g., surface plasmon resonance. KD is calculated as the ratio of kott/kon.
As used herein, the term “muscle mass” refers to the primary component of lean mass. Muscle mass can be measured experimentally by measuring muscle weight.
As used herein, the term “polypeptide” describes a single polymer in which the monomers are amino acid residues which are covalently conjugated together through amide bonds. A polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.
As used herein, the term “homodimer” refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. The two identical monomers may form a homodimer by covalent bonds or non-covalent bonds. For example, an Fc domain may be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a homodimer through the interaction of two Fc domain monomers, which form an Fc domain in the homodimer.
As used herein, the term “heterodimer” refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. The two monomers may form a heterodimer by covalent bonds or non-covalent bonds. For example, a polypeptide described herein including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a heterodimer through the interaction of two Fc domain monomers, each fused to a different extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof, which form an Fc domain in the heterodimer.
As used herein, the term “host cell” refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express proteins from their corresponding nucleic acids. The nucleic acids are typically included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). A host cell may be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or a HEK293 cell). As used herein, the terms “effective amount” and “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector described herein refer to a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a subject having DMD, it is an amount of the composition, polypeptide, nucleic acid, or vector sufficient to achieve a treatment response as compared to the response obtained without administration of the composition, polypeptide, nucleic acid, or vector. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a composition, polypeptide, nucleic acid, or vector of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition, polypeptide, nucleic acid, or vector of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response.
As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical formulation that includes an active ingredient as well as excipients and diluents to enable the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention includes pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in tablet or capsule form for oral administration or in aqueous form for intravenous or subcutaneous administration.
As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier or excipient must provide adequate pharmaceutical stability to a polypeptide, the nucleic acid molecule(s) encoding the polypeptide, or a vector containing such nucleic acid molecule(s). The nature of the carrier or excipient differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.
As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
As used herein “Duchenne muscular dystrophy” (DMD), refers to a disease caused by mutations in the X-linked dystrophin gene and characterized by progressive muscle degeneration and weakness in all skeletal muscles. DMD affects approximately one in 3,500 newborn boys. The disorder is characterized by progressive muscle degeneration and wasting, along with the emergence of respiratory failure and cardiac complications, ultimately leading to premature death. Most mutations underlying DMD are genomic out-of-frame deletions that induce a premature truncation in the open reading frame that results in the absence of the dystrophin protein.
“Dystrophin” is a rod-shaped cytoskeletal protein and a vital part of the dystrophin associated glycoprotein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. In various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD), muscle cells produce no dystrophin at all, or an altered and functionally defective form of dystrophin, respectively, mainly due to mutations in the gene sequence that lead to incorrect splicing. The predominant expression of the defective dystrophin protein, or the complete lack of dystrophin or a dystrophin-like protein, leads to rapid progression of muscle degeneration.
As used here, the term “activin A and myostatin signaling inhibitor” refers to an agent that reduces or prevents the interaction of activin A and myostatin with their receptors (e.g., ActRIIA and/or ActRIIB), by either binding to the ligand or to the receptor. An activin A and myostatin signaling inhibitor may be an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap. The activin A and myostatin signaling inhibitor may be a single agent that reduces the signaling of both activin A and myostatin (e.g., an ActRII antibody or an antigen binding fragment thereof or an ActRII ligand trap) or a combination of agents that together reduce activin A and myostatin signaling (e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein)).
The term “exon” as used herein refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after a portion of a pre-processed (or precursor) RNA has been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.
“Exon skipping” as used herein refers generally to the process by which an entire exon, or a portion thereof, is excluded (skipped) from the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein.
A “dystrophin exon skipping therapy” is an exon skipping therapy that can be administered to patients with DMD, which results in the production of an internally deleted dystrophin protein. In certain embodiments, the exon being skipped is an aberrant exon from the human dystrophin gene, which may contain a mutation or other alteration in its sequence that otherwise causes aberrant splicing. In certain embodiments, the exon being skipped is exon 2, 44, 45, 50, 51 , or 53 of the human dystrophin gene. Exon 44 skipping therapies include NS-089/NCNP-02, AOC 1044, and ENTR-601 -44. Exon 45 skipping therapies include casimersen and renadirsen. Exon 50 skipping therapies include NS-050/NCNP-03. Exon 51 skipping therapies include eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, and BMN-351 . Exon 53 skipping therapies include golodirsen, viltolarsen, and WVE-N531 . Exon 2 skipping therapies include scAAV9.U7.ACCA. Dystrophin exon skipping therapies include those described in International Patent Application Publication Nos. W02006000057, WO2022232478, W02009054725, WO2018118662, W02020004675, WO2018118627, WO2015194520, WO2021132591 , WO2023196400, WO2023168014, WO2019217784, W02022020107, WO2023034817, WO2011057350, WO2022192749, and WO2022067257 and US Patent Application Publication No. US20230045002, each of which is incorporated herein by reference.
As used herein, the term “amenable” refers to a subject that will likely be responsive to a particular treatment. For example, a subject having DMD who is amenable to exon 51 skipping has one or more mutations in the dystrophin gene which, absent the skipping of exon 51 of the dystrophin pre- mRNA, causes the reading frame to be out-of-frame, thereby disrupting translation of the pre-mRNA and leading to an inability of the subject to produce functional or semi-functional dystrophin. Determining whether a patient has a mutation in the dystrophin gene that is amenable to exon skipping is well within the purview of one of skill in the art (see, e.g., Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299; Gurvich et al., Hum Mutat. 2009; 30(4) 633-640; and Fletcher et al. (2010) Molecular Therapy 18(6) 1218-1223).
As used herein, the term “satellite cells” refers to quiescent mononucleated myogenic cells located between the sarcolemma and basement membrane of terminally differentiated muscle fibers. Satellite cells are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells.
As used herein, the terms “subject” and “patient” refer to a human.
Brief Description of the Drawings
FIG. 1 is a graph showing body weight measurements during an 8-week treatment regimen of nine-week-old male D2MDX mice with either Tris-buffered-saline (TBS, Vehicle) (n=10), Chimera 1 /2b- mFc (SEQ ID NO: 917 fused to a mouse Fc domain) (n=10), PMO-1 (a phosphorodiamidate morpholino oligomer having the sequence of SEQ ID NO: 1244) (n=16), or PMO-1 in combination with Chimera 1 /2b- mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1 /2b- mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Data are shown as a percentage of body weight change from baseline measurements. Data are shown as mean ± SEM.
FIG. 2 is a graph showing lean mass measurements during an 8-week treatment regimen of nine- week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO-1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT). The lean mass was determined by nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec) on days -1 , 25, and 55 of the study. Data are shown as a percentage of lean mass change from baseline measurements. From left to right at each timepoint: WT: Vehicle, D2MDX: Vehicle, D2MDX: Chimera 1/2b-mFc, D2MDX: PMO-1 , D2MDX: PMO-1 + Chimera 1/2b-mFc. Data are shown as mean ± SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test. * P<0.05, ** P<0.01 , *** P<0.001 , **** P<0.0001 .
FIG. 3 is a graph showing grip strength measurements during an 8-week treatment regimen of nine-week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO-1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). The grip strength of mice's forearms was measured using the Grip Strength Meter (Columbus Instruments) on days -1 (baseline) and 55 (terminal) of the study. Data are shown as absolute force in kg. From left to right at each timepoint: WT: Vehicle, D2MDX: Vehicle, D2MDX: Chimera 1/2b-mFc, D2MDX: PMO-1 , D2MDX: PMO-1 + Chimera 1/2b-mFc. Data are shown as mean ± SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test. * P<0.05, ** P<0.01 , *** P<0.001 , **** PcO.0001 , ns= not significant.
FIG. 4 is a graph showing dystrophin expression measurements during an 8-week treatment regimen of nine-week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO- 1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Mice were sacrificed on day 58 and total RNA was extracted from the quadriceps of the mice. The dystrophin mRNA levels were determined by qRT-PCR (QuantStudio 7 Pro, ThermoFisher Scientific). Data are shown as relative expression. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a T ukey’s multiple comparison test. * P<0.05, ** P<0.01 , *** P<0.001 , **** P<0.0001.
FIG. 5 is a series of images showing ex vivo CT scans of the lumbar spine after an 8-week treatment regimen of nine-week-old male D2MDX mice with either Vehicle (n=10), PMO-1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer). Representative images of L4 lumbar spine vertebral body are shown as single coronal section (top row) or 3D reconstructed trabecular bone (bottom row).
FIG. 6 is a graph showing trabecular bone volume fraction after an 8-week treatment regimen of nine-week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO-1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer). Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ±SEM. *** P <0.001 , **** P <0.0001 .
FIG. 7 is a graph showing trabecular pattern factor after an 8-week treatment regimen of nine- week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO-1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer). Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ±SEM. *** P <0.001 , **** P <0.0001 .
FIG. 8 is a graph showing minimum moment of inertia in Z axis after an 8-week treatment regimen of nine-week-old male D2MDX mice with either Vehicle (n=10), Chimera 1/2b-mFc (n=10), PMO- 1 (n=16), or PMO-1 in combination with Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally. The dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10). Mice were sacrificed on day 58 and ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer). Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ±SEM. *** P <0.001 , **** P <0.0001 .
FIG. 9 is a graph showing tricep weight after a treatment regimen of 60-day-old (P60) male D2MDX mice with either vehicle (TBS) (n=5), PMO-1 (n=5), or PMO-1 in combination with Chimera 1 /2b- mFc (n=5). DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5). Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA for the treatment factor (p<0.0001 , F(3,44)=17.04, R2=0.5375) and post-hoc Tukey’s multiple comparisons test among treatment groups (WT : Vehicle vs. D2MDX: Vehicle, ****p<0.0001 ; WT : Vehicle vs. D2MDX: PMO-1 , ****p<0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****p<0.0001 ; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ***p=0.0006).
FIG. 10 is a graph showing tricep myofiber cross-sectional area after a treatment regimen of P60 male D2MDX mice with either vehicle (TBS) (n=5), PMO-1 (n=5) , or PMO-1 in combination with Chimera 1/2b-mFc (n=5). DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5). Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA for the treatment factor (p<0.0001 , F(3,32)=25.99, R2=0.7090) and post-hoc Tukey's multiple comparisons test among treatment groups (WT : Vehicle vs. D2MDX: Vehicle, ****p<0.0001 ; WT : Vehicle vs. D2MDX: PMO-1 , ****p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ***p=0.0003; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, **p=0.0027).
FIGS. 11A-11B are a series of graphs showing dystrophin exon skipping efficiency after a treatment regimen of P60 male D2MDX mice with either vehicle (TBS) (n=5), PMO-1 (n=5), or PMO-1 in combination with Chimera 1/2b-mFc (n=5). DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5). Data in FIG. 11 A are shown as a percentage of exon skipping, as mean ± SEM. Statistical significance was determined using one-way ANOVA for the treatment factor (p<0.0001 , F(3,22)=245.1 , R2=0.9710) and post-hoc Tukey's multiple comparisons test among treatment groups (WT: Vehicle vs. D2MDX: PMO-1 , p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 , p<0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, p<0.0001 ; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, **p=0.0005). FIG. 11 B shows a representative image of a gel showing exon skipping.
FIG. 12 is a graph showing dystrophin protein expression after a treatment regimen of P60 male D2MDX mice with either vehicle (TBS) (n=5), PMO-1 (n=5), or PMO-1 in combination with Chimera 1 /2b- mFc (n=5). DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5). Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA for treatment (F(3, 44) = 75.74, P < 0.0001 , R2 = 0.8378) and post-hoc Tukey's multiple comparisons test among treatment groups (WT: Vehicle vs. D2MDX: Vehicle, ****P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 , P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, P < 0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ***P = 0.0018; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, **P = 0.0045).
FIGS. 13A-13B are a graph and a series of images showing dystrophin protein localization within the muscle after a treatment regimen of P60 male D2MDX mice with either vehicle (TBS) (n=5), PMO-1 (n=5), or PMO-1 in combination with Chimera 1/2b-mFc (n=5). DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5). FIG. 13A is a graph showing percentage of dystrophin-positive fibers as mean ± SEM. Statistical significance was determined using one-way ANOVA for treatment (F(3, 33) = 79.20, P < 0.0001 , R2 = 0.8781 ) and post-hoc Tukey's multiple comparisons test among treatment groups (WT : Vehicle vs. D2MDX: Vehicle, ****P < 0.0001 ; WT : Vehicle vs. D2MDX: PMO-1 , ****P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****P < 0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 , **P = 0.0052; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****P < 0.0001 ; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, *P = 0.0371 ). FIG. 13B is a series of representative images showing dystrophin protein localization.
FIGS. 14A-14B are a series of graphs showing satellite cell population and markers of satellite cell differentiation after treatment of ten-week-old male wild-type C57BI/6 mice with a single dose of 10 mg/kg Chimera 1/2b-mFc or vehicle. Chimera 1/2b-mFc and vehicle were administered intraperitoneally. Muscles were dissected and processed to obtain single cell suspensions on day 1 , day 2, and day 4 (n=5), stained for markers of satellite cells (CD31 , Sca.1 , CD34, a7 integrin, and CD106) and analyzed by flow cytometry. FIG. 14A is a graph showing % of CD31 - Sca.1 - cells. FIG. 14B is a series of graphs showing relative expression of Pax7, Myf5, and MyoD. Data are shown as mean ± SEM. Statistical significance was determined using a 2-way ANOVA and Sidak's multiple comparisons test (* P<0.05, ** P<0.01 , **** PcO.0001 , ns= not significant). Pax7 = paired box 7; Myf5 = myogenic factor 5 ; MyoD = myoblast determination protein 1 .
Detailed Description of the Invention
The invention features methods of co-administering a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor. The activin A and myostatin signaling inhibitor can be an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap. Exemplary dystrophin exon skipping therapies include NS-089/NCNP-02, AOC 1044, ENTR-601 -44, casimersen, renadirsen, NS-050/NCNP-03, eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN- 351 , golodirsen, viltolarsen, WVE-N531 , and scAAV9.U7.ACCA. These methods can be used to treat Duchenne muscular dystrophy (DMD). These methods can also increase lean mass, muscle mass, muscle strength, and/or dystrophin expression in a subject having DMD.
Activin A and myostatin signaling
Activin type II receptors are single transmembrane domain receptors that modulate signals for ligands in the transforming growth factor p (TGF-p) superfamily. Ligands in the TGF-p superfamily are involved in a host of physiological processes, such as muscle growth, vascular growth, cell differentiation, homeostasis, and osteogenesis. Examples of ligands in the TGF-p superfamily include, e.g., activin, inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activins are known to play a role in the regulation of skeletal muscle growth. For example, mice without myostatin show a large increase in skeletal muscle mass. Methods that reduce or inhibit activin A and myostatin signaling could, therefore, be used in the treatment of DMD.
The present invention is based, in part, on the discovery by the present inventors that the combination of an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy further increased muscle strength and dystrophin expression in a mouse model of DMD compared to administration of the activin A and myostatin signaling inhibitor or the dystrophin exon skipping therapy alone and further increased lean mass compared to administration of the dystrophin exon skipping therapy alone. Without wishing to be bound by theory, administration of the activin A and myostatin signaling inhibitor may lead to increased numbers of satellite cells, which may allow for increased cellular uptake of the dystrophin exon skipping therapy.
These data suggest that co-administration of an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy could increase lean mass, muscle mass (e.g., skeletal muscle mass), muscle strength, and dystrophin expression in subjects with DMD. Activin A and myostatin signaling inhibitors
Activin A and myostatin signaling inhibitors are agents that reduce or prevent the interaction of activin A and myostatin with their receptors (e.g., ActRIIA and/or ActRIIB), by either binding to these ligands or to the receptor. Activin A and myostatin signaling inhibitors for use in the methods described herein are provided herein below.
Activin A antibodies and anti-myostatin proteins
In some embodiments, the activin A and myostatin signaling inhibitor is an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein).
In some embodiments, the activin A antibody is garetosmab (also known as REGN-2477). Additional activin A antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2015017576, WO2013074557, W02008031061 , and WO2023147107; US Patent Application Publication No. US20150359850; and US Patent Nos. 9,718,881 , 10,526,403, 8,309,082, 8,753,627, and 10,100,109, each of which is incorporated herein by reference.
In some embodiments, the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) and a light chain variable region (LCVR) listed in Table 1 (e.g., an HCVR and an LCVR from the same row of Table 1 ). In some embodiments, the activin A antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , such as any one of SEQ ID NOs: 5, 7, 9, 10, 11 , 13, 15, 17, 18, 19, 21 , 23, 25, 27, 29, 31 , 32, 34, 36, 37, 39, 41 , 43, 45, 47, 48, 50, 52, and 54) and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 , such as any one of SEQ ID NOs: 6, 8, 12, 14, 16, 20, 22, 24, 26, 28, 30, 33, 35, 38, 40, 42, 44, 46, 49, 51 , and 53. In some embodiments, the activin A antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 1 . In some embodiments, the activin A antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 1 . In some embodiments, the activin A antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 1 .
