Compositions for targeted delivery to muscle tissue

Abstract

The present disclosure includes, among other things, compositions comprising cyclic peptides with selectivity for muscle tissue. The present disclosure includes, among other things, the recognition that the cyclic peptides with selectivity for muscle tissue can used in targeted delivery of a payload (e.g., an antisense oligonucleotide, or a small interfering RNA) to muscle tissue by conjugating such peptides to the payload. The present disclosure further includes, among other things, compositions comprising cyclic peptide conjugates and their use in the treatment of conditions of muscle tissue.

Description

COMPOSITIONS FOR TARGETED DELIVERY TO MUSCLE TISSUECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 550,793, filed February 7, 2024, and U.S. Provisional Application No. 63 / 738,685, filed December 24, 2024, the content of each of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Muscle tissues contribute to biological functions including movement, posture, temperature homeostasis, joint stabilization, and storage of molecules such as amino acids and carbohydrates. Impairment of muscle tissue functions has been associated with various diseases. Delivery of agents to muscle tissue can be useful in the treatment of such diseases.SUMMARY

[0003] The present disclosure provides, among other things, compositions comprising cyclic peptides that target muscle tissues, as well as the use of such cyclic peptides in the treatment of disease. Without wishing to be bound by any particular scientific theory, the present disclosure includes the unexpected finding that cyclic peptides disclosed herein can selectively target receptors present in muscle tissue. The present disclosure therefore provides, among other things compositions including cyclic peptides that selectively target muscle tissue, conjugates that include a cyclic peptide of the present disclosure and a therapeutic or diagnostic agent, pharmaceutical compositions, and the treatment of disease (e.g., treatment of diseases associated with muscle tissue) using agents disclosed herein. In various embodiments, the muscle tissue is, e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue. In various embodiments, the muscle tissue is skeletal muscle tissue.

[0004] The present disclosure therefore specifically provides, among other things, conjugates comprising a cyclic peptide disclosed herein associated with a therapeutic or diagnostic agent, and use of such conjugates in the treatment of disease. Without wishing to be bound by any particular scientific theory, the present disclosure is based at least in part on the recognition that conjugates comprising cyclic peptides can selectively deliver associated therapeutic or diagnostic agents to muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., skeletal muscle tissue). As disclosed herein, cyclic peptides of the present disclosure can be associated with a therapeutic or diagnostic agent thatcan be, without limitation, a nucleic acid, peptide, or chemical agent. A nucleic acid agent can be, e.g., an inhibitory nucleic acid or a nucleic acid (e.g., a transgene) that encodes an expression product. Inhibitory nucleic acids can be selected from, without limitation, small interfering RNA (siRNA), microRNA (miRNA), and inhibitory antisense oligonucleotides (ASOs).

[0005] In at least one aspect, the present disclosure provides a composition including a cyclic peptide that selectively targets muscle tissue, where the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from a sequence selected from SEQ ID NOs: 1-62, where each amino acid difference is independently selected from an insertion of an amino acid, a deletion of an amino acid, or a substitution of an amino acid. In some embodiments, the muscle tissue is skeletal muscle tissue. In certain embodiments, the cyclic peptide includes an amino acid sequence selected from SEQ ID NOs: 1-62. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from any one of SEQ ID NOs: 1, 3, and 4. In certain embodiments, the cyclic peptide includes an amino acid sequence according to any one of SEQ ID NOs: 1, 3, and 4. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 1. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 1. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 3. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 3. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 4. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 4.

[0006] In at least one aspect, the present disclosure provides a peptide conjugate including a cyclic peptide including an amino acid sequence having no more than three amino acid differences from a sequence selected from SEQ ID NOs: 1-62, where each amino acid difference is independently selected from an insertion of an amino acid, a deletion of an amino acid, or a substitution of an amino acid, and an agent associated with the cyclic peptide. In certain embodiments, the cyclic peptide includes an amino acid sequence selected from SEQ ID NOs: 1-62. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from any one of SEQ ID NOs: 1, 3, and 4. In certain embodiments, the cyclic peptide includes an amino acid sequence according to any one of SEQ ID NOs: 1, 3, and 4. In certain embodiments, the cyclic peptideincludes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 1. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 1. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 3. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 3. In certain embodiments, the cyclic peptide includes an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 4. In certain embodiments, the cyclic peptide includes an amino acid sequence according to SEQ ID NO: 4.

[0007] In various embodiments, the agent is a diagnostic agent or a therapeutic agent. In certain embodiments, the agent is an inhibitory nucleic acid. In certain embodiments, the agent is an antisense oligonucleotide. In certain embodiments, the agent is an siRNA or a miRNA. In certain embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO) or a peptide nucleic acid (PNA). In certain embodiments, the agent is an adeno-associated virus (AAV). In certain embodiments, the agent is a lipid nanoparticle (LNP). In certain embodiments, the agent is non-covalently associated with the cyclic peptide. In certain embodiments, the agent is covalently associated with the cyclic peptide. In certain embodiments, the agent is directly covalently associated with the cyclic peptide. In certain embodiments, the agent is indirectly covalently associated with the cyclic peptide. In certain embodiments, the agent is indirectly covalently associated with the cyclic peptide via a linker. In certain embodiments, the linker includes a thioether bond, a disulfide bond, an oxime, a thiazolidine, a hydrazone, an amide bond, an azide bond, or a maleimide bond. In certain embodiments, the linker includes a C1-C30 alkyl, a C2-C20 alkyl, a C3-C12 alkyl, a C6-C12 alkyl, or a Ce alkyl group. In certain embodiments, the linker includes a Ce alkyl amine. In certain embodiments, the linker is a cleavable linker.

[0008] In certain embodiments, the peptide conjugate selectively targets muscle tissue. In certain embodiments, the muscle tissue is skeletal muscle tissue. In certain embodiments, the peptide conjugate binds at least one integrin or integrin subtype, optionally where the integrin or integrin subtype is selected from selected from avpi, avP3, avP5, otvP6, avP8, a8pi, a5pi, and allbp3, optionally where the integrin or integrin subtype is selected from avpi, avP6, and avP8, optionally where the peptide conjugate binds the P transmembrane subunit of the least one integrin or integrin subtype. In certain embodiments, the amino acid sequence of thecyclic peptide includes the amino acid sequence RGD. In certain embodiments, the RGD amino acid sequence binds at least one integrin or integrin subtype, optionally where the integrin or integrin subtype is selected from selected from avpi, avP3, avP5, otvP6, avP8, a8pi, a5pi, and allbp3, optionally where the integrin or integrin subtype is selected from avpi, av 6, and avP8, optionally where the RGD amino acid sequence binds the P transmembrane subunit of the least one integrin or integrin subtype.

[0009] In at least one aspect, the present disclosure provides a pharmaceutical composition including a peptide conjugate disclosed herein and a pharmaceutically acceptable carrier. In at least one aspect, the present disclosure provides a method of delivering an agent to muscle tissue in a subject, the method including administering to the subject a peptide conjugate disclosed herein or a pharmaceutical composition disclosed herein. In at least one aspect, the present disclosure provides a method of diagnosing, preventing, and / or treating a disease related to muscle tissue, the method including administering to the subject a peptide conjugate disclosed herein or a pharmaceutical composition disclosed herein. In certain embodiments, the disease is selected from Myasthenia gravis, Botulism, Lambert-Eaton myasthenic syndrome (LEMS), Duchenne muscular dystrophy, Becker muscular dystrophy, Congenital muscular dystrophies, Myotonic muscular dystrophy, Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, Emery-Dreifuss muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, dermatomyositis, polymyositis, necrotizing myopathy, inclusion body myositis, rhabdomyolysis, and muscle atrophy. In certain embodiments, the muscle atrophy is primary muscle atrophy or secondary muscle atrophy.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a graph showing the expression and knockdown of Malatl following delivery of an exemplified antisense oligonucleotide (ASO)-cyclic peptide conjugate. FIG. 1 provides quantification of Malatl expression in skeletal muscle tissue following in vivo delivery in mice of an ASO conjugated to an exemplified targeting cyclic peptide having an amino acid sequence of SEQ ID NO: 1. The ASO, conjugated to a control peptide, was delivered as non-targeted control. Expression was normalized to Malatl expression levels in animals administered with PBS.

[0011] FIG. 2 is a graph showing the expression and knockdown of Malatl following delivery of an exemplified antisense oligonucleotide (ASO)-cyclic peptide conjugate. FIG. 2 provides quantification of Malatl knockdown in various tissues by the exemplified ASO- cyclic peptide conjugate relative to non-targeted control.

[0012] FIG. 3 is a graph showing the expression and knockdown of ALDH2 following delivery of an exemplified small interfering RNA (siRNA)-cyclic peptide conjugate at 1 mg / kg. FIG. 3 provides quantification of expression of the gene targeted by the siRNA (ALDH2) in indicated tissues following subcutaneous delivery in mice. For each tissue shown on the X axis, data include administration of a PBS control (left) or the conjugated cyclic peptide (right). The siRNA conjugate included a targeting cyclic peptide having the amino acid sequence of SEQ ID NO: 1. Expression was normalized to expression of the target gene in the animals administered with PBS.DETAILED DESCRIPTION

[0013] The present disclosure provides, among other things, compositions comprising cyclic peptides that selectively target muscle tissue, conjugates of cyclic peptides with therapeutic or diagnostic agents, and use of such peptides and conjugates to deliver agents to muscle tissue, e.g., for treatment or diagnosis of disease. Cyclic peptides of the present disclosure can be associated with a variety of agents, including without limitation nucleic acids, peptides, or chemical agent. A nucleic acid agent can be, e.g., an inhibitory nucleic acid or a nucleic acid (e.g., a transgene) that encodes an expression product. Inhibitory nucleic acids can be selected from, without limitation, inhibitory antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and microRNA (miRNA).Definitions

[0014] A, An, The'. As used herein, “a”, “an”, and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element.

[0015] About: As used herein, term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” mayencompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0016] Acyl. The term “acyl” is a term of art and as used herein refers to any group or radical of the form RCO- where R is any organic group, e.g., alkyl, aryl, heteroaryl, aralkyl, and heteroaralkyl. Representative acyl groups include acetyl, benzoyl, and malonyl.

[0017] Administration or administering. As used herein, the term “administration” or “administering” typically refers to administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition.

[0018] Alkenoxy, or alkenoxyl. The term “alkenoxy” or “alkenoxyl” means an alkenyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkenoxyl include, but are not limited to, 2-propen-l-oxyl (i.e., CH2=CH-CH2-O-) and vinyloxy (i.e., CH2=CH-O-).

[0019] Alkoxy . The term “alkoxy” as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0020] Alkoxycarbonyl The term “alkoxycarbonyl” means an alkoxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, represented by -C(=O)-, as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxy carbonyl, and Zc / 7-butoxy carbonyl.

[0021] Alkylcarbonyl. The term “alkylcarbonyl”, as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1 -oxopropyl, 2,2-dimethyl-l -oxopropyl, 1 -oxobutyl, and 1 -oxopentyl.

[0022] Alkylcarbonyloxy, or arylcarbonyloxy . The terms “alkylcarbonyloxy” and “arylcarbonyloxy”, as used herein, mean an alkylcarbonyl or arylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethyl carbonyl oxy, and / c / 7-butylcarbonyloxy. Representative examples of aryl carbonyloxy include, but are not limited to phenyl carbonyl oxy.

[0023] Aralkyl, or arylalkyl. The term “aralkyl” or “arylalkyl” is a term of art and as used herein refers to an alkyl group substituted with an aryl group, wherein the moiety is appended to the parent molecule through the alkyl group.

[0024] Arylcarbonyl. The term “arylcarbonyl”, as used herein, means an aryl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of arylcarbonyl include, but are not limited to, benzoyl and (2-pyridinyl)carbonyl.

