Cardiomyocyte-specific molecular guidance system peptides and uses thereof

Abstract

Embodiments are directed to Molecular Guidance System (MGS) peptides, MGS compounds comprising an MGS peptide, MGS-cargo conjugates comprising an MGS peptide conjugated to a cargo, and methods associated therewith. An example MGS peptide is selected from SEQ ID NOs: 2-6 and 8-26. In some embodiments, the MGS peptide may selectively bind to cardiomyocytes.

Description

CARDIOMYOCYTE-SPECIFIC MOLECULAR GUIDANCE SYSTEMPEPTIDES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This PCT Application claims benefit to U.S. Provisional Application No.63 / 647,525, filed May 14, 2024, and U.S. Provisional Application No. 63 / 691,084, filed September 5, 2024, both entitled “Cardiomyocyte- Specific Molecular Guidance System Peptides and Uses Thereof’, the entireties of which are each incorporated herein by reference.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (S1647192111_SequenceListing.xml; Size: 51,500 bytes; and Date of Creation: May 7, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Cardiomyopathy refers to any of several diseases of the heart muscle, e.g., the myocardium. Primary cardiomyopathies are characterized by morphological and functional abnormalities of the myocardium in the absence of any other disease that is sufficient, by itself, to cause such abnormalities. Types of cardiomyopathy include hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, left ventricular noncompaction, and Takotsubo cardiomyopathy. Secondary cardiomyopathies also occur as a result of another disease, such as thyroid disease, or treatment with a drug such as doxorubicin. The myocardium comprises cardiomyocytes which are muscle cells of the heart that are responsible for contraction of the heart. Cardiomyopathy may cause the heart to have difficulty pumping blood to the body, which can lead to various symptoms including heart failure and other heart conditions. In some instances, the heart muscles may become thicker, stiffer, or larger than normal, which can weaken the heart, cause irregular heartbeats, heart failure, or even cardiac arrest. Many patients first experience little to no symptoms, but may experience shortness of breath, tiredness, dizziness, orchest pain as the disease progresses. Cardiomyopathy can affect people at any age, race, or sex, and is estimated to affect about 1 in 500 people.

[0004] Molecular Guidance System (MGS) peptides are a class of intracellular targeting peptides which may be used to intracellularly deliver cargo. MGS combination therapeutics can create an entirely new market impacting virtually every disease state and creating a major breakthrough in modern medicine. Delivering cargo to cardiomyocytes may be useful for treatment of cardiomyopathy.SUMMARY

[0005] The present invention relates to Molecular Guidance System (MGS) peptides, MGS compounds, and MGS-cargo conjugates for the selective delivery of cargo to cardiomyocytes.

[0006] In some aspects, MGS peptides are a class of safe and efficacious peptides which may be used to intracellularly deliver cargo, such as therapeutics to cells. In some aspects, the present disclosure is directed to MGS compounds comprising an MGS peptide for the selective targeting of cardiomyocytes and the intracellular delivery of a cargo conjugated thereto, directly or indirectly. In certain aspects, MGS compounds are formed of at least one or at least two MGS peptides and a protecting group conjugated to the N-terminus of each of the at least one or at least two MGS peptides, a linker conjugated to the C-terminus of each of the at least one or at least two MGS peptides, and a cargo is conjugated, directly or indirectly through an additional linker, to at least one of the linkers. In some such aspects, each of the at least one or at least two MGS peptides comprise an N-terminus protecting group. In certain aspects of an MGS compound, such additional linkers may include reactive groups which may chemically react with and conjugate to the cargo and may also include branch linkers which are conjugated to at least two of the linkers.

[0007] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) peptide selected from SEQ ID NOs: 2-6 and 8-26. In some aspects, the MGS peptide selectively binds to cardiomyocytes.

[0008] In some aspects, the MGS peptide is selected from SEQ ID NOs: 2-6 and 13-19, and the MGS peptide further comprise a protecting group on the N-terminus.

[0009] In some aspects, the protecting group is selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0010] In some aspects, the protecting group is selected from -COCH3, -COH, -CO- (CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, -CO- (CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-polyethylene glycol (PEG)X-(CH)2- NH2, - CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

[0011] Some aspects of the present disclosure are directed to an MGS compound comprising at least two (e.g., two, three, four, or more) copies of an MGS peptide each independently selected from SEQ ID NOs: 1-38. Some aspects of the present disclosure are directed to an MGS compound comprising at least two copies of an MGS peptide selected from SEQ ID NOs: 1-38. In some aspects, each MGS peptide is conjugated to a protecting group at the N-terminus, each MGS peptide is conjugated to a linker at the C- terminus, and a cargo is conjugated to at least one of the linkers. In some such aspects, the linker may comprise multiple linkers, such as reactive groups, branch linkers, polyethylene glycol (PEG) linkers, and / or amino acid-maleimide linkers.

[0012] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide of the invention, wherein the MGS peptide is conjugated to a cargo, directly or indirectly through a linker.

[0013] In some aspects, the MGS peptide further comprises a linker.

[0014] In some aspects, the linker is selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof. In some aspects, the PEG linker comprises PEGn or PEG12.

[0015] In some aspects, the linker is conjugated to the C-terminus of the MGS peptide, directly or indirectly.

[0016] Various aspects of the present disclosure are directed to Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide conjugated to a cargo, directly or indirectly through a linker, wherein the MGS peptide is selected from SEQ ID NOs: 2-26.

[0017] In some aspects, the MGS peptide is selected from SEQ ID NOs: 2-6 and 13-19, and the MGS peptide further comprises a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0018] In some aspects, the MGS peptide comprises a first MGS peptide and a second MGS peptide. In some aspects, the first and second MGS peptides are the same MGS peptide. In some aspects, the first and second MGS peptides are different from one another.

[0019] In some aspects, the MGS-cargo conjugate comprises a dimer including the first MGS peptide and the second MGS peptide which are each independently selected from SEQ ID NOs: 2-26.

[0020] In some aspects, wherein the dimer comprises a linker selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0021] In some aspects, the dimer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly or indirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

[0022] In some aspects, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

[0023] In some aspects, the branch linker comprises a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0024] In some aspects, the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and in some such aspects, the reactive group is conjugated to the cargo.

[0025] In some aspects, the reactive group is selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with NoAcetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0026] In some aspects, the first and second PEG linkers comprise PEGn or PEG12.

[0027] In some aspects, the linker structure comprises:wherein Z and Z’ comprise reactive groups.

[0028] In some aspects, the dimer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide and the second MGS peptide; and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

[0029] In some aspects, the MGS peptide comprises a first MGS peptide, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide. In some aspects, each of the first, second, third, and fourth MGS peptides are the same MGS peptide. In some aspects, at least two of the first, second, third, and fourth MGS peptides are different from one another.

[0030] In some aspects, the MGS-cargo conjugate comprises a tetramer comprising the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide, wherein the first, second, third, and fourth MGS peptides are each independently selected from SEQ ID NOs: 2-26.

[0031] In some aspects, the tetramer comprises a linker selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0032] In some aspects, the tetramer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly orindirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; a third PEG linker conjugated to the third MGS peptide, directly or directly; a fourth PEG linker conjugated to the fourth MGS peptide; and branch linkers. The branch linkers being respectively conjugated, directly or indirectly: to the first PEG linker and to the second PEG linker; to the second PEG linker and to the third PEG linker; and to the third PEG linker and to the fourth PEG linker.

[0033] In some aspects, the linker structure further comprises additional linkers comprising reactive groups. And, the first PEG linker, the second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups.

[0034] In some aspects, the linker structure comprises additional PEG linkers and reactive groups, and wherein the branch linkers are indirectly conjugated to the first PEG linker and to the second PEG linker, to the second PEG linker and to the third PEG linker, and to the third PEG linker and to the fourth PEG linker through the additional PEG linkers and the reactive groups.

[0035] In some aspects, the branch linkers are each a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0036] In some aspects, the linker structure further comprises a reactive group conjugated to at least one of the branch linkers and conjugated to the cargo, directly or indirectly.

[0037] In some aspects, the reactive group is selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, am anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0038] In some aspects, each of the first, second, third, and fourth PEG linkers comprisePEGn or PEGn.

[0039] In some aspects, the linker structure comprises:wherein Z and Z’ comprise reactive groups.

[0040] In some aspects, the tetramer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptides; and lysine branch linkers conjugated to the respective PEG linkers, directly or indirectly.

[0041] In some aspects, the cargo is selected from a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, a nanoparticle, and a combination thereof.

[0042] In some aspects, the cargo comprises the nucleic acid selected from ribonucleic acid, deoxyribonucleic acid, and a combination thereof. In some aspects, the cargo comprises a small interfering ribonucleic acid (siRNA).

[0043] In some aspects, the MGS peptide specifically binds to cardiomyocytes and internalizes the cargo.

[0044] In some aspects, the MGS-cargo conjugate comprise the structure of:wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups.

[0045] In some aspects, the MGS-cargo conjugate comprise the structure of:wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups.

[0046] In some aspects, Z and Z’ are each independently selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0047] In some aspects, at least one of Z and Z’ are configured to react with and are conjugated to the cargo.

[0048] In some aspects, one of Z and Z’ react with and is conjugated to the cargo, and the other of Z and Z’ is H.

[0049] In some aspects, the cargo includes first cargo and second cargo, and Z is conjugated to the first cargo and Z’ is conjugated to the second cargo, wherein the first cargo and second cargo are the same or different.

[0050] Various aspects of the present disclosure are directed to Molecular Guidance System (MGS) compound comprising at least two MGS peptides, wherein the at least two MGS peptides are each independently selected from SEQ ID NOs: 2-26.

[0051] In some aspects, the at least two MGS peptides are each independently selected from SEQ ID NOs: 2-6 and 13-19, and each of the MGS peptides comprise a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0052] In some aspects, the at least two MGS peptides comprise two MGS peptides.

[0053] In some aspects, the at least two MGS peptides comprise four MGS peptides.

[0054] In some aspects, the at least two MGS peptides are each the same MGS peptide.

[0055] In some aspects, at least two of the at least two MGS peptides are different from one another.

[0056] In some aspects, the MGS compound further comprises a linker selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0057] In some aspects, the MGS compound further comprises a reactive group conjugated to the linker, directly or indirectly, and to allow for conjugation to a cargo.

[0058] In some aspects, the reactive group is selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0059] In some aspects, the at least two MGS peptides comprise two MGS peptides and the MGS compound comprises a dimer comprising the two MGS peptides.

[0060] In some aspects, the dimer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to a first MGS peptide of the two MGS peptides, directly or indirectly; a second PEG linker conjugated to a second MGS peptide of the two MGS peptides, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

[0061] In some aspects, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

[0062] In some aspects, the branch linker comprises a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0063] In some aspects, the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and to allow for conjugation to a cargo.

[0064] In some aspects, the first and second PEG linkers each comprise PEGn or PEG12.

[0065] In some aspects, the linker structure comprises:wherein Z and Z’ comprise reactive groups.

[0066] In some aspects, the dimer further comprises a polyethylene glycol (PEG) linker on the C-terminus of each of the two MGS peptides and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

[0067] In some aspects, the at least two MGS peptides comprise four MGS peptides and the MGS compound comprises a tetramer comprising the four MGS peptides.

[0068] In some aspects, the tetramer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to a first MGS peptide of the four MGS peptides, directly or indirectly; a second PEG linker conjugated to a second MGSpeptide of the four MGS peptides, directly or indirectly; a third PEG linker conjugated to a third MGS peptide of the four MGS peptides, directly or indirectly; a fourth PEG linker conjugated to a fourth MGS peptide of the four MGS peptides, directly or indirectly; and branch linkers. The branch linkers being respectively conjugated, directly or indirectly: to the first PEG linker and to the second PEG linker; to the second PEG linker and to the third PEG linker; and to the third PEG linker and to the fourth PEG linker.

[0069] In some aspects, the linker structure further comprises additional linkers comprising reactive groups and the first PEG linker, the second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups.

[0070] In some aspects, the linker structure comprises additional PEG linkers and reactive groups, and wherein the branch linkers are indirectly conjugated to the first PEG linker and to the second PEG linker, to the second PEG linker and to the third PEG linker, and to the third PEG linker and to the fourth PEG linker through the additional PEG linkers and the reactive groups.

[0071] In some aspects, the branch linkers are each a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0072] In some aspects, the linker structure further comprises a reactive group conjugated to at least one of the branch linkers, directly or indirectly, and to allow for conjugation to a cargo.

[0073] In some aspects, the first, second, third, and fourth PEG linkers each comprise PEGn or PEG12.

[0074] In some aspects, the linker structure comprises:wherein Z and Z’ comprise reactive groups.

[0075] In some aspects, the tetramer further comprises a polyethylene glycol (PEG) linker on the C-terminus of each of the four MGS peptides, and lysine branch linkers conjugated to, directly or indirectly, the respective PEG linkers.

[0076] In some aspects, the at least two MGS peptides selectively bind to cardiomyocytes.

[0077] In some aspects, the MGS compound comprises the structure of:wherein each X comprises one of the at least two MGS peptides, and Z and Z’ comprise reactive groups.

[0078] In some aspects, the MGS compound comprises the structure of:wherein each X comprises one of the at least two MGS peptide, and Z and Z’ comprise reactive groups.

[0079] In some aspects, the Z and Z’ are each independently selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0080] In some aspects, at least one of Z and Z’ are configured to react with a cargo.

[0081] In some aspects, one of Z and Z’ is configured to react with a cargo, and the other of Z and Z’ is H.

[0082] In some aspects, Z is configured to react with a first cargo and Z’ is configured to react with a second cargo, wherein the first cargo and second cargo are the same or different.

[0083] Various aspects of the present disclosure are directed to a method of treating a subject having a cardiomyopathy or suspected of having a cardiomyopathy comprising administering an MGS-cargo conjugate of the invention to a subject diagnosed with or having the cardiomyopathy, wherein the cargo of the MGS-cargo conjugate is selected to have a prophylactic, therapeutic, ameliorative effect for the cardiomyopathy, suchwhen internalized in cardiomyocytes. In certain such aspects, the MGS-cargo conjugate is an MGS-cargo conjugate of any one of claims 13-41 to a subject diagnosed with or having cardiomyopathy. In some aspects, the MGS peptide of the MGS-cargo conjugate preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes. In some aspects, the cargo retains functional activity inside the cardiomyocytes sufficient to have a therapeutic effect. In some aspects, administering the MGS-cargo conjugate comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS- cargo conjugate.

[0084] Various aspects of the present disclosure are directed to a method of targeting cardiomyocytes in a subject comprising administering the MGS-cargo conjugate of any one of the claims to a subject. In some aspects, the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes. In some aspects, the cargo retains functional activity inside the cardiomyocytes, such as being sufficient to have a therapeutic effect. In some aspects, administering comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

[0085] Various aspects of the present disclosure are directed to a method of decreasing gene expression of a gene of interest comprising administering the MGS- cargo conjugate of any one of the claims to a subject. In some aspects, wherein the cargo comprises a nucleic acid configured to bind to ribonucleic acid (RNA) transcribed from the gene of interest. In some aspects, the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes. In some aspects, the cargo retains functional activity inside the cardiomyocytes, such as being sufficient to have a therapeutic effect. In some aspects, administering comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

[0086] Various aspects of the present disclosure are directed to a method of expressing a gene of interest comprising administering the MGS-cargo conjugate of any one of the claims to a subject. In some aspects, wherein the cargo comprises a nucleicacid encoding the gene of interest, the gene being associated with a protein and the protein being expressed in response to internalization of the MGS-cargo conjugate.

[0087] In some aspects, the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes. In some aspects, the cargo retains functional activity inside the cardiomyocytes, such as being sufficient to have a therapeutic effect. In some aspects, administering comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

[0088] Various aspects of the present disclosure are directed to an MGS peptide that comprises W1B1SEAGPVVTVAB2B3RGTGSW (SEQ ID NO: 27), wherein: Wi is selected from W and a modified W; Bi is selected from L, I, V, Nle, and a-t- butylglycine; B2 is selected from A and 2-Aminoisobutyric acid (Aib); and B3 is selected from A, L, I, and Nle.

[0089] In some aspects, the MGS peptide is selected from: WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 2), WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 3), WISEAGPVVTVAALRGTGSW (SEQ ID NO: 4), WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 5), WNleSEAGPVVTVAALRGTGSW (SEQ ID NO: 6), WVSEAGPVVTVAAIRGTGSW (SEQ ID NO: 13), WISEAGPVVTVAAIRGTGSW (SEQ ID NO: 14), WLSEAGPVVTVAANleRGTGSW (SEQ ID NO: 15), WVSEAGPVVTVAAibLRGTGSW (SEQ ID NO: 16), WNleSEAGPVVTVAANleRGTGSW (SEQ ID NO: 17), W(a-t-butylglycine)SEAGPVVTVAALRGTGSW (SEQ ID NO: 18), and W*VSEAGPVVTVAAIRGTGSW (SEQ ID NO: 19), wherein W* is the modified W selected from a -CN, a -OH, a -Cl, a -F, a -CH3, and a -OCH3 modification in position 5, 6, or 7 of the indole ring.

[0090] In some aspects, the MGS peptide further comprises a protecting group on the N- terminus.

[0091] In some aspects, the protecting group is selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0092] In some aspects, the protecting group is selected from -COCH3, -COH, -CO- (CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, -CO- (CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-polyethylene glycol (PEG)X-(CH)2- NH2, - CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

[0093] Various aspects of the present disclosure are directed to Molecular Guidance System (MGS) compound comprising an MGS peptide, wherein the MGS peptide comprises SEQ ID NO: 1, wherein the MGS peptide further comprises a protecting group on the N-terminus.