Table 1. Exemplary HCVR and LCVR sequences of activin A antibodies
In some embodiments, the activin A antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 2 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the activin A antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 2, such as any one of SEQ ID NOs: 58, 64, 70, 76, 82, 88, 100, 119, 130, 141 , and 147; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 59, 65, 71 , 77, 83, 92, 101 , 110, 114, 120, 125, 131 , 142, and 148; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 2, such as any one of SEQ ID NOs: 60, 66, 72, 78, 84, 89, 93, 95, 102, 105, 1 1 1 , 1 15, 121 , 126, 132, 137, 143, 149; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 2, such as any one of SEQ ID NOs: 55, 61 , 67, 73, 79, 85, 90, 94, 96, 98, 103, 107,
1 12, 1 16, 122, 127, 134, 138, 144, and 150; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 2, such as any one of SEQ ID NOs: 56, 62, 68, 74, 80, 86, 91 , 97, 99, 104, 106, 108, 1 13, 1 17, 123, 128, 133, 135, 139, 145, and 151 ; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 2, such as any one of SEQ ID NOs: 57, 63, 69, 75, 81 , 87, 109, 1 18, 124, 129, 136, 140, and 146. In some embodiments, the activin A antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 2.
Table 2. Exemplary CDR sequences of activin A antibodies
In some embodiments, the activin A antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 3. In some embodiments, the activin A antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 3. In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 1094 and 1095; 1096 and 1097; 1098 and 1099; 1100 and 1101 ; 1102 and 1095; 1103 and 1104; 1105 and 1106; 1107 and 1106; 1108 and 1109; 1110 and 1111 ; 1112 and 1113; 1114 and 1115; 1116 and 1117; 1118 and 1117; 1119 and 1120; 1121 and 1122; 1123 and 1124; or 1125 and 1126 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair).
Table 3. Exemplary heavy and light chain sequences of activin A antibodies
In some embodiments, the anti-myostatin protein is a myostatin antibody or an antigen binding fragment thereof. In some embodiments, the myostatin antibody is domagrozumab (also known as PF- 06252616), landogrozumab (also known as LY2495655), trevogrumab (also known as REGN-1033), or apitegromab (SRK-015). Additional myostatin antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. W02007047112, W02007044411 , W02006116269, WO2012024242, WO2016073853, WO2013186719, W02009058346, WO2011150008, WO2016168613, W02007024535, WO2016098357, WO2022093724, WO2017049011 , and WO2017120523, US Patent Application Publication Nos. US20070178095 and US20210246198; and US Patent Nos. 10,000,560, 10,738,111 , 7,632,499, 8,066,995, 7,635,760, 7,745,583, 7,807,159, 8,999,343, 10,307,480, 8,992,913, 9,751 ,937, 9,409,981 , 9,850,301 , 8,840,894, 9,890,212, 9,260,515, 10,934,349, 8,871 ,209, 10,400,036, 7,888,486, and 8,372,625, each of which is incorporated herein by reference.
In some embodiments, the myostatin antibody or an antigen binding fragment thereof has a HCVR and a LCVR listed in Table 4 (e.g., an HCVR and an LCVR from the same row of Table 4). In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, such as any one of SEQ ID NOs: 152, 154, 156, 158, 160-166, 168-173, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 225, 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249, 251 , 253, 255, 257, 259, 261 , 263, 265, 267, 269, 271 , 274, 276, 278, 280-284, 286, 287, 289-291 , 293, 295, 297, 299, 301 , 303, 305-317, 319, 325, 327, 329, 331 , 333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , and 353, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4, such as any one of SEQ ID NOs: 153, 155, 157, 159, 167, 174-179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209,
211 , 213, 215, 217, 219, 221 , 223, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,
252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 273, 275, 277, 279, 285, 288, 292, 294, 296, 298,
300, 302, 304, 318, 320, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, and 354. In some embodiments, the myostatin antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 4. In some embodiments, the myostatin antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 4. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 4. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes an HCVR sequence of any one of SEQ ID NOs: 278, 280-284, 286, 287, 289-291 , 293, 295, 297, 299, 301 , 303, and 305-316 and an LCVR sequence of any one of SEQ ID NOs: 279, 285, 288, 292, 294, 296, 298, 300, 302, and 304.
Table 4. Exemplary HCVR and LCVR sequences of myostatin antibodies
In some embodiments, the myostatin antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 5, 6, or 7 (i.e. , a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 358, 363, 368, 374, 377, 389, 395, 400, 405, 413, 419, 425, 431 , 436, 442, 445, 449, 454, 458, 466, 469, 473, 476, 478, 516, and 517-519; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 364, 369, 375, 390, 396, 406, 414, 420, 437, 450, 459, 463, 470, and 1257-1262; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 5 or Table 7, such as any one of SEQ ID NOs: 359, 365, 370, 376, 381 , 385, 391 , 397, 407, 415, 421 , 427, 433, 438, 444, 451 , 460, 464, 471 , 480, and 510-515; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 355, 360, 366, 371 , 378, 382, 386, 392, 398, 402, 409, 410, 416, 422, 428, 439, 446, 452, 455, 461 , 465, 474, 500, 503, 506, and 1263; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 356, 361 , 367, 372, 379, 383, 387, 393, 399, 403, 408, 411 , 417, 423, 429, 434, 440, 447, 453, 456, 467, 472, 472, 477, 501 , and 504; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR3 sequence in Table 5 or Table 6, such as any one of SEQ ID NOs: 357, 362, 373, 380, 384, 388, 394, 404, 412, 418, 424, 430, 435, 441 , 448, 457, 462, 468, 479, 499, 502, 505, and 507-509. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 516; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1260; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 510; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 500; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity SEQ ID NO: 501 ; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 499. In some embodiments, the myostatin antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 5.
Table 5. Exemplary CDR sequences of myostatin antibodies
Table 6. Exemplary heavy chain CDR sequences of myostatin antibodies Table 7. Exemplary light chain CDR sequences of myostatin antibodies
In some embodiments, the myostatin antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 8. In some embodiments, the myostatin antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 8. In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 520 and 521 ; 522 and 523; 524 and 525; 526 and 527; 528 and 529; 530 and 531 ; 532 and 533; 534 and 535; 536 and 537; 538 and 539; 540 and 541 ; 542 and 543; 544 and 545; 546 and 547; or 548 and 549 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair).
Table 8. Exemplary heavy and light chain sequences of myostatin antibodies
In some embodiments, the myostatin antibody is a bi-specific antibody that also binds to activin A. Exemplary bi-specific myostatin antibodies that may be used in the methods described herein include those described in US Patent Nos. 9,718,881 , 10,526,403, 10,400,036 and 8,871 ,209, the disclosures of which are incorporated herein by reference. In some embodiments, the bi-specific antibody includes an activin A HCVR and LCVR from Table 1 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 1 , such as any one of SEQ ID NOs: 5, 7, 9, 10, 11 , 13, 15, 17, 18, 19, 21 , 23, 25, 27, 29, 31 , 32, 34, 36, 37, 39, 41 , 43, 45, 47, 48, 50, 52, and 54, and a LCVR sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 1 , such as any one of SEQ ID NOs: 6, 8, 12, 14, 16, 20, 22, 24, 26, 28, 30, 33, 35, 38, 40, 42, 44, 46, 49, 51 , and 53) and a myostatin HCVR and LCVR from Table 4 (e.g., a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 4, such as any one of SEQ ID NOs: 152, 154, 156, 158, 160-166, 168-173, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 225,
227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249, 251 , 253, 255, 257, 259, 261 , 263, 265, 267,
269, 271 , 274, 276, 278, 280-284, 286, 287, 289-291 , 293, 295, 297, 299, 301 , 303, 305-317, 319, 325,
327, 329, 331 , 333, 335, 337, 339, 341 , 343, 345, 347, 349, 351 , and 353, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 4, such as any one of SEQ ID NOs: 153, 155, 157, 159, 167, 174- 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209, 211 , 213, 215, 217, 219,
221 , 223, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,
262, 264, 266, 268, 270, 272, 273, 275, 277, 279, 285, 288, 292, 294, 296, 298, 300, 302, 304, 318, 320,
326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, and 354). In some embodiments, the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 2 (e.g., an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 2) and a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from Table 5 (e.g., a myostatin heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 from the same row of Table 5). In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 5 and LCVR of SEQ ID NO: 6 and a myostatin HCVR of SEQ ID NO: 152 and LCVR of SEQ ID NO: 153. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 5 and LCVR of SEQ ID NO: 6 and a myostatin HCVR of SEQ ID NO: 229 and LCVR of SEQ ID NO: 230. In some embodiments, the bi-specific antibody includes an activin A HCVR of SEQ ID NO: 5 and LCVR of SEQ ID NO: 6 and a myostatin HCVR of SEQ ID NO: 233 and LCVR of SEQ ID NO: 234. In some embodiments, the bi- specific antibody includes an activin A HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12 and a myostatin HCVR of SEQ ID NO: 152 and LCVR of SEQ ID NO: 153. In some embodiments, the bi- specific antibody includes an activin A HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12 and a myostatin HCVR of SEQ ID NO: 229 and LCVR of SEQ ID NO: 230. In some embodiments, the bi- specific antibody includes an activin A HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12 and a myostatin HCVR of SEQ ID NO: 233 and LCVR of SEQ ID NO: 234. In some embodiments, the bi- specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 55-60 and a myostatin heavy chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 355-357, a light chain CDR1 of SEQ ID NO: 358, a CDR2 of sequence TTS, and a CDR3 of SEQ ID NO:359. In some embodiments, the bi-specific antibody includes an activin A heavy chain CDR1 , CDR2, and CDR3 and a light chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 61 -66 and a myostatin heavy chain CDR1 , CDR2, and CDR3 of SEQ ID NOs: 355-357, a light chain CDR1 of SEQ ID NO: 358, a CDR2 of sequence TTS, and a CDR3 of SEQ ID NO:359.
In some embodiments, the anti-myostatin protein is an anti-myostatin adnectin recombinant protein. Anti-myostatin adnectin recombinant proteins bind specifically to myostatin and reduce myostatin signaling.
Anti-myostatin adnectin recombinant proteins in clinical trials include taldefgrobep alfa (phase 3). Taldefgrobep alfa (also known as BMS-986089, RG6206, RO7239361 , and BHV-2000) is anti-myostatin adnectin recombinant protein dimer having the sequence of SEQ ID NO: 1810 that binds to the C- terminus of mature myostatin and to the ActRIIB-myostatin complex. Taldefgrobep alfa is described in International Patent Application Publication No. WO2014043344 and Muntoni et al. (Neurol. Ther. 13(1 ) :183-219, 2024), each of which is incorporated herein by reference.
Additional anti-myostatin adnectin recombinant proteins that may be used in the methods described herein include those described in International Patent Application Publication No. WQ2014043344, which is incorporated herein by reference. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence listed in Table 9. In some embodiments, the anti- myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 9, such as any one of SEQ ID NOs: 1810-1896. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 9 from which the His6 tag has been removed (e.g., the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1812-1867 from which the C-terminal His6 tag has been removed). In some embodiments, the anti-myostatin adnectin recombinant protein has a sequence provided in Table 9. In some embodiments, the anti-myostatin adnectin recombinant protein includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 9 fused to an Fc domain by way of a linker (e.g., a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1811 -1867 and 1889-1896 fused to an Fc domain). In some embodiments, an anti-myostatin adnectin recombinant protein of Table 9 fused to an Fc domain forms a dimer (e.g., a homodimer or heterodimer, such as a dimer of any one of SEQ ID NOs: 1810 and 1868-1888, e.g., a homodimer of SEQ ID NO: 1810).
Table 9. Exemplary anti-myostatin adnectin recombinant proteins
ActRII antibodies
In some embodiments, the activin A and myostatin signaling inhibitor is ActRII antibody or an antigen binding fragment thereof. There exist two types of activin type II receptors: ActRIIA and ActRIIB. In some embodiments, the ActRII antibody is an ActRIIA antibody or an antigen binding fragment thereof. In some embodiments, the ActRII antibody is an ActRIIB antibody or an antigen binding fragment thereof. In some embodiments, the ActRII antibody or an antigen binding fragment thereof binds to both ActRIIA and ActRIIB. In some embodiments, the ActRII antibody is bimagrumab (also known as BYM338), CSJ089, CQI876, or CDD861 (described in Morvan et al., PNAS 114:12448-12453 (2017)). Additional ActRII antibodies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2010125003, WO2012064771 , WO2017156488, WO2013063536, WO2018175460, WO2021044287, WO2013188448, and WO2020243448; US Patent Application Publication Nos. US20180066061 , US20180230221 , US20180111991 , US20200181271 , US20210309749, and US20160200818; and US Patent Nos. 9,453,080, 10,266,598, 10,981 ,999, 10,307,455, 11 ,000,565, 10,982,000, 9,969,806, 9,365,651 , 8,388,968, 8,551 ,482, 9,493,556, 8,765,385, and 9,624,301 , each of which is incorporated herein by reference.
In some embodiments, the ActRII antibody or an antigen binding fragment thereof has a HCVR and a LCVR listed in Table 10 (e.g., an HCVR and an LCVR from the same row of Table 10). In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a HCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a HCVR sequence in Table 10, such as any one of SEQ ID NOs: 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584-589, 591 , 592, 594, 595, 597, 599-603, 605, 607, 610, 613, and 615, and a LCVR sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a LCVR sequence in Table 10, such as any one of SEQ ID NOs: 551 , 553, 555, 557, 559, 561 , 563, 565, 567, 569, 571 , 573, 575, 577, 579, 581 , 583, 590, 593, 596, 598, 604, 606, 608, 609, 611 , 612, 614, and 616. In some embodiments, the ActRII antibody or an antigen binding fragment thereof, apart from the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3, has a HCVR and LCVR sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or more sequence identity) to a HCVR and LCVR sequence listed in Table 10. In some embodiments, the ActRII antibody or an antigen binding fragment thereof has the light chain CDR1 , CDR2, and CDR3 and the heavy chain CDR1 , CDR2, and CDR3 sequences of an HCVR sequence and an LCVR sequence in Table 10. In some embodiments, the ActRII antibody or antigen binding fragment thereof includes an HCVR sequence and an LCVR sequence from the same row of Table 10. Table 10. Exemplary HCVR and LCVR sequences of ActRII antibodies
In some embodiments, the ActRII antibody or an antigen-binding fragment thereof, has the CDR sequences described in Table 1 1 (i.e., a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3). In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a light chain variable CDR1 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR1 sequence in Table 1 1 , such as any one of SEQ ID NOs: 620, 626, 632, 649, 655, 661 , 667, 675, 682, 690, 696, 705, 726, 732, 735, 741 , 744, 750, and 756; a light chain variable CDR2 sequence having at least 90% (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR2 sequence in Table 11 , such as any one of SEQ ID NOs: 621 , 627, 633, 650, 656, 662, 668, 676, 683, 691 , 697, 706, 727, 733, 736, 742, 745, 751 , and 757; a light chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a light chain variable CDR3 sequence in Table 11 , such as any one of SEQ ID NOs: 622, 628, 635, 636, 637, 638, 651 , 657, 663, 669, 677, 684, 692, 698, 707, 728, 734, 737, 744, 746, 752, and 758; a heavy chain variable CDR1 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a heavy chain variable CDR1 sequence in Table 11 , such as any one of SEQ ID NOs: 617, 623, 629, 646, 652, 658, 664, 671 , 672, 680, 686, 693, 701 , 702, 711 -714, 721 -723, 729, 738, 747, and 753; a heavy chain variable CDR2 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a heavy chain variable CDR2 sequence in Table 11 , such as any one of SEQ ID NOs: 618, 624, 630, 635, 639-645, 647, 653, 659, 665, 670, 673, 679, 685, 688, 694, 700, 703, 708-710, 718-720, 724, 730, 739, 748, and 754; and a heavy chain variable CDR3 sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence Identity to a heavy chain variable CDR3 sequence in Table 11 , such as any one of SEQ ID NOs: 619, 625, 631 , 648, 654, 660, 666, 674, 678, 681 , 689, 695, 699, 704, 715-717, 725, 731 , 740, 749, and 755. In some embodiments, the ActRII antibody or antigen binding fragment thereof includes a light chain CDR1 , CDR2, and CDR3 sequence and a heavy chain CDR1 , CDR2, and CDR3 sequence from the same row of Table 11 .
