Peptide dendrons and methods of use thereof

Abstract

The present specification relates to peptide dendrons comprising one or more residues derived from a modified lysine of formula (I), pharmaceutical delivery systems comprising these peptide dendrons, pharmaceutical compositions containing them, and their use in therapy. [Formula 1] TIFF2024537869000046.tif26161

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 262,269, filed October 8, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0002] The present specification relates to peptide dendrons comprising one or more residues derived from modified lysine, and the use of these peptide dendrons for delivering pharmaceutically active agents, particularly genetic material, to cells. The present specification also relates to the use of these peptide dendrons in therapy. [Background technology]

[0003] Gene therapy is a field of medicine that focuses on the therapeutic delivery of (foreign) genetic material (such as DNA and RNA) into a patient's cells to treat disease. To date, several gene therapies have received regulatory approval for a number of different medical conditions, including Luxturna® (blindness due to RPE65 mutations) and Kymriah® (chimeric antigen receptor T-cell therapy).

[0004] Gene delivery is a process used to introduce genetic material into cells. To be successful, the genetic material must remain stable during transport and ultimately be internalized by the target cell. When the genetic material is DNA, the DNA must be internalized within the target cell and delivered into the nucleus. Gene delivery requires a vector, and suitable vectors generally fall into two categories: viral and non-viral.

[0005] Viral-mediated gene delivery utilizes the ability of viruses to inject DNA into host cells. Genetic material is packaged into replication-deficient viral particles to form viral vectors. Viral methods are highly efficient but can induce immune responses. Furthermore, they can only deliver very small pieces of genetic material into cells, and the process of producing them is labor-intensive and involves the risk of random insertion sites, cytopathic effects, and mutagenesis.

[0006] Synthetic vectors offer several advantages over viruses for gene delivery applications in terms of structural diversity and scalability, and they can be individually and specifically designed to achieve a desired goal. These materials can be designed to package genetic material into genetically engineered nanoparticles or vesicles to overcome biological barriers associated with cellular uptake, transport into the cytoplasm, and (if desired) delivery into the nucleus. A common approach is to package genetic material into multimolecular assemblies with materials such as polymers, peptides, or lipids that contain the positive charge associated with anionic nucleic acids. Electrostatic interactions between positive and negative charges drive self-assembly into nanoparticle or microparticle structures, and the size and shape of these particles can be controlled by the type of material and condensation conditions (Non-Patent Document 1).

[0007] For example, formulation of DNA into a suitable vector, such as a nanoparticle, significantly improves the cellular uptake of DNA compared to uptake of unformulated DNA. Because DNA is large and negatively charged, it cannot be taken up by itself into cells (which also have a net negative charge) by passive processes such as diffusion across the cell membrane. Unformulated DNA also tends to provoke an immune response that leads to its degradation. By formulating DNA into a suitable vector, the negative charge of the DNA can be neutralized, protecting the DNA from degradation in the extracellular space.

[0008] For a vector to be effectively internalized, it must be transported into the cell by a process called endocytosis. During this process, the vector is surrounded by a region of the plasma membrane that subsequently buds inside the cell, forming an endosome. The vector must be designed to allow this process to occur but mitigate the possibility of subsequent lysosomal uptake, i.e., sequestration within the acidic membrane-bound lysosomal compartment. One way to achieve this is to ensure endosome rupture before lysosomal trafficking can occur. This can be achieved by the vector buffering the endosomal pH (which becomes increasingly acidic after endocytosis) until the resulting osmotic gradient causes the endosome to rupture, liberating the genetic material into the cytoplasm, where it can be made available for transcription / translation in situ. Suitable vector materials with efficient buffering capacity in the endosomal buffer range (pH 7.4–pH 5.0) can delay endosomal acidification by accepting protons, which triggers the influx of additional protons and counterions from the cytosol.

[0009] Current synthetic gene delivery systems are limited in vivo by poor stability, high toxicity, and inefficient cytoplasmic entry of the genetic material. The process of engineering multifunctional materials suitable for gene delivery that achieve an optimal balance between formulation properties (size, charge, etc.), stability, buffering capacity, and toxicity while maintaining high delivery efficiency has proven difficult and complex, but the resulting formulations would be greatly simplified.

[0010] Peptide dendrons (PDs) are three-dimensional structures containing amino acid residues, where one side chain of the residue, e.g., the ε-amine of lysine, is utilized to form branches or generations, building the molecule into a 3D macromolecule. Peptide dendrons are typically generated using solid-phase peptide synthesis, allowing precise control of the amino acid residue sequence and geometric arrangement within well-defined structures. The final product is monodisperse and has highly tunable properties (i.e., hydrophobicity, charge density, and molecular weight), allowing for multifunctional optimization and flexible application.