[0094] In some aspects, the protecting group is selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0095] In some aspects, the protecting group is selected from -COCH3, -COH, -CO- (CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, -CO- (CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-polyethylene glycol (PEG)X-(CH)2- NH2, - CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

[0096] In some aspects, the MGS peptide comprises a first MGS peptide and a second MGS peptide and MGS-cargo conjugate comprises a dimer of the first MGS peptide and the second MGS peptide and both the first MGS peptide and the second MGS peptide are SEQ ID NO: 1.

[0097] In some aspects, the dimer comprises a linker selected from a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0098] In some aspects, the dimer comprises a linker structure comprising a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly or indirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

[0099] In some aspects, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

[0100] In some aspects, the branch linker comprises a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0101] In some aspects, the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and wherein the reactive group is conjugated to a cargo.

[0102] In some aspects, the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with no acetylenic hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0103] In some aspects, the first and second PEG linkers comprise PEGn or PEG12.

[0104] In some aspects, the dimer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide and the second MGS peptide; and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

[0105] Various aspects of the present disclosure are directed to a Molecular Guidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 7).

[0106] Various aspects of the present disclosure are directed to a MolecularGuidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 8).

[0107] Various aspects of the present disclosure are directed to a MolecularGuidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 9).

[0108] Various aspects of the present disclosure are directed to a MolecularGuidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 10).

[0109] Various aspects of the present disclosure are directed to a MolecularGuidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 11).

[0110] Various aspects of the present disclosure are directed to a MolecularGuidance System (MGS) compound comprising:wherein: Z comprises H, Z’ comprises CH2SH, and each X comprises:(SEQ ID NO: 12).BRIEF DESCRIPTION OF THE DRAWINGSVarious example embodiments may be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, in which:

[0111] FIGs. 1 A-1E illustrate example MGS-cargo conjugates and MGS compounds comprising an MGS peptide, in accordance with the present disclosure.

[0112] FIGs. 2A-2B show analytical RP-HPLC and MALDI-TOF Mass Spectrometry results of the MGS_PCM_V2-2_B structure.

[0113] FIGs. 3A-3B show analytical RP-HPLC and MALDI-TOF Mass Spectrometry results of the MGS_PCM_V2-2_AF647 structure.

[0114] FIGs. 4A-4B show analytical RP-HPLC and electrospray iononization Mass Spectrometry results of the MGS_PCM_V2-4_AF647 structure.

[0115] FIGs. 5A-5B are brightfield and confocal fluorescence images of isolated primary mouse cardiomyocytes.

[0116] FIGs. 6A-6C are confocal images of samples of isolated primary mouse cardiomyocytes after exposure to scPCM_V2-2_B, MGS_ PCM_V2-1_B, and MGS PCM V2-2 B.

[0117] FIGs. 7A-7D are fluorescent images of samples of primary mouse cardiomyocytes after exposure to AF647, scPCM_V2-2_AF647, MGS PCM V2- 2_AF647, and MGS_PCM_V2-4CON_AF647.

[0118] FIGs. 8A-8B show the results of a semi-quantitative microscopy assay for assessing binding and internalization of MGS_PCM_V2-1_B-SA-AF647 and MGS_PCM_V2-2_B-SA-AF647 to primary cardiomyocytes.

[0119] FIGs. 9A-9E show the results of a semi-quantitative microscopy assay for assessing binding and internalization of MGS dimers and MGS tetramers to primary cardiomyocytes.

[0120] FIG. 10 is a graph showing the results of a semi-quantitative microscope assay for specificity of MGS_PCM_V2-2_AF647 and scPCM_V2-2_AF647 to cardiomyocytes.

[0121] FIGs. 11A-11C show results of assessment for MGS_PCM_V2-2 binding to MOVAS cells.

[0122] FIGs. 12A-12C show results of assessment for MGS_PCM_V2-2 binding to AML12 cells.

[0123] FIGs. 13A-13B show results of assessment for MGS_PCM_V2-2 binding to NIH 3T3 cells.

[0124] FIGs. 14A-14E are graphs showing the results of a semi-quantitative microscopy assay for assessing specificity of MGS_PCM_V2-2 to MOVUS cells, AML12 cells, and cardiomyocytes.

[0125] FIGs. 15A-15F are confocal images of samples of isolated untreated primary human cardiomyocytes and samples of isolated primary human cardiomyocytes after exposure to AF647, MGS_PCM_V2-2_AF647, scPCM_V2-2_AF647, SA_AF647, and MGS_PCM_V2-1_B-SA_AF647 at a concentration of 200 nM for 2 hours.

[0126] FIGs. 16A-16F are further confocal images of samples of isolated human mouse cardiomyocytes after exposure to MGS_PCM_V2-2_AF647 and scPCM_V2- 2_AF647.

[0127] FIG. 17 is a graph showing the results of a semi-quantitative microscopy assay for assessing specificity of MGS_PCM_V2-2_AF647 and scPCM_V2-2_AF647 to human cardiomyocytes.

[0128] FIGs. 18A-18B show an example conjugation scheme for PCM_V2-2_LDHA siRNA_AF647 and resulting analytical RP-HPLC.

[0129] FIGs. 19A-19E show the results from an untreated (control) mouse, and mice treated with siRNA- AF647, MGS_PCM_V2-2-AF647, and MGS_PCM_V2-2-AF647- LDHA siRNA conjugate.

[0130] FIG. 20 is an image taken 48 hours after injecting a mouse with MGS_PCM_V2-2_LEE.

[0131] FIGs. 21A-21B are images taken at 120 hour timepoints including an untreated mouse (control) and a mouse injected with MGS_PCM_V2-2_LEE.

[0132] FIG. 22 includes brightfield, mCardinal, and merged brightfield and mCardinal images taken 120 hours after injecting mice with MGS_PCM_V2-2_LEE and untreated control.

[0133] FIG. 23 shows a rapid assay developed for assessing peptide interaction with different versions of the MGS_PCM peptide.

[0134] FIG. 24 is a microscopy image after performing the rapid assay of FIG. 23 on freshly isolated primary cardiomyocytes.

[0135] FIG. 25 includes further microscopy images after performing the rapid assay of FIG. 23.

[0136] FIG. 26 is a graph showing the resulting RP-HPLC of MGS_PCM_V2- 2_AF647 incubated in human serum over a time period of 6 hours.

[0137] FIGs. 27A-27E show the results from an untreated mouse (control) and mice treated with MGS_PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10- 2_AF647, and MGS_PCM_V11-2_AF647.

[0138] FIGs. 28A-28B are graphs showing the radiant intensity (ROI) of MGS_PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V1 1-2_AF647 and the fold improvement of MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 compared to MGS_PCM_V2-2_AF647.

[0139] FIGs. 29A-29F illustrate example MGS peptide structures.

[0140] FIG. 30 is a graph showing MGS_PCM_V2-2_AF647, MGS_PCM_V9- 2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 are internalized by rat and mice cardiomyocytes and not internalized by HEK293 cells.

[0141] FIGs. 31A-31B are graphs showing internalization of PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11- 2_AF647 by mouse cardiomyocytes and by rat cardiomyocytes over time.DETAILED DESCRIPTION

[0142] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure may be practiced. It is to be understood that other examples may be utilized, and various changes may be made without departing from the scope of the disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0143] As described above, cardiomyopathy is a disease of the heart muscle that is estimated to impact a large number of humans and other subjects. Typical treatment for cardiomyopathy involves lifestyle changes (e.g., reducing salt and alcohol intake, reducing stress, changes to physical activity), medications, as well as surgical procedures including an implanted medical device to maintain heart rhythm, ablation procedures, and heart transplants. For many of these treatments, the goal of the treatment is to slow down the disease, control symptoms, and prevent sudden death. Surgical treatments, including implanted medical devices, ablation procedures, and heart transplants can result in complications and even death. Some recently developed treatments involve peptides and polymers which are targeted to primary cardiomyocytes for treatment of cardiomyopathy by delivering cargo to the primary cardiomyocytes. However, prior peptides and polymers used to deliver cargo to primary cardiomyocytes resulted in loss of specificity to the primary cardiomyocytes, lacked improvement of delivery of the cargo to the primary cardiomyocytes, and / or lost peptide functionality once the conjugates were formed. In some instances, the cargos that could be delivered were limited in size and other versatility.

[0144] Embodiments in accordance with the present disclosure are directed to MGS peptides which specifically bind to cardiomyocytes. The MGS peptides are capable of carrying cargo to and mediating internalization of the cargo to the cardiomyocytes. In some embodiments, multimer forms of the MGS peptides (e.g., dimers, trimers, or tetramers) showed greater uptake in cardiomyocytes than monomeric forms.Additionally, the MGS peptides are capable of carrying and mediating internalization of a variety of different cargos.

[0145] Disclosed are materials, compositions, and components that may be used for, may be used in conjunction with, may be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a composition is disclosed and discussed and a number of modifications that may be made to a number of molecules including the MGS peptide are discussed, each and every combination and permutation of the compositions and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that may be performed it is understood that each of these additional steps may be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0146] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0147] It must be noted that as used herein and in the claims, the singular forms "a", "an", and "the", as well as “one or more” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "an MGS peptide" includes a plurality of such MGS peptides, such as two, three, four, or more, reference to "the nucleic acid" is a reference to one or more nucleic acid sequences and equivalents thereof known to those skilled in the art, and so forth.

[0148] As used herein, “treat” is meant to mean administer a composition of the invention to a subject, such as a human or other mammal (for example, an animal model), that has a disease or condition, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease or condition. In some embodiments, the disease or condition is cardiomyopathy. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment comprises delivery of a MGS peptide, conjugate, and / or compound to a subject.

[0149] As used herein, “prevent” is meant to mean minimize the chance that a subject who has an increased susceptibility for developing disease, disorder or condition will develop the disease, disorder, or condition.

[0150] As used herein, the term "subject" refers to or includes the target of administration, e.g., a human. Thus, the subject of the disclosed methods may be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In some embodiments, a subject is a mammal. In some embodiments, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent, and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0151] As used herein, the term "patient" refers to or includes a subject afflicted with a disease or disorder. For example, the term "patient" includes human and veterinary subjects. In some embodiments, the “patient” has been diagnosed with a need for treatment prior to the administering step.

[0152] As used herein, the term "amino acid sequence" refers to or includes a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; and Nle, norleucine. Amino acids may include modified forms of W, G, and A. The modified forms of W, G, and A may include, but are not limited to, a-t-butylglycine, 2- Aminoisobutyric acid (Aib), and modified tryptophan, including -CN, -OH, -Cl, -F, -CH3, or -OCH3 modifications in position 5, 6, or 7 of the indole ring, among other modifications on the indole ring.

[0153] “Polypeptide", as used herein, refers to or includes any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0154] In addition, as used herein, the term "peptide" refers to or includes amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 standard protein building block amino acids. The peptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications may occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given peptide. Also, a given polypeptide may have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotidederivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins — Structure and Molecular Properties 2nd Ed., T.E.Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)). N-terminus of a peptide (or amino acid or other molecule) refers to or includes an end with a free amino group. The C-terminus of a peptide (or amino acid or other molecule) refers to or includes an end with a free carboxyl group.

[0155] As used herein, “MGS peptide” refers to or includes a peptide which specifically binds to target cells and is capable of being conjugated to cargo and mediating internalization of the cargo to the target cell. “Cargo” refers to or includes molecules, compounds, or other structures which are capable of being conjugated to the MGS peptide and being delivered to the target cells. Cargo may be interchangeably referred to as “a cargo moiety”, “a cargo molecule”, or “a cargo compound”. Example cargo include, without limitation, a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, a nanoparticle, or any combination thereof.

[0156] The phrase “nucleic acid” or “nucleic acid sequence”, as used herein, refers to or includes a naturally occurring or synthetic oligonucleotide or polynucleotide, whether deoxyribonucleic acid (DNA) or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention may also include nucleotide analogs (e.g., BrdU), and non-phosphodiester intemucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids may include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, siRNA, mRNA, miRNA, or any combination thereof. 1

[0157] As used herein, a “reactive group” refers to or includes an atom, group of atoms, molecule, compound, or other structure that is capable of undergoing a chemical reaction with another group (e.g., moiety). In some embodiments, a reactive group may be or form part of a linker. In some embodiments, a reactive group may facilitate conjugation of a particular cargo and may be chosen for such facilitation. The reactive group(s) may be used to link MGS peptides to other linkers, linkers to linkers, and / or linkers to cargo. A “linker” refers to or includes a molecule, compound, or other structure which may be used to link (e.g., by covalent bond) at least two components of a composition. As used herein, the reactive group may be interchangeably referred to as “a reactive moiety”, “a reactive molecule”, or “a reactive compound”. The linker may be interchangeably referred to as “a linker moiety” or “a linker molecule”.

[0158] As used herein, “MGS compound” refers to or includes a compound comprising an MGS peptide and at least one additional molecule. Example MGS compounds include an MGS peptide and a linker or linker structure, at least two MGS peptides, and at least two MGS peptides and a linker or linker structure, among other examples. “MGS-cargo conjugate” refers to or includes a compound comprising least one MGS peptide and cargo which are joined together (e.g., conjugated) either directly or indirectly through a linker. Unless expressly stated otherwise, when used herein, “conjugated to” refers to both direct and indirect conjugations, whether or not “directly or indirectly” is expressly recited.

[0159] As used herein, “effective amount” of a compound or conjugate refers to or includes a sufficient amount of the compound or conjugate to provide the desired effect. The exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound or conjugate used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0160] As used herein, “selectively binds”, “specifically binds”, and “preferentially binds” refers to or includes a nucleic acid (e.g., cargo) or MGS peptide recognizes andphysically interacts with its target (for example, a specific cell type) and does not significantly recognize and interact with other targets.

[0161] The term "percent (%) homology" is used interchangeably herein with the term "percent (%) identity" and refers to or includes the level of nucleic acid or amino acid sequence identity when aligned with a wild type sequence or sequence of interest using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence. Example levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., any of the MGS peptide sequences, as described herein. Example computer programs which may be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul, et al., 1990 and Altschul, et al., 1997. Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program may be used for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, S. E., et al., Nucleic Acids Res.25:3389-3402, 1997). A preferred alignment of selected sequences in order to determine "% identity" between two or more sequences, is performed using for example, the CLUSTAL-W program in Mac Vector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0162] Substitutions, deletions, insertions, or any combination thereof may be used to arrive at a final derivative, variant, or analog. Generally, these changes are done on a few nucleotides to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.

[0163] Generally, the nucleotide identity between individual variant sequences may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant sequence” may be one with the specified identity to the parent or reference sequence (e.g., wild-type sequence) of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence. For example, a “variant sequence” may be a sequence that contains 1, 2, or 3, 4 nucleotide base changes as compared to the parent or reference sequence of the invention, and shares or improves biological function, specificity and / or activity of the parent sequence. Thus, a “variant sequence” may be one with the specified identity to the parent sequence of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent sequence. The variant sequence may also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of a reference sequence (e.g., an MGS peptide sequence).

[0164] As used herein, "modulate" is meant to mean to alter, by increasing or decreasing.

[0165] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0166] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of eachof the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0167] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0168] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising at one step or operation it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0169] Disclosed herein are MGS peptides, MGS compounds, and MGS-cargo conjugates comprising MGS peptide(s) that specifically bind to cardiomyocytes. Forexample, the MGS peptides of the present disclosure selectively bind to cardiomyocytes. Example MGS peptides include, but are not limited to, the MGS sequences in Table 1. Other examples are directed to methods comprising at least one MGS peptide and methods of using example MGS peptides, MGS compounds, and / or MGS -cargo conjugates.

[0170] In some embodiments, an MGS peptide comprises W1B1SEAGPVVTVAB2B3RGTGSW (SEQ ID NO: 27). In some embodiments, Wi is selected from W and a modified W. In some embodiments, Bi is selected from L, I, V, Nle, and a-t-butylglycine. In some embodiments, B2 is selected from A and Aib. And, in some embodiments, B3 is selected from A, L, I, and Nle. In some embodiments, the modified W (sometime herein referred to as “W*”) is selected from a -CN, a -OH, a -Cl, a -F, a -CH3, and a -OCH3 modification in position 5, 6, or 7 of the indole ring of W, among other modifications on the indole ring. As may be appreciated, 7-CN, 7-OH, 7- Cl, 7-F, 7-CH3, and 7-OCH3 respectively refer to or include the addition of CN, OH, Cl, F, CH3, or OCH3 at the 7thposition of the indole ring of W, such that CN, OH, Cl, F, CH3, or a OCH3 are substituted for H at position 7. Similar substitutions may be made at the 5thor 6thposition of the indole ring. As may be further appreciated, below is an illustration of the indole ring of W and the positions of the indole ring:

[0171] In some embodiments, the MGS peptide is selected from WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 2), WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 3), WISEAGPVVTVAALRGTGSW (SEQ ID NO: 4), WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 5), WNleSEAGPVVTVAALRGTGSW (SEQ ID NO: 6), WVSEAGPVVTVAAIRGTGSW (SEQ ID NO: 13), WISEAGPVVTVAAIRGTGSW (SEQ ID NO: 14), WLSEAGPVVTVAANleRGTGSW (SEQ ID NO: 15), WVSEAGPVVTVAAibLRGTGSW (SEQ ID NO: 16), WNleSEAGPVVTVAANleRGTGSW (SEQ ID NO: 17), W(a-t- butylglycine)SEAGPVVTVAALRGTGSW (SEQ ID NO: 18), andW*VSEAGPVVTVAAIRGTGSW (SEQ ID NO: 19), wherein W* is a modified W comprising a -CN, a -OH, a -Cl, a -F, a -CH3, or a -OCH3 modification in position 5, 6, or 7 of the indole ring of W, among other modifications on the indole ring of W.