Table 11. Exemplary CDR sequences of ActRII antibodies
In some embodiments, the ActRII antibody or an antigen-binding fragment thereof, has a heavy chain and light chain sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to a heavy chain and light chain sequence provided in Table 12. In some embodiments, the ActRII antibody or an antigen binding fragment thereof, has a heavy chain and light chain sequence from the same row of Table 12. In some embodiments, the heavy chain and light chain have the sequence of SEQ ID NOs: 759 and 760; 761 and 762; 763 and 764; 765 and 766; 767 and 768; 769 and 770; 771 and 772; 773 and 774; 775 and 776; 777 and 778; 779 and 780; or 781 and 782 (e.g., the heavy chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the first SEQ ID
NO: in each pair and the light chain has at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100% sequence identity) to the sequence of the second SEQ ID NO: in each pair). Table 12. Exemplary heavy and light chain sequences of ActRII antibodies
ActRII ligand traps
In some embodiments, the activin A and myostatin signaling inhibitor is an ActRII ligand trap. ActRII ligand traps are polypeptides that contain an extracellular portion of ActRIIA and/or ActRIIB or a variant or chimera thereof that are capable of binding to one or more ActRII ligands (e.g., activin A and myostatin). The extracellular portion of ActRIIA and/or ActRIIB or a variant or chimera thereof may be fused to an Fc domain monomer by way of a linker and may form a dimer through the interaction between two Fc domain monomers. ActRII ligand traps can reduce or inhibit the binding of ActRII ligands to endogenous activin type II receptors, thereby reducing ActRII signaling. As the ActRII ligand traps contain the extracellular portion of the receptor, they will be soluble and able to bind to and sequester ligands (e.g., activin A and myostatin) without activating intracellular signaling pathways.
In some embodiments, the ActRII ligand trap is an ActRIIA ligand trap. The ActRIIA ligand trap may contain an extracellular portion of wild-type ActRIIA (e.g., human or murine ActRIIA) or may contain an extracellular portion of wild-type ActRIIA that contains one or more amino acid substitutions relative to the wild-type human extracellular ActRIIA. The wild-type amino acid sequence of the extracellular portion of human ActRIIA is shown below.
Human ActRIIA, extracellular portion (SEQ ID NO: 2):
GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS
An ActRIIA ligand trap may contain the sequence of SEQ ID NO: 2 or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap contains a portion of SEQ ID NO: 2 (e.g., a contiguous portion that is shortened by the removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIA ligand trap contains the sequence of SEQ ID NO: 2 or a portion thereof with additional amino acids at the C-terminus from the wild-type sequence of ActRIIA (SEQ ID NO: 1 ). An exemplary sequence of a portion of wild-type ActRIIA protein that is shortened at the N-terminus and includes additional amino acids from SEQ ID NO: 1 at the C-terminus that can be included in an ActRIIA ligand trap is provided below: ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGC WLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP (SEQ ID NO: 783)
Studies have shown that BMP9 binds ActRIIB with about 300-fold higher binding affinity than ActRIIA (see, e.g., Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA-Fc is known to have a longer half-life compared to ActRIIB-Fc. Described herein below are ActRIIA ligand traps containing extracellular ActRIIA variants that are constructed by introducing amino acid residues of ActRIIB to ActRIIA, with the goal of imparting physiological properties conferred by ActRIIB, while also maintaining beneficial physiological and pharmacokinetic properties of ActRIIA. The optimum peptides increase lean mass, muscle mass, and/or muscle strength while retaining low binding-affinity to BMP9 and longer serum half-life as an Fc fusion protein, for example. The preferred ActRIIA variants also exhibit similar or improved binding to activins and/or myostatin compared to wild-type ActRIIA, which allows them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat DMD by increasing lean mass, muscle mass, muscle strength, and/or dystrophin expression. In some embodiments, amino acid substitutions may be introduced to an extracellular ActRIIA variant to reduce or remove the binding affinity of the variant to BMP9.
ActRIIA ligand traps described herein can include an extracellular ActRIIA variant having at least one amino acid substitution relative to the wild-type extracellular ActRIIA having the sequence of SEQ ID NO: 2. Possible amino acid substitutions at 27 different positions may be introduced to an extracellular ActRIIA variant (Table 13). In some embodiments, an extracellular ActRIIA variant may have at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) amino acid sequence identity to the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 2). An extracellular ActRIIA variant may have one or more (e.g., 1 -27, 1 -25, 1 -23, 1 -21 , 1 -19, 1 -17, 1 -15, 1 -13, 1 -1 1 , 1 -9, 1 -7, 1 -5, 1 -3, or 1 -2; e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27) amino acid substitutions relative the sequence of a wild-type extracellular ActRIIA (SEQ ID NO: 2). In some embodiments, an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having a sequence of SEQ ID NO: 784) may include amino acid substitutions at all of the 27 positions as listed in Table 13. In some embodiments, an extracellular ActRIIA variant may include amino acid substitutions at a number of positions, e.g., at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 out of the 27 positions, as listed in Table 13.
Amino acid substitutions can worsen or improve the activity and/or binding affinity of the ActRIIA variants of the invention. To maintain polypeptide function, it is important that the lysine (K) at position X17 in the sequences shown in Tables 13 and 14 (SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) be retained. Substitutions at that position can lead to a loss of activity. For example, an ActRIIA variant having the sequence GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYD RTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 1091 ) has reduced activity in vivo, indicating that the substitution of alanine (A) for lysine (K) at X17 is not tolerated. ActRIIA variants of the invention, including variants in Tables 13 and 14 (e.g., SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), therefore, retain amino acid K at position X17.
The ActRIIA variants of the invention preferably have reduced, weak, or no substantial binding to BMP9. BMP9 binding is reduced in ActRIIA variants (e.g., reduced compared to wild-type ActRIIA) containing the amino acid sequence TEEN (SEQ ID NO: 1092) at positions X23, X24, X25, and X26, as well as in variants that maintain the amino acid K at position X24 and have the amino acid sequence TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26. The sequences TEEN (SEQ ID NO: 1092) and TKEN (SEQ ID NO: 1093) can be employed interchangeably in the ActRIIA variants (e.g., the variants in Tables 13 and 14, e.g., SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) of the invention to provide reduced BMP9 binding.
Table 13. Amino acid substitutions in an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 784-788 The ActRIIA variants of the invention may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one or more additional amino acids at the C-terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) to any of the variants shown in Tables 13 and 14 (e.g., SEQ ID NOs: 784-853 (e.g., SEQ ID NOs: 789-853)). The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA. One potential C-terminal extension that can be included in the ActRIIA variants of the invention is amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 854 (e.g., SEQ ID NO: 852 with a C-terminal extension of NP). Another exemplary C-terminal extension that can be included in the ActRIIA variants of the invention is amino acid sequence NPVTPK (SEQ ID NO: 1090). For example, a sequence including the C-terminal extension NPVTPK (SEQ ID NO: 1090) is SEQ ID NO: 855 (e.g., SEQ ID NO: 852 with a C-terminal extension of NPVTPK (SEQ ID NO: 1090)).
In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 784 or 785, X3 is E, Xs is R, Xn is D, X12 is K, X13 is R, X16 is K or R, X17 is K, X19 is W, X20 is L, X21 is D, and X22 is I or F. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 784, X2 is Y; X4 is L; Xs is E; X9 is E; Xu is L; Xis is K; X23 is T; X25 is E; X26 is N; and X27 is Q. These substitutions in SEQ ID NO: 784 can also be made in SEQ ID NOs: 785-788. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 784, Xi is F or Y; X2 is Y; X4 is L; X5 is D or E; X7 is P or R; Xs is E; X9 is E; X10 is K or Q; Xu is L; X15 is F or Y; X is K or R; X is K; X22 is I or F; X23 is T; X24 is K or E; X25 is E; X26 is N; and X27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 784, Xi is F or Y; X2 is Y; X3 is E; X4 is L; X5 is D or E; Xs is R; X7 is P or R; Xs is E; X9 is E; X10 is K or Q; Xn is D; X12 is K; X13 is R; Xu is L; X15 is F or Y; X16 is K or R; X17 is K; Xis is K; X19 is W; X20 is L; X21 is D; X22 is I or F; X23 is T; X24 is K or E; X25 is E; X26 is N; and X27 is Q. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NO: 784 or 785, X17 is K. In some embodiments of the extracellular ActRIIA variant having the sequence of SEQ ID NOs: 784-786, X17 is K, X23 is T, X24 is E, X25 is E, and X26 is N. In some embodiments of the extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784- 788, X17 is K, X23 is T, X24 is K, X25 is E, and X26 is N.
In some embodiments, an ActRIIA ligand trap described herein includes an extracellular ActRIIA variant having a sequence of any one of SEQ ID NOs: 789-855 (Table 14).
Table 14. Extracellular ActRIIA variants having the sequences of SEQ ID NOs: 789-855
In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) has amino acid K at position X17. Altering the amino acid at position X17 can result in reduced activity. For example, an ActRIIA variant having the sequence
GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYD RTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 1091 ) has reduced activity in vivo, indicating that the substitution of A for K at X17 is not tolerated. In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) with the sequence TEEN (SEQ ID NO: 1092) at positions X23, X24, X25, and X26 can have a substitution of the amino acid K for the amino acid E at position X24. In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855)) with the sequence TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26 can have a substitution of the amino acid E for the amino acid K at position X24. ActRIIA variants having the sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26 have reduced or weak binding to BMP9 (e.g., reduced binding to BMP9 compared to BMP9 binding of wild-type ActRIIA).
In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant (e.g., any one of SEQ ID NOs: 784-853 (e.g., SEQ ID NOs: 789-853)) may further include a C-terminal extension (e.g., one more additional amino acids at the C-terminus of the ActRIIA variant). The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA. In some embodiments, the C- terminal extension is amino acid sequence NP. For example, a sequence including the C-terminal extension NP is SEQ ID NO: 854 (e.g., SEQ ID NO: 852 with a C-terminal extension of NP). In some embodiments, the C-terminal extension is amino acid sequence NPVTPK (SEQ ID NO: 1090). For example, a sequence including the C-terminal extension NPVTPK (SEQ ID NO: 1090) is SEQ ID NO: 855 (e.g., SEQ ID NO: 852 with a C-terminal extension of NPVTPK (SEQ ID NO: 1090)). The C-terminal extension can add one or more amino acids at the C-terminus of the ActRIIA variant (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids).
In some embodiments, an ActRIIA ligand trap including an extracellular ActRIIA variant may further include an Fc domain monomer, which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIA variant by way of a linker or other covalent bonds. A polypeptide including an extracellular ActRIIA variant fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.
Furthermore, in some embodiments, an ActRIIA ligand trap described herein (e.g., an ActRIIA variant-Fc fusion protein) has a serum half-life of at least 7 days in humans. The ActRIIA ligand trap may bind to activin A with a KD of 10 pM or higher. In some embodiments, the ActRIIA ligand trap does not bind to BMP9 or activin A. In some embodiments, the ActRIIA ligand trap binds to activin A, activin B, and/or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., reduced BMP9 binding compared to BMP9 binding of wild-type ActRIIA). In some embodiments, the ActRIIA ligand trap that has reduced or weak binding to BMP9 has the sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X23, X24, X25, and X26. In some embodiments, the ActRIIA ligand trap does not substantially bind to human BMP9.
In some embodiments, the ActRIIA ligand trap may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM). In some embodiments, the ActRIIA ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM). In some embodiments, the ActRIIA ligand trap is sotatercept (also known as ACE-011 ). Additional ActRIIA ligand traps that may be used in the methods described herein include those described in International Patent Application Publication No. W02007062188 and US Patent Nos. 7,709,605, 9,138,459, 7,612,041 , 8,067,360, 8,629,109, 9,572,865, 9,163,075, 10,071 ,135, 11 ,013,785, and 7,951 ,771 , each of which is incorporated herein by reference.
In some embodiments, the ActRII ligand trap is an ActRIIB ligand trap. The ActRIIB ligand trap may contain an extracellular portion of wild-type ActRIIB (e.g., human or murine ActRIIB) or may contain an extracellular portion of wild-type ActRIIB that contains one or more amino acid substitutions relative to the wild-type human extracellular ActRIIB. The wild-type amino acid sequence of the extracellular portion of human ActRIIB is shown below.
Human ActRIIB, extracellular portion (SEQ ID NO: 4): GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWL DDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT
An ActRIIB ligand trap may contain the sequence of SEQ ID NO: 4 or a variant thereof that contains one or more amino acid substitutions. In some embodiments, the ActRIIB ligand trap contains a portion of SEQ ID NO: 4 (e.g., a contiguous portion that is shortened by the removal of amino acids from the N-terminus, C-terminus, or both) or a variant thereof that contains one or more amino acid substitutions. For example, the ActRIIB ligand trap can include the sequence of SEQ ID NO: 4 with an L60D substitution. In another example, the ActRIIB ligand trap can include the sequence of SEQ ID NO: 4 with a substitution at position E9 (e.g., an E9W, E9A, E9F, E9Q, E9V, E9I, E9L, E9M, E9K, E9H, or E9Y substitution), an S25T substitution, and/or an R45A substitution. In some embodiments, the ActRIIB ligand trap is BIIB110 (previously known as ALG-801 ), ALG-802, luspatercept (REBLOZYL®, also known as ACE-536), Ramatercept (also known as ACE-031 ), or ACE-2494. Additional ActRIIB ligand traps that may be used in the methods described herein include those described in International Patent Application Publication Nos. WQ2010062383, WO2015192127, WQ2019140283, and WQ2021189010; US Patent Application Publication Nos. US20110250198 and US20200407415; and US Patent Nos. 10,913,782, 8,058,229, 8,216,997, 8,703,927, 9,439,945, 9,932,379, 10,131 ,700, 10,689,427, 10,889,626, 10,829,532, 10,829,533, 8,361 ,957, 9,505,813, 10,377,996, 9,617,319, 8,710,016, 7,709,605, 8,252,900, 7,842,663, 8,343,933, 9,399,669, 10,259,861 , 8,138,142, 8,178,488, 8,293,881 , 9,181 ,533, 9,745,559, 10,358,633, 11 ,066,654, 9,610,327, 9,284,364, 8,067,562, 8,614,292, 7,947,646, 8,716,459, 8,501 ,678, 8,999,917, 9,447,165, 9,809,638, 10,407,487, 8,410,043, 9,273,114, and 10,308,704, each of which is incorporated herein by reference.
In some embodiments, the ActRIIB ligand trap contains an ActRIIB variant having the sequence of SEQ ID NO: 856 shown in Table 15. Table 15. Amino acid substitutions in an extracellular ActRIIB variant having a sequence of SEQ ID NO: 856
In some embodiments, the ActRIIB variant has the sequence of any one of SEQ ID NOs: 857-870 (Table 16).
Table 16. Extracellular ActRIIB variants having the sequences of SEQ ID NOs: 857-870
In some embodiments, the extracellular ActRIIB variant has an N-terminal truncation of 1 -7 amino acids (e.g., 1 , 2, 3, 4, 5, 6, or 7 amino acids). An N-terminal truncation can be produced by removing 1 -7 amino acids from the N-terminus of an of an ActRIIB variant shown in Tables 15 and 16. The N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE) are retained in the N-terminally truncated ActRIIB variants). Additional ActRIIB variants having an N-terminal truncation are provided below:
ETRECIYYNANWELERTNQSGLERCYGDKDKRRHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ
ID NO: 871 )
ETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDI NCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 872) ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT (SEQ ID NO: 873)
ETRWCIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 874)
ETRWCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDD FNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 875)
ETRYCIYYNANWELERTNQTGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDF NCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 876)
In some embodiments, an ActRIIB ligand trap including an ActRIIB variant may further include an Fc domain monomer, which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIB variant by way of a linker or other covalent bonds. An ActRIIB ligand trap including an extracellular ActRIIB variant fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.
Furthermore, in some embodiments, an ActRIIB ligand trap described herein has a serum half-life of at least 7 days in humans. The ActRIIB ligand trap may bind to bone morphogenetic protein 9 (BMP9) with a KD of 200 pM or higher. The ActRIIB ligand trap may bind to activin A with a KD of 10 pM or higher. In some embodiments, the ActRIIB ligand trap does not bind to BMP9 or activin A. In some embodiments, the ActRIIB ligand trap binds to activin and/or myostatin and exhibits reduced (e.g., weak) binding to BMP9.
Additionally, in some embodiments, the ActRIIB ligand trap may bind to human BMP9 with a KD of about 200 pM or higher (e.g., a KD of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or higher, e.g., a KD of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or higher, e.g., a KD of between about 200 pM and about 50 nM). In some embodiments, the ActRIIB ligand trap does not substantially bind to human BMP9. In some embodiments, the ActRIIB ligand trap may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM). In some embodiments, the ActRIIB ligand trap may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 200 pM) The ActRIIB ligand trap may also bind to growth and differentiation factor 11 (GDF-11 ) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher).