[0025] Aryloxy. The term “aryloxy” as used herein means an aryl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0026] Agent. As used herein, the term “agent” may refer to any chemical entity, including, without limitation, any of one or more of an atom, molecule, compound, amino acid, polypeptide, nucleotide, nucleic acid, protein, protein complex, liquid, solution, saccharide, polysaccharide, lipid, or combination or complex thereof.

[0027] Alkyl. The term “alkyl” as used herein is a term of art and refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and alternatively, about 20 or fewer, or 10 or fewer. In certain embodiments, the term “alkyl” refers to a C1-C10 straight-chain alkyl group. In certain embodiments, the term “alkyl” refers to a Ci-Cs straight-chain alkyl group. In certain embodiments, the term “alkyl” refers to a C3-C12 branched-chain alkyl group. In certain embodiments, the term “alkyl” refers to a C3- Cs branched-chain alkyl group. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.

[0028] Alkenyl. The term “alkenyl” as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbons and containing at least one carboncarbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l -heptenyl, and 3-decenyl. The unsaturated bond(s) of the alkenyl group can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s).

[0029] Alkylene. The term “alkylene” is art-recognized, and as used herein pertains to a diradical obtained by removing two hydrogen atoms of an alkyl group, as defined above. In one embodiment an alkylene refers to a disubstituted alkane, i.e., an alkane substituted at two positions with substituents such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluromethyl), cyano, or the like. That is, in one embodiment, a “substituted alkyl” is an “alkylene”.

[0030] Alkylphosphoryl. The term “alkylphosphoryl” as used herein refers to a phosphoryl group substituted with at least one alkyl group, as defined herein; for example, - P(O)(OH)Me.

[0031] Alkylthio. The term “alkylthio” as used herein refers to alkyl-S-.

[0032] Alkynyl. The term “alkynyl” as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbon atoms and containing at least one carboncarbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

[0033] Amido. The term “amido”, as used herein, means -NHC(=O)-, wherein the amido group is bound to the parent molecular moiety through the nitrogen. Examples of amido include alkylamido such as CH3C(=O)N(H)- and CH?>CH2CX-O)N(H)-.

[0034] Amino: The term “amino” is a term of art and as used herein refers to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:wherein Ra, Rb, and Rc each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)x-Rd, or Ra and Rb, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rd represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a polycyclyl; and x is zero or an integer in the range of 1 to 8. In certain embodiments, only one of Ra or Rb may be a carbonyl, e.g., Ra, Rb, and the nitrogen together do not form an imide. In other embodiments, R and Rb (and optionally Rc) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)x-Rd. In certain embodiments, the term “amino” refers to -NH2.

[0035] Amino acid: As used herein, the term “amin acid,” in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, anamino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L- amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with a typical or canonical amino acid structure. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, sulfation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification can, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” can be used to refer to a free amino acid; in some embodiments it can be used to refer to an amino acid residue of a polypeptide. In the context of any amino acid present in a polypeptide, or which is contemplated or discussed in the context of its presence or potential presence in a polypeptide, the terms amino acid and residue are interchangeable as used herein.

[0036] Aminoacyl: The term “aminoacyl” is a term of art and as used herein refers to an acyl group substituted with one or more amino groups.

[0037] Aminoalkyl: The term “aminoalkyl” as used herein refers to an alkyl group substituted with one or more one amino groups. In one embodiment, the term “aminoalkyl” refers to an aminomethyl group.

[0038] Aminophosphoryl: The term “aminophosphoryl” as used herein refers to a phosphoryl group substituted with at least one amino group, as defined herein; for example, - P(O)(OH)NMe2.

[0039] Aminothionyl: The term “aminothionyl” as used herein refers to an analog of an aminoacyl in which the O of RC(O)- has been replaced by sulfur, hence is of the form RC(S)-.

[0040] Aryl: The term “aryl” is a term of art and as used herein refers to includes monocyclic, bicyclic and polycyclic aromatic hydrocarbon groups, for example, benzene, naphthalene, anthracene, and pyrene. The aromatic ring may be substituted at one or morering positions with one or more substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluromethyl), cyano, or the like. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is an aromatic hydrocarbon, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. In certain embodiments, the term “aryl” refers to a phenyl group. In certain embodiments, “aryl” has from 6 to 10 carbon atoms.

[0041] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact (e.g., covalently or non-covalently), directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.

[0042] Azide, or azido. The term “azide” or “azido”, as used herein, means an -N3 group.

[0043] Between or From’. As used herein, the term “between” refers to content that falls between indicated upper and lower, or first and second, boundaries (or “bounds”), inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.

[0044] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a biological source is or includes an organism, such as an animal or human. In some embodiments, a biologicalsample is or includes biological tissue or fluid. In some embodiments, a biological sample can be or include cells, tissue, or bodily fluid. In some embodiments, a biological sample can be or include blood, blood cells, cell-free DNA, free floating nucleic acids, ascites, biopsy samples, surgical specimens, cell-containing body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow. In some embodiments, a biological sample is or includes cells obtained from a single subject or from a plurality of subjects. A sample can be a “primary sample” obtained directly from a biological source, or can be a “processed sample” (e.g., a sample prepared from a primary sample, e.g., by a process such as isolation, e.g., of mRNA, DNA, or protein, by a process that modifies the primary sample’s chemical structure, and / or by a process that produces a new or different composition that represents one or more components or properties of the primary sample). A biological sample can also be referred to as a “sample.”

[0045] Carbocyclyl. The term “carbocyclyl” as used herein means a monocyclic or multicyclic (e.g., bicyclic, tricyclic, etc.) hydrocarbon radical containing from 3 to 12 carbon atoms that is completely saturated or has one or more unsaturated bonds, and for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system (e.g., phenyl). Examples of carbocyclyl groups include 1 -cyclopropyl, 1-cyclobutyl, 2- cyclopentyl, 1 -cyclopentenyl, 3-cyclohexyl, 1 -cyclohexenyl and 2-cyclopentenylmethyl.

[0046] Carbonyl. The term “carbonyl” as used herein refers to -C(=O)-.

[0047] Carboxy. The term “carboxy”, as used herein, means a -CO2H group.

[0048] Conservative Substitution: A conservative substitution is one in which an amino acid has been replaced by a non-identical residue having appropriately similar structural and / or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a conservative substitution. Certain non-limiting amino acid categorizations are summarized below. Those of skill in the art will be aware of the categorizations and relationships among amino acids that can be used to identify a conservative substitution.

[0049] Cycloalkyl. The term “cycloalkyl” means mono- or bicyclic or bridged saturated carbocyclic rings, each having from 3 to 12 carbon atoms. Certain cycloalkyls have from 5-12 carbon atoms in their ring structure, and may have 6-10 carbons in the ring structure. Preferably, cycloalkyl is (C3-C?)cycloalkyl, which represents a monocyclic saturated carbocyclic ring, having from 3 to 7 carbon atoms. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems include bridged monocyclic rings and fused bicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form -(Ctb)™-, where w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. Cycloalkyl groups are optionally substituted. In certain embodiments, the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted.

[0050] Cyano. The term “cyano” is a term of art and as used herein refers to -CN.

[0051] Cycloalkylalkyl. The term “cycloalkylalkyl” as used herein refers to an alkyl group substituted with one or more cycloalkyl groups. An example of cycloalkylalkyl is cyclohexylmethyl group.

[0052] Gene As used herein, the term “gene” refers to a DNA sequence that is or includes a coding sequence (i.e., a DNA sequence that encodes an expression product, such as an RNA product and / or a polypeptide product), optionally together with some or all of the regulatory sequences that control expression of the coding sequence.

[0053] Expression: As used herein, “expression” refers individually and / or cumulatively to one or more biological process that result in production from a nucleic acid sequence of an encoded agent (i.e., an expression product), such as an RNA and / or a polypeptide. Expression specifically includes either or both of transcription and translation. A nucleic acid or cell that produces the encoded agent can be said to express the encoded agent.

[0054] Halo: The term “halo” is a term of art and as used herein refers to -F, -Cl, - Br, or -I.

[0055] Haloalkyl: The term “haloalkyl” as used herein refers to an alkyl group, as defined herein, wherein some or all of the hydrogens are replaced with halogen atoms.

[0056] Heteroaryl: The term “heteroaryl” is a term of art and as used herein refers to a monocyclic, bicyclic, and polycyclic aromatic group having 3 to 12 total atoms including one or more heteroatoms such as nitrogen, oxygen, or sulfur in the ring structure. Exemplary heteroaryl groups include azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl,isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl, pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl, thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl, thienyl, thiomorpholinyl, triazolyl or tropanyl, and the like. The “heteroaryl” may be substituted at one or more ring positions with one or more substituents such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluromethyl), cyano, or the like. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is an aromatic group having one or more heteroatoms in the ring structure, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.

[0057] Heteroaralkyl, or heteroarylalkyl. The term “heteroaralkyl” or “heteroarylalkyl” is a term of art and as used herein refers to an alkyl group substituted with a heteroaryl group, appended to the parent molecular moiety through the alkyl group.

[0058] Heteroaryloxy . The term “heteroaryl oxy” as used herein means a heteroaryl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0059] Heteroatom. The term “heteroatom” is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium, and alternatively oxygen, nitrogen or sulfur.

[0060] Heterocyclyl. The term “heterocyclyl” as used herein refers to a radical of a non-aromatic ring system, including, but not limited to, monocyclic, bicyclic, and tricyclic rings, which can be completely saturated or which can contain one or more units of unsaturation, for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system, and having 3 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, the following are examples of heterocyclic rings: aziridinyl, azirinyl, oxiranyl, thiiranyl, thiirenyl, dioxiranyl, diazirinyl, diazepanyl, 1,3- dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, azetyl, oxetanyl, oxetyl, thietanyl, thietyl, diazetidinyl, dioxetanyl, dioxetenyl, dithietanyl, dithietyl, dioxalanyl, oxazolyl, thiazolyl, triazinyl, isothiazolyl, isoxazolyl, azepines, azetidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, quinuclidinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. A heterocyclyl group is optionally substituted by one or more substituents as described below.

[0061] Heterocycloalkylalkyl. The term “heterocycloalkylalkyl” as used herein refers to an alkyl group substituted with one or more heterocycloalkyl (i.e., heterocyclyl) groups.

[0062] Hydroxy . The term “hydroxy” is a term of art and as used herein refers to -OH

[0063] Hydroxyalkyl. The term “hydroxyalkyl”, as used herein, means at least one hydroxy group, as defined herein, is appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3 -hydroxypropyl, 2,3-dihydroxypentyl, and 2- ethyl-4-hydroxyheptyl .

[0064] Improve, increase, inhibit, decrease or reduce: As used herein, the terms “improve”, “increase”, “inhibit”, “decrease” and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0065] Linker: As used herein, “linker” is used to refer to that portion of a multielement agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide including a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123).

[0066] Nucleic acid. As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, the term nucleic acid refers to an individual nucleic acid residue (e g., a nucleotide and / or nucleoside), and in some embodiments refers to a polynucleotide chain including a plurality of individual nucleic acid residues. A nucleic acid can be or include DNA, RNA, or any combination thereof. A nucleic acid can include natural nucleic acid residues, nucleic acid analogs, and / or synthetic residues. In some embodiments, a nucleic acid includes natural nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine). In some embodiments, a nucleic acid is or includes of one or more nucleotide analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, a nucleic acid includes one or more genes. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid can include one or more peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600,700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, a nucleic acid is partly or wholly single stranded, or partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence including at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0067] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable,” as applied to one or more, or all, component s) for formulation of a composition as disclosed herein, means that each component must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0068] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates formulation of an agent (e.g., a pharmaceutical agent), modifies bioavailability of an agent, or facilitates transport of an agent from one organ or portion of a subject to another. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0069] Pharmaceutical composition or formulation'. As used herein, the term“pharmaceutical composition” or “formulation” refers to a composition in which a therapeutic agent is formulated together with one or more pharmaceutically acceptable carriers.