[0172] As further described below, any of the described MGS peptides may be modified. For example, the MGS peptide of W1B1SEAGPVVTVA B2B3RGTGSW (SEQ ID NO: 27) may be modified at the N-terminus by acetylation or other alkylation, cyclization, amino-alkylation, methylation, succinylation, pegylation, and combinations thereof. In any such embodiment, the MGS peptide may be referred to as being acetylated, alkylated, cyclated, amino-alkylated, methylated, succinylated, and / or pegylated. In such embodiments, the MGS peptide comprises a protecting group on the N-terminus. In some embodiments, the N-terminal protecting group may comprise an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

[0173] In some embodiments, the protecting group on the N-terminus of the MGS peptide may comprise -COCH3, -COH, -CO-(CH)n-CH3, -CO-(CH)n-NH2, y- Aminobutyric acid, pyro-glutamic acid, methyl, -CO-(CH)2-CO-OH, -CO-(CH)2-CO- COH3, -CO-PEGX-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, or -CO-PEGx-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36. For example, the protecting group may be selected from:a, , , , , ,1-13, 16, or 24,such as 2 or4. In the above-listed and non-limiting example protecting groups, the -NH group (on right) represents the free amino group of the MGS peptide and the -NH does not form part of the protecting group.

[0174] In some embodiments, the MGS peptide may comprise any of SEQ ID NOs: 1-38 as shown below in Table 1. In some embodiments, the MGS peptide comprises any of SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 2-12, or SEQ ID NOs: 7-12, among other combinations.Table 1: Example MGS peptide sequences

[0175] In some embodiments, the MGS peptide selectively binds to cardiomyocytes. For example, MGS peptides of the present disclosure may have high affinity and specificity for cardiomyocytes to allow for delivery of cargo into the cardiomyocytes. In some embodiments, the MGS peptides comprising any of SEQ ID NO: 1-27 showed high affinity to cardiomyocytes and internalized into the cardiomyocytes.

[0176] Various embodiments are directed to MGS-cargo conjugates and MGS compounds comprising an MGS peptide. In some embodiments, the MGS cargoconjugates and / or MGS compounds comprising the MGS peptide further comprise a linker, such as further illustrated by the conjugates and compounds illustrated by FIGs. 1A-1E. Example linkers include a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, and a reactive group, as well as combinations thereof. In some embodiments, the linker is conjugated to the C-terminus of the MGS peptide. Accordingly, in some embodiments, the MGS-cargo conjugate and / or MGS compound comprising the MGS peptide may comprise any of the sequences set forth in SEQ ID NOs: 1-27, wherein SEQ ID NOs: 1-27 may be conjugated to PEG and / or another linker on the C-terminus of the MGS peptide.

[0177] In some embodiments, the linker may be any of those as further described herein. For example, a linker may be any length that allows conjugation of an MGS peptide with something else and prevents steric hindrance.

[0178] In some embodiments, the linker comprises a PEG linker. A PEG linker, as used herein, refers to or includes a linker containing at least one PEG unit. In some embodiments, the PEG linker may comprise a plurality of PEG units. For example, the number of PEG units in the PEG linker may be between 1-24 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the PEG linker comprises PEGn or PEG12. In any such embodiment, the MGS peptide may be referred to as being pegylated.

[0179] In some embodiments, the MGS peptide may be covalently attached to a cargo, directly or indirectly through a linker. Such cargo may include nucleic acids, small molecules, antibodies, proteins, lipids, imaging agents, dyes, radionuclides, carbohydrates, nanoparticles, and other cargo which may be used for therapeutic purposes. For example, in embodiments including a PEG linker, the number of PEG units may be of sufficient length to separate the MGS peptide from the cargo to prevent any steric interference between the MGS peptide and the cargo.

[0180] In some embodiments, the versatile chemistry of the MGS peptide sequence allows for modification(s) that enhances the cardiomyocyte-targeting sensitivity and specificity, as well as the combination with cargo. In some embodiments, the MGS peptide has the capability to induce high cardiomyocyte targeting sensitivity and specificity. Thus, the MGS peptide may be used as a guiding system for targeting cardiomyocytes.

[0181] Table 1 illustrates monomer MGS peptides. In some embodiments, the MGS peptides shown in Table 1 may be used to form multimers, such as dimers, trimers, and tetramers. As sometimes used herein, adding a “-2” at the end of the MGS peptide name refers to a dimer and adding a “-4” at the end of the MGS peptide name refers to a tetramer. For example, MGS_PCM_V2-2 refers to the dimeric version of the CH3CO-WLSEAGPVVTVRALRGTGSW(SEQ ID NO: 7) sequence. As another example, MGS_PCM_V2-4 refers to the tetrameric version of the CH3CO-WLSEAGPVVTVRALRGTGSW(SEQ ID NO: 7) sequence. As additionalexamples, the MGS_PCM_V2-2-AS3 refers to the dimeric version of the CH3CO-WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 8) sequence and MGS_PCM_V2-4-AS3 refers to the tetrameric version of the CH3CO-WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 8) sequence. The same may be true for CH3CO-WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 9), CH3CO-WISEAGPVVTVAALRGTGSW (SEQ ID NO: 10), CH3CO-WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 11), and CH3CO-WNleSEAGPVVTVAALRGTGSW (SEQ ID NO: 12), where MGS_PCM_V6- 2, MGS_PCM_V9-2, MGS_PCM_V10-2, and MGS_PCM_Vll-2 refer to dimers and MGS_PCM_V6-4, MGS_PCM_V9-4, MGS_PCM_V10-4, and MGS_PCM_Vll-4 refer to tetramers. If MGS_PCM_V2, MGS_PCM_V2 AS3, MGS_PCM_V6, MGS_PCM_V9, MGS_PCM_V10, and MGS_PCM_V11 are the monomeric forms of the peptides shown in Table 1, then MGS_PCM_V2-2, MGS_PCM_V2-AS3-2, MGS_PCM_V6-2, MGS_PCM_V9-2, MGS_PCM_V10-2, and MGS_PCM_Vll-2 are the dimer forms, respectively, and MGS_PCM_V2-4, MGS_PCM_V2-4-AS3, MGS_PCM_V6-4, MGS_PCM_V9-4, MGS_PCM_V10-4, and MGS_PCM_Vll-4 are the tetramer forms. Embodiments are not limited to multimers which comprise copies of the same MGS peptides. In some embodiments, a multimer may include a copy or copies of at least two MGS peptides. For example, a dimer may include a first MGS peptide and a second MGS peptide that is different than the first MGS peptide. As another example, a tetramer may include a first MGS peptide, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide, wherein each of the first, second, third, and fourth MGS peptides are different from one another. As a further example, a tetramer may include two copies of a first MGS peptide and two copies of a second MGS peptide that is different from the first MGS peptide. As a further example, a tetramer may include three copies of a first MGS peptide and one copy of a second MGS peptide that is different from the first MGS peptide. Embodiments include other multimers, such as trimers, pentamers, hexamers, and heptamers, among others.

[0182] As further described herein, in some embodiments, a linker structure comprising multiple linkers may conjugate at least two MGS peptides together to formthe multimer. Example multimers and linker structures are illustrated in connection with at least FIGs. 1B-1E.

[0183] In some embodiments, the MGS peptide has a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences set forth in SEQ ID NOs: 1-38, such as any sequences set forth in sequences 1-27. In some embodiments, the MGS peptide 100 has 100% identity in the active portion of the peptide, wherein the active portion is the portion that retains its ability to target cardiomyocytes. Thus, in some embodiments, the at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the MGS peptides occurs outside of the active portion.

[0184] In some embodiments, the MGS peptide may be or is modified to optimize and / or stabilize the MGS peptide. Optimized MGS peptides may be obtained by applying modifications to the individual parental peptide sequences. These modifications may be used to identify the amino acids within the parental sequence that are required for specific binding and internalization to cardiomyocytes. These modifications may be obtained by a combination of alanine scanning and truncations of the amino-terminal region and C-terminal region of the parental peptide. PEG may provide protection of the C-terminus of the MGS peptide, provide a spacer between the peptide and the cargo attached through the amino acid at the C-terminus, and enhance solubility of the MGS peptide. Modification at the N-terminus by acetylation (CH3CO-) or other acylation, cyclization, amino-alkylation, methylation, succinylation, pegylation, and / or d-amino acids, such as d(L) may protect against degradation. There is not a uniform length of optimized peptide that may be applied to all MGS peptides and all changes may be tested to confirm the effect on peptide uptake and stability. Thus, in some embodiments, the MGS peptide has an N-terminal protecting group, such as those described above. For example, as shown by Table 1, respective MGS peptides disclosed herein are modified by acetylation on the N-terminus. Accordingly, in some embodiments, the N-terminal protecting group comprises an acetyl group (Ac = CH3CO). In some embodiments, the N-terminal protecting group may be, but is not limited to, an acyl group, a cyclic group, an amin- alkyl group, a succinyl group, a PEG group, Formyl, methyl, CH3-(CH)n-CO, Fluorophore, Fatty acid, alkyl amine, arylgroups, carbohydrates, sulfonamide, or carbamate, among other groups such as those illustrated and described above. As may be appreciated, “Ac” is sometimes herein interchangeably used to refer to acetylation or an acetyl group (CH3CO). In some embodiments, the MGS peptide 100 may be or is modified by adding amino acids or replacing an amino acid(s) to the individual parental peptide sequences. For example, as least one amino acid of the parental peptide sequence may be replaced with another (such as another amino acid, a modified amino acid, or an isomer, such as Norleucine (Nle)). In some embodiments or in addition, as least one amino acid may be added to the parental peptide sequence, such as the addition of amino acid(s) within the sequence.

[0185] FIGs. 1A-1E illustrate example MGS-cargo conjugates and MGS compounds comprising an MGS peptide, in accordance with the present disclosure. In some embodiments, the MGS-cargo conjugates and MGS compounds 220, 230, 240, 260 of FIGs. 1A-1E may include at least one MGS peptide as previously described.

[0186] As further described herein, FIG. 1A depicts an MGS-cargo conjugate 220 with a single MGS peptide 200 conjugated to a cargo 222, directly or indirectly through an optional linker 224. FIG. IB depicts an MGS compound 230 with multiple MGS peptides 200A, 200B...200N conjugated to a (common) linker 232 and a reactive group 234 for binding to a cargo. FIG. 1C depicts an example linker structure 241 for an MGS compound 231 with multiple MGS peptides 200A, 200B...200N. FIG. ID depicts an MGS compound 240 comprising two MGS peptides 200A, 200B conjugated to a linker structure 242 where the linker structure 242 comprises multiple linkers 244A, 244B, 246, 248A, 248B including PEG linkers 244A, 244B conjugated to the MGS peptides 200A, 200B and to a branch linker 246 and the branch linker 246 is conjugated to two reactive groups 248A, 248B for attachment of cargo or other moieties. FIG. IE depicts an MGS compound 260 comprising four MGS peptides 200A, 200B, 200C, 200D conjugated to a linker structure 262 where the linker structure 262 comprises multiple linkers 244A, 244B, 244C, 244D, 246A, 246B, 246C, 248 A, 248B (as well as optional linkers 244E, 244F, 244G, 244H, 264A, 264B, 264C, 264D) including PEG linkers 244A, 244B, 244C, 244D conjugated to the MGS peptides 200A, 200B, 200C, 200D and also to branch linkers 246 A, 246B, 246C and the branch linkers 246 A, 246B, 246Cconjugated to each other and to reactive groups 248A, 248B for attachment of cargo or other moieties.

[0187] FIG. 1A illustrates an example MGS-cargo conjugate 220 comprising an MGS peptide 200 conjugated to a cargo 222, directly or indirectly through a linker 224. The MGS peptide 200 may include an implementation of and / or at least some of the same features and attributes as the MGS peptides as previously described. In some embodiments, the MGS peptide 200 may be selected from WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 1), WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 2), WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 3), WISEAGPVVTVAALRGTGSW (SEQ ID NO: 4), WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 5), and WNleSEAGPVVTVAALRGTGSW(SEQ ID NO: 6), CH3CO-WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 7), CH3CO-WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 8), CH3CO-WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 9), CH3CO-WISEAGPVVTVAALRGTGSW (SEQ ID NO: 10), CH3CO-WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 11), CH3CO-WNleSEAGPVVTVAALRGTGSW(SEQ ID NO: 12), WVSEAGPVVTVAAIRGTGSW (SEQ ID NO: 13), WISEAGPVVTVAAIRGTGSW (SEQ ID NO: 14), WLSEAGPVVTVAANleRGTGSW (SEQ ID NO: 15), WVSEAGPVVTVAAibLRGTGSW (SEQ ID NO: 16), WNleSEAGPVVTVAANleRGTGSW (SEQ ID NO: 17), W(a-t-butylglycine)SEAGPVVTVAALRGTGSW (SEQ ID NO: 18), W*VSEAGPVVTVAAIRGTGSW (SEQ ID NO: 19), WVSEAGPVVTVAAIRGTGSW (SEQ ID NO: 20), CH3CO-WISEAGPVVTVAAIRGTGSW (SEQ ID NO: 21), CH3CO-WLSEAGPVVTVAANleRGTGSW (SEQ ID NO: 22), CH3CO-WVSEAGPVVTVAAibLRGTGSW (SEQ ID NO: 23), CH3CO-WNleSEAGPVVTVAANleRGTGSW (SEQ ID NO: 24), CH3CO-W(a-t-butylglycine)SEAGPVVTVAALRGTGSW (SEQ ID NO: 25), and CH3CO-W*VSEAGPVVTVAAIRGTGSW (SEQ ID NO: 26), wherein W* is a modified W selected from a -CN, a -OH, a -Cl, a -F, a -CH3, and a -OCH3 modificationin position 5, 6, or 7 of the indole ring of W, among other modifications on the indole ring.

[0188] In some embodiments, the MGS peptide 200 is selected from SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1-12, or SEQ ID NOs: 2-12, among other combinations. In some embodiments, the MGS peptide 200 comprises SEQ ID NO: 27.

[0189] In some embodiments, as previously described, the MGS peptide 200 may include a protecting group. For example, the MGS peptide 200 may comprise SEQ ID NOs: 2-6 and 13-19 and further comprises a protecting group on the N-terminus selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a PEG group, a methyl group, and a combination thereof.

[0190] In some embodiments, the MGS peptide 200 specifically binds to cardiomyocytes and internalize the cargo 222 into the cardiomyocytes. Example cargo 222 includes a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, and a nanoparticle, among other molecules and compounds and combinations thereof. In some embodiments, the cargo 222 includes a nucleic acid selected from ribonucleic acid, deoxyribonucleic acid, and a combination thereof. In some embodiments, the cargo 222 includes a small interfering ribonucleic acid (siRNA), among other types of RNA and / or DNA sequences.

[0191] In some embodiments, the MGS-cargo conjugate 220 may comprise multimers, such as further illustrated by MGS compounds of FIGs. 1B-1E. For example, the MGS- cargo conjugate 220 may comprise two MGS peptides including a first MGS peptide 200 and a second MGS peptide (not illustrated by FIG. 1 A). In some such embodiments, the MGS-cargo conjugate 220 may be referred to as a dimer. In some embodiments, the two MGS peptides are the same MGS peptide (e.g., two copies of the same MGS peptide). In other embodiments, the two MGS peptides are different from one another. In some embodiments, the two MGS peptides are indirectly conjugated through a linker structure, as further described herein.

[0192] In some embodiments, the MGS-cargo conjugate 220 comprises four MGS peptides including a first MGS peptide 200, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide (not illustrated by FIG. 1 A). In some such embodiments, the MGS-cargo conjugate 220 may be referred to as a tetramer. In some embodiments, the four MGS peptides are the same MGS peptide (e.g., four copies of the same MGS peptide). In other embodiments, at least two of the four MGS peptides are different from one another. For example, two, three, or four of the MGS peptides may be different from one another. In some embodiments, the four MGS peptides are conjugated through a linker structure, as further described herein.

[0193] In various embodiments, linkers may conjugate the at least two MGS peptides together, conjugate respective linkers together, or conjugate a linker or MGS peptide to a cargo. In some embodiments, the linker structure may be used to conjugate the at least two MGS peptides and at least a cargo 222 together. The linker structure may comprise a plurality of linkers, such as but not limited to, PEG linker(s), branch linker(s), and at least one reactive group. As previously described, example linkers include a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amide linker, a thiol linker, an amine linker, an aryl linker, and a reactive group, among others.

[0194] In some embodiments, the MGS-cargo conjugate 220 comprises a dimer comprising the first MGS peptide 200 and the second MGS peptide. The first MGS peptide 200 and the second MGS peptide may be each independently selected from SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, , SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1-12, or SEQ ID NOs: 2-12, among other combinations. In some embodiments, the first and second MGS peptide each comprise a sequence of SEQ ID NO: 27.

[0195] In some embodiments, the dimer further comprises a linker 224. The linker 224 may include any of the above-described linkers and combinations thereof. In some embodiments, the dimer comprises a PEG linker on the C-terminus of each of the first MGS peptide 200 and the second MGS peptide and a lysine branch linker that links the PEG linkers.

[0196] In some embodiments, the dimer comprises a linker structure, which may include the linker 224. The linker structure may comprise a first PEG linker conjugated to the first MGS peptide 200 (directly or indirectly), a second PEG linker attached to the second MGS peptide (directly or indirectly), and a branch linker conjugated to the first PEG linker and to the second PEG linker together, directly or indirectly. In such embodiments, the branch linker may indirectly link the first and second MGS peptides together, and the directly or indirectly link the first PEG linker and the second PEG linker. For example, the first PEG linker may be indirectly conjugated to the second PEG linker through the branch linker. In some embodiments, the first and second PEG linkers comprise PEGn or PEG12, although embodiments may include other numbers of PEG units. As previously described, the PEG linkers may be a sufficient length to separate the first MGS peptide 200 from the cargo 222 to prevent any steric interference between the first MGS peptide 200 (and the second MGS peptide) and the cargo 222. A branch linker, as used herein, refers to or includes a linker that connects MGS chains which comprise at least an MGS peptide and a linker, such as a PEG linker that connects the MGS peptide to the branch linker.