In some embodiments, the ActRII ligand trap is an ActRII chimera ligand trap. The ActRII chimera ligand traps contain portions of extracellular ActRIIA (e.g., human ActRIIA) and extracellular ActRIIB (e.g., human ActRIIB). In some embodiments, the ActRII chimera ligand traps described herein contain an N- terminal portion of extracellular ActRIIB (SEQ ID NO: 4 shown above) joined to a C-terminal portion of extracellular ActRIIA (SEQ ID NO: 2 shown above) such that the sequences are contiguous (e.g., the ActRIIA sequence continues where the ActRIIB sequence left off, starting with the next the amino acid located in the corresponding position of ActRIIA). In some embodiments, the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the six amino acids found at the N-terminus of extracellular ActRIIA joined to the fifth amino acid of extracellular ActRIIB. In some embodiments, the N- terminus of the ActRII chimera included in the ActRII chimera ligand trap begins with the first amino acid located at the N-terminus of extracellular ActRIIB. Accordingly, in some embodiments, the N-terminal portion of ActRIIB begins with the amino acid in the fifth position of SEQ ID NO: 4 (A), while in other embodiments (e.g., in embodiments in which the six amino acids found at the N-terminus of extracellular ActRIIA are not included in the chimera), the N-terminal portion of ActRIIB begins with the amino acid in the first position of SEQ ID NO: 4 (G). In some embodiments, the N-terminus of the ActRII chimera included in the ActRII chimera ligand trap includes the first ten amino acids found at the N-terminus of extracellular ActRIIA joined to the ninth amino acid of extracellular ActRIIB, in which case the N-terminal portion of ActRIIB begins with the amino acid in the ninth position of SEQ ID NO: 4. The extracellular ActRII chimera included in the ActRII chimera ligand trap may also include one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIB compared to wildtype extracellular ActRIIB (e.g., SEQ ID NO: 4 shown above), and/or one or more amino acid substitutions in the portion of the chimera that corresponds to the sequence of ActRIIA compared to wildtype extracellular ActRIIA (e.g., SEQ ID NO: 2 shown above). Amino acid substitutions at 9 different positions may be introduced into an extracellular ActRII chimera (Table 17). An extracellular ActRII chimera may have one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions relative to the sequence of a wild-type sequence (e.g., relative to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 4) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIB, or relative to the sequence of wild-type extracellular ActRIIA (SEQ ID NO: 2) if the portion of the chimera corresponds to a region of wild-type extracellular ActRIIA). The positions at which amino acid substitutions may be made, as well as the amino acids that may be substituted at these positions, are listed in Table 17. ActRII chimera ligand traps that may be used in the methods described herein include those described in US Patent Application Publication No. US20230079602, the disclosure of which is incorporated herein by reference.
Amino acid substitutions can alter the activity and/or binding affinity of the extracellular ActRII chimeras. In some embodiments, the extracellular ActRII chimeras bind to activin A, activin B, myostatin, and/or GDF11 with sufficient affinity to compete with endogenous activin receptors for binding to one or more of these ligands. In some embodiments, the extracellular ActRII chimeras have reduced, weak, or no substantial binding to BMP9 (e.g., compared to wild-type ActRIIB). BMP9 binding may be reduced in extracellular ActRII chimeras containing the amino acid sequence TEEN (SEQ ID NO: 1092) or TKEN (SEQ ID NO: 1093) at positions X3, X4, X5, and Xe. In some embodiments, BMP9 binding is reduced in extracellular ActRII chimeras containing the amino acid sequence KKDS (SEQ ID NO: 1245) or TKDS (SEQ ID NO: 1246) at positions X3, X4, X5, and Xe. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919)) with the sequence TEEN (SEQ ID NO: 1092) at positions X3, X4, X5, and Xe can have a substitution of the amino acid K for the amino acid E at position X4. In some embodiments, a polypeptide including an extracellular ActRII chimera (e.g., any one of SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919)) with the sequence TKEN (SEQ ID NO: 1093) at positions X3, X4, X5, and Xe can have a substitution of the amino acid E for the amino acid K at position X4. The sequences TEEN (SEQ ID NO: 1092) and TKEN (SEQ ID NO: 1093) can be used interchangeably in the extracellular ActRII chimeras (e.g., the chimeras in Tables 17 and 18, e.g., SEQ ID NOs: 877-919 (e.g., SEQ ID NOs: 898-919)).
Table 17. Amino acid substitutions in an extracellular ActRII chimera having a sequence of any one of SEQ ID NOs: 877-897 GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWKNISGSIEIVKQGCWLDDX2X3C
YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 886)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGSIEIVKQGCWLDDX2X3C
YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 887)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIEIVKQGCWLDDX2X3C
YDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 888)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2X3
CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 889)
GRGEAETRECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2X3
CYDRQECVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 890)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRRHCFATWKNISGSIEIVKQGCWLDDX2X3
CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 891 )
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCFATWKNISGSIEIVKQGCWLDDX2X3
CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 892)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWKNISGSIEIVKQGCWLDDX2X3 CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 893)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGSIEIVKQGCWLDDX2
X3CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 894)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIEIVKQGCWLDDX2
X3CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 895)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2
X3CYDRTDCVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 896)
GAILGRSETQECIYYNANWELERTNQSGLERCEGEQX1KRLHCYASWRNSSGTIELVKKGCWLDDX2
X3CYDRQECVX4X5X6X7X8PX9VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 897)
In some embodiments, in ActRII chimeras of SEQ ID NOs: 877-897 (shown in Table 17), Xi is D, X2 is I, F, or E, X3 is N or T, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xs is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 877-897, Xi is D, X2 is I or F, X3 is N, X4 is A or E, X5 is T or K, Xe is E or K, X7 is E or D, Xs is N or S, and X9 is E or Q. In some embodiments, in the extracellular ActRII chimeras of SEQ ID NOs: 877-897, Xi is D, X2 is F, X3 is N, X4 is E, X5 is K, Xe is K, X7 is D, Xs is S, and X9 is Q.
In some embodiments, ActRII chimera ligand trap contains the sequence of any one of SEQ ID NOs: 898-919 (Table 18).
Table 18. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 898-919
In some embodiments, a polypeptide containing an ActRII chimera of Table 17 or 18 may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 30 pM). In some embodiments, the polypeptide containing an ActRII chimera of Table
17 or 18 may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM) The polypeptide containing an ActRII chimera of Table 17 or 18 may also bind to growth and differentiation factor 11 (GDF-11 ) with a KD of approximately 5 pM or higher (e.g., a KD of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or higher). In some embodiments, the ActRII chimera included in the ActRII chimera ligand trap results from the substitution of one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more intervening sequence (e.g., a sequence between the p-sheets), from one ActRII protein (e.g., ActRIIB) into the corresponding position of the other ActRII protein (e.g., ActRIIA). For example, an ActRII chimera may be produced by replacing one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more or an intervening sequence, in ActRIIB with an amino acid sequence corresponding to the p-sheet and, optionally, the intervening sequence, from ActRIIA. An ActRII chimera may also be produced by replacing one or more amino acid sequence corresponding to a p-sheet and, optionally, one or more intervening sequence, in ActRIIA with an amino acid sequence corresponding to the p-sheet and, optionally, the intervening sequence, from ActRIIB. In the ActRII chimeras, a p-sheet and, optionally, an intervening sequence from one protein is replaced with the corresponding p-sheet and, optionally, the corresponding intervening sequence from the other protein (e.g., the 5th p-sheet from ActRIIA (PSA) can be replaced with the 5th p-sheet from ActRIIB (PSB)). Each ActRII protein has seven p- sheets (pi -p?) and eight intervening sequences (Xi-Xs). The ActRII chimeras include at least one of pia, p2a, p3a, p4a, psa, or p7a and at least one of p , p2b, p3b, p4b, psb, or p7b. Accordingly, an ActRII chimera included in the ActRII chimera ligand trap may have one to five p-sheet substitutions (e.g., 1 , 2, 3, 4, or 5 of pi , p2, p3, p4, ps, and p7 from one ActRII protein may be substituted with the corresponding p-sheet sequence from the other ActRII protein). The ActRII chimera may also have one to seven intervening sequence substitutions (e.g., 1 , 2, 3, 4, 5, 6, or 7 of Xi , X2, X3, Xs, Xe, X7, and Xs from one ActRII protein may be substituted with the corresponding intervening sequence from the other ActRII protein). In some embodiments, the p-sheet sequence that is substituted is a minimal p-sheet sequence (e.g., at least HCFATWK (SEQ ID NO: 931 ), which is a portion of RHCFATWKNI (p3a) (SEQ ID NO: 930); at least HCYASWR (SEQ ID NO: 933), which is a portion of LHCYASWRNS (p3b) (SEQ ID NO: 932); at least EIVKQGCW (SEQ ID NO: 935), which is a portion of SIEIVKQGCW (p4a) (SEQ ID NO: 934); at least ELVKKGCW (SEQ ID NO: 937), which is a portion of TIELVKKGCW (p4b) (SEQ ID NO: 936); at least VE, which is a portion of VEK (psa); at least V, which is a portion of VAT (psb); at least SYF, which is a portion of KFSYF (p7a) (SEQ ID NO: 945); or at least T, which is a portion of RFTHL (p7b) (SEQ ID NO: 946)). The extracellular ActRII chimeras are the same length (e.g., have the same number of amino acids) as wild-type extracellular ActRIIA and ActRIIB, therefore, in embodiments in which minimal p-sheet sequences are substituted, contiguous amino acids from ActRIIA or ActRIIB are used to connect the minimal p-sheet to the neighboring intervening sequences to maintain the length (e.g., the number of amino acids) of the ActRII chimeras (e.g., to prevent the extracellular ActRII chimeras from having fewer amino acids than the corresponding regions of extracellular ActRIIA and ActRIIB). Exemplary ActRII chimera sequences that can be included in an ActRII chimera ligand trap are provided in Table 19. ActRII chimera ligand traps that may be used in the methods described herein include those described in International Patent Application Publication No. WQ2022235620 and International Patent Application Publication No. WQ2024102906, the disclosures of which is incorporated herein by reference. Table 19. Extracellular ActRII chimera sequences
In some embodiments, the ActRII chimera has the sequence of an ActRII chimera listed in Table , below. Table 20. Extracellular ActRII chimeras having the sequences of SEQ ID NOs: 1128-1158
In some embodiments, a polypeptide containing an extracellular ActRII chimera of Table 20 may bind to human activin A with a KD of about 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 300 pM and about 1 pM). In some embodiments, the polypeptide containing an extracellular ActRII chimera of Table 20 may bind to human activin B with a KD of 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 200 pM and about 1 pM, or a KD of less than 1 pM). The polypeptide containing an extracellular ActRII chimera of Table 20 may also bind to growth and differentiation factor 11 (GDF-11 ) with a KD of approximately 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 200 pM and about 1 pM, or a KD of less than 1 pM). The polypeptide containing an extracellular ActRII chimera of Table 20 may bind to GDF-8 with a KD of approximately 800 pM or less (e.g., a KD of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a KD of between about 800 pM and about 5 pM). In some embodiments, the polypeptide containing an extracellular ActRII chimera of Table 20 may bind to human BMP9 with a KD of about 1 pM or higher (e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nM or higher, e.g., a KD of 1 nM or higher). The polypeptide containing an extracellular ActRII chimera of Table 20 may also bind to human BMP10 with a KD of about 1 pM or higher (e.g., a KD of about 1 , 5, 15, 30, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pM or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM or higher).
In some embodiments, the extracellular ActRII chimeras described herein have an N-terminal truncation of 1 -9 amino acids (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). The N-terminal truncation can involve the removal of 1 -9 amino acids from the N-terminus of any of the chimeras shown in Tables 17- 20. The N-terminal truncation can remove amino acids up two to amino acids before the first cysteine (e.g., the two amino acids before the first cysteine (RE or QE) are retained in the N-terminally truncated ActRII chimera ligand traps).
The extracellular ActRII chimera ligand traps may further include a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension can add one or more additional amino acids at the C-terminus (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) to any of the chimeras shown in Tables 17-20. The C-terminal extension may correspond to sequence from the same position in wild-type ActRIIA or ActRIIB. For example, C-terminal extensions that can be included in the extracellular ActRII chimera ligand traps of the invention are the amino acid sequence NP and the amino acid sequence NPVTPK (SEQ ID NO: 1090), which correspond to sequence found in the same position in wild-type ActRIIA. In some embodiments, a polypeptide containing an extracellular ActRII chimera may further include an Fc domain monomer, which may be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRII chimera by way of a linker or other covalent bonds. An ActRII chimera ligand trap including an extracellular ActRII chimera fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.
In some embodiments, a polypeptide containing an extracellular ActRII chimera described herein (e.g., an ActRII chimera-Fc fusion protein) has a serum half-life of at least 7 days in humans. The polypeptide containing an ActRII chimera described herein may bind to activin A with a KD of 1 pM or higher (e.g., 10 pM or higher). In some embodiments, the polypeptide containing an ActRII chimera described herein binds to activin A, activin B, and/or myostatin and exhibits reduced (e.g., weak) binding to BMP9 (e.g., compared to wild-type extracellular ActRIIB). In some embodiments, the polypeptide containing an ActRII chimera described herein does not substantially bind to human BMP9.
Dystrophin exon skipping therapies
An activin A and myostatin signaling inhibitor described herein can be administered to a subject having DMD in combination with a dystrophin exon skipping therapy. In some embodiments, the dystrophin exon skipping therapy is an exon 2, 44, 45, 50, 51 , or 53 skipping therapy.
In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the dystrophin gene (called the DMD gene) that is amenable to exon 44 skipping in combination with a dystrophin exon 44 skipping therapy.
Dystrophin exon 44 skipping therapies in clinical trials include NS-089/NCNP-02 (phase 2), AOC 1044 (phase 1/2), and ENTR-601 -44 (phase 1 ). NS-089/NCNP-02 (also known as brogidirsen) is a PMO having the sequence of SEQ ID NO: 949 and is described in International Patent Application Publication No. WO2015194520 and Watanabe et al. (Mol. Ther. Nucleic Acids 34, 2023), each of which is incorporated herein by reference. Additional dystrophin exon 44 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2015194520, WO2023196400, and WO2023034817, each of which is incorporated herein by reference.
In some embodiments, the exon 44 skipping therapy includes 18-30 consecutive nucleotides of SEQ ID NO: 984, in which the first nucleotide starts at position 1 , 2, 3, 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, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70,
71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98,
99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, or 131 of SEQ ID NO: 984.
In some embodiments, the exon 44 skipping therapy includes 18-30 consecutive nucleotides of SEQ ID NO: 985, in which the first nucleotide starts at position 1 , 2, 3, 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, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70,
71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, or 131 of SEQ ID NO: 985.
In some embodiments, the exon 44 skipping therapy includes a sequence listed in Table 21 . In some embodiments, the exon 44 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table
21 , such as any one of SEQ ID NOs: 949-990, 1264-1431 , or 1794-1796. In some embodiments, the exon 44 skipping therapy has the sequence of any of the sequences listed in Table 21 .
Table 21. Exemplary exon 44 skipping therapies
In some embodiments, in any of the exon 44 skipping therapies provided in Table 21 , any one or more of the thymine bases (T) may optionally be a uracil base (U).
In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 45 skipping in combination with a dystrophin exon 45 skipping therapy. Casimersen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 45 skipping (under brand name AMONDYS 45™). It can be administered as an intravenous infusion over 35 to 60 minutes via an in-line 0.2 micron filter at a dose of 30 milligrams per kilogram of body weight once weekly. Casimersen is a PMO having the sequence of SEQ ID NO: 991 .
Exon 45 skipping therapies in clinical trials include renadirsen (phase 2). Renadirsen (also known as DS-5141 b) is a 2’-O,4’-C-ethylene-bridged phosphorothioate modified oligonucleotide having the sequence of SEQ ID NO: 992 and is described in International Patent Application Publication No.
W02022020107 and Ito et al. (Curr. Issues Mol. Biol. 43:1267-1281 , 2021 ), each of which is incorporated herein by reference. Additional dystrophin exon 45 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos.
WO2018118627 and W02022020107, each of which is incorporated herein by reference. In some embodiments, the exon 45 skipping therapy includes a sequence listed in Table 22. In some embodiments, the exon 45 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 22, such as any one of SEQ ID NOs: 991 , 992, 1432-1545, or 1797-1801 . In some embodiments, the exon 45 skipping therapy has a sequence provided in Table 22.
Table 22. Exemplary exon 45 skipping therapies
In some embodiments, in any of the exon 45 skipping therapies provided in Table 22, any one or more of the thymine bases (T) may optionally be a uracil base (U). In some embodiments, nucleotide position 16 of SEQ ID NO: 992 contains U in place of T. In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 50 skipping in combination with a dystrophin exon 50 skipping therapy.
Exon 50 skipping therapies in clinical trials include NS-050/NCNP-03 (phase 1/2).
Additional dystrophin exon 50 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication No. WO2021132591 A1 , which is incorporated herein by reference. In some embodiments, the exon 50 skipping therapy includes a sequence listed in Table 23. In some embodiments, the exon 50 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 23, such as any one of SEQ ID NOs: 993-999, 1546-1580, or 1802-1804. In some embodiments, the exon 50 skipping therapy has a sequence provided in Table 23. Table 23. Exemplary exon 50 skipping therapies
In some embodiments, in any of the exon 50 skipping therapies provided in Table 23, any one or more of the thymine bases (T) may optionally be a uracil base (U).
In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 51 skipping in combination with a dystrophin exon 51 skipping therapy.
Eteplirsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 51 skipping (under brand name EXONDYS 51 ™). It can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly. Eteplirsen is a PMO having the sequence of SEQ ID NO: 1000.