[0070] Phosphoryl. The term “phosphoryl” is a term of art and as used herein may in general be represented by the formula:wherein Q50 represents S or O, and R59 represents hydrogen, a lower alkyl or an aryl; for example, -P(O)(OMe)- or -P(0)(0H)2. When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl may be represented by the general formulas:wherein Q50 and R59, each independently, are defined above, and Q51 represents O, S or N; for example, -O-P(O)(OH)OMe or -NH-P(0)(0H)2. When Q50 is S, the phosphoryl moiety is a “phosphorothioate.”

[0071] Polypeptide: As used herein, “polypeptide” refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through human intervention. In some embodiments, a polypeptide may be or include of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may be or include only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide can include D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may include only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., one or more amino acid side chains, e.g., at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, at non-terminal amino acids, or at any combination thereof. In some embodiments, such pendant groups or modifications may be selected from acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may include a cyclic portion.

[0072] In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure to indicate a class of polypeptides that share a relevant activity or structure. For such classes, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acidsequences and / or functions are known. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class. For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e g., a conserved region that can in some embodiments be or include a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and in some instances up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide can be or include a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may be or include a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0073] Reference: As used herein, “reference” refers to a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof, is compared with a reference, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is an established standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative of qualitative. Typically, as would be understood by those of skill in the art, a reference and the value to which it is compared represents measure under comparable conditions. Those of skill in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison. In some embodiments, an appropriate reference may be an agent, sample, sequence, subject, animal, or individual, or population thereof, under conditions those of skill in the art will recognize ascomparable, e.g., for the purpose of assessing one or more particular variables (e.g., presence or absence of an agent or condition), or a measure or characteristic representative thereof.

[0074] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest. In some embodiments, the term “sample” refers to a preparation that is obtained by processing of a primary sample (e.g., by removing one or more components of and / or by adding one or more agents to a primary sample). Such a “processed sample” can include, for example cells, nucleic acids, or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as isolation and / or purification of certain components.

[0075] Silyl: The term “silyl”, as used herein, includes hydrocarbyl derivatives of the silyl (HiSi-) group (i.e., (hydrocarbyl)?, Si-), wherein a hydrocarbyl groups are univalent groups formed by removing a hydrogen atom from a hydrocarbon, e.g., ethyl, phenyl. The hydrocarbyl groups can be combinations of differing groups which can be varied in order to provide a number of silyl groups, such as trimethyl silyl (TMS), tert-butyldiphenyl silyl (TBDPS), tert-butyldimethyl silyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2- (trimethylsilyl)ethoxy]methyl (SEM).

[0076] Silyloxy . The term “silyloxy”, as used herein, means a silyl group, as defined herein, is appended to the parent molecule through an oxygen atom.

[0077] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0078] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease,disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual.” A subject administered an agent associated with treatment of a disease, disorder, or condition with which the subject is associated can be referred to as a subject in need of the agent, i.e., as a subject in need thereof.

[0079] Substitution, or substituted with. It will be understood that “substitution” or “substituted with,” as used herein with respect to molecular structures, includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, fragmentation, decomposition, cyclization, elimination, or other reaction, except in such instances as cyclization is desired in accordance with the present disclosure.

[0080] Substituted. The term “substituted,” as used herein with respect to molecular structures, is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0081] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certainage group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition. In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0082] Therapeutic regimen: As used herein, “therapeutic regimen” refers to a dosing regimen whose administration across a relevant population may be correlated with a desired or beneficial therapeutic outcome.

[0083] Therapeutically effective amount: As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that a therapeutically effective amount does not necessarily achieve successful treatment in every particular treated individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0084] Thiocarbonyl. The term “thiocarbonyl” as used herein refers to -C(=S)-.

[0085] Treatment. As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, suchtreatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition. Alternatively or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. For the avoidance of doubt, the terms “disease,” “disorder,” and “condition,” and any equivalents thereto, can be used interchangeably, such that use of any one likewise constitutes the use of its alternatives unless otherwise indicated.Cyclic Peptides

[0086] The present disclosure includes the discovery of compositions comprising cyclic peptides that selectively target the muscle tissue, as set forth herein. The present disclosure includes the recognition that, in various embodiments, cyclic peptides provided herein are useful for delivering an associated agent to muscle tissue, e.g., for treatment and / or diagnosis of a disease (e.g., a disease associated with muscle tissue).

[0087] In various embodiments, a cyclic peptide of the present disclosure is or includes a sequence selected from SEQ ID NOs: 1-62 as set forth herein (e.g., in Table 1), or a variant thereof. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from a sequence selected from SEQ ID NOs: 1- 62 by one or more amino acid sequence differences. In various embodiments, an amino acid difference refers to a deletion (the deletion of one amino acid), an insertion (an insertion of one amino acid), or a substitution (a substitution of one amino acid for another different amino acid). In various embodiments including multiple amino acid differences, each amino acid difference can be independently selected from, e.g., a deletion, an insertion, or a substitution. Sequences that have one or more amino acid differences from a sequence disclosed herein (e.g., a reference sequence and / or a sequence set forth in Table 1) can be referred to, e.g., as “variants” or “mutants.”

[0088] In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from a sequence selected from SEQ ID NOs: 1-62 by at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises apolypeptide that differs from a sequence selected from SEQ ID NOs: 1-62 by exactly 1, exactly 2, exactly 3, exactly 4, or exactly 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from a sequence selected from SEQ ID NOs: 1-62 by 1 to 5 amino acid differences, 1 to 4 amino acid differences, 1 to 3 amino acid differences, or 1 to 2 amino acid differences.

[0089] In various embodiments, a cyclic peptide of the present disclosure is or includes a sequence according to SEQ ID NO: 1 as set forth herein (e.g., in Table 1), or a variant thereof. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 1 by one or more amino acid sequence differences.

[0090] In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 1 by at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 1 by exactly 1, exactly 2, exactly 3, exactly 4, or exactly 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 1 by 1 to 5 amino acid differences, 1 to 4 amino acid differences, 1 to 3 amino acid differences, or 1 to 2 amino acid differences.

[0091] In various embodiments, a cyclic peptide of the present disclosure is or includes a sequence according to SEQ ID NO: 3 as set forth herein (e.g., in Table 1), or a variant thereof. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 3 by one or more amino acid sequence differences.

[0092] In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 3 by at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 3 by exactly 1, exactly 2, exactly 3, exactly 4, or exactly 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 3 by 1 to 5 amino acid differences, 1 to 4 amino acid differences, 1 to 3 amino acid differences, or 1 to 2 amino acid differences.

[0093] In various embodiments, a cyclic peptide of the present disclosure is or includes a sequence according to SEQ ID NO: 4 as set forth herein (e.g., in Table 1), or a variant thereof. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 4 by one or more amino acid sequence differences.

[0094] In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 4 by at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 4 by exactly 1, exactly 2, exactly 3, exactly 4, or exactly 5 amino acid sequence differences. In various embodiments, the present disclosure provides a cyclic peptide that is or comprises a polypeptide that differs from SEQ ID NO: 4 by 1 to 5 amino acid differences, 1 to 4 amino acid differences, 1 to 3 amino acid differences, or 1 to 2 amino acid differences.

[0095] In various embodiments, the present disclosure provides a cyclic peptide that is or includes a polypeptide having a sequence selected from SEQ ID NOs: 1-62, and / or a cyclic peptide that is or includes a variant of a polypeptide having a sequence selected from SEQ ID NOs: 1-62 (e.g., that includes at least one amino acid sequence difference as compared to a sequence selected from SEQ ID NOs: 1-62), where the N-terminal amino acid of the cyclic peptide is a cysteine residue, the C-terminal amino acid of the cyclic peptide is a cysteine residue, and / or both the N-terminal amino acid and the C-terminal amino acid of the cyclic peptide are cysteine residues.

[0096] In various embodiments, the present disclosure provides a cyclic peptide that is or includes a polypeptide having a sequence selected from SEQ ID NOs: 1-62, and / or a cyclic peptide that is or includes a variant of a polypeptide having a sequence selected from SEQ ID NOs: 1-62 (e.g., that includes at least one amino acid sequence difference as compared to a sequence selected from SEQ ID NOs: 1-62), where the cyclic peptide binds (e.g., selective binds) with an integrin. In various embodiments, the present disclosure provides a cyclic peptide that is or includes a polypeptide having a sequence selected from SEQ ID NOs: 1-62, and / or a cyclic peptide that is or includes a variant of a polypeptide having a sequence selected from SEQ ID NOs: 1-62 (e.g., that includes at least one amino acid sequence difference as compared to a sequence selected from SEQ ID NOs: 1-62), where the cyclic peptide includes the amino acid sequence RGD (also referred to herein as an arginine-glycine-aspartic acid motif or an RGD motif). Without wishing to be bound by anyparticular scientific theory, an RGD motif is a ligand for, and binds (e.g., selectively binds), integrin receptors, where the interaction of RGD in a cyclic peptide with integrin can facilitate targeted delivery of an agent disclosed herein to muscle tissue. Integrins (e.g., RGD- binding integrins) include heterodimeric cell surface receptors formed by an a transmembrane subunit and a 0 transmembrane subunit. In various embodiments, integrins (e.g., RGD-binding integrins) include av01, a 03, av05, av06, av08, a801, a.501, and allb03. In various embodiments, integrins (e.g., RGD-binding integrins) include otv01, av06, and av08.

[0097] In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype selected from av01, av03, av05, av06, av08, a801, a501, and allb03. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype selected from av01, av06, and av08. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) av01. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) av06. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) av08. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) av06 and / or av08.

[0098] In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does not selectively bind one or more integrins or integrin subtypes selected from av01, av03, av05, av06, av08, a801, a501, and allb03. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does not selectively bind one or more integrins or integrin subtypes selected from av01, av06, and av08. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does notselectively bind avpi. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does not selectively bind avp6. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does not selectively avp8. In certain embodiments, a cyclic peptide of the present disclosure or a variant thereof (e.g., where the cyclic peptide or variant thereof includes an RGD motif) can bind (e.g., selectively bind) an integrin or integrin subtype but does not bind or does not selectively bind one or more integrins or integrin subtypes selected from avP6 and / or avP8.

[0099] In various embodiments, a cyclic peptide of the present disclosure that includes an RGD motif can exhibit selective binding with one or more integrins or integrin subtypes as compared to one or more distinct integrins or integrin subtypes, e.g., due to differential selective binding of such integrins and / or integrin subtypes by the cyclic peptide, e.g., by an RGD motif present in a cyclic peptide. In various embodiments, a cyclic peptide of the present disclosure or variant thereof can bind and / or selectively bind muscle (e.g., one or more of skeletal muscle, cardiac muscle, and / or smooth muscle as disclosed herein), as disclosed herein. Without wishing to be bound by any particular scientific theory, in various embodiments a cyclic peptide of the present disclosure or variant thereof that binds integrin or an integrin subtype can bind a P transmembrane subunit of integrin or of the integrin subtype.

[0100] In various embodiments, a peptide and / or a cyclic peptide of the present disclosure can include multiple RGD motifs, e.g., 1, 2, or 3 RGD motifs. In various embodiments, an RGD motif can be positioned at the second amino acid position of a cyclic peptide of the present disclosure (where the second amino acid of the cyclic peptide is the arginine amino acid of the RGD motif, optionally where the first amino acid is a cysteine residue). In various embodiments, an RGD motif can be positioned at the second or third amino acid position of a cyclic peptide of the present disclosure (where the second or third amino acid of the cyclic peptide is the arginine amino acid of the RGD motif, optionally where the first amino acid is a cysteine residue). In various embodiments, an RGD motif can be positioned at the second, third, or fourth amino acid position of a cyclic peptide of the present disclosure (where the second, third, or fourth amino acid of the cyclic peptide is the arginine amino acid of the RGD motif, optionally where the first amino acid is a cysteine residue).