[0197] The branch linker may be conjugated to the first MGS peptide 200 and to the second MGS peptide, directly or indirectly, such that the branch linker links the first and second MGS peptides. In some embodiments, the branch linker may include a modified amino acid. For example, the branch linker may be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid, among other modified amino acids.

[0198] In some embodiments, the linker structure further comprises a reactive group conjugated to the branch linker and conjugated to the cargo 222. Example reactive groups include carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (with No Acetylenic Hydrogen), amides, imides, amines, thiols, phosphines, pyridines, anhydrides, azo compounds, diazo compounds, azido compounds, hydrazine, azide compounds, carbamates, epoxides, esters, sulfate esters, phosphate, thiophosphate esters, borate esters, halogenated organic compounds, isocyanates, isothiocyanates, ketones, oximes, sulfides (Organic), lipids, hydrogen, and combinations thereof, among other reactive moieties. In various embodiments, thereactive group is conjugated to the C-terminal side of the branch linker which comprises an amino acid.

[0199] In some embodiments, the linker structure of the dimer may comprise additional linkers, such as additional reactive groups and as further illustrated in connection with FIG. ID. In some embodiments, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups, such as -NH.

[0200] In some embodiments, the linker structure of a dimer may comprise:wherein Z and Z’ comprise reactive groups. In some embodiments, the Z and / or Z’ reactive groups are configured to react with a cargo, as further described herein.

[0201] In some embodiments, the MGS-cargo conjugate 220 comprises a tetramer comprising a first MGS peptide 200, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide. The first, second, third, and fourth MGS peptides may be each independently selected from SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1-12, or SEQ ID NOs: 2-12, among other combinations. In some embodiments, the four MGS peptides each comprise a sequence of SEQ ID NO: 27. In some embodiments, the tetramer further comprises a linker 224. The linker 224 may include any of the above described linkers and combinations thereof. In some embodiments, the tetramer comprises a PEG linker on the C-terminus of each of the MGS peptide 200, the second MGS peptide, the third MGS peptide, and the fourth MGS peptides, and lysine branch linkers that link the PEG linkers.

[0202] In some embodiments, the tetramer comprises a linker structure, which may include the linker 224. The linker structure may comprise a first PEG linker conjugated to the MGS peptide 200 (directly or indirectly), a second PEG linker conjugated to thesecond MGS peptide (directly or indirectly), a third PEG linker conjugated to the third MGS peptide (directly or indirectly), and a fourth PEG linker conjugated to the fourth MGS peptide (directly or indirectly). The linker structure may further comprise branch linkers that are respectively conjugated, directly or indirectly: (i) to the first PEG linker and to the second PEG linker, (ii) to the second PEG linker and to the third PEG linker, and (iii) to the third PEG linker and to the fourth PEG linker. In such embodiments, the branch linkers may indirectly link the first, second, third, and fourth MGS peptides together, and directly or indirectly link the first PEG linker and the second PEG linker, the second PEG linker and the third PEG linker, and the third PEG linker and the fourth PEG linker. In some embodiments, the first, second, third, and fourth PEG linkers may be indirectly conjugated to each other via the branch linkers and additional linkers, such as additional PEG linkers and amino acid linker and / or other reactive groups, as further described herein. In some embodiments, the first, second, third, and fourth PEG linkers (each) comprise PEGn or PEG12, although embodiments may include other numbers of PEG units.

[0203] In some embodiments, the branch linker may include a modified amino acid. For example, the branch linker may be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid.

[0204] In some embodiments, the linker structure further comprises a reactive group conjugated to at least one of the branch linkers and conjugated to the cargo 222. The reactive group may include any of the previously described reactive groups. In various embodiments, the reactive group is conjugated to the C-terminal side of the branch linker, wherein the branch linkers comprise amino acids.

[0205] In some embodiments, the linker structure of the tetramer may comprise additional linkers, such as additional PEG linkers and reactive groups and as further illustrated in connection with FIG. IE. In some embodiments, the first PEG linker, the second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups, such as -CONH.

[0206] In some embodiments, the linker structure of a tetramer may comprise:wherein Z and Z’ comprise reactive groups. In some embodiments, the Z and / or Z’ reactive groups are configured to react with a cargo.

[0207] In some embodiments, the MGS-cargo conjugate 220 comprises the structure of:wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups. At least one of the reactive groups may be configured to react with a cargo. In some embodiments, the above illustrated structure may be referred to as a “dimer core”.

[0208] In some embodiments, the MGS-cargo conjugate 220 comprises the structure of:wherein each X comprises the MGS peptide, and Z and Z’ comprise reactive groups. In some embodiments, at least one of the reactive groups may be configured to react with a cargo. In some embodiments, the above illustrated structure may be referred to as a “tetramer core”.

[0209] In some embodiments, Z and Z’ are each independently selected from carboxylic acids, acyl halides, sulfonyl halides, chloroformates, aldehydes, alkynes, alkynes (with No Acetylenic Hydrogen), amides, imides, amines, thiols, phosphines, pyridines, anhydrides, azo compounds, diazo compounds, azido compounds, hydrazine, azide compounds, carbamates, epoxides, esters, sulfate esters, phosphate, thiophosphate esters, borate esters, halogenated organic compounds, isocyanates, isothiocyanates, ketones, oximes, sulfides (Organic), lipids, hydrogen, and combinations thereof, among other reactive moieties.

[0210] In various embodiments, at least one of Z and Z’ are configured to react with and are conjugated to the cargo or a portion thereof. In some embodiments, one of Z and Z’ is conjugated to the cargo and the other of Z and Z’ is H, which may not be reacted with a cargo. Embodiments are not so limited and the other of Z and Z’ which is not conjugated to a cargo may be conjugated to another type of moiety. In some embodiments, the cargo includes a first cargo and a second cargo, and Z is conjugated to the first cargo and Z’ is conjugated to the second cargo. The first cargo and second cargo may include the same type or different type of cargos.

[0211] FIG. IB illustrates an example of an MGS compound that comprises a multimer. For example, the MGS compound 230 comprises at least two MGS peptides 200A, 200B...200N. In some embodiments, the at least two MGS peptides 200A, 200B...200N are each independently selected from SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1- 12, or SEQ ID NOs: 2-12, among other combinations. In some embodiments, each MGS peptide 200A, 200B...200N comprises SEQ ID NO: 27.

[0212] In some embodiments, the at least two MGS peptides 200A, 200B...200N comprise two MGS peptides 200A, 200B, such as further illustrated and described by the dimer of FIG. ID. In some embodiments, the at least two MGS peptides 200A, 200B comprise four MGS peptides, such as further illustrated and described by the tetramer of FIG. IE.

[0213] In some embodiments, the at least two MGS peptides 200A, 200B...200N are each the same MGS peptide sequence (e.g., multiple copies of the same MGS peptide). In other embodiments, at least two of the at least two MGS peptides 200A, 200B...200N are different from one another. In some embodiments, all of the at least two MGS peptides 200A, 200B ...200N are different from one another.

[0214] In some embodiments, the at least two MGS peptides 200A, 200B...200N may include protecting groups, as previously described. For example, the at least two MGS peptides 200A, 200B...200N may each comprise protecting group on the N-terminus, the protecting groups each being independently selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a PEG group, a methyl group, and a combination thereof. In some such embodiments, the at least two MGS peptides 200A, 200B...200N are each independently selected from SEQ ID NOs: 1-6 and 13-19 or from SEQ ID NOs: 2-6 and 13-19.

[0215] In some embodiments, the MGS compound 230 further comprises at least one linker 232. The linker 232 may comprise a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof. In some embodiments, the linker 232 comprises multiple linkers forming a linker structure,as further illustrated by FIGs. 1C-1E. For example, the linker structure may include first linkers conjugated to each of the at least two MGS peptides 200A, 200B...200N, directly or indirectly, and a branch linker conjugated to the first linkers, directly or indirectly.

[0216] In some embodiments, the MGS compound 230 further comprises a reactive group 234 conjugated to the linker 232, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 222 illustrated by FIG. 1A. The reactive group 234 may include any of the previously described reactive groups.

[0217] In some embodiments, the MGS compound 230 comprises the structure of:wherein each X comprises one of the at least two MGS peptides 200A, 200B...200N, and Z and Z’ comprise reactive groups (e.g., 234). As previously described, Z and / or Z’ may be configured to react with a cargo. In some embodiments, the above structure may include a dimer 240 of FIG. ID.

[0218] In some embodiments, the MGS compound 230 comprises the structure of:wherein each X comprises one of the at least two MGS peptides 200A,200B...200N, and Z and Z’ comprise reactive groups (e.g., 234). In someembodiments, Z and / or Z’ may be configured to react with a cargo. In some embodiments, the above structure may include a tetramer 260 of FIG. IE.

[0219] In some embodiments, Z and Z’ may include any of the above-described reactive groups, which are not repeated for ease of reference.

[0220] FIG. 1C illustrates an example of a linker structure 241 of an MGS compound 231. As shown, the MGS compound 231 comprises at least two MGS peptides 200A, 200B...200N, as previously described in connection with FIG. IB, the common features not being repeated for ease of reference and as illustrated by the common reference numerals.

[0221] As shown by FIG. 1C, the linker structure 241 comprises chains 245A, 245B ....245N of linkers which are each directly and / or indirectly conjugated to a respective one of the at least two MGS peptides 200A, 200B...200N and to branch linker(s) 246A...246N-1 which conjugate the at least two MGS peptides 200A, 200B...200N together. At least one branch linker 246A...246N-1 is conjugated to a reactive group 248 which may react with and conjugate to a cargo. The chains 245A, 245B ....245N of linkers comprise at least PEG linkers 244A, 244B ...244N which are between the respective MGS peptide 200A, 200B ...200N and the branch linker 246A...246N-1. In some embodiments, the PEG linkers 244A, 244B...244N comprise between one PEG unit and 24 PEG units or more, which is sometimes herein referred to as PEG1-PEG24. The PEG linkers 244A, 244B...244N between the MGS peptides 200A, 200B...200N and the branch linker(s) 246A...246N-1 may provide solubility and separate the MGS peptides 200A, 200B ...200N from the cargo(s) to prevent steric interference between the MGS peptides 200A, 200B...200N and the cargo(s).

[0222] As previously described and further illustrated by the MGS compounds 240, 260 of FIGs. ID- IE, the linker structure 241 may include additional linkers. For example, the chains 245 A, 245B...245N of linkers may include reactive groups which are directly conjugated to the MGS peptides 200A, 200B ...200N and branch linker(s) 246A...246N-1. In some embodiments, the chains 245 A, 245B...245N of linkers may include additional PEG linkers and reactive groups between the PEG linkers 244A, 244B...244 and the branch linker(s) 246A...246N-1. In some embodiments, the linker structure 241 may include additional reactive groups conjugated to at least one of branchlinker(s) 246A...246N-1, such that multiple cargos may be conjugated to the linker structure 241.

[0223] The reactive group 248 may react with the cargo using a variety of techniques. For example, the cargo may react with the reactive group 248 using amide chemistry, maleimide chemistry, click chemistry, and / or hydrozone linkers, among other techniques. In some embodiments, the linker structure 241 includes multiple reactive groups conjugated to the branch linker, and one reactive group may be reacted with and conjugated to cargo and the other may be reactive with and conjugated to other cargo, such as a lipid, or may not be further reacted.

[0224] FIG. ID illustrates an example MGS compound 240 comprising a dimer that includes two MGS peptides 200A, 200B, herein generally referred to as “dimer 240” for ease of reference. In some embodiments, MGS peptides 200A, 200B are each individually selected from SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1-12, or SEQ ID NOs: 2-12, as previously described. In some embodiments, the two MGS peptides 200A, 200B each comprise SEQ ID NO: 27.

[0225] In some embodiments, the dimer 240 comprises a linker structure 242. In some embodiments, the linker structure 242 comprises a first PEG linker 244A conjugated to a first MGS peptide 200A of the two MGS peptides 200A, 200B (directly or indirectly), a second PEG linker 244B conjugated to a second MGS peptide 200B of the two MGS peptides 200A, 200B (directly or indirectly), and a branch linker 246 conjugated to the first PEG linker 244A and to the second PEG linker 244B (directly or indirectly). The first and second PEG linkers 244A, 244B may comprise PEGn or PEG 12, among other numbers of PEG units. In some embodiments, dimer 240 comprises a PEG linker 244A, 244B on the C-terminus of each of two MGS peptides 200A, 200B and a lysine branch linker (e.g., linker 246) conjugated to the PEG linkers 244A, 244B.

[0226] In some embodiments, the branch linker 246 comprises an amino acid. For example, the branch linker 246 may comprise a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0227] In some embodiments, the linker structure 242 further comprises a reactive group 248A, 248B conjugated to the branch linker 246, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 222 illustrated by FIG. 1A. For example, at least one reactive group 248A, 248B may be conjugated on the C-terminal side of the branch linker 246 comprising an amino acid.

[0228] In some embodiments, the linker structure 242 may include additional linkers. For example, the additional linkers may include reactive groups 243A, 243B between the MGS peptides 200A, 200B and the PEG linkers 244A, 244B. In some embodiments, the reactive groups 243A, 243B between the MGS peptides 200A, 200B and the PEG linkers 244A, 244B may include -NH, but embodiments are not so limited and other reactive groups may be used. In some such embodiments, the first PEG linker 244A and the second PEG linker 244B are indirectly conjugated to, respectively, the first MGS peptide 200A and the second MGS peptide 200B through the reactive groups 243A, 243B.

[0229] In some embodiments, the linker structure 242 of the dimer 240 comprises:wherein Z and Z’ comprise reactive groups. In some embodiments, at least one of the Z and Z’ groups are configured to react with a cargo or a portion thereof. In some embodiments, one of Z and Z’ is configured to react the with cargo, and the other of Z and Z’ is H. In some embodiments, Z is configured to react with a first cargo and Z’ is configured to react with a second cargo, wherein the first cargo and the second cargo are the same or different (e.g., different types of cargos). For example, the first cargo may include a lipid and the second cargo may include a nucleic acid, among other combinations. In some embodiments, one of Z and Z’ is a lipid, although embodiments are not so limited.

[0230] As previously described, the cargo may be, but is not limited to, a dye, an imaging agent, a therapeutic, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a small molecule, a radionuclide, carbohydrate, or a nanoparticle.

[0231] FIG. IE illustrates an example MGS compound 260 comprising a tetramer that includes four MGS peptides 200A, 200B, 200C, 200D, herein generally referred to as “tetramer 260” for each of reference. In some embodiments, MGS peptide 200A, 200B, 200C, 200D are each individually selected from SEQ ID NOs: 1-27, SEQ ID NOs: 1-26, SEQ ID NOs: 2-26, SEQ ID NOs: 1-6 and 13-19, SEQ ID NOs: 2-6 and 8-26, SEQ ID NOs: 2-6 and 13-19, SEQ ID NOs: 7-12 and 20-26, SEQ ID NOs: 8-12 and 20-26, SEQ ID NOs: 1-12, or SEQ ID NOs: 2-12, as previously described. In some embodiments, each MGS peptide 200A, 200B, 200C, 200D comprises SEQ ID NO: 27.

[0232] In some embodiments, the tetramer 260 comprises a linker structure 262. In some embodiments, the linker structure 242 comprises a first PEG linker 244A conjugated to a first MGS peptide 200A of the four MGS peptides 200A, 200B, 200C, 200D (directly or indirectly), a second PEG linker 244B conjugated to a second MGS peptide 200B of the four MGS peptides 200A, 200B, 200C, 200D (directly or indirectly), a third PEG linker 244C conjugated to a third MGS peptide 200C of the four MGS peptides 200A, 200B, 200C, 200D (directly or indirectly), and a fourth PEG linker 244C conjugated to a fourth MGS peptide 200D of the four MGS peptides 200A, 200B, 200C, 200D (directly or indirectly). The linker structure 242 may further comprise branch linkers 246 A, 246B, 246C that respectively are conjugated (directly or indirectly): (i) to the first PEG linker 244A and to the second PEG linker 244B, (ii) to the second PEG linker 244B and to the third PEG linker 244C, and (iii) to the third PEG linker 244C and to the fourth PEG linker 244D. The first, second, third, and fourth PEG linkers 244A, 244B, 244C, 244D may comprise PEGn or PEG12, among other numbers of PEG units. In some embodiments, tetramer 260 further comprises a PEG linker 244A, 244B, 244C, 244D on the C-terminus of each of the four MGS peptides 200A, 200B, 200C, 200D and lysine branch linkers (e.g., 246A, 246B, 246C) conjugated to the respective PEG linkers 244A, 244B, 244C, 244D, directly or indirectly through additional linkers.

[0233] In some embodiments, the branch linkers 246A, 246B, 246C may each comprise an amino acid. For example, each of branch linkers 246 A, 246B, 246C may comprise a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0234] In some embodiments, the linker structure 262 further comprises a reactive group 248A, 248B conjugated to at least one of branch linkers 246A, 246B, 246C, directly or indirectly, and to allow for conjugation to a cargo, such as the cargo 222 illustrated by FIG. 1A. For example, at least one reactive group 248A, 248B may be on the C-terminal side of the branch linker 246B comprising an amino acid.