Exon 51 skipping therapies in clinical trials include vesleteplirsen (phase 2), drisapersen (phase 3), DYNE-251 (phase 1/2), PGN-EDO51 (phase 2), suvodirsen (phase 2/3), and BMN-351 (phase 1/2). Vesleteplirsen is a PMO including the sequence of SEQ ID NO: 1001 conjugated to a cell-penetrating peptide and is described in International Patent Application Publication No. WO2022232478A1 , which is incorporated herein by reference. Drisapersen is a 2'-O-methyl phosphorothioate modified oligonucleotide having the sequence of SEQ ID NO: 1002 and is described in International Patent Application Publication No. WQ2009054725A2. PGN-EDO51 is a PMO conjugated to a peptide and is described in International Patent Application Publication No. WO2022192749, which is incorporated herein by reference. Suvodirsen (also known as WVE-210201 ) is a 2’-fluoro-2'-deoxy-modified phosphorothioate (Sp)/phosphodiester modified oligonucleotide having the sequence of SEQ ID NO: 1028 and is described in International Patent Application Publication No. WQ2019217784A1 , which is incorporated herein by reference. BMN-351 is an oligonucleotide having the sequence of SEQ ID NO: 1897, in which the cytosine nucleotides are 5-methylcytosine, the guanine and cytosine nucleotides are locked nucleic acids (LNA), a tri-ethylene glycol (TEG) group is attached to the 5' terminus of the oligonucleotide via a phosphate group, the internucleoside linkages are phosphorothioate linkages, and the non-LNA nucleotides are 2'-OMe nucleotides, as described in U.S. Patent Application Publication No. US20230416741 A1 , which is incorporated herein by reference. Additional dystrophin exon 51 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WQ2006000057A1 , WO2022232478A1 , WQ2009054725A2, WQ2022192749, and WO2019217784A1 , and US Patent Application Publication No. US20230045002A1 , each of which is incorporated herein by reference.
In some embodiments, the exon 51 skipping therapy includes a sequence listed in Table 24. In some embodiments, the exon 51 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 24, such as any one of SEQ ID NOs: 1000-1028, 1247-1256, 1581 -1745, 1805, or 1897. In some embodiments, the exon 51 skipping therapy has a sequence provided in Table 24.
Table 24. Exemplary exon 51 skipping therapies
In some embodiments, in any of the exon 51 skipping therapies provided in Table 24, any one or more of the thymine bases (T) may optionally be a uracil base (U). In some embodiments, any one of SEQ ID NOs: 1002-1028 or 1247-1256 contains U in place of T.
In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 53 skipping in combination with a dystrophin exon 53 skipping therapy.
Golodirsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping (under brand name VYONDYS 53™). It can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly. Golodirsen is a PMO having the sequence of SEQ ID NO: 1029.
Viltolarsen is a dystrophin exon skipping therapy FDA approved for the treatment of patients with DMD who have a confirmed mutation of the DMD gene that is amenable to exon 53 skipping (under brand name VILTEPSO™). It can be administered as an intravenous infusion over 60 minutes at a dose of 80 mg/kg once weekly. Viltolarsen is a PMO having the sequence of SEQ ID NO: 1030.
Exon 53 skipping therapies in clinical trials include WVE-N531 (phase 1 b/2a). WVE-N531 is a 2’- F and 2’-OMe modified oligonucleotide having the sequence of SEQ ID NO: 1031 with 17 stereodefined internucleotide linkages, 14 of which are Sp phosphorothioate linkages, and three as Rp N-(1 ,3- dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages, and is described in WO2023168014A2, which is incorporated herein by reference.
Additional dystrophin exon 53 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication Nos. WO2018118662A1 , W02020004675A1 , and WO2023168014A2, each of which is incorporated herein by reference.
In some embodiments, the exon 53 skipping therapy includes a sequence listed in Table 25. In some embodiments, the exon 53 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table 25, such as any one of SEQ ID NOs: 1029-1031 , 1746-1793, or 1806-1809. In some embodiments, the exon 53 skipping therapy has a sequence provided in Table 25.
Table 25. Exemplary exon 53 skipping therapies
In some embodiments, in any of the exon 53 skipping therapies provided in Table 25, any one or more of the thymine bases (T) may optionally be a uracil base (U). In some embodiments, SEQ ID NO: 1031 contains U in place of T. In some embodiments, an activin A and myostatin signaling inhibitor described herein is administered to a subject having a confirmed mutation of the DMD gene that is amenable to exon 2 skipping in combination with a dystrophin exon 2 skipping therapy.
Exon 2 skipping therapies in clinical trials include scAAV9.U7.ACCA (phase 1/2). scAAV9.U7.ACCA (SEQ ID NO: 1032) contains four copies of the U7snRNA in a self-complementary genome, with two copies targeting the splice acceptor site and two targeting the splice donor, encapsulated in AAV9 (scAAV9.U7.ACCA), as described in International Patent Application Publication No. WO2022067257A1 , which is incorporated herein by reference.
In some embodiments, the exon 2 skipping therapy includes a sequence listed in Table 26. In some embodiments, the exon 2 skipping therapy includes a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence in Table
26, such as SEQ ID NO: 1032. Additional dystrophin exon 2 skipping therapies that may be used in the methods described herein include those described in International Patent Application Publication No. WO2022067257A1 , which is incorporated herein by reference.
In some embodiments, the exon 2 skipping therapy has the sequence in Table 26. Table 26. Exemplary exon 2 skipping therapies
In some embodiments, in any of the exon 2 skipping therapies provided in Table 26, any one or more of the thymine bases (T) may optionally be a uracil base (U).
The dystrophin exon-skipping therapies disclosed herein may contain naturally occurring (e.g., DNA or RNA) and/or modified nucleotides (e.g., naturally occurring nucleotides with one or more modifications in order to increase the stability and/or therapeutic efficiency in vivo). Modifications that will improve the efficacy of an exon-skipping therapy of the disclosure, such as a stabilizing modification and/or a modification that reduces RNase H activation in order to avoid degradation of the targeted transcript are known in the art (see, e.g., Bennett and Swayze, Annu. Rev. Pharmacol. Toxicol. 50:259- 293, 2010; and Juliano, Nucleic Acids Res. 19;44(14):6518-48, 2016). The dystrophin exon-skipping therapies may include one or more modifications, each of which is independently a backbone modification, nucleobase modification, sugar modification, or conjugation. In some embodiments, the modification is a backbone modification. Examples of oligonucleotides with backbone modifications include morpholinos, phosphorodiamidate morpholino oligomers (PMO), peptide nucleic acid (PNA), phosphorothioate (PS) oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, stereochemically pure phosphorothioate (PS) oligonucleotides, phosphoramidite oligonucleotides, P-ethoxy oligonucleotides, boranephosphate oligonucleotides; thiophosphoramidate oligonucleotides, and methylphosphonate oligonucleotides.
In some embodiments, the modification is a nucleobase modification. Examples of modified nucleobases include bicycle modified oligonucleotides, Bicyclic Nucleic Acid (BNA), tricycle modified oligonucleotides, tricyclo-DNA-antisense oligonucleotides (ASOs), 5-methyl substitution on pyrimidine nucleobases (e.g., 5-methylcytosine), 5-substituted pyrimidine analogues, 2-Thio-thymine modified oligonucleotides, and purine modified oligonucleotides.
In some embodiments, the modification is a sugar modification. Examples of oligonucleotides with sugar modifications include Locked Nucleic Acid (LNA) oligonucleotides, 2’,4’-Methyleneoxy Bridged Nucleic Acid (BNA), ethylene-bridged nucleic acid (ENA) oligonucleotides, constrained ethyl (cEt) oligonucleotides, oligonucleotides with modifications at the 2' position of the sugar (e.g., 2’-0-Me RNA (2’- OMe), 2’-O-Methoxyethyl (MOE), and 2’-Fluoro), and oligonucleotides with modifications at the 4' position of the sugar (e.g., 4’-Thio).
In some embodiments, the modification is a conjugation. In this embodiment, the oligonucleotides may be conjugated to another molecule, such as tri-ethylene glycol (TEG) (e.g., a TEG group attached at the 5’ terminus of the oligonucleotide via a phosphate group), N-acetyl galactosamine (GalNAc) oligonucleotide conjugates such as 5’-GalNAc and 3’-GalNAc oligonucleotides conjugates, lipid oligonucleotide conjugates, cell penetrating peptide (CPP) oligonucleotide conjugates, targeted oligonucleotide conjugates, antibody-oligonucleotide conjugates, and polymer-oligonucleotide conjugate such as with PEGylation or targeting ligands.
In some embodiments, the exon-skipping therapy of any one of SEQ ID NOs: 949-991 , 1000, 1001 , 1003-1027, 1029, 1030, and 1247-1256, may be a phosphorodiamidate morpholino oligomer (PMO), as described in International Patent Application Publication Nos. W02006000057A1 , WO2022232478A1 , WO2018118662A1 , W02020004675A1 , WO2018118627A1 , WO2011057350A1 , WO2015194520A1 , WO2023196400A2, US20230045002A1 , WO2023034817A1 , and WO2022192749, each of which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of SEQ ID NO: 1002 may be a 2'-O-methyl phosphorothioate oligomer, as described in International Patent Application Publication No. W02009054725A2, which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of SEQ ID NO: 1031 may be a 2’-F and 2’-OMe modified oligonucleotide with 17 stereodefined internucleotide linkages, 14 of which are identified as Sp phosphorothioate linkages, and three as Rp N-(1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkages. Its internucleotide linkages can be illustrated as: 5’-SSRSSRSSOSSSOSSSRSS-3’, where ‘S’, ‘R’, and ‘O’ represent Sp phosphorothioate linkage, Rp N-(1 ,3-dimethylimidazolidin-2-ylidenyl) phosphoramidate linkage, and phosphate linkages, respectively, as described in International Patent Application Publication No. WO2023168014A2, which is incorporated herein by reference. In some embodiments, the exon-skipping therapy of SEQ ID NO: 1028 may be a 2'-fluoro-2'- deoxy-modified phosphorothioate (Sp)/phosphodiester modified oligonucleotide, as described in International Patent Application Publication No. WO2019217784A1 , which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of SEQ ID NO: 992 may be 2'-O,4'-C-ethylene- bridged nucleic acid phosphorothioate modified oligonucleotide, as described in International Patent Application Publication No. W02022020107A1 , which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of any one of SEQ ID NO: 984-990, 1001 , or 1247-1256, may be a PMO conjugated to a peptide (e.g., a cell-penetrating peptide, a muscle-targeting peptide, a cyclic peptide, or an endosomal escape vehicle), as described in International Patent Application Publication Nos. WO2022232478A1 , WQ2022192749, and WQ2023034817A1 , each of which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of any one of SEQ ID NOs: 950-983 or 1003- 1027 may be a PMO conjugated to an antibody or antigen-binding fragment thereof (e.g., a transferrin antibody or antigen-binding fragment thereof), as described in International Patent Application Publication No. WQ2023196400A2 and US Patent Application Publication No. US20230045002A1 , each of which is incorporated herein by reference.
In some embodiments, the exon-skipping therapy of SEQ ID NO: 1897 may include the following modifications: cytosine nucleotides are 5-methylcytosine, guanine and cytosine nucleotides are locked nucleic acids (LNA), a tri-ethylene glycol (TEG) group is attached to the 5' terminus via a phosphate group, the internucleoside linkages are phosphorothioate linkages, and the non-LNA nucleotides are 2'- OMe nucleotides, as described in U.S. Patent Application Publication No. US20230416741 A1 , which is incorporated herein by reference.
Fc domains
In some embodiments, ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer of an immunoglobulin or a fragment of an Fc domain to increase the serum half-life of the polypeptide. An ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may form a dimer (e.g., homodimer or heterodimer) through the interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is the protein structure that is found at the C-terminus of an immunoglobulin. An Fc domain includes two Fc domain monomers that are dimerized by the interaction between the CH3 antibody constant domains. A wild-type Fc domain forms the minimum structure that binds to an Fc receptor, e.g., FcyRI, FcyRlla, FcyRllb, FcyRllla, FcyRlllb, FcyRIV. In some embodiments, an Fc domain may be mutated to lack effector functions, typical of a “dead” Fc domain. For example, an Fc domain may include specific amino acid substitutions that are known to minimize the interaction between the Fc domain and an Fey receptor. In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions L234A, L235A, and G237A. In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions D265A, K322A, and N434A. The aforementioned amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )). The Kabat numbering of amino acid residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Furthermore, in some embodiments, an Fc domain does not induce any immune system- related response. For example, the Fc domain in a dimer of an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and an Fey receptor. The sequence of an Fc domain monomer that may be included in an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below (SEQ ID NO: 1033):
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
The sequence of a wild-type Fc domain monomer that may be fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof is shown below in SEQ ID NO: 1034:
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
In some embodiments, the Fc domain monomer fused to an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof lacks a terminal lysine. An exemplary sequence for a wild-type Fc domain monomer lacking the terminal lysine is provided below (SEQ ID NO: 1035):
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions L12A, L13A, and G15A, relative to the sequence of SEQ ID NO: 1033. In some embodiments, an Fc domain is from an IgG 1 antibody and includes amino acid substitutions D43A, K100A, and N212A, relative to the sequence of SEQ ID NO: 1033. In some embodiments, the terminal lysine is absent from the Fc domain monomer having the sequence of SEQ ID NO: 1033 or SEQ ID NO: 1034. In some embodiments, an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) may be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 1033, SEQ ID NO: 1034, or SEQ ID NO: 1035) through conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof. The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain monomer can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). In some embodiments, the Fc domain monomer is an IgG 1 Fc domain monomer (e.g., a human lgG1 Fc domain monomer).
In some embodiments, an ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain contains a hinge domain. The Fc domain can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). The Fc domain can also be a non-naturally occurring Fc domain, e.g., a recombinant Fc domain.
Methods of engineering Fc domains that have reduced dimerization are known in the art. In some embodiments, one or more amino acids with large sidechains (e.g., tyrosine or tryptophan) may be introduced to the CH3-CH3 dimer interface to hinder dimer formation due to steric clash. In other embodiments, one or more amino acids with small sidechains (e.g., alanine, valine, or threonine) may be introduced to the CH3-CH3 dimer interface to remove favorable interactions. Methods of introducing amino acids with large or small sidechains in the CH3 domain are described in, e.g., Ying et al. {J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. US20060074225, U.S. Patent Nos. 8,216,805 and 5,731 ,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26- 35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681 , 1998), all of which are incorporated herein by reference in their entireties.
In yet other embodiments, one or more amino acid residues in the CH3 domain that make up the CH3-CH3 interface between two Fc domains are replaced with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable depending on the specific charged amino acids introduced. Methods of introducing charged amino acids in the CH3 domain to disfavor or prevent dimer formation are described in, e.g., Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication Nos. US20060074225, US20120244578, and US20140024111 , all of which are incorporated herein by reference in their entireties.
In some embodiments, an Fc domain includes one or more of the following amino acid substitutions: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351 H, L351 N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I, relative to the sequence of human IgG 1 . In some embodiments, the terminal lysine is absent from the Fc domain amino acid sequence. In one particular embodiment, an Fc domain includes the amino acid substitution T366W, relative to the sequence of human IgG 1 .
Linkers
An ActRII ligand trap described herein may include an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer by way of a linker. In some embodiments, the Fc domain monomer increases stability of the polypeptide. In the present invention, a linker between an Fc domain monomer (e.g., the sequence of SEQ ID NO: 1033, SEQ ID NO: 1034, or SEQ ID NO: 1035) and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)), can be an amino acid spacer including 1 -200 amino acids. Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 1039), GGGS (SEQ ID NO: 1040), GGGG (SEQ ID NO: 1041 ), GGGGA (SEQ ID NO: 1042), GGGGS (SEQ ID NO: 1043), GGGGG (SEQ ID NO: 1044), GGAG (SEQ ID NO:
1045), GGSG (SEQ ID NO: 1046), AGGG (SEQ ID NO: 1047), or SGGG (SEQ ID NO: 1048). In some embodiments, a spacer can contain 2 to 12 amino acids including motifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 1049), GSGS (SEQ ID NO: 1050), GAGAGA (SEQ ID NO: 1051 ), GSGSGS (SEQ ID NO: 1052), GAGAGAGA (SEQ ID NO: 1053), GSGSGSGS (SEQ ID NO: 1054), GAGAGAGAGA (SEQ ID NO: 1055), GSGSGSGSGS (SEQ ID NO: 1056), GAGAGAGAGAGA (SEQ ID NO: 1057), and GSGSGSGSGSGS (SEQ ID NO: 1058). In some embodiments, a spacer can contain 3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 1059), GGSGGS (SEQ ID NO: 1060), GGAGGAGGA (SEQ ID NO: 1061 ), GGSGGSGGS (SEQ ID NO: 1062), GGAGGAGGAGGA (SEQ ID NO: 1063), and GGSGGSGGSGGS (SEQ ID NO: 1064). In yet some embodiments, a spacer can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 1045), GGSG (SEQ ID NO:
1046), GGAGGGAG (SEQ ID NO: 1065), GGSGGGSG (SEQ ID NO: 1066), GGAGGGAGGGAG (SEQ ID NO: 1067), and GGSGGGSGGGSG (SEQ ID NO: 1068). In some embodiments, a spacer can contain motifs of GGGGA (SEQ ID NO: 1042) or GGGGS (SEQ ID NO: 1043), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 1069) and GGGGSGGGGSGGGGS (SEQ ID NO: 1070). In some embodiments of the invention, an amino acid spacer between an Fc domain monomer and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) may be GGG, GGGA (SEQ ID NO: 1039), GGGG (SEQ ID NO: 1041 ), GGGAG (SEQ ID NO: 1071 ), GGGAGG (SEQ ID NO: 1072), or GGGAGGG (SEQ ID NO: 1073).