[0101] In various embodiments, a variant of a cyclic peptide sequence disclosed herein (e.g., of a sequence disclosed in Table 1 and / or selected from SEQ ID NOs: 1-62) targets muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., skeletal muscle tissue). In various embodiments, a variant of a cyclic peptide sequence disclosed herein (e.g., of a sequence disclosed in Table 1 and / or selected from SEQ ID NOs: 1-62) targets muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., skeletal muscle tissue) at a level comparable to that of a reference sequence (e.g., a reference sequence selected from SEQ ID NOs: 1-62). Muscle targeting can be determined according to any binding assay or functional activity assay disclosed herein or otherwise known in the art. To provide one non-limiting example, muscle targeting can be measured and / or compared according to delivery and / or function of an exemplary inhibitory nucleic acid as demonstrated herein.Table 1. Muscle Tissue-Selective Cyclic Peptide Sequences^denotes cysteine residues capable of forming disulfide bonds, e.g., with each other. In cyclized peptides, the two cysteine residues marked with * can form a disulfide bond.

[0102] For the avoidance of doubt, the present disclosure includes peptide sequences that are larger than one or more of the cyclic peptides disclosed in SEQ ID NOs: 1-62 and / or that are larger than one or more variants thereof. In various embodiments, the sequence of a cyclic peptide of the present disclosure, or a variant thereof, can be present in a larger polypeptide that is partially or entirely cyclic (e.g., for which a contiguous sequence or backbone is partially or entirely cyclic). In various such embodiments in which a sequence selected from SEQ ID NOs: 1-62, or of a variant thereof, is present in a larger polypeptide that is partially cyclic, the sequence selected from SEQ ID NOs: 1-62, or the variant thereof, is present in the cyclic portion of the polypeptide. It is to be appreciated from the present disclosure that a sequence selected from SEQ ID NOs: 1-62, or of a variant thereof, can be referred to as “cyclic” even when present as a portion of a larger cyclic sequence.Accordingly, the present disclosure includes embodiments in which the “terminal” amino acids of a sequence selected from SEQ ID NOs: 1-62, or the variant thereof, are directly bound to each other, as well as other embodiments in which additional amino acids of a larger cyclic polypeptide are positioned between such “terminal” amino acids, acting as a linker.

[0103] The present disclosure therefore contemplates and include embodiments in which a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length of, e.g., 3 to 40, or more, amino acids. In certain embodiments, a cyclic peptide of the present disclosure can have a length of 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, or more amino acids. In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length of, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids. In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length of, e.g., about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 amino acids.

[0104] In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length that is between 5 and 40 amino acids, 5 and 35 amino acids, 5 and 30 amino acids, 5 and 25 amino acids, 5 and 20 amino acids, 5 and 19 amino acids, 5 and 18 amino acids, 5 and 17 amino acids, 5 and 16 amino acids, 5 and 15 amino acids, 5 and 14 amino acids, 5 and 13 amino acids, 5 and 12 amino acids, 5 and 11 aminoacids, 5 and 10 amino acids, 5 and 9 amino acids, 5 and 8 amino acids. In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length that is between 6 and 40 amino acids, 6 and 35 amino acids, 6 and 30 amino acids, 6 and 25 amino acids, 6 and 20 amino acids, 6 and 19 amino acids, 6 and 18 amino acids, 6 and 17 amino acids, 6 and 16 amino acids, 6 and 15 amino acids, 6 and 14 amino acids, 6 and 13 amino acids, 6 and 12 amino acids, 6 and 11 amino acids, 6 and 10 amino acids, 6 and 9 amino acids, 6 and 8 amino acids. In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length that is between 7 and 40 amino acids, 7 and 35 amino acids, 7 and 30 amino acids, 7 and 25 amino acids, 7 and 20 amino acids, 7 and 19 amino acids, 7 and 18 amino acids, 7 and 17 amino acids, 7 and 16 amino acids, 7 and 15 amino acids, 7 and 14 amino acids, 7 and 13 amino acids, 7 and 12 amino acids, 7 and 11 amino acids, 7 and 10 amino acids, 7 and 9 amino acids, 7 and 8 amino acids. In certain embodiments, a cyclic peptide of the present disclosure, or a cyclic portion of a peptide, can have a length that is between 8 and 40 amino acids, 8 and 35 amino acids, 8 and 30 amino acids, 8 and 25 amino acids, 8 and 20 amino acids, 8 and 19 amino acids, 8 and 18 amino acids, 8 and 17 amino acids, 8 and 16 amino acids, 8 and 15 amino acids, 8 and 14 amino acids, 8 and 13 amino acids, 8 and 12 amino acids, 8 and 11 amino acids, 8 and 10 amino acids, 8 and 9 amino acids, 8 and 8 amino acids.

[0105] In various embodiments, preparation of cyclic peptides of 40 or more amino acids can be more challenging than preparation of cyclic peptides having fewer than 40 amino acids. Certain advantageous properties of cyclic peptides disclosed herein can also be more pronounced for cyclic peptide shaving fewer than 40 amino acids, such as cyclic peptides of 4 to 30 or 4 to 20 amino acids. In various embodiments, cyclic peptides disclosed herein have a length less than 40 amino acids, length less 30 amino acids, or length less 20 amino acids. In various embodiments, cyclic peptides disclosed herein have a length less than 15 amino acids. In various embodiments, cyclic peptides disclosed herein have a length from 9 to 11 amino acids.

[0106] In various embodiments, a cyclic peptide of the present disclosure has a number of amino acids between two cysteine residues (e.g., two cysteine residues indicated herein as forming a disulfide bond) that is between 3 and 30 amino acids. In various embodiments, a cyclic peptide of the present disclosure has a number of amino acids between two cysteine residues (e.g., two cysteine residues indicated herein as forming a disulfide bond) that is equal to or about 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, or 30 amino acids. In various embodiments, a cyclic peptide ofthe present disclosure has a number of amino acids between two cysteine residues (e.g., two cysteine residues indicated herein as forming a disulfide bond) that is between 3 and 20 amino acids, 3 and 15 amino acids, 3 and 14 amino acids, 3 and 13 amino acids, 3 and 12 amino acids, 3 and 11 amino acids, 3 and 10 amino acids, 3 and 9 amino acids, 3 and 8 amino acids, 3 and 7 amino acids, 3 and 6 amino acids, 3 and 5 amino acids, and / or 3 and 4 amino acids. In various embodiments, a cyclic peptide of the present disclosure has a number of amino acids between two cysteine residues (e.g., two cysteine residues indicated herein as forming a disulfide bond) that is between 5 and 20 amino acids, 5 and 15 amino acids, 5 and 14 amino acids, 5 and 13 amino acids, 5 and 12 amino acids, 5 and 11 amino acids, 5 and 10 amino acids, 5 and 9 amino acids, 5 and 8 amino acids, 5 and 7 amino acids, 5 and 6 amino acids, 5 and 5 amino acids, and / or 5 and 4 amino acids. In various embodiments, a cyclic peptide of the present disclosure has a number of amino acids between two cysteine residues (e.g., two cysteine residues indicated herein as forming a disulfide bond) that is between 7 and 20 amino acids, 7 and 15 amino acids, 7 and 14 amino acids, 7 and 13 amino acids, 7 and 12 amino acids, 7 and 11 amino acids, 7 and 10 amino acids, 7 and 9 amino acids, 7 and 8 amino acids, 7 and 7 amino acids, 7 and 6 amino acids, 7 and 5 amino acids, and / or 7 and 4 amino acids. In various embodiments, cyclic peptides disclosed herein include 5 to 9 amino acids between cysteine residues of the cyclic peptides that form the one or more disulfide bridges. In various embodiments, cyclic peptides disclosed herein include 7 amino acids between cysteine residues of the cyclic peptides that form the one or more disulfide bridges.

[0107] Peptide cyclization and methods of obtaining cyclic peptides are known in the art. The cyclic peptides according to embodiments of the present invention may be prepared by procedures known to those of skill in the art. For example, the cyclic peptides may be prepared using standard solid-phase peptide synthesis (SPPS) techniques as described for example, in Joo. (2012) Biomol Ther, 20(1): 19-26, which is incorporated herein by reference with respect to preparation of cyclic peptides. In various embodiments, cyclic peptides disclosed herein can be peptides in which a bridge or a link is formed between two amino acids that are part of the peptide or constitute the peptide. The bridge can be formed between amino acids having a reactive group (other than the amino and the carboxyl group that are essential for the respective amino acid), e.g., a thiol group. As will be appreciated by those of skill in the art, peptides comprising two or more amino acids having such a reactive group can be cyclized. For example, a peptide comprising two amino acids that have a thiol group can be cyclized under conditions wherein a disulfide bridge between the thiol groups of the two amino acids containing a thiol group is formed. Examples of amino acids having a thiolgroup and thus being capable of forming a bridge, i.e. a disulfide bridge include, but are not limited to, cysteine. In various embodiments, cyclic peptides disclosed herein include two or more cysteine residues. In various embodiments, cyclic peptides disclosed herein can include 2, 3, 4, 5, 6, 7, or 8 cysteine residues. In various embodiments, cyclic peptides disclosed herein include one or more disulfide bridges formed between the thiol groups of amino acids (e.g., cysteines). In various embodiments, cyclic peptides disclosed herein can include 1, 2, 3, or 4 disulfide bridges formed between the thiol groups of amino acids (e.g., cysteines). As will be appreciated by those of skill in the art, cyclic peptides disclosed herein can be cyclized using any bridge, linkage or methodology known in the art or described herein. In various embodiments, cyclic peptides disclosed herein are cyclized due to the presence of naturally occurring amino acids (e g., cysteines). In various embodiments, cyclic peptides disclosed herein are cyclized due to the presence of one or more non-naturally occurring amino acids. In various embodiments, cyclic peptides disclosed herein can be cyclized with a lactam linkage.

[0108] In various embodiments, a cyclic peptide or a conjugate of the present disclosure does not target and / or does not significantly target a tissue that is not a muscle tissue. In various embodiments, a cyclic peptide or a conjugate of the present disclosure does not target and / or does not significantly target an adipose tissue, a brain tissue, a heart tissue, or a kidney tissue. In various embodiments, a cyclic peptide or a conjugate of the present disclosure targets muscle tissue at a higher level as compared to a tissue that is not a muscle tissue. In various embodiments, a cyclic peptide or a conjugate of the present disclosure targets skeletal muscle tissue at a higher level as compared to a tissue that is not skeletal muscle tissue (e.g., adipose tissue, smooth muscle tissue, and / or cardiac muscle tissue). In various embodiments, the level is about 10-fold, about 9-fold, about 8-fold, about 7-fold, about 6-fold, about 5-fold, about 4-fold, about 3-fold, about 2-fold, about 1.5-fold, about 1.4- fold, about 1.3 -fold, about 1.2-fold, about 1.1 -fold higher. In various embodiments, a cyclic peptide or a conjugate of the present disclosure does not selectively target one or more of adipose tissue, smooth muscle tissue, and / or cardiac muscle tissue.Conjugation of Cyclic Peptides with Therapeutic and / or Diagnostic Agents

[0109] The present disclosure includes cyclic peptide covalently or non-covalently associated with one or more agents, such as one or more therapeutic agents and / or diagnostic agents. As disclosed herein, cyclic peptide covalently or non-covalently associated with oneor more agents can be referred to, e.g., as “conjugated” or by grammatical equivalents thereof.