[0235] In some embodiments, the linker structure 262 may include additional linkers, such as linkers between the PEG linkers 244A, 244B, 244C, 244D on the C-terminal side of each of the four MGS peptides 200A, 200B, 200C, 200D and the branch linkers 246A, 246B, 246C. In some embodiments, the additional linkers may include modified cysteine and maleimide linkers (e.g., cysteine-maleimide linkers) 264A, 264B, 264C, 264D and additional PEG linkers 244E, 244F, 244G, 244H. In some embodiments, each of the additional PEG linkers 244E, 244F, 244G, 244H may include the same length or different length of PEG units as the PEG linkers 244A, 244B, 244C, 244D. In such embodiments, the linker structure 262 comprises, for each MGS peptide 200A, 200B, 200C, 200D, at least two PEG linkers (e.g., 244A and 244E) and a reactive group (e.g., cysteine-maleimide linker 264A) between the at least two PEG linkers, with the reactive groups (e.g., 264A, 264B, 264C, 264D) connecting the at least two PEG linkers. For example, additional PEG linkers 244E, 244F, 244G, 244H may each include PEGn or PEG12 in some embodiments.

[0236] Accordingly, in some embodiments, the linker structure 262 comprises additional PEG linkers 244E, 244F, 244G, 244H and reactive groups (e.g., 264A, 264B, 264C, 264D), wherein the branch linkers 246A, 246B, 246C are indirectly conjugated to the first PEG linker 244A and to the second PEG linker 244B, to the second PEG linker 244B and to the third PEG linker 244C, and to the third PEG linker 244C and to the fourth PEG linker 244D through the additional PEG linkers 244E, 244F, 244G, 244H and reactive groups (e.g., 264A, 264B, 264C, 264D).

[0237] In some such embodiments, the linker structure 262 further comprises a first reactive group (e.g., cysteine-maleimide linker 264A) and a fifth PEG linker 244E, the first reactive group (e.g., cysteine-maleimide linker 264A) being between the first PEG linker 244A linker and the fifth PEG linker 244E, and a first of the branch linkers 246A being indirectly conjugated to the first PEG linker 244A through the first reactive group (e.g., cysteine-maleimide linker 264A) and the fifth PEG linker 244E. The linker structure 262 may further comprise a second reactive group (e.g., cysteine-maleimide linker 264B) and a sixth PEG linker 244F, the second reactive group (e.g., cysteine- maleimide linker 264B) being between the second PEG linker 244B and the sixth PEG linker 244F, and the first and a second of the branch linkers 246A, 246B being indirectly conjugated to the second PEG linker 244B through the second reactive group (e.g., cysteine-maleimide linker 264B) and the sixth PEG linker 244F. The linker structure 262 may further comprise a third reactive group (e.g., cysteine-maleimide linker 264C) and a seventh PEG linker 244G, the third reactive group (e.g., cysteine-maleimide linker 264C) being between the third PEG linker 244C and seventh PEG linker 244G, and the second and a third of the branch linkers 246B, 246C being indirectly conjugated to the third PEG linker 244C through the third reactive group (e.g., cysteine-maleimide linker 264C) and the seventh PEG linker 244G. And, the linker structure 262 may further comprise a fourth reactive group (e.g., cysteine-maleimide linker 264D) and eighth PEG linker 244H, the fourth reactive group (e.g., cysteine-maleimide linker 264D) being between the fourth PEG linker 244D and eighth PEG linker 244H, and the third of the branch linkers 246C being indirectly conjugated to the fourth PEG linker 244D through the fourth reactive group (e.g., cysteine-maleimide linker 264D) and the eighth PEG linker 244H.

[0238] In some embodiments, the additional linkers may include reactive groups 243A, 243B, 243C, 243D between the four MGS peptides 200A, 200B, 200C, 200D and the PEG linkers 244A, 244B, 244C, 244D. In some embodiments, the reactive groups 243A, 243B, 243C, 243D between the four MGS peptides 200A, 200B, 200C, 200D and the PEG linkers 244A, 244B, 244C, 244D may include -CONH, but embodiments are not so limited and other reactive groups may be used. In some such embodiments, the first PEG linker 244 A, the second PEG linker 244B, the third PEG linker 244C, and thefourth PEG linker 244D are indirectly conjugated to, respectively, the first MGS peptide 200A, the second MGS peptide 200B, the third MGS peptide 200C, and the fourth MGS peptide 200D through the reactive groups 243A, 243B, 243C, 243D.

[0239] In some embodiments, the linker structure 262 of the tetramer 260 comprises:wherein Z and Z’ comprise reactive groups. In some embodiments, at least one of Z and Z’ are configured to react with a cargo.

[0240] In some embodiments, the Z and / or Z’ reactive groups may be configured to react with a cargo or a portion thereof. As previously described, a cargo may be, but is not limited to, a dye, an imaging agent, a therapeutic, a protein, a nucleic acid, an amino acid, a peptide, a lipid, an antibody, a radionuclide, carbohydrate or a nanoparticle. In some embodiments, one of Z and Z’ is conjugated to a cargo and the other of Z and Z’ is H, which may not be further reacted with a cargo. In some embodiments, both of Z and Z’ are independently conjugated to cargo, which may be the same or different cargo. For example, the first cargo may include a lipid and the second cargo may include a nucleic acid, such as RNA, among other combinations. In some embodiments, one of Z and Z’ is a lipid, although embodiments are not so limited.

[0241] In some embodiments, the MGS compound 240 of FIG. ID including the linker structure 242 used to conjugate the two MGS peptides 200A, 200B together and the MGS compound 260 of FIG. IE including the linker structure 262 used to conjugate the four MGS peptides 200A, 200B, 200C, 200D together may be respectively referred to as a “dimer core” (if linking two MGS peptides) and a “tetramer core” (if linking fourMGS peptides). The linker structures 242, 262 are examples of linkers used in dimer and tetramer cores. In other embodiments, PEG linkers of other lengths may be used. For example, PEG linkers of between 1-5000 PEG units may be used in place of the illustrated PEG12 linkers.

[0242] In some embodiments, linker structures 242, 262 comprise at least one reactive group configured to react with and conjugate to a C-terminus of an MGS peptide and at least one additional reactive group configured to react with and conjugate to a cargo. The reactive group configured to conjugate to a C-terminus of the MGS peptide may include -NH or -CONH, among other groups. For example, the dimer core may include an -NH group directly attached to the C-terminus of each MGS peptide and which are directly attached to the PEG linkers. For the tetramer core, in some embodiments, a -CONH group may be directly attached to the C-terminus of each MGS peptide and which are directly attached to the (first) PEG linkers. In some embodiments, the dimer core may be linear. In some embodiments, the tetramer core may be non-linear.

[0243] As previously described, the linker structures 242, 262 may include multiple linkers which respectively link at least two MGS peptides and link the at least two MGS peptides to a cargo. In some embodiments, a PEG linker may be used to conjugate an MGS peptide to the cargo, directly or indirectly. In some embodiments, the linker between the MGS peptide and the cargo may be longer than the linker between two MGS peptides.

[0244] In some embodiments, a linker structure comprises at least two PEG linkers and a reactive group between at least two of the PEG linkers, such as illustrated by the linker structure 262 of FIG. IE. In some embodiments, the reactive group connects at least two PEG linkers.

[0245] Example linkers of linker structures 242, 262 include an amino acid, a peptide, an alkyl, a maleimide, an amino acid-maleimide linker, a thiol, hydrazone, amide, and a reactive group, among other groups. In some embodiments, the amino acid may be a modified amino acid. For example, a modified amino acid may be a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, or a functionalized aspartic acid, among other modified amino acids. In some embodiments, a linker of the linker structure 242, 262 comprises biotin.

[0246] Embodiments are not limited to the MGS peptides of SEQ ID NOs: 1-27. In various embodiments, the MGS peptide and / or composition including an MGS peptide may include an MGS of any of SEQ ID NOs: 1-38. For example, an MGS compound may comprise at least two MGS peptides which are each independently selected from SEQ ID NO: 1-38. Further, MGS compounds are not limited to multimers and may include single MGS peptides.

[0247] In some embodiments, an MGS compound comprises an MGS peptide, wherein the MGS peptide comprises SEQ ID NO: 1 and wherein the MGS peptide further comprises a protecting group on the N-terminus.

[0248] As previously described, the protecting group may be selected from an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a PEG group, a methyl group, and a combination thereof.

[0249] In some embodiments, the protecting group is selected from -COCH3, -COH, - CO-(CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyro-glutamic acid, methyl, - CO-(CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-PEGX-(CH)2- NH2, -CO-PEGX-(CH)2- COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

[0250] In some embodiments, the MGS peptide comprises a first MGS peptide and a second MGS peptide, and the MGS compound comprises a dimer of the first MGS peptide and the second MGS peptide, and both the first MGS peptide and the second MGS peptide are SEQ ID NO: 1.

[0251] The dimer may further comprise a linker selected from a PEG linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

[0252] In some embodiments, the dimer comprises a linker structure comprising a first PEG linker conjugated to the first MGS peptide, directly or indirectly, a second PEG linker conjugated to the second MGS peptide, directly or indirectly, and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

[0253] In some embodiments, the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

[0254] In some embodiments, the branch linker comprises a modified amino acid selected from a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

[0255] In some embodiments, the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and wherein the reactive group is conjugated to a cargo. In some such embodiments, the reactive group is selected from a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with no acetylenic hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

[0256] In some embodiments, the first and second PEG linkers comprise PEGn or PEG12.

[0257] In some embodiments, the dimer further comprises a PEG linker on the C- terminus of each of the first MGS peptide and the second MGS peptide, and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

[0258] Various embodiments are directed to MGS compounds comprising particular structures including specific MGS peptides.

[0259] FIGs. 29A-29F illustrate example MGS peptide structures. Various embodiments are directed to MGS compounds that comprise the specific structures including specific linkers and MGS peptides.

[0260] Some embodiments are directed to an MGS compound comprising:wherein Z comprises H and Z’ comprises CH2SH.

[0261] In some embodiments, as illustrated by FIG. 29A, each X comprises:(SEQ ID NO: 7).

[0262] In some embodiments, as illustrated by FIG. 29B, each X comprises:(SEQ ID NO: 8).

[0263] In some embodiments, as illustrated by FIG. 29C, each X comprises:(SEQ ID NO: 9).

[0264] In some embodiments, as illustrated by FIG. 29D, each X comprises:(SEQ ID NO: 10).

[0265] In some embodiments, as illustrated by FIG. 29E, each X comprises:(SEQ ID NO: 11).

[0266] In some embodiments, as illustrated by FIG. 29F, each X comprises:(SEQ ID NO: 12).

[0267] Various embodiments are directed to methods of using any of the above described MGS peptides, MGS compounds, and MGS-cargo conjugates.

[0268] In the methods described herein, delivery (or administration) of the MGS peptides, MGS compounds, or MGS-cargo conjugates disclosed herein may be via a variety of mechanisms. Any of the above-described MGS compounds and / or MGS- cargo conjugates comprising an MGS peptide may be used to produce a composition which may also include a carrier such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions comprising the MGS peptides disclosed herein, and a pharmaceutically acceptable carrier.

[0269] For example, the compositions described herein may comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Examples of carriers include dimyristoylphosphatidyl choline (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol:peptide or PC:peptide may be used as carriers in this invention.Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution may be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles,e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0270] Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, nucleic acid, vector of the invention is not compromised. Pharmaceutical compositions may also include an active ingredient (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, and anesthetics, among others. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0271] Preparations of parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0272] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0273] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some ofthe compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0274] The disclosed delivery techniques may be used not only for the disclosed compositions but also the disclosed nucleic acid sequences and vectors.

[0275] Various methods of the present disclosure are directed to transporting a cargo to the cardiomyocytes of a subject by administering a composition disclosed herein to a subject in need thereof. An example method for targeting cardiomyocytes in a subject or for targeting cardiomyopathy in a subject comprises administering any of the abovedescribed MGS compounds or MGS-cargo conjugates to a subject, such as a subject diagnosed with or having cardiomyopathy. In some embodiments, administering is an intravenous, intrathecal, subcutaneous, intramuscular, intraperitoneal, intradermal, or intracardiac administration. As previously described, in some embodiments, the MGS peptide specifically binds to cardiomyocytes and mediates internalization of the cargo to the cardiomyocytes. The cargo may retain functional activity inside cardiomyocytes. For example, the MGS peptide of the MGS-cargo conjugate may bind to the outer surface of the cardiomyocyte which triggers uptake of the MGS-cargo conjugate into the cardiomyocyte, and the cargo is the active component once inside the cell.

[0276] Other embodiments are directed to methods of decreasing gene expression or expressing a gene of interest comprising administering any of the above-described MGS compounds and MGS-cargo conjugates to a subject or to a cell (e.g., a cardiomyocyte). The cargo may include a nucleic acid. In some embodiments, the nucleic acid binds to RNA transcribed from the gene of interest. In some embodiments, the cargo includes a nucleic acid encoding a gene of interest associated with a protein that is expressed in response to internalization of the MGS-cargo conjugate.

[0277] Disclosed are dosing regimens comprising administering a single dose of any of the disclosed MGS peptides, MGS compounds, or MGS-cargo conjugates to a subject in need thereof, wherein the single dose comprises an amount effective to target cardiomyocytes.

[0278] Disclosed are dosing regimens comprising administering at least two doses of any of the disclosed MGS peptides, MGS compounds, or MGS-cargo conjugates to a subject in need thereof, wherein each dose is the same concentration. In some embodiments, each dose after a first dose may be decreased. In some embodiments, each dose after a first dose may be increased.

[0279] In some embodiments, a single dose may be a continuous administration. In some embodiments, a continuous administration may be hours, days, weeks, or months. In some embodiments, there may be two or more doses. In some embodiments, the two or more doses may be administered days, weeks, or months apart.

[0280] The materials described above as well as other materials may be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits comprising at least one of the disclosed MGS peptides, MGS compounds, MGS- cargo conjugates, compositions, linkers, or combinations thereof.

[0281] Reference throughout the specification to “examples”, “an example”, “some examples”, and so forth, means that a particular element (e.g., feature, structure, characteristic) described in connection with the example is included in the example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any embodiment may be combined in any suitable manner in various embodiments unless the context clearly dictates otherwise.

[0282] Although specific embodiments have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. This application is intended to cover adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.EXPERIMENTAL EMBODIMENTS

[0283] A number of experimental embodiments were conducted to generate MGS peptides that selectively bind to cardiomyocytes and mediate internalization of a cargo thereto, as well as compositions (e.g., compounds and conjugates) formed therefrom. Experiments were directed to assessing the cardiomyocyte specificity and uptake in vitro and in vivo.Example 1: Dimer and Tetramer Core Formation

[0284] Below is an example of a structure of a dimer core representing MGS_primary cardiomyocyte (PCM) variants in accordance with various experimental embodiments:where each X comprises an MGS peptide, and Z and Z’ comprise reactive groups. When X is CH3O-WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 7), the dimer structure is referred to as MGS_PCM_V2-2. Similar structures were made with X being SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, among other MGS peptide sequences as further described herein. The use of the PEG linker, in various experiments, provides solubility.

[0285] Below is an example of a structure of a tetramer core representing MGS_PCM variants in accordance with various experimental embodiments:where each X comprises an MGS peptide, and each Z and Z’ comprise reactive groups. When X is CH30-WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 7), the structure is referred to as MGS_PCM_V2-4. Similar structures were made with X being SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, among other MGS peptide sequences as further described herein.

[0286] In various experiments, Z’ was:and Z was:

[0287] Table 2 below summarizes the different dimers formed and assessment thereof.Table 2: Example MGS Peptide DimersThe modification to peptide sequences from MGS_PCM_V2-2 in Table 2 improved cardiomyocyte binding and / or serum stability. The same modifications were and / or can be made to tetramer forms of the peptides.

[0288] Table 3 below summarizes the different dimers and tetramers formed and assessment thereof.Table 3: Example Assessment of MGS Peptides* “B” stands for biotin, “AF” stands for Alex Fluor, DBCO stands for Dibenzo cyclooctaltrazole, and ED2 stands for lactate dehydrogenase A (EDHA) siRNA in Table 3 and throughout various experiments. All MGS peptides included CH3CO-WESEAGPVVTVRAERGTGSW (SEQ ID NO: 7). The tetramers were made by convergent synthesis, sometimes referred to herein as “CON”, as further described below. For example, MGS_PCM_V2-4CON_AF647 refers to a tetramer formed using four MGS_PCM_V2 and made by convergent synthesis and conjugated to AF647. The following provide further guidance on the naming convention used herein for the peptides, multimers, and conjugates. The “V#” after MGS_PCM or scPCM is the peptide name (e.g., MGS_PCM_V2) and refers to the peptide sequence (e.g., V2 refers to SEQ ID NO: 7). As described above, the number after “V#” in the peptide name (e.g., the “1” in V2-1) refers to the number of peptides, thereby identifying the composition as a monomer or multimer. Any additional components, which may be used to form conjugates, are listed after “V#-#”. For example, MGS_PCM_V2-1_B refers to the MGS-cargo conjugate that is specific to cardiomyocytes and includes the MGS peptide of CH3C0-WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 7) in a monomer form and is conjugated to biotin. MGS_PCM_V2-2_B refers to the MGS-cargo conjugate that isspecific to cardiomyocytes and includes MGS peptide of CH3CO- WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 7) in a dimer form and is conjugated to biotin. As further described herein, scrambled forms of the MGS sequence(s) may be referred to as “scPCM”.