In some embodiments, a spacer can also contain amino acids other than glycine, alanine, and serine, e.g., AAAL (SEQ ID NO: 1074), AAAK (SEQ ID NO: 1075), AAAR (SEQ ID NO: 1076), EGKSSGSGSESKST (SEQ ID NO: 1077), GSAGSAAGSGEF (SEQ ID NO: 1078), AEAAAKEAAAKA (SEQ ID NO: 1079), KESGSVSSEQLAQFRSLD (SEQ ID NO: 1080), GENLYFQSGG (SEQ ID NO: 1081 ), SACYCELS (SEQ ID NO: 1082), RSIAT (SEQ ID NO: 1083), RPACKIPNDLKQKVMNH (SEQ ID NO: 1084), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 1085), AAANSSIDLISVPVDSR (SEQ ID NO: 1086), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 1087). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 1088). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of proline-rich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1 -5, and PAPAP (SEQ ID NO: 1089).
The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.
In some embodiments, the linker between an Fc domain monomer (e.g., the sequence of SEQ ID NO: 1033, SEQ ID NO: 1034, or SEQ ID NO: 1035) and an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)), is an amino acid spacer having the sequence GGG. For example, an ActRII ligand trap of the invention can contain an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer (e.g., SEQ ID NO: 1034 or SEQ ID NO: 1035) by a GGG linker. Exemplary polypeptides containing an ActRII variant, an Fc domain monomer, and a linker are provided in Table 27, below. In some embodiments, the terminal lysine is absent from the Fc domain amino acid sequence. The C-terminal lysine of the Fc domain monomer of the polypeptides provided in Table 27 (i.e., the C-terminal lysine in each polypeptide sequence) may or may not be present, without affecting the structure or stability of the polypeptide. The disclosure specifically contemplates SEQ ID NOs: 1159-1243 that do not include the C-terminal lysine at the end of the polypeptide sequence. The polypeptides of SEQ ID NOs: 1159-1243 may be expressed including a C- terminal lysine, which then may be proteolytically cleaved upon expression of the polypeptide (e.g., the polypeptides of SEQ ID NOs: 1159-1243 are expressed using nucleic acid constructs encoding the polypeptide including a C-terminal lysine residue). The polypeptides of SEQ ID NOs: 1159-1243 may also be expressed without including the C-terminal lysine residue. Table 27. Polypeptides containing an ActRII variant fused to an Fc domain monomer by way of a linker
Vectors, host cells, and protein production
The activin A and myostatin signaling inhibitors described herein can be produced from a host cell. A host cell refers to a vehicle that includes the necessary cellular components, e.g., organelles, needed to express the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acids may be included in nucleic acid vectors that can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, or the like). The choice of nucleic acid vectors depends in part on the host cells to be used. Generally, preferred host cells are of either eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.
Nucleic acid vector construction and host cells
A nucleic acid sequence encoding the amino acid sequence of a polypeptide described herein may be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, ligation, and overlap extension PCR. A nucleic acid molecule encoding a polypeptide described herein may be obtained using standard techniques, e.g., gene synthesis. Alternatively, a nucleic acid molecule encoding a wild-type extracellular ActRIIA or ActRIIB may be mutated to include specific amino acid substitutions using standard techniques in the art, e.g., QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.
A nucleic acid sequence encoding a polypeptide described herein may be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purpose of the invention. Each vector may include various components that may be adjusted and optimized for compatibility with the particular host cell. For example, the vector components may include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding protein of interest, and a transcription termination sequence. In some embodiments, mammalian cells may be used as host cells for the invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, E. co// cells may also be used as host cells for the invention. Examples of E. co// strains include, but are not limited to, E. coli 294 (ATCC® 31 ,446), E. coli h 1776 (ATCC®31 ,537, E. coli BL21 (DE3) (ATCC® BAA- 1025), and E. coli RV308 (ATCC® 31 ,608). Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems may be chosen to ensure the correct modification and processing of the polypeptide expressed. The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vectors are introduced into host cells for protein production, host cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Methods for expression of therapeutic proteins are known in the art, see, for example, Paulina Baibas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012.
Protein production, recovery, and purification
Host cells used to produce the polypeptides described herein may be grown in media known in the art and suitable for culturing of the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) plus necessary supplements, such as a selection agent, e.g., ampicillin. Host cells are cultured at suitable temperatures, such as from about 20 °C to about 39 °C, e.g., from 25 °C to about 37 °C, preferably 37 °C, and CO2 levels, such as 5 to 10%. The pH of the medium is generally from about 6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
In some embodiments, depending on the expression vector and the host cells used, the expressed protein may be secreted from the host cells (e.g., mammalian host cells) into the cell culture media. Protein recovery may involve filtering the cell culture media to remove cell debris. The proteins may be further purified. A polypeptide described herein may be purified by any method known in the art of protein purification, for example, by chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. For example, the protein can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column (e.g., POROS Protein A chromatography) with chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultra filtration, salting-out and dialysis procedures. In other embodiments, host cells may be disrupted, e.g., by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, cell debris may be removed by centrifugation or filtration. In some instances, a polypeptide can be conjugated to marker sequences, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His- tag), which binds to nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).
Alternatively, the polypeptides described herein can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector, and alphaviral vector)) containing a nucleic acid molecule encoding the polypeptide. The vector, once inside a cell of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.) will promote expression of the polypeptide, which is then secreted from the cell. If treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood may be collected from the subject and the protein purified from the blood by methods known in the art.
Pharmaceutical compositions and preparations
The invention features pharmaceutical compositions that include the activin A and myostatin signaling inhibitors described herein. In some embodiments, a pharmaceutical composition of the invention includes an ActRII ligand trap including an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-853 (e.g., SEQ ID NOs: 789-853) or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) with a C-terminal extension (e.g., 1 , 2, 3, 4, 5, 6 or more additional amino acids) as the therapeutic protein. In some embodiments, a pharmaceutical composition of the invention includes an ActRII ligand trap including an extracellular portion of ActRIIA, ActRIIB, a variant thereof, or a chimera thereof described herein (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer as the therapeutic protein. In some embodiments, a pharmaceutical composition of the invention including a polypeptide described herein may be used in combination with other agents (e.g., therapeutic biologies and/or small molecules) or compositions in a therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art. In some embodiments, a pharmaceutical composition of the invention includes a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding a polypeptide described herein, or a vector containing such a nucleic acid molecule.
Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, arginine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol. Pharmaceutical compositions of the invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco’s Modified Eagle Medium (DMEM), a-Modified Eagles Medium (a-MEM), F-12 medium). Formulation methods are known in the art, see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor & Francis Group, CRC Press (2015).
The pharmaceutical compositions may be prepared in microcapsules, such as hydroxylmethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule. The pharmaceutical compositions of the invention may also be prepared in other drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy 22nd edition (2012). The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
The pharmaceutical compositions may also be prepared as a sustained-release formulation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides described herein. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and y ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™, and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over a few months, e.g., one to six months, while other formulations release pharmaceutical compositions of the invention for shorter time periods, e.g., days to weeks.
The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a polypeptide described herein, included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01 - 100 mg/kg of body weight).
The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a slow-release matrix in which the gene delivery vehicle is imbedded. If hydrodynamic injection is used as the delivery method, the pharmaceutical composition containing a nucleic acid molecule encoding a polypeptide described herein or a vector (e.g., a viral vector) containing the nucleic acid molecule is delivered rapidly in a large fluid volume intravenously. Vectors that may be used as in vivo gene delivery vehicle include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara), adeno-associated viral vectors, and alphaviral vectors. Routes, dosage, and administration
Pharmaceutical compositions that include the polypeptides described herein as the therapeutic proteins may be formulated for, e.g., intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable formulations, various effective pharmaceutical carriers are known in the art. See, e.g., ASHP Handbook on Injectable Drugs, Toissel, 18th ed. (2014).
In some embodiments, a pharmaceutical composition that includes a nucleic acid molecule encoding a polypeptide described herein or a vector containing such nucleic acid molecule may be administered by way of gene delivery. Methods of gene delivery are well-known to one of skill in the art. Vectors that may be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviral vectors. In some embodiments, mRNA molecules encoding polypeptides described herein may be administered directly to a subject.
In some embodiments of the present invention, nucleic acid molecules encoding a polypeptide described herein or vectors containing such nucleic acid molecules may be administered using a hydrodynamic injection platform. In the hydrodynamic injection method, a nucleic acid molecule encoding a polypeptide described herein is put under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). The plasmid is often delivered rapidly in a large fluid volume intravenously. Hydrodynamic injection uses controlled hydrodynamic pressure in veins to enhance cell permeability such that the elevated pressure from the rapid injection of the large fluid volume results in fluid and plasmid extravasation from the vein. The expression of the nucleic acid molecule is driven primarily by the liver. In mice, hydrodynamic injection is often performed by injection of the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding a polypeptide described herein may be administered using hydrodynamic injection.
The dosage of the pharmaceutical compositions of the invention depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. A pharmaceutical composition may include a dosage of a polypeptide described herein ranging from 0.01 to 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 0.3 to about 30 mg/kg. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.
The pharmaceutical compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The pharmaceutical compositions are administered in a variety of dosage forms, e.g., intravenous dosage forms, subcutaneous dosage forms, and oral dosage forms (e.g., ingestible solutions, drug release capsules). Generally, therapeutic proteins are dosed at 0.1 -100 mg/kg, e.g., 0.5-50 mg/kg. Pharmaceutical compositions that include a polypeptide described herein may be administered to a subject in need thereof, for example, one or more times (e.g., 1 -10 times or more) daily, weekly, biweekly, every four weeks, monthly, bimonthly, quarterly, biannually, annually, or as medically necessary. In some embodiments, pharmaceutical compositions that include a polypeptide described herein may be administered to a subject in need thereof weekly, biweekly, every four weeks, monthly, bimonthly, or quarterly. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may increase as the medical condition improves or decrease as the health of the patient declines.
Methods of treatment
The activin A and myostatin signaling inhibitors described herein (e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap) can reduce or inhibit the activity of negative regulators of skeletal muscle (e.g., activin A and myostatin). Therefore, the activin A and myostatin signaling inhibitors can be used to treat diseases in which muscle cells have increased susceptibility to damage and death, such as DMD. Based on the discovery that administration of an activin A and myostatin signaling inhibitor described herein (e.g., a polypeptide including an ActRII chimera) to DMD mice in combination with a dystrophin exon skipping therapy further increased muscle strength and dystrophin expression compared to administration of the activin A and myostatin signaling inhibitor or the dystrophin exon skipping therapy alone and further increased lean mass compared to administration of the dystrophin exon skipping therapy alone, the activin A and myostatin signaling inhibitors described herein can be used to treat a subject having DMD in combination with a dystrophin exon skipping therapy (e.g., to increase lean mass (e.g., muscle mass), muscle strength, and/or dystrophin expression, or to ameliorate (e.g., reduce) muscle loss).
An activin A and myostatin signaling inhibitor described herein (e.g., an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody or an antigen binding fragment thereof, or an ActRII ligand trap) can be used to treat a subject having DMD in combination with a dystrophin exon skipping therapy. In some embodiments, treatment with the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy is started concurrently (e.g., the subject begins treatment with both agents at approximately the same time, e.g., begins treatment with both agents during the same day, week, or month). In some embodiments, the subject has been undergoing treatment with the dystrophin exon skipping therapy prior to co-administration of an activin A and myostatin signaling inhibitor described herein (e.g., the subject has been undergoing treatment with the dystrophin exon skipping therapy for at least two weeks (e.g., 2 weeks or longer, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or more weeks, or 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more months) prior to co-administration of an activin A and myostatin signaling inhibitor described herein). The method can further include evaluating body weight, lean mass, muscle mass, muscle strength, and/or dystrophin expression after administration of an activin A and myostatin signaling inhibitor described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an activin A and myostatin signaling inhibitor described herein). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 44 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 - 44, or a dystrophin exon 44 skipping therapy provided in Table 21 ). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 45 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 50 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 51 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 53 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy provided in Table 25). In some embodiments, the subject has a confirmed mutation of the DMD gene that is amenable to exon 2 skipping and is administered an activin A and myostatin signaling inhibitor in combination with a dystrophin exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26).
Administration of an activin A and myostatin signaling inhibitor described herein can maintain or increase lean mass (e.g., muscle mass, such as skeletal muscle mass) in a subject undergoing treatment with a dystrophin exon skipping therapy. The activin A and myostatin signaling inhibitor may increase lean mass as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone. In some embodiments, co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in lean mass compared to administration of either agent alone. In some embodiments, administration of an activin A and myostatin signaling inhibitor described herein increases muscle strength. The activin A and myostatin signaling inhibitor may increase muscle strength as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone. In some embodiments, administration of the activin A and myostatin signaling inhibitor leads to an amelioration of muscle loss as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone. In some embodiments, co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater amelioration of muscle loss compared to administration of either agent alone. In some embodiments, co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle strength compared to administration of either agent alone. In some embodiments, administration of the activin A and myostatin signaling inhibitor leads to an increase in dystrophin expression (e.g., increase in dystrophin mRNA levels and/or increase in dystrophin protein levels) as compared to baseline measurements prior to treatment initiation with the activin A and myostatin signaling inhibitor or as compared to treatment with a dystrophin exon skipping therapy alone. In some embodiments, coadministration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in dystrophin expression compared to administration of either agent alone.
In some embodiments, the methods described herein (e.g., co-administration of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor) increase lean mass, increase muscle mass (e.g., skeletal muscle mass), ameliorate muscle loss, increase muscle strength, and/or increase dystrophin expression. The methods described herein may increase lean mass, increase muscle mass, ameliorate muscle loss, increase muscle strength, or increase dystrophin expression (e.g., increase dystrophin mRNA levels and/or increase dystrophin protein levels) compared to measurements obtained prior to treatment (e.g., treatment with the combination of the dystrophin exon skipping therapy and the activin A and myostatin signaling inhibitor) or compared to measurements obtained from subjects treated with a dystrophin exon skipping therapy or an activin A and myostatin signaling inhibitor alone. In some embodiments, the methods described herein (e.g., co-administration of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor) slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications (e.g., beathing difficulties, shortness of breath, cardiomyopathy, or respiratory and/or heart failure) (e.g., compared to outcomes in subjects treated with a dystrophin exon skipping therapy or an activin A and myostatin signaling inhibitor alone). The method can further include evaluating body weight, lean mass, muscle mass, muscle loss, muscle strength, dystrophin expression (e.g., dystrophin mRNA levels and/or dystrophin protein levels), or other DMD symptoms (e.g., ambulation, respiratory function, or cardiac function) after administration of an activin A and myostatin signaling inhibitor described herein (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1 , 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 months or more after the start of treatment with an activin A and myostatin signaling inhibitor described herein in combination with a dystrophin exon skipping therapy).
In any of the methods described herein, the activin A and myostatin signaling inhibitor may be an activin A antibody or an antigen-binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or an antigen-binding fragment thereof or an anti-myostatin adnectin recombinant protein) (i.e., a combination of an activin A antibody and an anti-myostatin protein). In any of the methods described herein, the activin A and myostatin signaling inhibitor may be an ActRII antibody or an antigen-binding fragment thereof. In some embodiments, the activin A and myostatin signaling inhibitor may be an ActRII ligand trap, such as an ActRII ligand trap including an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877- 919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)). In some embodiments, the activin A and myostatin signaling inhibitor is administered at a dosage ranging from 0.01 to 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.75, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 0.3 to about 30 mg/kg. In any of the methods described herein, an ActRII ligand trap including an extracellular ActRIIA variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-854 (e.g., SEQ ID NOs: 789-854) or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., 898-919)) that further includes a C-terminal extension of one or more amino acids (e.g., 1 , 2, 3, 4, 5, 6 or more amino acids) may be used as the therapeutic protein. In any of the methods described herein, a dimer (e.g., homodimer or heterodimer) formed by the interaction of two Fc domain monomers that are each fused to a polypeptide including an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784- 855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) may be used as the therapeutic protein. In any of the methods described herein, an extracellular ActRII variant (e.g., an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919)) fused to an Fc domain monomer may be used as the therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing said nucleic acids can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition.
In some embodiments, the dystrophin exon skipping therapy is an exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy provided in Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22), an exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23), an exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24), an exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy provided in Table 25), or an exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26). In some embodiments, the dystrophin exon skipping therapy is eteplirsen, golodirsen, viltolarsen, or casimersen.