[0011] Peptide dendrons have been widely explored, but their clinical success has been hindered by the high production costs and poor proteolytic stability. Kwok et al. (2011) explored peptide dendrimer / lipid hybrid systems as transfection agents for DNA and RNA, but were limited to dipeptide branches due to synthetic limitations, limited material functionality, and in vivo efficacy.

[0012] This application describes specific peptide dendrons containing one or more residues derived from modified lysine. These peptide dendrons: i) possess high buffering capacity tuned to allow protonation during pH transitions that occur during cellular internalization and lysosomal trafficking; ii) exhibit high stability due to increased nucleic acid binding through electrostatic and non-electrostatic (e.g., π-π stacking) interactions; iii) possess high biocompatibility when core units based on naturally occurring metabolites are used, which are unlikely to be toxic or immunogenic; and / or iv) achieve rapid nucleic acid release through specific intracellular enzymatic degradation (cathepsin B). They form nanoparticles containing multiple nucleic acid morphologies, including plasmid DNA, mRNA, siRNA, and antisense oligonucleotides (ASOs) (approximately 25-100 nm, spherical, rod-like, and toroidal), and exhibit high serum stability and prolonged blood circulation after intravenous injection in mice without associated toxicity. They have been shown to have the ability to successfully deliver and / or co-deliver a wide range of nucleic acid forms, including DNA, mRNA, siRNA, and ASO, and to protect the encapsulated genetic material by not readily dissociating into cells. Furthermore, co-delivery of RNA / DNA within the same nanoparticle allows for control of protein expression kinetics, expression of synergistic therapeutic agents, and expands the application of nanoparticles to areas where multiple components are required, such as CRISPR. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Park et al.Adv Drug Del Rev,2006,58(4):467-86 [Non-patent document 2] Kwok et al ACS Nano,2013 May 28;7(5):4668-82,doi:10.1021 / nn400343z [Non-patent document 3] Kwok et al ChemBioChem,2016,17(23),2223-2229 Summary of the Invention [Means for solving the problem]

[0014] This specification describes, in part, peptide dendrons that include one or more residues derived from a modified lysine of formula (I): [ka] (In the formula, A is a bond, C is 1~6 alkylene, carbocyclyl, or heterocyclyl; wherein said carbocyclyl or heterocyclyl optionally has one or more R 2 when said heterocyclyl contains an -NH- moiety, the nitrogen may optionally be substituted with R A may be substituted with a group selected from Q is a bond, carbocyclyl, or heterocyclyl; wherein said carbocyclyl or heterocyclyl optionally has one or more R 3 when said heterocyclyl contains an -NH- moiety, the nitrogen may optionally be substituted with R B may be substituted with a group selected from Ring B is morpholinyl or thiomorpholinyl; where said morpholinyl or thiomorpholinyl contains an —NH— moiety, the nitrogen is optionally R C may be substituted with a group selected from R 1 , R 2 and R 3are each independently selected from halo, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, and N-methyl-N-ethylsulfamoyl; n is 0 to 4; R A , R B and R C are independently selected from methyl, ethyl, propyl, isopropyl, acetyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, carbamoyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, and N-methyl-N-ethylcarbamoyl.

[0015] This specification also describes, in part, peptide dendrons described herein for use in delivering pharmaceutically active agents intracellularly.

[0016] This specification also describes, in part, pharmaceutical compositions that include one or more peptide dendrons described herein and a pharmaceutically active agent.

[0017] This document also describes, in part, a method of gene therapy comprising administering to the animal an effective amount of a pharmaceutical composition described herein. DETAILED DESCRIPTION OF THE INVENTION

[0018] Many embodiments are detailed throughout this specification and will be apparent to those skilled in the art, and the present disclosure should not be construed as limited to any of the embodiments listed.

[0019] "A" means "at least one." In any embodiment where "a" is used to refer to a given substance or element, "a" can mean one.

[0020] "Comprising" means that a given substance or element may contain other substances or elements. In any embodiment where "comprising" is mentioned, the given material or element may be made up of at least 1% w / w, at least 5% w / w, at least 10% w / w, at least 20% w / w, at least 30% w / w, or at least 40% w / w of that material or element. "Comprising" can also mean "consisting of" (or "consist of") or "consisting essentially of" (or "consist essentially of") the given material or element.

[0021] "Consisting of" or "consist of" means that a given material or element is formed entirely from that material or element. In any embodiment where "consisting of" or "consist of" is mentioned, the given material or element may be formed from 100% w / w of that material or element.

[0022] "Consisting essentially of" or "consist essentially of" means that a given material or element is made up almost entirely of that material or element. In any embodiment where "consisting essentially of" or "consist essentially of" is mentioned, the given material or element may be made up of at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or at least 99% w / w of that material or element.

[0023] In any embodiment where "is" or "may be" is used to define a material or element, "is" or "may be" can mean that the material or element "consists of" or "consists essentially of" the material or element.