[0289] All MGS peptides were made by fluorenylmethoxycarbonyl protecting group (Fmoc) solid phase peptide synthesis. PEG linkers (e.g., PEG12) were incorporated between the MGS peptide and the linker structure. The peptides were purified by reversed-phased high performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry. Analytical RP-HPLC was used to determine purity. The analytical RP-HPLC conditions included the use of Agilent 1220 instrument, column of Jupitor-C4 (5 micrometer (pm), Phenomenex), Eluent A of 0.1% Trifluoroacetic acid (TFA), 99.9% water and Eluent B of 0.1% TFA, 99.9% acetonitrile, flow rate of 1 millimeter (ml) / minute, gradient of 20-70% B (30 minutes), column temperature of room temperature, and UV absorbance of 210 nanometers (nm).

[0290] In various experiments, the following MGS dimers were prepared:which respectively include MGS_PCM_V2-2_B and MGS_PCM_V2-2_AF647. The dimers were synthesized by linear Fmoc solid phase peptide chemistry and tags were added by the addition of a thiol group with maleimide biotin or AF647.

[0291] FIGs. 2A-2B show the analytical RP-HPLC (FIG. 2A) and MALDLTOF Mass Spectrometry (FIG. 2B) results of the MGS_PCM_V2-2_B structure. FIGs. 3A-3B show the analytical RP-HPLC (FIG. 3A) and MALDLTOF Mass Spectrometry (FIG. 3B) results of the MGS_PCM_V2-2_AF647 structure.

[0292] In various experiments, the following MGS tetramer was prepared:The above structure is MGS_PCM_V2-4_AF647. The monomeric MGS_PCM_V2 contained a thiol at the C-terminus and was synthesized and purified by RP-HPLC. Trilysine core containing an acetamidomethy (ACM) protected cysteine before the branch linker was synthesized and all amines capped with maleimide, followed by deprotection of the core cysteine structure to form the tetramer which was labeled with AF647. As described above, the tetramer was made by convergent synthesis and exhibited solubility in PBS at around 10 microMolar (pM).

[0293] FIGs. 4A-4B show the analytical RP-HPLC (FIG. 4A) and electrospray iononization Mass Spectrometry (FIG. 4B) results of the MGS_PCM_V2-4_AF647 structure.Example 2: Dimer and Tetramer Core Assessed for Cardiomyocyte Binding

[0294] Primary mouse cardiomyocytes were isolated from BALB / c mice using a method at least substantially similar to and / or adapted from Ackers- Johnson et al., Circulation Research, 119:909-920 (2016), which is incorporated herein by reference in its entirety for its teachings. Collagenase 2 digestion of mouse hearts in the presence of BDM or blebbistatin was used to obtain single cell suspension. The cell were cultured using Celprogen Cardiomyocyte Media (DMEM-based), and using tissue culture plastic and slides coated with cardiomyocyte extracellular matrix (ECM). The isolation method resulted in good recovery (800,000 viable cells) and purity of cells (no other cell types observed in preparation). Characteristic cardiomyocyte appearance observed in brightfield and confocal fluorescence microscopy. FIGs. 5A-5B are a brightfield (FIG. 5A) and confocal fluorescence (FIG. 5B) images of isolated primary mouse cardiomyocytes.

[0295] In various experiments, the isolated primary mouse cardiomyocytes were exposed to MGS peptides to assess for cardiomyocyte binding.

[0296] FIGs. 6A-6C are confocal images of samples of isolated primary mouse cardiomyocytes after exposure to scPCM_V2-2_B (FIG. 6A), MGS_ PCM_V2-1_B (FIG. 6B), and MGS_PCM_V2-2_B (FIG. 6C). As used herein, “sc” stands for scrambled and refers to or includes a peptide including the same amino acids as MGS_PCM_V2 but with the order of the amino acids being rearranged (e.g., in a different order but including the same amino acids). The isolated primary mouse cardiomyocytes were cultured on ECM-coated chamber slide with the biotinylated MGS peptides conjugated to Streptavidin (SA) AF647 1:1 at a concentration of 200 nanoMolar (nM) peptides and for 24 hours. The cells were washed and formalin-fixed prior to imaging on a Zeiss confocal microscope. The initial peptide sequence (scPCM_V2-2_B) was inactive and the monomer MGS-cargo conjugate (MGS_PCM_V2-1_B) did not mediate update in cardiomyocytes. Somewhat surprisingly, the dimer (MGS_PCM_V2-2_B) bound to the primary mouse cardiomyocytes.

[0297] An additional experiment was conducted to assess MGS_PCM_V2-2 and MGS_PCM_V2-4CON binding to primary cardiomyocytes as a free peptide. In suchexperiments, the MGS peptides were directly conjugated to AF647 and exposed to the primary cardiomyocytes at a concentration of 200 nM of peptides and for 24 hours of incubation. The resulting samples were imaged using a Leica Fluorescent microscope, as shown by the images of FIGs. 7A-7D.

[0298] FIGs. 7A-7D are fluorescent images from samples of primary mouse cardiomyocytes exposed to AF647 (FIG. 7A), scPCM_V2-2_AF647 (FIG. 7B), MGS_PCM_V2-2_AF647 (FIG. 7C) and MGS_PCM_V2-4CON_AF647 (FIG. 7D). As shown, no binding above background was observed when no MGS (FIG. 7 A) was used or the scrambled version of PCM_V2-2 was conjugated to AF647 labeled SA (FIG. 7B), sometimes herein referred to as SA-AF647. Significant binding was observed for MGS_PCM_V2-2 (FIG. 7C), demonstrating acetylation of the sequence is tolerated. MGS_PCM_V2-4CON (FIG. 7D) binding to cardiomyocytes was observed but not as pronounced as MGS_PCM_V2-2 (FIG. 7C). This demonstrated, surprisingly, that further increasing valency from the dimeric form did not improve binding specificity to cardiomyocytes.Example 3: Semi-Quantitative Microscopy Assay for Cardiomyocytes

[0299] PCMs are large in size and have irregular shapes, which prohibited use of standard flow cytometry assay. In addition, background fluorescent confounded measurement made by the plate reader, and the density of the cardiomyocytes made it difficult to distribute cells evenly to wells. This resulted in the normalization of cell counts using LDS or Cell Titer-Gio not being sensitive enough for accurate reading. Based on this, an assay was developed that allowed for the quantification of MGS peptide uptake and comparison across multiple cell types.

[0300] As noted above, a semi-quantitative microscope assay was developed and used to assess primary cardiomyocyte binding for MGS peptide optimization and for determining MGS peptide specificity. The assay allowed for the generation of histograms based on pixel intensity to overcome the difficulty of cell size that limits flow cytometry and to account for uptake / cell. For example, the semi-quantitative microscopy assay translated the brightness of a cell image into information about the total area of the cell and how bright (e.g., intense) the fluorescence of AF647 is withinthe cell boundary. The brighter the fluorescence indicates uptake of the AF647 dye that is linked to the peptide. Cells with more areas that are bright, took in more peptide. In the histogram graphs, the peak of the frequency of the bright area shifts to the right when the cell has internalized more peptide.

[0301] FIGs. 8A-8B show the result of a semi-quantitative microscopy assay for assessing binding and internalization of MGS_PCM_V2-1_B-SA-AF647 and MGS_PCM_V2-2_B-SA-AF647 to primary cardiomyocytes. scPCM_V2-2_B-SA- AF647, MGS_PCM_V2-1_B-SA-AF647, and MGS_PCM_B-SA-V2-2_AF647 were exposed to primary mouse cardiomyocytes at a peptide concentration of 200 nM and incubated for 24 hours, and then assayed. FIG 8A is a histogram showing the resulting uptake for scPCM_V2-2_B-SA-AF647 (top row of FIG. 8A, control peptide), MGS_PCM_V2-1_B-SA-AF647 (middle row of FIG. 8A), and MGS_PCM_V2-2_B- SA-AF647 (bottom row of FIG. 8A). More specifically, the left column of FIG. 8A are confocal images of the samples after 24 hours of incubation and the right column of FIG. 8A are histograms illustrating the intensity distribution in a single cell (which is in the box in the images of the left column). The shift to the right in the bottom row histogram demonstrates there is more peptide internalized in cells treated with the dimer. FIG. 8B is a graph showing the AF647 average (at least 10 cells / treatment) of pixel intensity and pixel frequency for each treatment of scPCM_V2-2_B-SA-AF647, MGS_PCM_V2-1_B-SA-AF647, and MGS_PCM_V2-2_B-SA-AF647. As shown by FIGs. 8A-8B, the monomer MGS peptide (MGS_PCM_V2-1_B-SA-AF647) did not mediate uptake in cardiomyocytes and the control peptide (scPCM_V2-2_B-SA-AF647) was inactive. The dimer MGS peptide (MGS_PCM_V2-2_B-SA-AF647) binds to primary mouse cardiomyocytes and was internalized. In particular, MGS_PCM_V2- 2_B-SA-AF647 showed an increase in frequency of high intensity pixels with a concomitant decrease in frequency in low intensity pixels.

[0302] FIGs. 9A-9E illustrate the results of a semi-quantitative microscopy assay for assessing binding and internalization of MGS dimers and MGS tetramers to primary cardiomyocytes. In the experiment, a control of AF647, scPCM_V2-2_AF647, MGS_PCM_V2-2_AF647, and MGS_PCM_V2-4_AF647 were exposed to primary mouse cardiomyocytes at a peptide concentration of 200 nM and incubated for 24 hours,and then assayed. FIGs. 9A-9D are histograms showing the resulting uptake of AF647 (no peptide, FIG. 9A), scPCM_V2-2_AF647 (FIG. 9B), MGS_PCM_V2-2_AF647 (FIG. 9C), and MGS_PCM_V2-4_AF647 (FIG. 9D). FIG. 9E is a graph showing the average of AF647 pixel intensity and pixel frequency for each group of cells treated with AF647, scPCM_V2-2_AF647, MGS_PCM_V2-2_AF647, and MGS_PCM_V2-4 AF647. As shown by FIGs. 9A-9E, tetramerization did not improve cellular binding as compared to dimerization. More specifically, MGS_PCM_V2-2 and MGS_PCM_V2-4 showed a similar profile with an increase in high intensity pixels with a concomitant decrease in frequency in low intensity pixels.

[0303] FIG. 10 is a graph showing the results of a semi-quantitative microscope assay for specificity of MGS_PCM_V2-2_AF647 and scPCM_V2-2_AF647 to cardiomyocytes. As shown, specific binding of MGS_PCM_V2-2 to cardiomyocytes was observed at 50 nM.Example 4: Semi-Quantitative Assay for Various Cell Types

[0304] To assess for specific binding to cardiomyocytes, binding for other types of cells was assessed. The MGS peptides were assessed for binding to mouse vascular aortic smooth muscle cells (MOVAS) cells, mouse hepatocytes of AML12 cells, and fibroblast cell line of NIH 3T3 cells, in addition to primary cardiomyocytes.

[0305] FIGs. 11A-11C show results of assessment for MGS_PCM_V2-2 binding to MOVAS cells. MOVAS cells were isolated, cultured, and then incubated with MGS_PCM_V2-2 that was directly conjugated to AF647 and scPCM_V2-2 that was directly conjugated to AF647 for 2 hours (FIG. 11 A) and for 24 hours (FIG. 1 IB) and assessed via flow cytometry, with concentrations of 50 nM, 100 nM, and 200 nM of peptide. FIG. 11 A is a graph showing the concentration of MGS peptides and molecules internalized per MOVAS cell after 2 hours of incubation. FIG. 1 IB is a graph showing the concentration of MGS peptides and molecules internalized per MOVAS cell after 24 hours of incubation. MOVAS cells were isolated, cultured, and then incubated with MGS_PCM_V2-2_B-SA-AF647, scPCM_V2-2_B-SA-AF647 and also with SA-AF647 at a concentration of 200 nM for 2 hours and then imaged using microscopy. FIG. 11C are confocal images of samples after MOVAS cells were incubated withMGS_PCM_V2-2_B-SA-AF647, scPCM_V2-2_B-SA-AF647, and also with SA- AF647 at a concentration of 200 nM for 2 hours. The samples were stained with DAPI nuclear stain, as shown in blue, and Wheat Germ Agglutinin (WGA)-488, which binds to membranes of cells and is shown in green. AF647 which was conjugated to SA and bound to biotinylated MGS and is shown in red. As shown by FIGs. 11A-11C, no specific binding was observed to MOVAS cells by MGS_PCM_V2-2.

[0306] FIGs. 12A-12C show results of assessment for MGS_PCM_V2-2 binding to AML 12 cells. AML 12 cells were seeded, cultured, and then incubated with MGS_PCM_V2-2 that was directly conjugated to AF647 and scPCM_V2-2 that was directly conjugated to AF647 for 2 hours (FIG. 12 A) and for 24 hours (FIG. 12B) and assessed via flow cytometry, with concentrations of 50 nM, 100 nM, and 200 nM of peptide. FIG. 12A is a graph showing the concentration of MGS peptides and molecules internalized per AML12 cell after 2 hours of incubation. FIG. 12B is a graph showing the concentration of MGS peptides and molecules internalized per AML 12 cell after 24 hours of incubation. AML12 cells were seeded, cultured, and then incubated with MGS_PCM_V2-2_B-SA-AF647, scPCM_V2-2_B-SA-AF647, and also with SA- AF647 at a concentration of 200 nM for 2 hours and then imaged using microscopy. FIG. 12C are confocal images of samples after AML12 cells were incubated with MGS_PCM_V2-2_B-SA-AF647, scPCM_V2-2_B-SA-AF647, and also with SA- AF647 at a concentration of 200 nM for 2 hours. The samples were stained with DAPI nuclear stain (blue), and WGA-488 (green, which binds to plasma membranes). AF647 was conjugated to SA and bound biotinylated MGS and is shown in red. As shown by FIGs. 12A-12C, minimal specific binding was observed to AML12 cells by MGS_PCM_V2-2 after 2 hours and no specific binding was observed after 12 hours.

[0307] FIGs. 13A-13B show results of assessment for MGS_PCM_V2-2 binding to NIH 3T3 cells. NIH 3T3 cells were isolated, cultured, and then incubated with MGS_PCM_V2-2 that was directly conjugated to AF647 and scPCM_V2-2 that was directly conjugated to AF647 for 2 hours (FIG. 13 A) and for 24 hours (FIG. 13B) and assessed via flow cytometry, with concentrations of 50 nM, 100 nM, and 200 nM of peptide. FIG. 13A is a graph showing the concentration of MGS peptides and molecules internalized per NIH 3T3 cell after 2 hours of incubation. FIG. 13B is a graph showingthe concentration of MGS peptides and molecules internalized per NIH 3T3 cell after 24 hours of incubation. As shown by FIGs. 13A-13B, no specific binding was observed to NIH 3T3 cells by MGS_PCM_V2-2. More specifically, minimal uptake of MGS_PCM_V2-2 was observed at 2 hours incubation at 50 nM and 100 nM and it was nonspecific. There was low uptake of MGS_PCM_V2-2 in NIH 3T3 cells at 2 hours incubation at 200 nM, but it was nonspecific, not MGS-peptide dependent. At 24 hours incubation, high background uptake was observed for all samples and specific uptake was low.

[0308] FIGs. 14A-14E are graphs showing the results of a semi-quantitative microscopy assay for assessing specificity of MGS_PCM_V2-2 to MOVAS cells, AML12 cells, and cardiomyocytes. 200 nM of MGS_PCM_V2-2_SA-AF647 and scPCM_V2-2_SA-AF647 were incubated for 2 hours with isolated MOVAS cells (FIG. 14A), isolated AML12 cells (FIG. 14B), and adherent PCM cells (FIG. 14C). After incubation, samples were fixed and semi-quantitative microscopy assays were performed. Samples included an average of a minimum of 10 separated cells / ROI. FIG. 14A is a graph showing the AF647 pixel intensity and fraction of pixels for each of SA- AF647, scPCM_V2-2_SA-AF647 and MGS_PCM_V2-2_SA-AF647 after MOVAS cells were incubated therewith for 2 hours. FIG. 14B is a graph showing the AF647 pixel intensity and fraction of pixels for each of scPCM_V2-2_SA-AF647 and MGS_PCM_V2-2_SA-AF647 after AML12 cells were incubated therewith for 2 hours. FIG. 14C is a graph showing the AF647 pixel intensity and fraction of pixels for each of scPCM_V2-2_SA-AF647 and MGS_PCM_V2-2_SA-AF647 after PCM cells were incubated therewith for 2 hours. The data from the semi-quantitative microscopy assays was consistent with the flow cytometry assays and confocal microscopy (e.g., FIGs. 11A-13B) and supports that MGS_PCM_V2-2 are specific to cardiomyocytes.

[0309] The experiment was repeated with 200 nM of MGS_PCM_V2-2_SA-AF647 and scPCM_V2-2_SA-AF647 incubated for 24 hours with isolated AML12 cells (FIG. 14D), and adherent PCM cells (FIG. 14E). After incubation, samples were fixed and semi-quantitative microscopy assays were performed. Samples included an average of a minimum of 10 separated cells / ROI. FIG. 14D is a graph showing the AF647 pixel intensity and fraction of pixels for each of scPCM_V2-2_SA-AF647 andMGS_PCM_V2-2_SA-AF647 after AML12 cells were incubated therewith for 24 hours. FIG. 14E is a graph showing the AF647 pixel intensity and fraction of pixels for each of scPCM_V2-2_SA-AF647 and MGS_PCM_V2-2_SA-AF647 after PCM cells were incubated therewith for 24 hours. The data from the semi-quantitative microscopy assays was consistent with the flow cytometry assays and confocal microscopy (e.g., FIGs. 11A-13B) and supports that MGS_PCM_V2-2 are specific to cardiomyocytes.Example 5: Assessment For Binding to Human Cardiomyocytes

[0310] In various experiments, the isolated primary human cardiomyocytes were exposed to MGS peptides to assess for cardiomyocyte binding. Cadaveric human primary cardiomyocytes were purchased from a commercial vendor. The cells were isolated by a process similar to the Ackers-Jackson protocol used to isolate cardiomyocytes from mouse and rat hearts, as described above. The cells were suspended in a proprietary “stasis buffer” for shipping. The cells were transferred to the same culture media used for mouse cardiomyocytes and cultured on slides coated with laminin 521 as adhesion matrix as suggested by the vendor. The isolated primary human cardiomyocytes were incubated on slides for 2 hours with 200 nM of AF647, MGS_PCM_V2-2_AF647, scPCM_V2-2_AF647, SA-AF647, and MGS_PCM_V2- 1_B-SA-AF647 in a primary mouse cardiomyocyte media.