[0110] Those of skill in the art will appreciate that the present disclosure includes the important discovery that cyclic peptides provided herein (e.g., cyclic peptides that are or include a sequence selected from SEQ ID NOs: 1-62, or a variant of such a sequence) are useful in targeting muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., skeletal muscle tissue), and therefore are characterized by certain general utility, e.g., in the delivery of agents to muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., skeletal muscle tissue). This discovery regarding disclosed cyclic peptides can therefore be applied in the delivery a wide variety of agents to muscle tissue, when those agents are conjugated with cyclic peptides. Moreover, because techniques for conjugation of agents to a peptide are well known in the art, it will be appreciated that a wide variety of agents or classes of agents can be readily selected by those of skill in the art for conjugation and / or delivery to muscle tissue. The present disclosure provides a broadly applicable vehicle for delivery of such agents or classes of agents, as may be conjugated with the cyclic peptide in any of a variety of appropriate means known in the art, to muscle tissue.[OHl] As disclosed herein and throughout, a cyclic peptide of the present disclosure can be conjugated with, e.g., up to 5 agents that are each independently selected from a therapeutic agent and / or a diagnostic agent, e g., with 1, 2, 3, 4, or 5 agents that are each independently selected from a therapeutic agent and / or a diagnostic agent. In various embodiments, a cyclic peptide of the present disclosure can be conjugated with 1 or 2, 1 to 3, 1 to 4, or 1 to 5 agents that are each independently selected from a therapeutic agent and / or a diagnostic agent. In some embodiments, the molar ratio of agent to cyclic peptide, either in a conjugation reaction or in a population of conjugated cyclic peptides, is from 1 : 10 to 10: 1, from 1:5 to 5:1, from 1 :4 to 4: 1, from 1:3 to 3:1, from 2:3 to 3:2, from 2: 1 to 1:2, about 4: 1, about 3: 1, about 1 :3, about 2:1, about 1 :2, about 3 :2, about 2:3, or about 1 : 1.

[0112] As disclosed herein and throughout, an agent (e.g., an ASO or other agent) can be conjugated with, e g., up to 5 cyclic peptides that are each independently selected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In various embodiments, an agent can be conjugated with, e.g., 1, 2, 3, 4, or 5 cyclic peptides that are each independently selected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In various embodiments, an agent can be conjugated with 1 or 2, 1 to 3, 1 to 4, or 1 to 5 cyclic peptides that are each independentlyselected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In various embodiments, an agent can be conjugated with 2 or 3, 2 to 4, or 2 to 5 cyclic peptides that are each independently selected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In various embodiments, an agent can be conjugated with 3 or 4, or 3 to 5 cyclic peptides that are each independently selected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In various embodiments, an agent can be conjugated with 4 to 5 cyclic peptides that are each independently selected from cyclic peptides of the present disclosure (e.g., from SEQ ID NOs: 1-62 and variants thereof). In certain embodiments, the molar ratio of agent to cyclic peptide is from 1 :1 to 1 : 10, from 1:1 to 1 :5, or from 1:2 to 1 :5. In some embodiments, the molar ratio of agent to cyclic peptide is about 1 :2. In certain embodiments, the molar ratio of agent to cyclic peptide is about 1 :3.

[0113] In some embodiments, an agent is covalently associated with a cyclic peptide. In certain embodiments, an agent is covalently associated with a functional group on a side chain of a cyclic peptide. In certain embodiments, an agent is covalently associated with the N-terminus of a cyclic peptide. In some embodiments, an agent is covalently associated with the C-terminus of a cyclic peptide.

[0114] In certain embodiments, an agent is directly covalently associated with a cyclic peptide. In certain embodiments, an agent is indirectly covalently associated with a cyclic peptide. In some embodiments, an agent is directly covalently associated with a functional group on a side chain of a cyclic peptide. In some embodiments, an agent is indirectly covalently associated with a functional group on a side chain of a cyclic peptide. In certain embodiments, an agent is directly covalently associated with the N-terminus of a cyclic peptide. In certain embodiments, an agent is indirectly covalently associated with the N-terminus of a cyclic peptide. In some embodiments, an agent is directly covalently associated with the C-terminus of a cyclic peptide. In some embodiments, an agent is indirectly covalently associated with the C-terminus of a cyclic peptide.

[0115] In some embodiments, an agent is covalently associated with a cyclic peptide via a thioether bond, a disulfide bond, an oxime, a thiazolidine, a hydrazone, an amide bond, an azide bond, or a maleimide bond. In some embodiments, the covalent association between a cyclic peptide and an agent is formed via a Diels- Alder reaction. In certain embodiments, the covalent association between a cyclic peptide and an agent is formed via click chemistry.

[0116] In certain embodiments, an agent is indirectly covalently associated with a cyclic peptide via a linker. In certain embodiments, the linker comprises an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl (i.e., heterocyclyl), an aryl, a heteroaryl, an aralkyl, a heteroaralkyl, a carboxy, a ketone, an aldehyde, an alkyl phosphoryl, an alkenyl, a heteroalkenyl, a cycloalkenyl, a cycloheteroalkenyl, an alkynyl, a sulfonyl, a hydroxy, a cyano, an azido, a carbonyl, a halo (e.g. -F, -Cl, -Br, or -I), a haloalkyl, a silyl, a silyloxy, an amine, an ether, a thioether, a phosphine, a phosphoramidate, a carboxamide, an ester, an imidoester, an amidine, a thioester, a sulfonamide, a carbamate, a urea, a guanidine, a thiourea, a disulfide, an oxime, a hydrazine, a hydrazides, a hydrazone, a diaza bond, a triazole, a triazoline, a tetrazine, an amino acid, or combinations thereof.

[0117] In some embodiments, a linker is cleavable linker. In some embodiments, the linker is a self-immolative linker. In certain embodiments, a linker comprises a para- aminobenzyl alcohol (PAB). In certain embodiments, a linker is bifunctional. In some embodiments, a linker is trifunctional.

[0118] In certain embodiments, a linker comprises a thioether bond, a disulfide bond, an oxime, a thiazolidine, a hydrazone, an amide bond, an azide bond, or a maleimide bond.

[0119] In certain embodiments, the linker comprises a C1-C30 alkyl, a C2-C20 alkyl, a C3-C12 alkyl, a C6-C12 alkyl, or a Ce alkyl. In some embodiments, shorter carbon chain linkers may be used to attach agents where proximity to a cyclic peptide poses no problem. In some embodiments, longer carbon chain linkers may be used where an agent must be adequately spaced from a cyclic peptide.

[0120] In certain embodiments, the linker comprises an alkyl amine. In some embodiments, the linker comprises an alkyl phosphate. In certain embodiments, the linker comprises an alkyl amide. In some embodiments, the linker comprises a Ce alkyl amine. In some embodiments, the linker comprises an amino Ce alkyl phosphate.

[0121] In some embodiments, the linker comprises the product of a Diels-Alder reaction, the product of a copper(I)-catalyzed 1,3-dipolar cycloaddition, or the product of a click chemistry reaction.

[0122] In certain embodiments, an agent is an antisense oligonucleotide (ASO) and a linker comprising a Ce alkyl amine connects a cyclic peptide to the 5’ end of the ASO.

[0123] In some embodiments, the peptide conjugate is prepared by solid-phase synthesis. In certain embodiments, the peptide conjugate is prepared using an automated synthesizer.

[0124] In certain embodiments, an agent is not covalently associated with a cyclic peptide. In some embodiments, a cyclic peptide and an agent are associated by anelectrostatic interaction (e.g. an ionic bond or a hydrogen bond). In certain embodiments, a cyclic peptide and an agent are associated by a hydrophobic interaction.

[0125] In some embodiments, the peptide conjugate comprises an admixture of a cyclic peptide and an agent. Accordingly, in certain embodiments, the peptide conjugate is prepared by a process comprising intermixing a cyclic peptide and an agent.

[0126] The present disclosure therefore includes covalent and non-covalent conjugation of agents with a cyclic peptide of the present disclosure. Therapeutic and diagnostic agents of the present disclosure can include, e.g., without limitation, nucleic acids, peptides, or chemical agent. In various embodiments, a therapeutic or diagnostic agent of the present disclosure can diagnose, monitor, or modulate (e.g., upregulate, or downregulate) the expression, production, and / or activity of one or more targets (e.g., target nucleic acids, genes, peptides, proteins, or compounds) provided in Table 2, e.g., in a tissue (e.g., muscle tissue) and / or disease disclosed herein.

[0127] Therapeutic agents of the present disclosure include, e.g., therapeutic nucleic acids, peptides, and chemical agents. Nucleic acids of the present disclosure include, e.g., inhibitory nucleic acids and nucleic acids (e.g., a transgenes) that encodes expression product. Examples of inhibitory nucleic acids include, without limitation, small interfering RNA (siRNA), microRNA (miRNA), and inhibitory antisense oligonucleotides (ASOs). In some embodiments, the nucleic acids disclosed herein comprise Adenosine Deaminase Acting on RNA- (ADAR-) recruiting oligonucleotides. Examples of expression products include, without limitation, inhibitory nucleic acids and proteins. Examples of peptide agents include, e.g., biologically active peptides, proteins, protein complexes, and peptidomimetics.Examples of chemical agents include, without limitation, small molecules, e.g. small molecules having a known biological activity. In various embodiments, a therapeutic agent can be, e.g., a chemotherapeutic agent. In various embodiments, a therapeutic agent can be, e.g., a radionuclide.

[0128] In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down-regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2). In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down- regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2) by at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%. In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down-regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2) by, e.g., at least about 40%. In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down-regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2) by, e.g., at least about 60%. In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down- regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2) by, e.g., about 20% to about 40%, about 20% to about 60%, about 20% to about 80%, about 20% to about 100%, about 40% to about 60%, about 40% to about 80%, about 40% to about 100%, about 60% to about 80%, or about 60% to aboutl00%. In certain embodiments, an inhibitory nucleic acid of the present disclosure inhibits or down-regulates, or is capable of inhibiting or down-regulating, expression of a gene (e.g., expression of a gene related to a disease or condition associated with muscle tissue, or of a gene according to Table 2, or a gene encoding a protein according to Table 2) by a percentage that is within a range having a lower boundary selected from about 20%, about 30%, about 40%, about 50%, or about 60%, and an upper boundary selected from 100%, about 80%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or about 30%.

[0129] In certain embodiments, an agent is an antisense oligonucleotide (ASO). In some embodiments, the ASO is covalently associated with a cyclic peptide via the 5’ end of the ASO. In some embodiments, the ASO is covalently associated with a cyclic peptide via the 3’ end of the ASO. In certain embodiments, the ASO is covalently associated with acyclic peptide via a heterocyclic base of the ASO. In various embodiments, an antisense oligonucleotide includes a phosphorodi ami date morpholino oligonucleotide (PMO), or a peptide nucleic acid (PNA). In various embodiments, an antisense oligonucleotide hybridizes to a target nucleic acid (e.g., a target nucleic acid in accordance with Table 2, or a target nucleic acid encoding a protein or RNA in accordance with Table 2) and effects modulation of gene expression activity or function, such as transcription, translation or splicing. The modulation of gene expression can be achieved by, for example, target degradation or occupancy -based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. In various embodiments, the antisense oligonucleotide can include one or more modifications. In various embodiments, the antisense oligonucleotide can include one or more modifications to reduce degradation. In various embodiments, the modification of the antisense oligonucleotide is a sugar modification. Sugar modifications include, but are not limited to, 2 '-O-(2 -methoxy ethyl), 2'-fluoro, locked nucleic acid and ethylene bridged nucleic acid. In various embodiments, the sugar modification is 2'-O-(2- methoxy ethyl). In various embodiments, the antisense oligonucleotide further includes phosphorothioate internucleoside linkages.