Compositions that can be administered to a subject according to the methods described herein are provided in Tables 28-31 , below.
Combination therapy
An activin A and myostatin signaling inhibitor described herein is to be administered to the subject in combination with a dystrophin exon skipping therapy. The dystrophin exon skipping therapy may be, e.g., an exon 44 skipping therapy (e.g., NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy provided in Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22), an exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23), an exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24), an exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE-N531 , or a dystrophin exon 53 skipping therapy provided in Table 25), or an exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26). In some embodiments, the dystrophin exon skipping therapy is eteplirsen, golodirsen, viltolarsen, or casimersen.
The dystrophin exon skipping therapy may be administered at the same time (e.g., administration of all agents occurs within 15 minutes, 10 minutes, 5 minutes, 2 minutes or less) as the activin A and myostatin signaling inhibitor. The agents can also be administered simultaneously via co-formulation. The activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy can also be administered sequentially, such that the action of the two overlaps and their combined effect is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy treatment can be performed by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, local routes, and direct absorption through mucous membrane tissues. The activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy can be administered by the same route or by different routes. For example, an activin A and myostatin signaling inhibitor may be administered by subcutaneous (e.g., for an ActRII ligand trap or an anti-myostatin adnectin recombinant protein) or intravenous (e.g., for an activin A antibody and a myostatin antibody or for an ActRII antibody) injection or infusion while the dystrophin exon skipping therapy can be administered by intravenous injection or infusion (e.g., for casimersen, eteplirsen, golodirsen, viltolarsen, NS-089/NCNP-02, AOC 1044, NS-050/NCNP-03, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , WVE-N531 , ENTR-601 -44, or scAAV9.U7.ACCA) or by subcutaneous injection (e.g., for renadirsen or drisapersen). The activin A and myostatin signaling inhibitor may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 1 1 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours or up to 1 -7, 1 -14, 1 -21 or 1 -30 days before or after the dystrophin exon skipping therapy. In some embodiments, the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy are administered at different frequencies. For example, the activin A and myostatin signaling inhibitor can be administered once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months and the dystrophin exon skipping therapy can be administered once weekly (e.g., for casimersen, eteplirsen, golodirsen, viltolarsen, NS-089/NCNP-02, renadirsen, NS- 050/NCNP-03, drisapersen, suvodirsen, or BMN-351 ), once every two weeks (e.g., for BMN-351 ), or once monthly (e.g., for vesleteplirsen, DYNE-251 , PGN-EDO51 , or WVE-N531 ). In some embodiments, the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy are administered at the same or at similar frequencies. For example, both the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy can be administered once a week, once every two weeks, once every four weeks, or once a month.
In some embodiments, the dystrophin exon skipping therapy is administered as indicated on the label. For example, casimersen can be administered as an intravenous infusion over 35 to 60 minutes (e.g., via an in-line 0.2 micron filter) at a dose of 30 milligrams per kilogram of body weight once weekly. Eteplirsen can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly. Golodirsen can be administered as an intravenous infusion over 35 to 60 minutes at a dose of 30 mg/kg once weekly. Viltolarsen can be administered as an intravenous infusion over 60 minutes at a dose of 80 mg/kg once weekly. The activin A and myostatin signaling inhibitor can be administered by subcutaneous or intravenous injection or infusion at a dose of from about 0.01 to about 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.325, 0.35, 0.375, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 0.3 to about 30 mg/kg, once a week, once every two weeks, once every four weeks, once a month, once bimonthly, once every three months, once every four months, or once every six months, or once a year.
In some embodiments, combination therapy with an activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy maintains or increases lean mass (e.g., muscle mass, such as skeletal muscle mass). In some embodiments, combination therapy ameliorates (e.g., reduces or inhibits) muscle loss. In some embodiments, combination therapy increases muscle strength. In some embodiments, combination therapy increases dystrophin expression (e.g., increases dystrophin mRNA levels and/or dystrophin protein levels). In some embodiments, combination therapy slows or inhibits the progression of DMD, reduces or inhibits muscle atrophy or wasting, preserves ambulation or slows the loss of ambulation, or reduces or inhibits respiratory and/or cardiac complications.
Kits
An activin A and myostatin signaling inhibitor and a dystrophin exon skipping therapy described herein can be provided in a kit for use in treating a subject with DMD. Each agent may be provided in unit dosage form, optionally in a pharmaceutically acceptable excipient (e.g., saline), in an amount sufficient to treat the subject. The kit can further include a package insert that instructs a user of the kit, such as a physician, to perform the methods described herein. The kit may optionally include a syringe or other device for administering the activin A and myostatin signaling inhibitor or dystrophin exon skipping therapy. Table 28
Table 29
Table 30 Table 31
Examples
The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
Example 1 - Effect of an ActRII chimera alone or in combination with a dystrophin exon skipping therapy in a mouse model of DMD
Nine-week-old male D2MDX mice underwent an 8-week treatment regimen with vehicle (Trisbuffered saline (TBS)) (n=10), Chimera 1/2b-mFc (SEQ ID NO: 917 fused to a mouse Fc domain monomer in the form of a homodimer) (n=10), PMO-1 (a phosphorodiamidate morpholino oligomer having the sequence of SEQ ID NO: 1244 (ggccaaacctcggcttacctgaaat)) (n=16), or a combination of PMO-1 and Chimera 1/2b-mFc (n=16). TBS and Chimera 1/2b-mFc were administered intraperitoneally and the dose of Chimera 1/2b-mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=10).
The body weight of the mice was measured twice a week right before Chimera 1/2b-mFc or vehicle administration. Body weight results are shown in FIG. 1 . Data are shown as a percentage of body weight change from baseline measurements. Data are shown as mean ± SEM.
The lean mass was determined by nuclear magnetic resonance (NMR) body composition analysis (Bruker Minispec) on days -1 , 25, and 55 of the study. Lean mass results are shown in FIG. 2. Data are shown as a percentage of lean mass change from baseline measurements. Data are shown as mean ± SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test, * P<0.05, ** P<0.01 , *** P<0.001 , **** P<0.0001 .
Forelimb grip strength was measured using the Grip Strength Meter (Columbus Instruments) on days -1 (baseline) and 55 (terminal) of the study. Grip strength results are shown in FIG. 3. Data are shown as absolute force in kg. Data are shown as mean ± SEM. Statistics are shown using 2-way ANOVA with a Tukey’s multiple comparison test. * P<0.05, ** P<0.01 , *“ P<0.001 , *“* P<0.0001 , ns= not significant.
Mice were sacrificed on day 58 and total RNA was extracted from the quadriceps of the mice. The dystrophin mRNA levels were determined by qRT-PCR (QuantStudio 7 Pro, ThermoFisher Scientific). Dystrophin expression results are shown in FIG. 4. Data are shown as relative expression to housekeeping genes. Data are shown as mean ± SEM. Statistics are shown using 1 -way ANOVA with a Tukey’s multiple comparison test. * P<0.05, ** P<0.01 , *“ P<0.001 , *“* P<0.0001 .
Ex vivo CT scans of the lumbar spine were conducted using GX2 pCT (5 mm FOV, 90 kV, 88 pA, 4 minutes, Perkin Elmer). Micro-structure of L4 vertebral body was evaluated using Analyze 14.0 Bone micro-architecture Analysis software (AnalyzeDirect). Representative images of L4 lumbar spine vertebral body are shown as single coronal section (FIG. 5A, top row) or 3D reconstructed trabecular bone (FIG. 5B, bottom row). Trabecular bone volume fraction (BV/TV), trabecular pattern factor (Tb.pf), and minimum moment of inertia in Z axis (MMI (Z)) results are shown in FIGS. 6-8, respectively. Statistical analysis was done by one-way ANOVA and individual comparisons shown from Tukey’s multiple comparison test. Data are shown as mean ±SEM *** P <0.001 , *“* P <0.0001 .
Dystrophic mice (vehicle) showed lower lean mass gain and grip strength compared to WT mice. PMO-1 treatment alone did not yield improvements in these parameters. However, combined Chimera 1/2b-mFc + PMO-1 treatment was comparable to WT in lean mass and grip strength, significantly outperforming vehicle and PMO-1 groups. Notably, Chimera 1/2b-mFc + PMO-1 treatment increased dystrophin mRNA levels 2.8-fold versus vehicle, with a 1 .8-fold increase over PMO-1 only, suggesting Chimera 1/2b-mFc enhances exon skipping and dystrophin expression by PMOs.
D2MDX mice exhibited lower trabecular bone volume fraction and minimum moment of inertia in Z axis and increased trabecular pattern factor. Chimera 1/2b-mFc and PMO-1 combination treatment significantly improved bone volume and bone structure associated with greater bone strength.
In summary, Chimera 1/2b-mFc ameliorated muscle loss in a DMD mouse model. The observation that Chimera 1/2b-mFc treatment augmented PMO-1 induced dystrophin expression highlighted the potential synergistic benefit of using an activin A and myostatin signaling inhibitor and dystrophin exon skipping therapy combination therapy for DMD.
Example 2 - Effect of an ActRII chimera in combination with a dystrophin exon skipping therapy in a mouse model of DMD
Sixty-day-old (P60) male D2MDX mice underwent an 8-week treatment regimen with vehicle (Tris-buffered saline (TBS)) (n=5), PMO-1 (a phosphorodiamidate morpholino oligomer having the sequence of SEQ ID NO: 1244 (ggccaaacctcggcttacctgaaat)) (n=5), or a combination of PMO-1 and Chimera 1/2b-mFc (SEQ ID NO: 917 fused to a mouse Fc domain monomer in the form of a homodimer) (n=5). TBS and Chimera 1/2b-mFc were administered intraperitoneally and the dose of Chimera 1 /2b- mFc was 10 mg/kg. PMO-1 was administered intravenously at a dose of 25 mg/kg. The treatment frequency of Chimera 1/2b-mFc and PMO-1 was twice weekly and once weekly, respectively. DBA2J mice treated with TBS intraperitoneal injection served as controls (WT) (n=5).
Mice were sacrificed after 8 weeks and tricep weight was measured. Tricep weight results are shown in FIG. 9. Data are shown as absolute tricep weight in grams. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with significant effects for the treatment factor (p<0.0001 , F(3,44)=17.04, R2=0.5375). Post-hoc Tukey's multiple comparisons test showed significant differences among groups (WT : Vehicle vs. D2MDX: Vehicle, ****p<0.0001 ; WT : Vehicle vs. D2MDX: PMO-1 , ****p<0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****p<0.0001 ; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ***p=0.0006). Notably, the combination of PMO-1 and Chimera 1/2b-mFc significantly increased muscle weight.
The tricep myofiber cross-sectional area (CSA) was determined. Triceps were snap-frozen, cross-sectioned on a cryostat (8 pm), and slide-mounted. The sections were stained with a Laminin primary antibody (ABCAM®, ab11576, Rat, lgG1 , 5 pg/mL) and an Alexa Fluor 555 secondary antibody (INVITROGEN®, A21434, Goat-anti-Rat, IgG, 4 pg/mL). Images were captured at 20x magnification using a ZEISS® Axioscan7. The files were exported, converted to .sis format, and analyzed using ARMS® Vision4D software (2023, Version 4.1 .2, Carl Zeiss AG). A cell-pose model was used for myofiber segmentation to determine cross-sectional area of individual fibers to determine mean CSA per sample. Creation parameters were as follows: cell pose-based segmenter using CPx model at 60 pm with a minimum diameter of 15 pm detection, segment morphology objects erosion by 5 pixels to disclude basal lamina and an object feature filter to only include objects greater than or equal to 50 pm2 for artifact removal. Data export Surface Area (Voxel) to .xls before compiling area of each individual fiber into a mean CSA per sample. Tricep myofiber cross-sectional area results are shown in FIG. 10. Data are shown as average CSA in pm2. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with significant effects for the treatment factor (p<0.0001 , F(3,32)=25.99, R2=0.7090). Post-hoc Tukey's multiple comparisons test showed significant differences among groups (WT: Vehicle vs. D2MDX: Vehicle, ****p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 , ****p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ***p=0.0003; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, **p=0.0027). Notably, the combination of PMO-1 and Chimera 1/2b-mFc significantly increased the tricep myofiber cross-sectional area.
The differences in tricep myofiber cross-sectional area were also visualized using a heat map image. Similar to patients with DMD, the D2MDX model showed a strong muscle degenerative phenotype. The group treated with the combination of PMO-1 and Chimera 1/2b-mFc not only showed overall increase of fiber size, but also the fiber size was more evenly distributed relative to vehicle or PMO-1 groups.
To delineate whether the improvement in muscle function was associated with improvements in PMO-mediated exon skipping, the exon skipping efficiency was determined. RNA was extracted from muscle tissue samples of WT and D2MDX mice treated with vehicle, PMO-1 , or PMO-1 in combination with Chimera 1/2b-mFc. The RNA was reverse transcribed into cDNA, followed by PCR amplification using primers designed to target a region encompassing the DMD mouse mutation in exon 23 of the dystrophin gene. The PCR products were then analyzed using gel electrophoresis to identify and compare the presence of full-length (unskipped) and truncated (skipped) dystrophin mRNA. Exon skipping efficiency results are shown in FIG. 1 1 A. Data are shown as a percentage of exon skipping. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with significant effects for the treatment factor (p<0.0001 , F(3,22)=245.1 , R2=0.9710). Post-hoc Tukey's multiple comparisons test showed significant differences among groups (WT: Vehicle vs. D2MDX: PMO- 1 , p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, p<0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 , p<0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, p<0.0001 ; D2MDX: PMO- 1 vs. D2MDX: PMO-1 + Chimera 1 /2b-mFc, **p=0.0005). A representative image of a gel showing exon skipping is provided in FIG. 1 1 B. In the WT : Vehicle group, there were no skipped exons, representing full-length dystrophin production. In the D2MDX: Vehicle group, no exon skipping was observed, consistent with the expected mutation profile without treatment. In the PMO-1 -treated group, partial exon skipping was observed, indicated by the presence of a band that migrated further on the gel compared to the full-length dystrophin band. These results demonstrate the efficacy of PMO-1 in inducing exon skipping. Notably, the group treated with the combination of PMO-1 and Chimera 1/2b-mFc exhibited a higher percentage of exon skipping, demonstrated by the more intense bands at the position corresponding to the skipped dystrophin, suggesting that the combination therapy enhanced exon skipping more effectively than PMO-1 alone.
Dystrophin protein expression was determined by enzyme-linked immunosorbent assay (ELISA). Dystrophin protein expression results are shown in FIG. 12. Data are shown as absolute dystrophin levels in pg/mL. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with significant effects for treatment (F(3, 44) = 75.74, P < 0.0001 , R2 = 0.8378). Post-hoc Tukey's multiple comparisons test showed significant differences among groups (WT: Vehicle vs. D2MDX: Vehicle, ****P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 , P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, P < 0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1 /2b-mFc, ***P = 0.0018; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, **P = 0.0045). Notably, the combination of PMO-1 and Chimera 1 /2b-mFc resulted in significantly higher dystrophin protein expression relative to Vehicle or PMO-1 alone groups.
The distribution of dystrophin protein localization within the muscle was determined by immunofluorescence imaging. Initially, the tricep tissues were embedded in optimal cutting temperature (OCT) compound and snap frozen in 2-methyl-butane cooled with liquid nitrogen. The embedded tissues were cryosectioned into 8 pm slices and mounted onto slides. On Day 1 , a hydrophobic barrier was created around each slide with a PAP pen and the slides were rehydrated in 1 X PBS. Antigen retrieval was performed using DAKO® Target Retrieval Solution in a water bath at 90°C for 20 minutes, followed by cooling to room temperature for 20 minutes. The slides were washed with PBS and permeabilized using 0.3% TritonX-100 for 20 minutes. After another series of PBS washes, the slides were blocked with a solution containing 0.1 % TritonX and 10% BSA in PBS for one hour at room temperature. The primary antibodies (anti-dystrophin (ABCAM® ab15277) rabbit IgG) were applied in a solution containing 4% BSA and 0.1 % TritonX, then the slides were incubated overnight at 4°C. On Day 2, the slides were washed to remove unbound primary antibodies and the secondary antibodies (Alexa Fluor™ 488 (A1 1034) goat antirabbit IgG) were applied in a similar blocking solution for one hour at room temperature in the dark. The slides were then washed again to remove unbound secondary antibodies, and nuclear staining was performed using a DAPI working solution. After a final series of PBS washes, the slides were mounted using PROLONG™ Glass Mountant, dried overnight at 2-8°C, sealed with nail polish, and cleaned thoroughly before imaging using a ZEISS® Axioscan7 slide scanner with a 20x objective with automated tile stitching.