[0311] FIGs. 15A-15F are confocal images of samples of isolated untreated primary human cardiomyocytes and samples of isolated primary human cardiomyocytes after exposure to AF647 (FIG. 15B), MGS_PCM_V2-2_AF647 (FIG. 15C), scPCM_V2- 2_AF647 (FIG. 15D), SA-AF647 (FIG. 15E), and MGS_PCM_V2-1_B-SA-AF647 (FIG. 15F) at a concentration of 200 nM for 2 hours. FIG. 15A is an image of a sample of isolated primary human cardiomyocytes which are untreated. The samples were stained with Hoechst 33342 (blue, which binds to nucleus of cells), and Calcein AM (green, which labels cytoplasm of cells). AF647 is conjugated to the peptides or SA and is shown in red). As shown by FIG. 15C, the MGS_PCM_V2-2_AF647 binds to the human cardiomyocytes.

[0312] FIGs. 16A-16F are further confocal microscopy images of samples of isolated human mouse cardiomyocytes after exposure to MGS_PCM_V2-2_AF647 (FIGs. 16A- 16C) and after exposure to scPCM_V2-2_AF647 (FIGs. 16D-16F).

[0313] FIG. 17 is a graph showing the results of a semi-quantitative microscopy assay for assessing specificity of MGS_PCM_V2-2_AF647 and scPCM_V2-2_AF647 to human cardiomyocytes (e.g., AF647 pixel intensity and pixel frequency for each of MGS_PCM_V2-2_AF647 and scPCM_V2-2_AF647). The semi-quantitative assay was performed using the techniques as described above.Example 6: Synthesis Scheme for PCM_V2-2_LD2_AF647

[0314] PCM_V2-2_LD2_AF647 conjugates were formed in various experiments. LD2 (which stands for and is referred to herein sometimes as “LDHA siRNA”) is an siRNA that blocks expression of the lactate dehydrogenase enzyme. More specifically, delivering LD2 to cells may result in blocking or decreasing expression of the lactate dehydrogenase enzyme via knock-down activity. Successfully delivering the siRNA demonstrated the capability of blocking protein expression using the MGS peptides as described herein.

[0315] FIGs. 18A-18B show an example conjugation scheme for PCM_V2- 2_LD2_AF647 and resulting analytical RP-HPLC. More specifically, FIG. 18A shows an example synthesis scheme for forming a conjugate including PCM_V2-2 and LD2_AF647. The MGS dimer of PCM_V2-2 was synthesized by solid-phase peptide synthesis (SPPS) with a 6-Azido-L-lysine in the dimer core. The formed dimer was then reacted in a solution of phosphate-buffered saline (PBS) buffer at pH of around 7.2 for around 16 hours at room temperature with DBCO-LD2-AF647. The MGS dimer was at a concentration of 24 pM and the DBCO-LD2-AF647 was at a concentration of 24 pM, with a molar ratio of MGS dimer: DBCO-LD2-AF647 of 1 : 1. The reaction volume was 0.5 ml. The resulting PCM_V2-2_LD2_AF647 was purified by RP-HPLC, using Clarity oligo column (10 pm, Phenomenex). Analytical RP-HPLC was used to determine purity. EIG. 18B shows the analytical RP-HPLC results of the PCM_V2-2_LD2_AE647 structure formed using the synthesis scheme of EIG. 18 A. The analytical RP-HPLC conditions included the use of Agilent 1220 instrument, column of Clarity oligo (3 pm,Phenomenex), Eluent A of 0.1M Triethlyamine Acetate (TEAA), 5% acetonitrile and Eluent B of 0.1M TEAA, 90% acetonitrile, flow rate of 1 ml / minute, gradient of 0-90% B (30 minutes), column temperature of room temperature, and UV absorbance of 280nm (peptide), 260nm (siRNA), and 650nm (AF647).Example 7: Cargo Delivery with MGS_PCM_V2-2_LD2 in vivo

[0316] Various experiments were conducted to assess for the ability of MGS_PCM_V2-2 to deliver cargo to cardiomyocytes in vivo. C57BL / 6 mice were intravenously injected with PCM_V2-2_LD2_AF647 at a dosage of 2 nanomoles (nmol) / mouse through the tail vein and then imaged after 96 hours.

[0317] FIGs. 19A-19E show the results from injecting mice with an untreated mouse (control) and mice treated with siRNA-AF647, MGS_PCM_V2-2-AF647, and MGS_PCM_V2-2_AF647-LD2 conjugate. For example, FIGs. 19A-19E are microscopy images of tissue harvested from the untreated control mouse (FIG. 19A) and mice treated with the siRNA-AF647 alone (FIG. 19B), MGS_PCM_V2-2-AF647 without cargo (FIG. 19C), and MGS_PCM_V2-2-AF647 conjugated to ED2 (FIG. 19D). As shown by FIG. 19C, MGS_PCM_V2-2 was delivered to the heart and, as shown by FIG. 19D, the MGS_PCM_V2-2_AF647-ED2 conjugate was effective in delivering the functional cargo of siRNA to cardiomyocytes. FIG. 19E is a graph showing relative gene expression of the untreated control, the siRNA, the MGS_PCM_V2-2, and the MGS_PCM_V2-2_ED2 96 hours after the injection as determined by quantitative polymerase chain reaction (qPCR).Example 8: EEE-mCardinal Expression at 48 hours in vivo

[0318] Various experiments were conducted to assess for the ability of MGS_PCM_V2-2 to deliver cargo of EEE-mCardinal to cardiomyocytes in mice in vivo. FEE signifies a linear double stranded DNA construct encoding the sequence of the mCardinal protein (e.g., the DNA encoding the protein sequence that the cell has to make). A C57BE / 6 mouse was intravenously injected with MGS_ PCM_V2-2_EEE through the tail vein at a dosage of 1.6 nmol and then imaged after 48 hours. The EEE- mCardinal construct was delivered as a cDNA and causes expression of a protein notnormally expressed in cardiomyocytes. mCardinal is a near infrared fluorescent protein derived from the organism Entacmaea quadricolor. Successfully delivering the cDNA and showing expression of LEE-mCardinal demonstrated gain of function capabilities of the MGS peptides described herein.

[0319] FIG. 20 is an image taken 48 hours after injecting a mouse with MGS_PCM_V2-2_LEE (right). An untreated (e.g., uninjected) control mouse is shown on the left of FIG. 20. As shown, the MGS_PCM_V2-2_LEE is expressed in the heart of the mouse, demonstrating MGS_PCM_V2-2-dependent internalization of the DNA encoding the protein by the cardiomyocytes. The control mouse (left) illustrated autofluorescence, which was believed to be due to food remaining in the digestive system. The mice were fed a purified diet during the experiment and fasted for 4 hours prior to imaging. It is believed a longer fasting time may eliminate the autofluorescence.

[0320] FIGs. 21A-21B are images taken at 120 hour timepoints including an untreated (control) mouse (FIG. 21A) and a mouse injected with MGS_PCM_V2-2_EEE- mCardinal (FIG. 21B). As shown by FIG. 21B, the MGS_PCM_V2-2_EEE treated heart tissue expresses the mCardinal protein, demonstrating MGS-PCM_V2-2- dependent uptake and expression of the construct by cardiomyocytes. The liver and muscle tissue are not expressing mCardinal protein, demonstrating that the MGS_PCM_V2-2 delivered the FEE construct specifically to heart tissue.

[0321] FIG. 22 includes brightfield (left column), mCardinal (middle column), and merged brightfield and mCardinal (right column) images taken 120 hours after injecting mice with MGS_PCM_V2-2_EEE (top) and untreated control (bottom).Example 9: Rapid Assay

[0322] An assay was developed to assess MGS peptide variant binding to freshly isolated primary cardiomyocytes.

[0323] FIG. 23 shows the rapid assay developed to assess cardiomyocyte interaction (e.g., specific binding) of different versions of the MGS_PCM peptide. As shown, untreated SA-coated beads were exposed to MGS-cargo conjugates of MGS_PCM_V2- 2_B for 30 minutes. The beads were SA A280 beads. After 30 minutes, the sample was quenched with excess biotin and then washed before being incubated for 30 minuteswith cardiomyocytes, followed by exposing the samples to magnetic fields using a magnet to separate bound cells from unbound fractions. After exposure to magnetic fields, bound and unbound fractions were separated and visualized under microscope.

[0324] FIG. 24 is a microscopy image after performing the rapid assay of FIG. 23 on freshly isolated primary cardiomyocytes. As shown by the image, cardiomyocytes were retained on beads having the MGS peptide. The cells (isolated from rats) were stained with H33342 (blue, binds to nucleus) and Calcein AM (green, binds to viable cells) and imaged using Evos Microscope, 20x Zoom.

[0325] FIG. 25 includes further microscopy images after performing the rapid assay of FIG. 23. The top row of FIG. 25 are images of beads with MGS peptides, with the left column including the fraction bound to the magnet and the middle and right columns including fractions not bound to the magnet. The bottom row of FIG. 25 are images of beads only, with the left column including the fraction bound to the magnet and the middle and right columns including fractions not bound to the magnet. All images were obtained using Evos Microscope, 20x Zoom. The binding is mediated by the MGS peptides attached to the beads, with the number cells retained on beads being correlated with binding activity of the peptides.Example 10: Serum Stability in Human Serum

[0326] Various experiments were directed to assessing the serum stability ofMGS_PCM_V2-2_AF647 in human serum. The structure of MGS_PCM_V2-2_AF647 is:

[0327] To assess for serum stability, MGS_PCM_V2-2_AF647 was placed in 100% human serum at a concentration of 10 pM at pH 7.3, 37 degrees C, and mixed at 120 rotations per minute (rpm). At indicated time points, a portion of the sample was removed and serum proteins were precipitated in ethanol. The stability of the dimer MGS_PCM_V2-2_AF647 was monitored on RP-HPLC under ultraviolet (UV) absorbance at 650nm.

[0328] FIG. 26 is a graph showing the resulting RP-HPLC of MGS_PCM_V2- 2_AF647 incubated in human serum over a time period of 6 hours. As previously described, the MGS_PCM_V2-2_AF647 was degraded in 100% human serum at 37 degrees C. Three cleavage cites (as shown by the bottom of FIG. 26) were identified by mass spectrometry, with cleavage occurring on both peptide branches.Example 11: Further Optimization of PCM_V2-2 and Assessment

[0329] The MGS_PCM_V2 (SEQ ID NO: 1) was modified for optimizing stability and cardiomyocyte binding. The optimization included the modification illustrated in Table 2 and Table 4. In some experimental embodiments, the modifications includes the addition of Alanine(s) in different locations of the MGS_PCM_V2 sequence. The different placements may be referred to as an “Alanine Substitution” (AS). Table 4 summarizes the cardiomyocyte binding assessment of various optimized MGS peptides.Table 4: Cardiomyocyte Binding of AS Optimized MGS Peptides

[0330] In various experiments, the optimized forms, including those from Table 2 and Table 4 were assessed for serum stability. Table 5 summarizes the serum stability assessment.Table 5: Serum Stability of Optimized MGS Peptides*MGS_PCM_V3-2, MGS_PCM_V4-2, MGS_PCM_V5-2, MGS_PCM_V7-2,MGS_PCM_V8-2 include dimers of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, respectfully. MS stands for Mouse Serum and HS stands for Human Serum in Table 5.

[0331] Table 6 summarizes binding of various optimized forms of the MGS peptides to cardiomyocytes.Table 6: Cardiomyocyte Binding of Optimized MGS PeptidesExample 12: Further In Vivo Assessment

[0332] Various experiments were conducted to assess the ability of MGS_PCM_V2- 2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 to deliver cargo to cardiomyocytes in vivo, such as previously described in connection with Examples 6-8. C57BL / 6 mice were intravenously injected with MGS_PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 at a dosage of 2 nmol / mouse through the tail vein and then imaged after 48 hours.

[0333] FIGs. 27A-27E show the results from an untreated mouse (control) and mice respectfully treated with MGS_PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647. For example, FIGs. 27 A- 27E are images of the untreated control mouse (FIG. 27 A) and mice treated with the MGS_PCM_V2-2_AF647 (FIG. 27B), MGS_PCM_V9-2_AF647 (FIG. 27C), MGS_PCM_V10-2_AF647 (FIG. 27D), and MGS_PCM_V11-2_AF647 (FIG. 27E). As shown, MGS_PCM_V9-2_AF647 (FIG. 27C), MGS_PCM_V10-2_AF647 (FIG. 27D), and MGS_PCM_V 11-2_AF647 (FIG. 27E) were delivered to the heart at greater uptake rates than MGS_PCM_V2-2_AF647 (FIG. 27B).

[0334] FIGs. 28A-28B are graphs showing the radiant intensity (ROI) of MGS_PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V1 1-2_AF647 (FIG. 28 A) and the fold improvement of MGS_PCM_V9- 2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 compared to MGS_PCM_V2-2_AF647 (FIG. 28B).

[0335] FIG. 30 is a graph showing MGS_PCM_V2-2_AF647, MGS_PCM_V9- 2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11-2_AF647 are internalized by rat and mice cardiomyocytes and not internalized by HEK293 cells. In such experiments, freshly isolated rat primary cardiomyocytes, mouse primary cardiomyocytes, and cultured HEK293 cells were incubated for two hours at 37 degrees C with 200 nM MGS peptide-AF647 conjugates. Cells were washed three times with PBS, two times with pH 2.2 acid wash, and rinsed with PBS before analysis by flow cytometry. Calibrated beads were used to create a standard line relating the number of AF647 molecules per cell with mean fluorescence intensity of gated single cells.

[0336] FIGs. 31A-31B are graphs showing internalization of PCM_V2-2_AF647, MGS_PCM_V9-2_AF647, MGS_PCM_V10-2_AF647, and MGS_PCM_V11- 2_AF647 by mouse cardiomyocytes (FIG. 31 A) and by rat cardiomyocytes (FIG. 3 IB) over time. In such experiments, freshly isolated rat primary cardiomyocytes and mouse primary cardiomyocytes were incubated respectively for two hours, for four hours, and for twenty-four hours at 37 degrees C with 200 nM MGS peptide-AF647 conjugates. Cells were washed three times with PBS, two times with pH 2.2 acid wash, and rinsed with PBS before analysis by flow cytometry. Calibrated beads were used to create a standard line relating the number of AF647 molecules per cell with mean fluorescence intensity of gated single cells.

[0337] Example embodiments in accordance with the present disclosure are directed to MGS peptides which specifically bind to cardiomyocytes and are capable of mediating internalization of cargo to the cardiomyocytes. In some experiments, multimers were formed in order to mediate internalization of the cargo, such as siRNA.

Claims

CLAIMS1. A Molecular Guidance System (MGS) peptide selected from: SEQ ID NOs: 2-6 and 8-26.

2. The MGS peptide of claim 1, wherein the MGS peptide selectively binds to cardiomyocytes.

3. The MGS peptide of claim 1, wherein the MGS peptide is selected from SEQ ID NOs: 2-6 and 13-19, and the MGS peptide further comprise a protecting group on the N- terminus.

4. The MGS peptide of claim 3, wherein the protecting group is selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

5. The MGS peptide of claim 3, wherein the protecting group is selected from: -COCH3, -COH, -C0-(CH)n-CH3, -C0-(CH)n-NH2, y-Aminobutyric acid, pyroglutamic acid, methyl, -C0-(CH)2-C0-0H, -CO-(CH)2-CO-COH3, -CO-poly ethylene glycol (PEG)X-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

6. A Molecular Guidance System (MGS)-cargo conjugate comprising the MGS peptide of any of claims 1-5, wherein the MGS peptide is conjugated to a cargo, directly or indirectly through a linker.

7. The MGS-cargo conjugate of claim 6, wherein the MGS peptide further comprises the linker.

8. The MGS-cargo conjugate of claim 7, wherein the linker is selected from:a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

9. The MGS-cargo conjugate of claim 8, wherein the PEG linker comprises PEGn or PEGn.

10. The MGS-cargo conjugate of claim 7, wherein the linker is conjugated to the C- terminus of the MGS peptide, directly or indirectly.

11. A Molecular Guidance System (MGS)-cargo conjugate comprising an MGS peptide conjugated to a cargo, directly or indirectly through a linker, wherein the MGS peptide is selected from SEQ ID NOs: 2-26.

12. The MGS-cargo conjugate of claim 11, wherein the MGS peptide is selected from SEQ ID NOs: 2-6 and 13-19, and the MGS peptide further comprise a protecting group on the N-terminus selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

13. The MGS-cargo conjugate of claim 11, wherein the MGS peptide comprises a first MGS peptide and a second MGS peptide.

14. The MGS-cargo conjugate of claim 13, wherein the first and second MGS peptides are the same MGS peptide.

15. The MGS-cargo conjugate of claim 13, wherein the first and second MGS peptides are different from one another.

16. The MGS-cargo conjugate of claim 13, wherein the MGS-cargo conjugate comprises a dimer including the first MGS peptide and the second MGS peptide which are each independently selected from SEQ ID NOs: 2-26.