[0130] In various embodiments, an agent is an adeno-associated virus (AAV). In certain embodiments, the AAV is not covalently associated with a cyclic peptide. In certain embodiments, the AAV is covalently associated with a cyclic peptide. In some embodiments, the AAV is directly covalently associated with a cyclic peptide. In certain embodiments, cyclic peptide of the present disclosure is present in (e.g., within, at the N-terminus of, or at the C-terminus of the sequence of) a protein of the AAV. In certain embodiments, cyclic peptide of the present disclosure is present in (e.g., within, at the N-terminus of, or at the C- terminus of the sequence of) a structural protein of the AAV, e.g., a capsid protein of the AAV). In certain embodiments, cyclic peptide of the present disclosure is present in (e.g., within, at the N-terminus of, or at the C-terminus of the sequence of) an AAV capsid protein selected from VP1, VP2, or VP3. In certain embodiments, cyclic peptide of the present disclosure is present in a hypervariable region the AAV capsid. Without wishing to be bound by any particular scientific theory, AAV capsids include 12 hypervariable regions exposed on the capsid surface. In certain embodiments, the AAV is indirectly covalently associated with a cyclic peptide via a linker. In certain embodiments, the AAV is covalently associated with a cyclic peptide via a functional group (e.g., a chemical handle) of the AAV capsid. In some embodiments, the AAV is covalently associated with a cyclic peptide via a lysine or arginineresidue on the AAV capsid. In some embodiments, the AAV capsid comprises an unnatural amino acid. The unnatural amino acid may serve as a chemical handle to facilitate covalent association to a cyclic peptide. Accordingly, in some embodiments, the AAV is covalently associated with a cyclic peptide via the unnatural amino acid. In certain embodiments, the unnatural amino acid comprises an azide. In some embodiments, the AAV is covalently associated with a cyclic peptide via an azide of an unnatural amino acid. In certain embodiments, a cyclic peptide comprises an alkyne. In some embodiments, a covalent association between the AAV and a cyclic peptide is formed via a click chemistry reaction. In certain embodiments, a covalent association between the AAV and a cyclic peptide is formed via a click chemistry reaction between an alkyne and an azide. In certain embodiments, a covalent association between the AAV and a cyclic peptide is formed via a click chemistry reaction between an alkyne of the cyclic peptide and an azide of the AAV capsid. In various embodiments, the AAV can be used as a vehicle, e.g., vehicle for the delivery of a nucleic acid (e.g., a nucleic acid provided in Table 2, or a nucleic acid encoding a protein or RNA provided in Table 2). In various embodiments, the AAV can be used as a vehicle, e.g., vehicle for the delivery of a transgene (e.g., a transgene provided in Table 2, or a transgene encoding a protein or RNA provided in Table 2).

[0131] In various embodiments, the agent is a lipid nanoparticle (LNP). LNPs can include one or more lipid components. LNPs may be used as carriers for therapeutic agents and can include synthetic ionizable or cationic lipids, phospholipids, cholesterol, and a polyethylene glycol (PEG) lipid. In certain embodiments, the LNP is not covalently associated with a cyclic peptide. In certain embodiments, the LNP is covalently associated with a cyclic peptide (e.g., via association of the cyclic peptide with an LNP component such as a synthetic ionizable or cationic lipid, phospholipid, cholesterol, or PEG lipid). In some embodiments, the LNP is directly covalently associated with a cyclic peptide. In certain embodiments, the LNP is indirectly covalently associated with a cyclic peptide via a linker. In some embodiments, the LNP is covalently associated with a cyclic peptide via a lipid of the LNP. In some embodiments, the LNP is covalently associated with a cyclic peptide via a carboxylic acid of the LNP. In certain embodiments, the LNP is covalently associated with a cyclic peptide via an amine of the cyclic peptide. In certain embodiments, the LNP is covalently associated with a cyclic peptide via an amide bond. In some embodiments, a covalent association between the LNP and a cyclic peptide is formed via an amidation reaction. In some embodiments, a covalent association between the LNP and a cyclic peptide is formed from an amine of the cyclic peptide and a carboxylic acid of the LNP.

[0132] In various embodiments, the nucleic acid agent is a small noncoding RNA (e.g., a noncoding RNA according to Table 2). In various embodiments, the small noncoding RNA includes without limitation a siRNA or a miRNA. In various embodiments, the siRNA includes any nucleic acid molecule capable of inhibiting or down regulating gene expression (e.g., of a gene according to Table 2, or a gene encoding a protein according to Table 2) or viral replication, for example by mediating RNA interference or gene silencing in a sequencespecific manner. In various embodiments, the miRNA includes any type of interfering RNA, including but not limited to, endogenous miRNA and artificial miRNA. Endogenous miRNA are small RNAs naturally present in the genome which are capable of modulating the productive utilization of mRNA. Artificial miRNA includes any type of RNA sequence, other than endogenous miRNA, which is capable of modulating the productive utilization of mRNA.

[0133] In various embodiments, the nucleic acid agent is a transgene (e.g., a transgene according to Table 2, or a transgene encoding a protein or RNA according to Table 2). In various embodiments, a transgene includes a nucleic acid (e.g., DNA or RNA) sequence encoding a protein or RNA (e.g., a functional non-coding RNA). In some embodiments, the transgene includes an open reading frame encoding a protein or RNA (e g., a functional noncoding RNA). A transgene may be isolated from an organism and introduced into a different organism of the same or different species to produce the transgene product (e.g., the protein or RNA). As used herein, in the context of a DNA encoding a protein, the term transgene may or may not include un-transcribed flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, terminators, promoters, or enhancers.

[0134] In various embodiments, a therapeutic agent includes a peptide agent (e.g., a peptide provided in Table 2). In various embodiments, the peptide agent includes a protein agent or a peptidomimetic agent. In various embodiments, the peptide or peptidomimetic agents of the present disclosure have a defined length. The peptide or peptidomimetic agents of the present disclosure can have, without limitation, a length of at most 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000 residues. In various embodiments, the peptide or peptidomimetic agents of the present disclosure have a length of at least 5, 10, 20, 30, 50, 100, 150, 200, 250, or 300 residues.

[0135] In various embodiments, a therapeutic agent includes a small molecule agent. In various embodiments, a small molecule agent includes DNA damaging agents, agents that inhibit DNA synthesis, microtubule and tubulin binding agents, anti-metabolites, inducers of oxidative damage, anti angiogenics, endocrine therapies, anti-estrogens, immuno-modulatorssuch as Toll-like receptor agonists or antagonists, histone deacetylase inhibitors, inhibitors of signal transduction such as inhibitors of kinases, inhibitors of heat shock proteins, retinoids, inhibitors of growth factor receptors, anti-mitotic compounds, anti-inflammatories, cell cycle regulators, transcription factor inhibitors, and apoptosis inducers, and any combination thereof.

[0136] In various embodiments, a therapeutic agent includes a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to methotrexate, daunomycin, mitomycin, cisplatin (cisplatinum or cis-dianminedichloroplatinum(II) (CCDP)), vincristine, epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside, aminopterin, bleomycin, mitomycin C, democolcine, etoposide, mithramycin, chlorambucil, melphalan, daunorubicin, doxorubicin, tamoxifen, paclitaxel, vincristine, vinblastine, camptothecin, actinomycin D, and cytarabine, combrestatin and its derivatives.

[0137] Diagnostic agents of the present disclosure can include, e.g., agents that label tissue (e.g., adipose tissue) and / or are useful in the diagnosis of one or more medical conditions. In various embodiments, an agent includes an imaging agent. Diagnostic agents of the present disclosure can include, e.g., without limitation, a fluorescent label, luminescent label, enzyme, or detectable tag. Examples of imaging agents include, but are not limited to, the following: radioisotopes (e.g.,3H,14C,35S,125I,131I) fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), MRI contrast agents (e g., Gadolinum chelates (Gd)) luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, betagalactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In various embodiments, an image agent includes any suitable radionuclide, including but not limited to227Ac,211At,131Ba,77Br,109Cd,51Cr,67Cu,165Dy,155Eu,153Gd,198Au,166Ho,113mIn,115mIn,123I,125I,131I,189Ir,191Ir,192Ir,194Ir,52Fe,55Fe,59Fe,177Lu,109Pd,32P,226Ra,186Re,188Re,153Sm,46Sc,47Sc,72Se,75Se,105Ag,89Sr,35S,177Ta,117mSn,121Sn,166Yb,169Yb,90Y,212Bi,119Sb,197Hg,100Pd,101mRh, and212Pb. In various embodiments, a radionuclide may also be useful in delivering a therapeutic dosage of radiation to a tissue or a cell.Formulation and Administration of Cyclic Peptides and Cyclic Peptide Conjugates

[0138] Cyclic peptides and peptide conjugates of the present disclosure are useful for various applications (e.g., therapeutic or diagnostic applications), including without limitation the treatment of diseases (e.g., diseases associated with the muscle tissue, e.g., diseases associated with the skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., diseases associated with the skeletal muscle tissue). The present disclosure includes the broad recognition that cyclic peptides and peptide conjugates of the present disclosure can be useful to deliver an agent to muscle tissue and to treat a disease (e.g., diseases associated with the muscle tissue, e g., diseases associated with the skeletal muscle tissue, smooth muscle tissue, or cardiac muscle tissue, e.g., diseases associated with the skeletal muscle tissue). Those of skill in the art will further appreciate from the present disclosure that cyclic peptides and peptide conjugates of the present disclosure are not limited in use to the treatment of diseases associated with muscle tissue, and can instead be used in the treatment of any condition in any tissue for which a cyclic peptide has selectivity.

[0139] The present disclosure includes pharmaceutical compositions for delivery of one or more agents to a subject. As disclosed herein, a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art. A suitable means of administration can be selected based on the age and condition of a subject.

[0140] Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms. Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application. Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0141] In some embodiments, the compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration. Such a unit dosage form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.

[0142] A pharmaceutical composition of the present disclosure can be in an injectable or infusible form. For example, the present disclosure includes sterile formulations for injection, which can be formulated in accordance with conventional pharmaceutical practices. Sterile injectable solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Injectable solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and / or a polyalcohol such as propylene glycol or polyethylene glycol, and / or a nonionic surfactant such as polysorbate 80™ or HCO-50, and the like. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. In particular instances, a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).

[0143] In various embodiments, a pharmaceutical composition of the present disclosure can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium.

[0144] In various instances, a pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Pharmaceutical composition of therapeutic agents of the present disclosure can include pharmaceutically acceptable salts, e.g., an acid addition salt or a base addition salt.

[0145] In certain embodiments, compositions can be formulated with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e.g., J. R. Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0146] Route of administration can be parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, or transcutaneous administration. Administration can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. Administration can be systemic or local. In certain embodiments, a composition described herein can be therapeutically delivered to a subject by way of local administration. As used herein, “local administration” or “local delivery,” can refer to delivery that does not rely upon transport of the composition or agent to its intended target tissue (e g., muscle tissue) or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. In certain embodiments, following local administration in the vicinity of a target tissue or site (e g., muscle tissue), the composition or agent, or one or more components thereof, may diffuse to an intended target tissue or site that is not the site of administration.

[0147] A pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, asoothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0148] In various embodiments, subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.

[0149] An injection system of the present disclosure may employ a delivery pen as described in U.S. Pat. No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can include at least one injection needle (e.g., a 31 gauge needle of about 5 to 8 mm in length), are typically prefilled with one or more therapeutic unit doses of a therapeutic solution, and are useful for rapidly delivering solution to a subject with as little pain as possible. One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received. The pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and / or indicate the dosage of medication that is injected into the user. See, e.g., U.S. Pat. No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kits include one or more disposable replacement needles. Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U.S. Pat. Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g., U.S. Pat. No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLY™, manufactured by Scandinavian Health Ltd.

[0150] In some embodiments, compositions can be formulated in a composition suitable for intrapulmonary administration (e.g., for administration via an inhaler or nebulizer) to a mammal such as a human. Methods for formulating such compositions are well known in the art. Dry powder inhaler formulations and suitable systems for administration of the formulations are also known in the art. Pulmonary administration may be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers. For example, a composition described herein can be administered to the lungs of a subject by way of a dry powder inhaler. These inhalers are propellant-free devices that deliver dispersible and stable dry powder formulations to the lungs. DPI devices have been used for pulmonaryadministration of polypeptides such as insulin and growth hormone. In some embodiments, a composition described herein can be intrapulmonarily administered by way of a metered dose inhaler. These inhalers rely on a propellant to deliver a discrete dose of a compound to the lungs.