Data are shown in FIG. 13A as percentage of dystrophin-positive fibers. Data are shown as mean ± SEM. Statistical significance was determined using one-way ANOVA with significant effects for treatment (F(3, 33) = 79.20, P < 0.0001 , R2 = 0.8781 ). Post-hoc Tukey's multiple comparisons test showed significant differences among groups (WT : Vehicle vs. D2MDX: Vehicle, ****P < 0.0001 ; VVT : Vehicle vs. D2MDX: PMO-1 , ****P < 0.0001 ; WT: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****P < 0.0001 ; D2MDX: Vehicle vs. D2MDX: PMO-1 , **P = 0.0052; D2MDX: Vehicle vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, ****P < 0.0001 ; D2MDX: PMO-1 vs. D2MDX: PMO-1 + Chimera 1/2b-mFc, *P = 0.0371 ). A series of representative images showing dystrophin protein localization is provided in FIG. 13B and highlights the increased expression and improved localization of dystrophin in the combined PMO-1 and Chimera 1/2b-mFc treatment group. Notably, the combination of PMO-1 and Chimera 1/2b-mFc treatment significantly increased dystrophin localization and percentage of dystrophin-positive fibers with an increase in the distribution of dystrophin across the muscle section.
In summary, improvements in muscle mass and structure were predominantly observed in the group receiving PMO-1 + Chimera 1/2b-mFc, underscoring its unique efficacy. The observation that Chimera 1/2b-mFc treatment augmented PMO-induced dystrophin expression highlights the potential synergistic benefit of using Chimera 1/2b-mFc and PMO combination therapy for DMD.
Example 3 - Effect of an ActRII chimera on satellite cells
Ten-week-old male wild-type C57BI/6 mice were treated with a single dose of 10 mg/kg Chimera 1/2b-mFc or vehicle. Chimera 1/2b-mFc and vehicle were administered intraperitoneally. Muscles were dissected and processed to obtain single cell suspensions on day 1 , day 2, and day 4 (n=5), stained for markers of satellite cells (CD31 , Sca.1 , CD34, a7 integrin, and CD106) and analyzed by flow cytometry.
Satellite cell population results are shown in FIG. 14A. Data are shown as percent of CD31 - Sca.1 - cells. Satellite cell differentiation marker results are shown in FIG. 14B. Data are shown as relative expression of paired box 7 (Pax7), myogenic factor 5 (Myf5), and myoblast determination protein 1 (MyoD). Data are shown as mean ± SEM. Statistical significance was determined using a 2-way ANOVA and Sidak's multiple comparisons test (* P<0.05, ** P<0.01 , **** P<0.0001 , ns= not significant). Treatment with Chimera 1/2b-mFc increased the pool of satellite cells in wild type mice. Molecular markers demonstrated commitment/differentiation of satellite cells to muscle.
In summary, treatment with Chimera 1/2b-mFc increased satellite cells in skeletal muscle.
Example 4 - Treatment of a subject having DMD by administration of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor
According to the methods disclosed herein, a physician of skill in the art can treat a subject, such as a human patient, having DMD so as to increase lean mass (e.g., muscle mass), increase muscle strength, increase dystrophin expression, slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications. To treat the subject, a physician of skill in the art can administer to the subject a composition containing an activin A and myostatin signaling inhibitor (e.g., an activin A antibody or antigen binding fragment thereof and an anti-myostatin protein (e.g., a myostatin antibody or antigen binding fragment thereof or an anti-myostatin adnectin recombinant protein), an ActRII antibody, an ActRII ligand trap (e.g., a polypeptide including an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919), such as an extracellular ActRIIA variant, ActRIIB variant, or ActRII chimera fused to an Fc domain or Fc domain monomer)) in combination with a dystrophin exon skipping therapy (e.g., an exon 44 skipping therapy (e.g.,NS-089/NCNP-02, AOC 1044, ENTR-601 -44, or a dystrophin exon 44 skipping therapy provided in Table 21 ), an exon 45 skipping therapy (e.g., casimersen, renadirsen, or a dystrophin exon 45 skipping therapy provided in Table 22), an exon 50 skipping therapy (e.g., NS-050/NCNP-03 or a dystrophin exon 50 skipping therapy provided in Table 23), an exon 51 skipping therapy (e.g., eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , or a dystrophin exon 51 skipping therapy provided in Table 24), an exon 53 skipping therapy (e.g., golodirsen, viltolarsen, WVE- N531 , or a dystrophin exon 53 skipping therapy provided in Table 25), or an exon 2 skipping therapy (e.g., scAAV9.U7.ACCA, or a dystrophin exon 2 skipping therapy provided in Table 26)). The composition containing the activin A and myostatin signaling inhibitor may be administered to the subject, for example, by parenteral injection (e.g., subcutaneous or intravenous injection) in combination with the dystrophin exon skipping therapy, which may be administered by intravenous injection or infusion (e.g., for casimersen, eteplirsen, golodirsen, viltolarsen, NS-089/NCNP-02, AOC 1044, NS-050/NCNP-03, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, BMN-351 , WVE-N531 , ENTR-601 -44, or scAAV9.U7.ACCA) or by subcutaneous injection (e.g., for renadirsen or drisapersen). The activin A and myostatin signaling inhibitor is administered in a therapeutically effective amount, such as from 0.01 to 500 mg/kg (e.g., 0.01 , 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg). In some embodiments, the activin A and myostatin signaling inhibitor (e.g., a polypeptide including an extracellular ActRIIA variant having the sequence of any one of SEQ ID NOs: 784-855 (e.g., SEQ ID NOs: 789-855), an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 856-876 (e.g., SEQ ID NOs: 857-876), or an extracellular ActRII chimera having the sequence of any one of SEQ ID NOs: 877-919 or 1128-1158 (e.g., SEQ ID NOs: 898-919), such as an extracellular ActRIIA variant, ActRIIB variant, or ActRII chimera fused to an Fc domain or Fc domain monomer) is administered bimonthly, once a month, once every four weeks, once every two weeks, or at least once a week or more (e.g., 1 , 2, 3, 4, 5, 6, or 7 times a week or more) and the dystrophin exon skipping therapy is administered once per week or once per month. The activin A and myostatin signaling inhibitor is administered in an amount sufficient to increase lean mass (e.g., skeletal muscle mass), increase muscle strength, increase dystrophin expression, slow or inhibit the progression of DMD, reduce or inhibit muscle atrophy or wasting, preserve ambulation or slow the loss of ambulation, or reduce or inhibit respiratory and/or cardiac complications. Following administration of the composition to a patient, a practitioner of skill in the art can monitor the patient’s improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient’s lean mass, muscle strength, or dystrophin expression using standard clinical tests. A finding that the patient exhibits increased lean mass, muscle strength, or dystrophin expression following administration of the composition compared to test results prior to administration of the composition indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
Other Embodiments While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth. All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Other embodiments are within the following claims.

Claims

1 . A method of treating a subject having Duchenne muscular dystrophy (DMD), the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
2. A method of increasing lean mass in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
3 A method of increasing muscle strength in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
4 A method of increasing dystrophin expression in a subject having DMD, the method comprising administering in combination to the subject effective amounts of a dystrophin exon skipping therapy and an activin A and myostatin signaling inhibitor.
5 The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 44 skipping.
6 The method of claim 5, wherein the dystrophin exon skipping therapy is an exon 44 skipping therapy.
7 The method of claim 6, wherein the exon 44 skipping therapy comprises the sequence of any one of SEQ ID NOs: 949-990, 1264-1431 , and 1794-1796.
8 The method of claim 6 or 7, wherein the exon 44 skipping therapy is NS-089/NCNP-02, AOC 1044, or ENTR-601 -44.
9 The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 45 skipping.
10 The method of claim 9, wherein the dystrophin exon skipping therapy is an exon 45 skipping therapy.
1 1 The method of claim 10, wherein the exon 45 skipping therapy comprises the sequence of any one of SEQ ID NOs: 991 , 992, 1432-1545, and 1797-1801 .
12 The method of claim 10 or 1 1 , wherein the exon 45 skipping therapy is casimersen or renadirsen.
13 The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 50 skipping.
14. The method of claim 13, wherein the dystrophin exon skipping therapy is an exon 50 skipping therapy.
15. The method of claim 14, wherein the exon 50 skipping therapy comprises the sequence of any one of SEQ ID NOs: 993-999, 1546-1580, and 1802-1804.
16. The method of claim 14 or 15, wherein the exon 50 skipping therapy is NS-050/NCNP-03.
17. The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 51 skipping.
18. The method of claim 17, wherein the dystrophin exon skipping therapy is an exon 51 skipping therapy.
19. The method of claim 18, wherein the exon 51 skipping therapy comprises the sequence of any one of SEQ ID NOs: 1000-1028, 1247-1256, 1581 -1745, 1805, and 1897.
20. The method of claim 18 or 19, wherein the exon 51 skipping therapy is eteplirsen, vesleteplirsen, drisapersen, DYNE-251 , PGN-EDO51 , suvodirsen, or BMN-351 .
21 . The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 53 skipping.
22. The method of claim 21 , wherein the dystrophin exon skipping therapy is an exon 53 skipping therapy.
23. The method of claim 22, wherein the exon 53 skipping therapy comprises the sequence of any one of SEQ ID NOs: 1029-1031 , 1746-1793, and 1806-1809.
24. The method of claim 22 or 23, wherein the exon 53 skipping therapy is golodirsen, viltolarsen, or WVE-N531 .
25. The method of any one of claims 1 -4, wherein the subject has a confirmed mutation of the DMD gene that is amenable to exon 2 skipping.
26. The method of claim 25, wherein the dystrophin exon skipping therapy is an exon 2 skipping therapy.
27. The method of claim 26, wherein the exon 2 skipping therapy comprises the sequence of SEQ ID NO: 1032.
28. The method of claim 26 or 27, wherein the exon 2 skipping therapy is scAAV9.U7.ACCA.
29. The method of any one of claims 1 -28, wherein the activin A and myostatin signaling inhibitor is an activin A antibody or an antigen binding fragment thereof and an anti-myostatin protein.
30. The method of claim 29, wherein the activin A antibody is garetosmab.
31 . The method of claim 29, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain variable region (HCVR) sequence having at least 90% sequence identity to a HCVR sequence in Table 1 and a light chain variable region (LCVR) sequence having at least 90% sequence identity to a LCVR sequence in Table 1 .
32. The method of claim 29 or 31 , wherein the activin A antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 2.
33. The method of claim 29, 31 or 32, wherein the activin A antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity to a heavy chain and light chain sequence provided in Table 3.
34. The method of claim 29, wherein the anti-myostatin protein is a myostatin antibody or an antigen binding fragment thereof.
35. The method of claim 34, wherein the myostatin antibody is domagrozumab, landogrozumab, trevogrumab, or apitegromab (SRK-015).
36. The method of claim 34, wherein the myostatin antibody or an antigen binding fragment thereof has a HCVR sequence having at least 90% sequence identity to a HCVR sequence in Table 4 and a LCVR sequence having at least 90% sequence identity to a LCVR sequence in Table 4.
37. The method of claim 34 or 36, wherein the myostatin antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 5, Table 6, or Table 7.
38. The method of claim 34, 36, or 37, wherein the myostatin antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity to a heavy chain and light chain sequence provided in Table 8.
39. The method of claim 29, wherein the anti-myostatin protein is an anti-myostatin adnectin recombinant protein.
40. The method of claim 39, wherein the anti-myostatin adnectin recombinant protein is taldefgrobep alfa.
41 . The method of claim 39, wherein the anti-myostatin adnectin recombinant protein comprises a sequence having at least 90% sequence identity to a sequence provided in Table 9.
42. The method of any one of claims 1 -28, wherein the activin A and myostatin signaling inhibitor is an ActRII antibody or an antigen binding fragment thereof.
43. The method of claim 42, wherein the ActRII antibody is bimagrumab, CSJ089, CQI876, or CDD861 .
44. The method of claim 42, wherein the ActRII antibody or an antigen binding fragment thereof has a HCVR sequence having at least 90% sequence identity to a HCVR sequence in Table 10 and a LCVR sequence having at least 90% sequence identity to a LCVR sequence in Table 10.
45. The method of claim 42 or 44, wherein the ActRII antibody or an antigen binding fragment thereof has a light chain CDR1 , CDR2, and CDR3 and a heavy chain CDR1 , CDR2, and CDR3 listed in Table 11 .
46. The method of claim 42, 44, or 45, wherein the ActRII antibody or an antigen binding fragment thereof has a heavy chain and light chain sequence having at least 90% sequence identity to a heavy chain and light chain sequence provided in Table 12.
47. The method of any one of claims 1 -28, wherein the activin A and myostatin signaling inhibitor is an ActRII ligand trap.
48. The method of claim 47, wherein the ActRII ligand trap is an ActRIIA ligand trap.
49. The method of claim 48, wherein the ActRIIA ligand trap is a composition of Table 28.
50. The method of claim 48, wherein the ActRIIA ligand trap comprises an extracellular portion of wildtype ActRIIA.
51 . The method of claim 48, wherein the ActRIIA ligand trap is sotatercept.
52. The method of claim 47, wherein the ActRII ligand trap is an ActRIIB ligand trap.
53. The method of claim 52, wherein the ActRIIB ligand trap comprises an extracellular portion of wildtype ActRIIB.
54. The method of claim 52, wherein the ActRIIB ligand trap is BIIB110, ALG-802, luspatercept, ramatercept, or ACE-2494.
55. The method of claim 52, wherein the ActRIIB ligand trap is a composition of Table 29.
56. The method of claim 52, wherein the ActRIIB ligand trap comprises the sequence of any one of SEQ ID NOs: 871 -876.
57. The method of claim 47, wherein the ActRII ligand trap is an ActRII chimera ligand trap.
58. The method of claim 57, wherein the ActRII chimera ligand trap is a composition of Table 30 or Table
31 .
59. The method of any one of claims 1 -58, wherein the method increases lean mass.
60. The method of any one of claims 1 -59, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in lean mass compared to administration of the dystrophin exon skipping therapy alone.
61 . The method of any one of claims 1 -60, wherein the method increases muscle mass.
62. The method of any one of claims 1 -61 , wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle mass compared to administration of the dystrophin exon skipping therapy alone.
63. The method of any one of claims 1 -62, wherein the method increases muscle strength.
64. The method of any one of claims 1 -63, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase in muscle strength compared to administration of either agent alone.
65. The method of any one of claims 1 -64, wherein the method increases dystrophin expression.
66. The method of any one of claims 1 -65, wherein co-administration of the activin A and myostatin signaling inhibitor and the dystrophin exon skipping therapy leads to a greater increase of dystrophin expression compared to administration of either agent alone.
67. The method of any one of claims 1 -66, wherein the subject is a human.
PCT/US2025/015776 2024-02-13 2025-02-13 Methods of using activin a and myostatin signaling inhibitors Pending WO2025174995A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202463552952P 2024-02-13 2024-02-13
US63/552,952 2024-02-13
US202463558423P 2024-02-27 2024-02-27
US63/558,423 2024-02-27
US202463662819P 2024-06-21 2024-06-21
US63/662,819 2024-06-21

Publications (1)

Publication Number Publication Date
WO2025174995A1 true WO2025174995A1 (en) 2025-08-21

Family

ID=96773506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/015776 Pending WO2025174995A1 (en) 2024-02-13 2025-02-13 Methods of using activin a and myostatin signaling inhibitors

Country Status (1)

Country Link
WO (1) WO2025174995A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210015807A1 (en) * 2018-03-23 2021-01-21 Cytoo Alk5 inhibitors as skeletal muscle hypertrophy inducers
US20220049257A1 (en) * 2014-06-17 2022-02-17 Nippon Shinyaku Co., Ltd. Antisense nucleic acids

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220049257A1 (en) * 2014-06-17 2022-02-17 Nippon Shinyaku Co., Ltd. Antisense nucleic acids
US20210015807A1 (en) * 2018-03-23 2021-01-21 Cytoo Alk5 inhibitors as skeletal muscle hypertrophy inducers

Similar Documents

Publication Publication Date Title
JP7757439B2 (en) Polypeptides and pharmaceutical compositions containing mutant activin type IIB receptors
US12350313B2 (en) Activin receptor type IIa variants and methods of use thereof
US9610362B2 (en) Antisense conjugates for decreasing expression of DMPK
EP3706777B1 (en) Activin receptor type iia variants and methods of use thereof
US20230348565A1 (en) Methods of using activin receptor type ii variants
WO2023023345A2 (en) Methods of using activin receptor type ii signaling inhibitors
EP4359075A2 (en) Methods of using activin receptor type ii signaling inhibitors
WO2025174995A1 (en) Methods of using activin a and myostatin signaling inhibitors
CN118475369A (en) Methods of using inhibitors of activin receptor type II signaling
HK40115905A (en) Activin receptor type iia variants and methods of use thereof
WO2025184284A1 (en) Methods of using activin receptor type ii signaling inhibitors
WO2025122830A1 (en) Methods of using activin receptor type ii signaling inhibitors
HK40037556B (en) Activin receptor type iia variants and methods of use thereof
HK40037556A (en) Activin receptor type iia variants and methods of use thereof
HK40015854A (en) Activin receptor type iia variants and methods of use thereof
HK40015854B (en) Activin receptor type iia variants and methods of use thereof
HK40015852B (en) Activin receptor type iia variants and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25755682

Country of ref document: EP

Kind code of ref document: A1