17. The MGS-cargo conjugate of claim 16, wherein the dimer comprises a linker selected from: a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

18. The MGS-cargo conjugate of claim 16, wherein the dimer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly or indirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

19. The MGS-cargo conjugate of claim 18, wherein the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

20. The MGS-cargo conjugate of claim 18, wherein the branch linker comprises a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

21. The MGS-cargo conjugate of claim 18, wherein the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and wherein the reactive group is conjugated to the cargo.

22. The MGS-cargo conjugate of claim 21, wherein the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, athiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

23. The MGS-cargo conjugate of claim 18, wherein the first and second PEG linkers comprise PEGn or PEG12.

24. The MGS-cargo conjugate of claim 18, wherein the linker structure comprises:wherein Z and Z’ comprise reactive groups.

25. The MGS-cargo conjugate of claim 16, wherein the dimer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide and the second MGS peptide; and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

26. The MGS-cargo conjugate of claim 11, wherein the MGS peptide comprises a first MGS peptide, a second MGS peptide, a third MGS peptide, and a fourth MGS peptide.

27. The MGS-cargo conjugate of claim 26, wherein each of the first, second, third, and fourth MGS peptides are the same MGS peptide.

28. The MGS-cargo conjugate of claim 26, wherein at least two of the first, second, third, and fourth MGS peptides are different from one another.

29. The MGS-cargo conjugate of claim 26, wherein the MGS-cargo conjugate comprises a tetramer comprising the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide, wherein the first, second, third, and fourth MGS peptides are each independently selected from SEQ ID NOs: 2-26.

30. The MGS-cargo conjugate of claim 29, wherein the tetramer comprises a linker selected from: a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

31. The MGS-cargo conjugate of claim 29, wherein the tetramer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly or indirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; a third PEG linker conjugated to the third MGS peptide, directly or directly; a fourth PEG linker conjugated to the fourth MGS peptide; and branch linkers respectively conjugated, directly or indirectly: to the first PEG linker and to the second PEG linker; to the second PEG linker and to the third PEG linker; and to the third PEG linker and to the fourth PEG linker.

32. The MGS-cargo conjugate of claim 31, wherein: the linker structure further comprises additional linkers comprising reactive groups; and the first PEG linker, the second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups.

33. The MGS-cargo conjugate of claim 31, wherein the linker structure comprises additional PEG linkers and reactive groups, and wherein the branch linkers are indirectly conjugated to the first PEG linker and to the second PEG linker, to the second PEG linker and to the third PEG linker, and to the third PEG linker and to the fourth PEG linker through the additional PEG linkers and the reactive groups.

34. The MGS-cargo conjugate of claim 31, wherein the branch linkers are each a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

35. The MGS-cargo conjugate of claim 31, wherein the linker structure further comprises a reactive group conjugated to at least one of the branch linkers and conjugated to the cargo, directly or indirectly.

36. The MGS-cargo conjugate of claim 35, wherein the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, am anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

37. The MGS-cargo conjugate of claim 31, wherein each of the first, second, third, and fourth PEG linkers comprise PEGn or PEG12.

38. The MGS-cargo conjugate of claim 31, wherein the linker structure comprises:wherein Z and Z’ comprise reactive groups.

39. The MGS-cargo conjugate of claim 29, wherein the tetramer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptides; and lysine branch linkers conjugated to the respective PEG linkers, directly or indirectly.

40. The MGS-cargo conjugate of any one of claims 11-39, wherein the cargo is selected from: a nucleic acid, a peptide, a protein, an antibody, a lipid, an imaging agent, a dye, a therapeutic, a small molecule, a radionuclide, a carbohydrate, a nanoparticle, and a combination thereof.

41. The MGS-cargo conjugate of any one of claims 11-39, wherein the cargo comprises the nucleic acid selected from: ribonucleic acid, deoxyribonucleic acid, and a combination thereof.

42. The MGS-cargo conjugate of any one of claims 11-39, wherein the cargo comprises a small interfering ribonucleic acid (siRNA).

43. The MGS-cargo conjugate of any one of claims 11-39, wherein the MGS peptide specifically binds to cardiomyocytes and internalizes the cargo.

44. The MGS-cargo conjugate of claim 11, wherein the MGS-cargo conjugate comprises the structure of:wherein: each X comprises the MGS peptide; and Z and Z’ comprise reactive groups.

45. The MGS-cargo conjugate of claim 11, wherein the MGS-cargo conjugate comprises the structure of:wherein: each X comprises the MGS peptide; and Z and Z’ comprise reactive groups.

46. The MGS-cargo conjugate of claim 44 or 45, wherein Z and Z’ are each independently selected from:a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

47. The MGS-cargo conjugate of claim 44 or 45, wherein at least one of Z and Z’ are configured to react with and are conjugated to the cargo.

48. The MGS-cargo conjugate of claim 44 or 45, wherein one of Z and Z’ react with and is conjugated to the cargo, and the other of Z and Z’ is H.

49. The MGS-cargo conjugate of claim 44 or 45, wherein the cargo includes first cargo and second cargo, and Z is conjugated to the first cargo and Z’ is conjugated to the second cargo, wherein the first cargo and second cargo are the same or different.

50. A Molecular Guidance System (MGS) compound comprising at least two MGS peptides, wherein the at least two MGS peptides are each independently selected from SEQ ID NOs: 2-26.

51. The MGS compound of claim 50, wherein the at least two MGS peptides are each independently selected from SEQ ID NOs: 2-6 and 13-19, and each of the MGS peptides comprise a protecting group on the N-terminus selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

52. The MGS compound of claim 50, wherein the at least two MGS peptides comprise two MGS peptides.

53. The MGS compound of claim 50, wherein the at least two MGS peptides comprise four MGS peptides.

54. The MGS compound of any one of claims 50-53, wherein the at least two MGS peptides are each the same MGS peptide.

55. The MGS compound of any one of claims 50-53, wherein at least two of the at least two MGS peptides are different from one another.

56. The MGS compound of any one of claims 50-53, wherein the MGS compound further comprises a linker selected from: a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

57. The MGS compound of any one of claims 56, wherein the MGS compound further comprises a reactive group conjugated to the linker, directly or indirectly, and to allow for conjugation to a cargo.

58. The MGS compound of claim 57, wherein the reactive group is selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

59. The MGS compound of claim 50, wherein the at least two MGS peptides comprise two MGS peptides and the MGS compound comprises a dimer comprising the two MGS peptides.

60. The MGS compound of claim 59, wherein the dimer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to a first MGS peptide of the two MGS peptides, directly or indirectly; a second PEG linker conjugated to a second MGS peptide of the two MGS peptides, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

61. The MGS compound of claim 60, wherein the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

62. The MGS compound of claim 60, wherein the branch linker comprises a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

63. The MGS compound of claim 60, wherein the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and to allow for conjugation to a cargo.

64. The MGS compound of claim 60, wherein the first and second PEG linkers each comprise PEGn or PEG12.

65. The MGS compound claim 60, wherein the linker structure comprises:wherein Z and Z’ comprise reactive groups.

66. The MGS compound of claim 60, wherein the dimer further comprises a polyethylene glycol (PEG) linker on the C-terminus of each of the two MGS peptides and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

67. The MGS compound of claim 50, wherein the at least two MGS peptides comprise four MGS peptides and the MGS compound comprises a tetramer comprising the four MGS peptides.

68. The MGS compound of claim 67, wherein the tetramer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to a first MGS peptide of the four MGS peptides, directly or indirectly; a second PEG linker conjugated to a second MGS peptide of the four MGS peptides, directly or indirectly; a third PEG linker conjugated to a third MGS peptide of the four MGS peptides, directly or indirectly; a fourth PEG linker conjugated to a fourth MGS peptide of the four MGS peptides, directly or indirectly; and branch linkers respectively conjugated, directly or indirectly: to the first PEG linker and to the second PEG linker; to the second PEG linker and to the third PEG linker; and to the third PEG linker and to the fourth PEG linker.

69. The MGS compound of claim 68, wherein the linker structure further comprises additional linkers comprising reactive groups and the first PEG linker, the second PEG linker, the third PEG linker, and the fourth PEG linker are indirectly conjugated to, respectively, the first MGS peptide, the second MGS peptide, the third MGS peptide, and the fourth MGS peptide through the reactive groups.

70. The MGS compound of claim 68, wherein the linker structure comprises additional PEG linkers and reactive groups, and wherein the branch linkers are indirectly conjugated to the first PEG linker and to the second PEG linker, to thesecond PEG linker and to the third PEG linker, and to the third PEG linker and to the fourth PEG linker through the additional PEG linkers and the reactive groups.

71. The MGS compound of claim 68, wherein the branch linkers are each a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

72. The MGS compound of claim 68, wherein the linker structure further comprises a reactive group conjugated to at least one of the branch linkers, directly or indirectly, and to allow for conjugation to a cargo.

73. The MGS compound of claim 68, wherein the first, second, third, and fourth PEG linkers each comprise PEGn or PEG12.

74. The MGS compound of claim 68, wherein the linker structure comprises:wherein Z and Z’ comprise reactive groups.

75. The MGS compound of claim 67, wherein the tetramer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the four MGS peptides; andlysine branch linkers conjugated to, directly or indirectly, the respective PEG linkers.

76. The MGS compound of any of claims 51-53, wherein the at least two MGS peptides selectively bind to cardiomyocytes.

77. The MGS compound of claim 51, wherein the MGS compound comprises the structure of:wherein: each X comprises one of the at least two MGS peptides; and Z and Z’ comprise reactive groups.

78. The MGS compound of claim 51, wherein the MGS compound comprises the structure of:wherein: each X comprises one of the at least two MGS peptides; and Z and Z’ comprise reactive groups.

79. The MGS compound of claim 77 or 78, wherein Z and Z’ are each independently selected from: a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with No Acetylenic Hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

80. The MGS compound of claim 77 or 78, wherein at least one of Z and Z’ are configured to react with a cargo.

81. The MGS compound of claim 77 or 78, wherein one of Z and Z’ is configured to react with a cargo, and the other of Z and Z’ is H.

82. The MGS compound of claim 77 or 78, wherein Z is configured to react with a first cargo and Z’ is configured to react with a second cargo, wherein the first cargo and second cargo are the same or different.

83. A method of treating a subject having a cardiomyopathy or suspected of having a cardiomyopathy comprising: administering the MGS-cargo conjugate of any one of claims 11-39 to a subject diagnosed with or having the cardiomyopathy, wherein the cargo of the MGS-cargo conjugate is selected to have a prophylactic, therapeutic, or ameliorative effect for the cardiomyopathy .

84. The method of claim 83, wherein the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes.

85. The method of claim 83, wherein the cargo retains functional activity inside the cardiomyocytes sufficient to have a therapeutic effect.

86. The method of claim 83, wherein administering comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

87. A method of targeting cardiomyocytes in a subject comprising: administering the MGS-cargo conjugate of any one of claims 11-39 to a subject.

88. The method of claim 87, wherein the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes.

89. The method of claim 87, wherein the cargo retains functional activity inside the cardiomyocytes.

90. The method of claim 87, wherein administering comprises an intravenous, intracardiac, subcutaneous, intramuscular, intraperitoneal, or intrathecal administration of a formulation comprising the MGS-cargo conjugate.

91. A method of decreasing gene expression of a gene of interest comprising: administering the MGS-cargo conjugate of any one of claims 11-39 to a subject.

92. The method of claim 91, wherein the cargo comprises a nucleic acid configured to bind to ribonucleic acid (RNA) transcribed from the gene of interest.

93. The method of claim 91, wherein the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes.

94. The method of claim 91, wherein the cargo retains functional activity inside the cardiomyocytes.

95. A method of expressing a gene of interest comprising: administering the MGS-cargo conjugate of any one of claims 11-39 to a subject.

96. The method of claim 95, wherein the cargo comprises a nucleic acid encoding the gene of interest, the gene being associated with a protein and the protein being expressed in response to internalization of the MGS-cargo conjugate.

97. The method of claim 95, wherein the MGS peptide preferentially binds to cardiomyocytes and internalizes the cargo to the cardiomyocytes.

98. The method of claim 95, wherein the cargo retains functional activity inside the cardiomyocytes.

99. A Molecular Guidance System (MGS) peptide comprising: W1B1SEAGPVVTVAB2B3RGTGSW (SEQ ID NO: 27), wherein:Wi is selected from W and a modified W;Bi is selected from L, I, V, Nle, and a-t-butylglycine;B2 is selected from A and 2- Aminoisobutyric acid (Aib); and B3 is selected from A, L, I, and Nle.

100. The MGS peptide of claim 99, wherein the MGS peptide is selected from: WLSEAGPVVTVAAARGTGSW (SEQ ID NO: 2),WLSEAGPVVTVAALRGTGSW (SEQ ID NO: 3), WISEAGPVVTVAALRGTGSW (SEQ ID NO: 4), WVSEAGPVVTVAALRGTGSW (SEQ ID NO: 5), WNleSEAGPVVTVAALRGTGSW (SEQ ID NO: 6), WVSEAGPVVTVAAIRGTGSW (SEQ ID NO: 13), WISEAGPVVTVAAIRGTGSW (SEQ ID NO: 14), WLSEAGPVVTVAANleRGTGSW (SEQ ID NO: 15), WVSEAGPVVTVAAibLRGTGSW (SEQ ID NO: 16), WNleSEAGPVVTVAANleRGTGSW (SEQ ID NO: 17), W(a-t-butylglycine)SEAGPVVTVAALRGTGSW (SEQ ID NO: 18), and W*VSEAGPVVTVAAIRGTGSW (SEQ ID NO: 19), wherein W* is the modified W selected from a -CN, a -OH, a -Cl, a -F, a -CH3, and a -OCH3 modification in position 5, 6, or 7 of the indole ring.

101. The MGS peptide of claim 99, wherein the MGS peptide further comprises a protecting group on the N-terminus.

102. The MGS peptide of claim 101, wherein the protecting group is selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

103. The MGS peptide of claim 101, wherein the protecting group is selected from:-COCH3, -COH, -CO-(CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyroglutamic acid, methyl, -CO-(CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-poly ethylene glycol (PEG)X-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

104. A Molecular Guidance System (MGS) compound comprising an MGS peptide, wherein the MGS peptide comprises SEQ ID NO: 1, wherein the MGS peptide further comprises a protecting group on the N-terminus.

105. The MGS compound of claim 104, wherein the protecting group is selected from: an acyl group, a cyclic group, an amino-alkyl group, a succinyl group, a polyethylene glycol (PEG) group, a methyl group, and a combination thereof.

106. The MGS compound of claim 104, wherein the protecting group is selected from:-COCH3, -COH, -CO-(CH)n-CH3, -CO-(CH)n-NH2, y-Aminobutyric acid, pyroglutamic acid, methyl, -CO-(CH)2-CO-OH, -CO-(CH)2-CO-COH3, -CO-poly ethylene glycol (PEG)X-(CH)2- NH2, -CO-PEGX-(CH)2-COH3, and -CO-PEGX-(CH)2-NH-CH3, wherein n = 1-20 and x = 1-36.

107. The MGS compound of claim 104, wherein the MGS peptide comprises a first MGS peptide and a second MGS peptide and the MGS compound comprises a dimer ofthe first MGS peptide and the second MGS peptide and both the first MGS peptide and the second MGS peptide are SEQ ID NO: 1.

108. The MGS compound of claim 107, wherein the dimer comprises a linker selected from: a polyethylene glycol (PEG) linker, an alkyl linker, a maleimide linker, an amino acid linker, an amino acid-maleimide linker, an amide linker, a thiol linker, an amine linker, an aryl linker, a reactive group, and a combination thereof.

109. The MGS compound of claim 107, wherein the dimer comprises a linker structure comprising: a first polyethylene glycol (PEG) linker conjugated to the first MGS peptide, directly or indirectly; a second PEG linker conjugated to the second MGS peptide, directly or indirectly; and a branch linker conjugated to the first PEG linker and to the second PEG linker, directly or indirectly.

110. The MGS compound of claim 109, wherein the first PEG linker and the second PEG linker are indirectly conjugated to, respectively, the first MGS peptide and the second MGS peptide through reactive groups.

111. The MGS compound of claim 109, wherein the branch linker comprises a modified amino acid selected from: a functionalized lysine, a functionalized cysteine, a functionalized glutamic acid, and a functionalized aspartic acid.

112. The MGS compound of claim 109, wherein the linker structure further comprises a reactive group conjugated to the branch linker, directly or indirectly, and wherein the reactive group is conjugated to a cargo.

113. The MGS compound of claim 112, wherein the reactive group is selected from:a carboxylic acid, an acyl halide, a sulfonyl halide, a chloroformate, an aldehyde, an alkyne, an alkyne (with no acetylenic hydrogen), an amide, an imide, an amine, a thiol, a phosphine, a pyridine, an anhydride, an azo compound, a diazo compound, an azido compound, a hydrazine, an azide compound, a carbamate, an epoxide, an ester, a sulfate ester, a phosphate, a thiophosphate ester, a borate ester, an halogenated organic compound, an isocyanate, an isothiocyanate, a ketone, an oxime, a sulfide (Organic), a lipid, a hydrogen, and a combination thereof.

114. The MGS compound of claim 109, wherein the first and second PEG linkers comprise PEGn or PEG12.

115. The MGS compound of claim 107, wherein the dimer further comprises: a polyethylene glycol (PEG) linker on the C-terminus of each of the first MGS peptide and the second MGS peptide; and a lysine branch linker conjugated to the PEG linkers, directly or indirectly.

116. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 7).

117. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 8).

118. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 9).

119. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 10).

120. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 11).

121. A Molecular Guidance System (MGS) compound comprising:wherein:Z comprises H;Z’ comprises CH2SH; and each X comprises:(SEQ ID NO: 12).

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