[0151] In some embodiments, compositions can be formulated for delivery to the eye, e.g., in the form of a pharmaceutically acceptable solution, suspension or ointment. A preparation for use in treating an eye can be in the form of a sterile aqueous solution containing, e.g., additional ingredients such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, and viscosity-increasing agents. A preparation as described herein can be administered topically to the eye of the subject in need of treatment (e.g., a subject afflicted with AMD) by conventional methods, e g., in the form of drops, or by bathing the eye in a therapeutic solution, containing one or more compositions.

[0152] A variety of devices for introducing drugs into the vitreal cavity of the eye may be appropriate, in certain embodiments, for administration of a composition as described herein. For example, U.S. Publication No. 2002 / 0026176 describes a pharmaceuticalcontaining plug that can be inserted through the sclera such that it projects into the vitreous cavity to deliver the pharmaceutical agent into the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantable device for introduction into a suprachoroidal space or an avascular region for sustained release of drug into the interior of the eye.

[0153] In some embodiments, a composition described herein can be locally administered to a joint (e.g., an articulated joint). For example, in embodiments where the disorder is arthritis, a therapeutically appropriate composition can be administered directly to a joint (e.g., into a joint space) or in the vicinity of a joint. Examples of intraarticular joints to which a composition described herein can be locally administered include, e.g., the hip, knee, elbow, wrist, sternoclavicular, temporomandibular, carpal, tarsal, ankle, and any other joint subject to arthritic conditions. A composition described herein can also be administered to bursa such as, e.g., acromial, bicipitoradial, cubitoradial, deltoid, infrapatellar, ischial, and any other bursa known in the art of medicine.

[0154] In some embodiments, a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11months, 1 year, 11 / 2 years, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0155] A pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in a pharmaceutical preparations is such that a suitable dose within the designated range can be administered to subjects. The dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.

[0156] Pharmaceutical compositions including certain therapeutic agents, e.g., cyclic peptide conjugates as disclosed herein, can be administered as a fixed dose, or in a milligram per kilogram (mg / kg) dose. While in no way intended to be limiting, an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg / kg, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight. Exemplary dosages of a composition described herein include, without limitation, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg. The present disclosure is not limited to such ranges or dosages.Muscle Tissue and Diseases Thereof

[0157] As used herein, muscle tissue refers to tissues characterized by the presence of muscle cells, and cells thereof. Muscle tissue contributes to biological functions including movement (e.g., voluntary movement and / or involuntary movement), energy metabolism (e.g., production of heat for maintenance of core temperature), and storage of molecules including amino acids and carbohydrates. Muscle tissue can also play an important role in, and / or contribute to, certain diseases.

[0158] Examples of muscle tissue include skeletal muscle tissue, smooth muscle tissue, and cardiac muscle tissue. In various embodiments, skeletal muscle tissue is characterized by the presence of skeletal muscle cells. In various embodiments, skeletalmuscle tissue can include skeletal muscle cells such as multinucleated muscle cells or cells of muscle fibers. Muscle cells can be classified based on various factors, including, without limitation, glycogen content, rate of fatigue, or speed of contraction. Examples of muscle cells include type I cells, type IIA cells, and type IIX cells. In various embodiments, type I cells include slow oxidative cells. In various embodiments, type I cells include muscle cells that have a low rate of fatigue, slow contractile speed, and low myosin ATPase activity. In various embodiments, type IIA cells include fast oxidative cells. In various embodiments, type IIA cells include muscle cells that have an intermediate rate of fatigue, fast contractile speed, and high myosin ATPase activity. In various embodiments, type IIX cells include fast glycolytic cells. In various embodiments, type IIX cells include muscle cells that have a high rate of fatigue, high contractile speed, and high myosin ATPase activity. In various embodiments, smooth muscle tissue is characterized by the presence of smooth muscle cells. In various embodiments, cardiac muscle tissue is characterized by the presence of cardiac muscle cells (i.e., cardiomyocytes). In various embodiments, muscle tissue can include muscle cells, immune cells, endothelial cells, satellite cells, and fibro-adipogenic progenitors. In certain embodiments, muscle tissue includes skeletal muscle cells. In certain embodiments, the muscle tissue includes smooth muscle cells. In certain embodiments, the muscle tissue includes cardiac muscle cells. In certain embodiments, the muscle tissue is skeletal muscle tissue.

[0159] Impairment of muscle tissue functions has been associated with various diseases. Delivery of agents disclosed herein to muscle tissue can be useful in the treatment of such diseases. The present disclosure includes, among other things, use of a cyclic peptide of the present disclosure to deliver an agent to muscle tissue (e.g., where the cyclic peptide is covalently or non-covalently conjugated with the agent). Delivery of a therapeutic or diagnostic agent disclosed herein to a tissue can be useful, e.g., for treatment or diagnosis of diseases associated with muscle tissue. In various embodiments, a disease for which delivery of a therapeutic or diagnostic agent disclosed herein to a tissue can be useful, e.g., for treatment or diagnosis of the disease, and / or diseases associated with muscle tissue, includes myopathy (i.e., a muscle disease). In various embodiments, a myopathy includes, without limitation, a myopathy associated with infectious, inflammatory, traumatic, neurological, genetic, neoplastic, and iatrogenic conditions. In various embodiments, a myopathy is a neuromuscular junction disease. In various embodiments, a neuromuscular junction disease includes, without limitation, Myasthenia gravis, Botulism, and Lambert-Eaton myasthenic syndrome (LEMS). In various embodiments, a myopathy is a muscular dystrophy. In variousembodiments, a muscular dystrophy includes, without limitation, Duchenne muscular dystrophy, Becker muscular dystrophy, Congenital muscular dystrophies, Myotonic muscular dystrophy, Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, Emery-Dreifuss muscular dystrophy, Distal muscular dystrophy, and Oculopharyngeal muscular dystrophy. In various embodiments, a myopathy is an idiopathic inflammatory myopathy. In various embodiments, an idiopathic inflammatory myopathy includes, without limitation, dermatomyositis, polymyositis, necrotizing myopathy, and inclusion body myositis. In various embodiments, a myopathy is rhabdomyolysis (e.g., direct injury to muscle tissue and release of intracellular contents). In various embodiments, a myopathy is atrophy. In various embodiments, the atrophy is muscle atrophy or muscle wasting. In various embodiments, the muscle atrophy is primary muscle atrophy or secondary muscle atrophy. In various embodiments, a myopathy is cardiomyopathy. In various embodiments, a cardiomyopathy myopathy includes Dilated cardiomyopathy, Hypertrophic cardiomyopathy, Restrictive cardiomyopathy, Arrhythmogenic right ventricular dysplasia, Transthyretin amyloid cardiomyopathy (ATTR-CM). In various embodiments, a myopathy is a disease associated with smooth muscle dysfunction. In various embodiments, a disease associated with smooth muscle dysfunction includes systemic and pulmonary hypertension, heart failure, asthma, chronic obstructive pulmonary disease (COPD), gastrointestinal dysmotility, inflammatory bowel diseases, impotence, genito-urinary and bladder dysfunction, uterine dysfunction, and multisystemic smooth muscle dysfunction syndrome (MSMDS).

[0160] In various embodiments, a disease or disorder can be treated by administration of a composition of the present disclosure, peptide conjugate of the present disclosure, or agent (e.g., an agent covalently or non-covalently associated with a cyclic peptide of the present disclosure and / or a therapeutic agent) that targets a nucleic acid or protein, e.g., a nucleic acid (e.g., a gene encoding a protein or RNA) associated with the disease, a protein associated with disease, or a nucleic acid encoding such a protein. In various embodiments, the target of the composition, the peptide conjugate, or the agent can be a target according to Table 2. In various embodiments, a target nucleic acid (e.g., a gene encoding a protein or RNA) can be a nucleic acid according to Table 2. In various embodiments, a target protein can be, or the protein encoded by a target nucleic acid, can be a protein according to Table 2. In various embodiments, a target nucleic acid (e.g., a gene encoding a protein or RNA), a target protein, or protein encoded by a target nucleic acid, can be a nucleic acid or protein according to Table 2 and the composition of the present disclosure, peptide conjugate of the present disclosure, or agent can be used to treat a corresponding disease indicated in Table 2.In some embodiments a disease can be muscle atrophy and / or muscle wasting, Duchenne muscular dystrophy (DMD), Myotonic dystrophy type 1 (DM1), Myotonic dystrophy type 2 (DM2), Facioscapulohumeral muscular dystrophy (FSHD), Centronuclear myopathy (CNM), Pompe disease, Inclusion body myopathy (IBM), Laing distal myopathy, Myofibrillar myopathy (MFM), Myotonia congenita, Oculopharyngeal muscular dystrophy (OPMD), Paramyotonia congenita (PMC), Friedreich's ataxia (FRDA), or Fibrodysplasia ossificans progressiva (FOP).

[0161] In various embodiments, a disease of the present disclosure (e.g., a disease associated with muscle tissue or impairment of muscle tissue functions) is associated with the expression, production, and / or activity of, and / or can be treated by reducing the expression production and / or activity of, one or more targets (e.g., target nucleic acids, genes, peptides, proteins, or compounds) according to Table 2. In various embodiments, the expression, production, and / or activity of the one or more targets in Table 2 is diagnosed, monitored, or modulated (e.g., upregulated or downregulated) with the delivery of a therapeutic or diagnostic agent disclosed herein.Table 2. List of Targets of Diseases Associated with Muscle TissueEXAMPLES

[0162] The present Examples demonstrate that cyclic peptides as set forth in the present disclosure are useful in delivering therapeutic agents to muscle tissue, of which presently exemplified antisense oligonucleotide payloads are representative. The present Examples provide data showing that a cyclic peptide of the present disclosure can effectively deliver a therapeutic agent to muscle tissue, as exemplified by delivery of representative payloads, such as an antisense nucleotide or a small interfering RNA that regulates gene expression in muscle tissue cells. As will be appreciated from the present Examples, those of skill in the art can select payloads that will provide therapeutic benefit in the treatment of disease.Example 1: Use of Cyclic Peptides of the Present Disclosure to Deliver a Representative Antisense Oligonucleotide (ASO) Payload to Muscle Tissue

[0163] The present Example confirms that a cyclic peptide of the present disclosure conjugated to a representative ASO payload can deliver the payload to target muscle tissue cells and regulate biological activity, evidencing utility for treatment of disease. In particular, the present Example demonstrates use of a cyclic peptide of the present disclosure to deliveran ASO to target muscle tissue cells, achieving downregulation of expression of a targeted gene.

[0164] The present disclosure utilized cyclic peptides of the present disclosure conjugated with an ASO targeting Malatl (SEQ ID NO: 63). Malatl is a long non-coding RNA transcript, the upregulation of which is correlated with the progression and development of a wide range of indications including cancers, diabetes, and inflammatory conditions (Biswas, S. etal. (2018) Sci Rep, 8: 6526.). The Malatl ASO contains phosphorothioate substitutions and 2' sugar modifications, as indicated in Table 3, to inhibit nuclease degradation and to facilitate vehicle-free delivery to cells, and may contain one or more additional modifications.

[0165] Conjugation between an exemplified cyclic peptide (SEQ ID NO: 1 as indicated in Table 1) and exemplified antisense oligonucleotides (SEQ ID NO: 63 as indicated in Table 3) was done with an amino C6 linker comprising an amino group and a six-carbon spacer. The C terminus of the cyclic peptide was functionalized with an azide and the 5’ end of the ASO was functionalized with a dibenzaocyclooctyl (DBCO). Following copper free click chemistry, the peptide oligo conjugate was constructed with a 1 : 1 ratio between cyclic peptide and ASO.

[0166] Conjugated cyclic peptides of the present Example, and non-targeting controls, pre-formulated in isotonic saline, as well as a PBS injection control, were delivered in vivo through tail vein injections in male BL6 mice of 8-10 weeks of age. Mice received a total of 8 injections across 24 days at 1.5 mg / kg. On day 27, 3 days after the last dose, mice were perfused to collect tissues, including adipose tissue, muscle tissues, brain tissue, heart tissues, and kidney tissues for RNA processing or histology processing. For each of the collected tissues, a tissue sample was flash frozen, homogenized with SPEX SamplePrep with steel beads for 3 minutes at max speed in the presence of BME. White adipose tissue (WAT) was processed with a Qiagen QIAzol kit, brain and kidney tissues were processed through a Qiagen RNeasy kit, and muscle and heart tissues were processed through a Qiagen RNeasy fibrous tissue kit.

[0167] Total RNA aliquots were prepped using High-Affinity cDNA Reverse Transcription mix by Applied Biosystems. cDNA samples were then processed for qPCR using Applied Biosystems Taqman reagents. Measurements were made for the target ASO gene (MALAT1) and housekeeping gene (GAPDH) based on dosing in each mouse group. Applied Biosystems Taqman primer probe IDs Mm01227912_sl (MALAT1) and Mm99999915_gl (GAPDH) were used. AACq analysis for all processed tissues wasperformed. Target gene mRNA expression profiles were visualized with respect to PBS control groups and non-targeting ASO control groups when relevant.Table 3. Exemplified Antisense Oligonucleotide Sequences* denotes positions of phosphorothioate bonds. M denotes 2'-O-methylation (addition of a methyl group to the 2’ hydroxyl of the ribose moiety of a nucleoside). For example, MG denotes 2'-O-methylated guanosine.

[0168] As shown in FIG. 1, delivery of Malatl ASO conjugated to an exemplified targeted cyclic peptide resulted in significantly decreased Malatl expression in skeletal muscle tissue compared to both PBS control and non-targeted control, where Malatl ASO was conjugated to a control peptide. When normalized to Malatl expression levels in nontargeted control across tissues, results in FIG. 2 demonstrate that the conjugation to an exemplified cyclic peptide resulted in increased Malatl downregulation in skeletal muscle tissue but not in adipose, brain, heart or kidney tissues. Taken together, these results suggest that a cyclic peptide of the present disclosure is useful in delivering therapeutic agents to muscle tissue. The results suggest that the cyclic peptide as set forth in the present disclosure, conjugated to a representative ASO payload targeting Malatl, could selectively target muscle tissue for Malatl expression knockdown. Therefore, the targeted cyclic peptides disclosed herein are useful in delivering a payload to target muscle tissue cells and regulate biological activity, evidencing utility for treatment of disease.Example 2: Use of Cyclic Peptides of the Present Disclosure to Deliver a Representative Small Interfering RNA (siRNA) Payload to Muscle Tissue

[0169] The present Example confirms that a cyclic peptide of the present disclosure conjugated to a representative siRNA payload can deliver the payload to target muscle tissue cells and regulate biological activity, evidencing utility for treatment of disease. In particular, the present Example demonstrates use of a cyclic peptide of the present disclosure to deliver an siRNA to target muscle tissue cells, achieving downregulation of expression of a targeted gene.

[0170] Peptides of the present disclosure conjugated with an siRNA targeting ALDH2 were tested as described herein. ALDH2 is a mitochondrial enzyme expressed in most human tissues, with high levels in the liver, heart, kidney, and muscle tissues (Mark J et al (1998)BBA, 1399: 181-186.). It belongs to the aldehyde dehydrogenase family of enzymes, the activity of which is correlated with a variety of indications including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer (Chen, C. H. etal. (2014) Physiol Rev, 94: 1-34.).

[0171] Conjugation between an exemplified cyclic peptide (SEQ ID NO: 1 as indicated in Table 1) and exemplified siRNA was carried out as described in Example 1, with an amino C6 linker comprising an amino group and a six-carbon spacer. The C terminus of the cyclic peptide was functionalized with an azide and the 5’ end of the siRNA was functionalized with a dibenzaocyclooctyl (DBCO). Following copper free click chemistry, the peptide-siRNA conjugate was constructed with a 1 : 1 ratio between cyclic peptide and siRNA.

[0172] Conjugated cyclic peptides of the present Example, as well as a PBS injection control, were delivered in vivo through subcutaneous injections in female C57BL / 6 (BL6) mice. Mice received a single dose of the conjugated cyclic peptide at 1 mg / kg, or PBS as control. On day 7 after the injection, mice were perfused to collect tissues, including liver tissue, kidney tissue, spleen tissue, adipose tissue, heat tissue, and muscle tissues, for RNA processing. Adipose tissue includes gonadal white adipose tissue (gWAT). Muscle tissues include gastrocnemius, tibialis anterior (TA), triceps, and quadriceps (Quad) tissues. RNA processing was carried out as described in Example 1. Target gene mRNA expression in treated mice was normalized to expression of the same gene in the PBS-treated control mice.

[0173] As shown in FIG. 3, delivery of a siRNA conjugate to an exemplified targeted cyclic peptide resulted in significantly decreased target gene expression in muscle tissues (gastrocnemius, TA, triceps, and Quad tissues) compared to PBS control. Target gene expression in muscle tissue decreased in the conjugate-treated mice by a greater percentage than in other evaluated tissues (e.g., than in cardiac / heart tissue).

[0174] These results suggest that a cyclic peptide of the present disclosure is useful in delivering various types of therapeutic agents to muscle tissue. The results suggest that the cyclic peptide as set forth in the present disclosure, conjugated with a representative siRNA payload, could selectively target muscle tissue for expression knockdown. Data therefore demonstrated that the targeted cyclic peptides disclosed herein are useful in delivering a payload to target muscle tissue cells and regulate biological activity, evidencing utility for treatment of disease.OTHER EMBODIMENTS

[0175] It will be appreciated that the scope of the present disclosure is to be defined by that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. Moreover, recitation of claim elements in connection with a particular independent claim support recitation of such elements in connection with other independent claims. Throughout the disclosure and claims, where compositions or methods are described as having, including, or comprising specific elements, compositions that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. All references cited herein are hereby incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a cyclic peptide that selectively targets skeletal muscle tissue, wherein the cyclic peptide comprises an amino acid sequence having no more than three amino acid differences from a sequence selected from SEQ ID NOs: 1-62, wherein each amino acid difference is independently selected from an insertion of an amino acid, a deletion of an amino acid, or a substitution of an amino acid.

2. The composition of claim 1, wherein the cyclic peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-62.

3. The composition of claim 1, wherein the cyclic peptide comprises an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 1.

4. The composition of claim 1, wherein the cyclic peptide comprises an amino acid sequence according to SEQ ID NO: 1.

5. A peptide conjugate comprising a cyclic peptide comprising an amino acid sequence having no more than three amino acid differences from a sequence selected from SEQ ID NOs: 1-62, wherein each amino acid difference is independently selected from an insertion of an amino acid, a deletion of an amino acid, or a substitution of an amino acid, and an agent associated with the cyclic peptide.

6. The peptide conjugate of claim 5, wherein the cyclic peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-62.

7. The peptide conjugate of claim 5, wherein the cyclic peptide comprises an amino acid sequence having no more than three amino acid differences from SEQ ID NO: 1.

8. The peptide conjugate of claim 5, wherein the cyclic peptide comprises an amino acid sequence according to SEQ ID NO: 1.

9. The peptide conjugate of any one of claims 5-8, wherein the agent is a diagnostic agent or a therapeutic agent, optionally wherein the therapeutic agent targets a target according to Table 2.

10. The peptide conjugate of any one of claims 5-9, wherein the agent is an inhibitory nucleic acid.

11. The peptide conjugate of any one of claims 5-9, wherein the agent is an antisense oligonucleotide.

12. The peptide conjugate of claim 10, wherein the agent is an siRNA or a miRNA.

13. The peptide conjugate of claim 11, wherein the antisense oligonucleotide is a phosphorodi ami date morpholino oligonucleotide (PMO) or a peptide nucleic acid (PNA).

14. The peptide conjugate of any one of claims 5-9, wherein the agent is an adeno- associated virus (AAV).

15. The peptide conjugate of any one of claims 5-9, wherein the agent is a lipid nanoparticle (LNP).

16. The peptide conjugate of any one of claims 5-15, wherein the agent is non-covalently associated with the cyclic peptide.

17. The peptide conjugate of any one of claims 5-15, wherein the agent is covalently associated with the cyclic peptide.

18. The peptide conjugate of claim 17, wherein the agent is directly covalently associated with the cyclic peptide.

19. The peptide conjugate of claim 17, wherein the agent is indirectly covalently associated with the cyclic peptide.

20. The peptide conjugate of claim 19, wherein the agent is indirectly covalently associated with the cyclic peptide via a linker.

21. The peptide conjugate of claim 20, wherein the linker comprises a thioether bond, a disulfide bond, an oxime, a thiazolidine, a hydrazone, an amide bond, an azide bond, or a mal eimide bond.

22. The peptide conjugate of claim 20 or 21, wherein the linker comprises a C1-C30 alkyl, a C2-C20 alkyl, a C3-C12 alkyl, a C6-C12 alkyl, or a Ce alkyl group.

23. The peptide conjugate of any one of claims 20-22, wherein the linker comprises a Ce alkyl amine.

24. The peptide conjugate of any one of claims 20-23, wherein the linker is a cleavable linker.

25. The peptide conjugate of any one of claims 5-24, wherein the peptide conjugate selectively targets muscle tissue.

26. The peptide conjugate of claim 25, wherein the muscle tissue is skeletal muscle tissue.

27. The peptide conjugate of claims 5-26, wherein the peptide conjugate binds at least one integrin or integrin subtype, optionally wherein the integrin or integrin subtype is selected from selected from avpi, avP3, avP5, avP6, avP8, a8pi, a5pi, and allbp3, optionally wherein the integrin or integrin subtype is selected from avpi, avP6, and avP8, optionally wherein the peptide conjugate binds the P transmembrane subunit of the least one integrin or integrin subtype.

28. The peptide conjugate of any one of claims 5-27, wherein the amino acid sequence of the cyclic peptide comprises the amino acid sequence RGD.

29. The peptide conjugate of claim 28, wherein the RGD amino acid sequence binds at least one integrin or integrin subtype, optionally wherein the integrin or integrin subtype is selected from selected from avpi, avP3, otvP5, avP6, avP8, a8pi, a5pi, and allbp3, optionally wherein the integrin or integrin subtype is selected from avpi, a.vP6, and avP8, optionally wherein the RGD amino acid sequence binds the P transmembrane subunit of the least one integrin or integrin subtype.

30. A pharmaceutical composition comprising the peptide conjugate of any one of claims 5-29 and a pharmaceutically acceptable carrier.

31. A method of delivering an agent to muscle tissue in a subject, the method comprising administering to the subject a peptide conjugate according to any one of claims 5-29 or a pharmaceutical composition of claim 30.

32. A method of diagnosing, preventing, and / or treating a disease related to muscle tissue, the method comprising administering to the subject a peptide conjugate according to any one of claims 5-29 or a pharmaceutical composition of claim 30, optionally wherein the disease is a disease provided in Table 2.

33. The method of claim 32, wherein the disease is selected from Myasthenia gravis, Botulism, Lambert-Eaton myasthenic syndrome (LEMS), Duchenne muscular dystrophy, Becker muscular dystrophy, Congenital muscular dystrophies, Myotonic muscular dystrophy, Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, Emery -Dreifuss muscular dystrophy, Distal muscular dystrophy, Oculopharyngeal muscular dystrophy, dermatomyositis, polymyositis, necrotizing myopathy, inclusion body myositis, rhabdomyolysis, and muscle atrophy.

34. The method of claim 33, wherein the muscle atrophy is primary muscle atrophy or secondary muscle atrophy.

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