[0024] In any embodiment herein where "about" is referenced, "about" can mean + / - 0 (i.e., no variation), + / - 0.01, + / - 0.05, + / - 0.1, + / - 0.5, + / - 1, + / - 2, + / - 5, + / - 10, or + / - 20 percent of the recited numerical value. Where a numerical value is recited, in another embodiment, this also refers to approximately the recited numerical value.

[0025] The claims are embodiments.

[0026] Disclosed herein are peptide dendrons comprising one or more residues derived from a modified lysine of formula (I): [ka] (In the formula, A, Q, B, R 1 and n are as described herein).

[0027] In one embodiment, A is a bond.

[0028] In one embodiment, A is C 1~6 It is alkylene.

[0029] In one embodiment, A is methylene.

[0030] In one embodiment, A is carbocyclyl; said carbocyclyl optionally having one or more R 2 may be substituted with

[0031] In one embodiment, A is heterocyclyl; said heterocyclyl optionally having one or more R 2 when said heterocyclyl contains an -NH- moiety, the nitrogen may optionally be substituted with R A It may be substituted with a group selected from:

[0032] In one embodiment, A is heterocyclyl.

[0033] In one embodiment, A is pyridyl.

[0034] In one embodiment, A is a bond, C 1~6 It is alkylene or heterocyclyl.

[0035] In one embodiment, A is a bond, methylene, or pyridyl.

[0036] In one embodiment, Q is a bond.

[0037] In one embodiment, Q is carbocyclyl; said carbocyclyl optionally has one or more R 3 may be substituted with

[0038] In one embodiment, Q is heterocyclyl; said heterocyclyl optionally having one or more R 3when said heterocyclyl contains an -NH- moiety, the nitrogen may optionally be substituted with R B It may be substituted with a group selected from:

[0039] In one embodiment, Ring B is morpholinyl.

[0040] In one embodiment, Ring B is morpholinyl; when said morpholinyl contains an —NH— moiety, the nitrogen is optionally R C It may be substituted with a group selected from:

[0041] In one embodiment, Ring B is thiomorpholinyl.

[0042] In one embodiment, Ring B is thiomorpholinyl; when said thiomorpholinyl contains an —NH— moiety, the nitrogen is optionally R C It may be substituted with a group selected from:

[0043] In one embodiment, R 1 is a halo.

[0044] In one embodiment, n is 0.

[0045] In one embodiment, n is 1.

[0046] In one embodiment, n is 2.

[0047] In one embodiment, n is 3.

[0048] In one embodiment, n is 4.

[0049] In one embodiment, a compound of formula (I), wherein: A is a bond, C is 1~6 alkylene or heterocyclyl; Q is a bond; Ring B is morpholinyl or thiomorpholinyl; and n is 0) Peptide dendrons are provided that include one or more residues derived from the modified lysine of

[0050] In one embodiment, a compound of formula (I), wherein: A is a bond, methylene or pyridyl; Q is a bond; Ring B is morpholinyl or thiomorpholinyl; and n is 0) Peptide dendrons are provided that include one or more residues derived from the modified lysine of

[0051] In one embodiment, the modified lysine of formula (I) is a modified lysine of formula (IA): [ka] (In the formula, A, Q, B, R 1 and n is as defined herein.) The modified lysine of formula (IA) may also be referred to as a modified D-lysine.

[0052] In one embodiment, the modified lysine of formula (I) is a modified lysine of formula (IB): [ka] (In the formula, A, Q, B, R 1 and n is as defined herein.) The modified lysine of formula (IB) may also be referred to as modified L-lysine.

[0053] In one embodiment, the modified lysine of formula (I) is 2-Amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid; 2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid; and 2-Amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid is selected from.

[0054] In one embodiment, the modified lysine of formula (I) is (R)-2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid; (R)-2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid; and (R)-2-Amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid is selected from.

[0055] In one embodiment, the modified lysine of formula (I) is (S)-2-Amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid; (S)-2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid; and (S)-2-Amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid is selected from.

[0056] As used herein, the term "substituted," when referring to a chemical group, means that the chemical group has had one or more hydrogen atoms removed and replaced by a substituent. As used herein, the term "substituent" has its ordinary meaning known in the art and refers to a chemical moiety that is covalently bonded to a parent group. As used herein, the term "optionally substituted" means that the chemical group may have no substituents (i.e., unsubstituted) or may have one or more substituents (i.e., substituted). It should be understood that substitution at a given atom is limited by valence. When optional substituents are selected from a list of groups, it should be understood that this definition includes all substituents selected from one of the specified groups or includes substituents selected from two or more specified groups. Two or more of the same substituents, e.g., R 2 When is selected from a list of groups, it should be understood that this definition includes all such substituents selected from one of the specified groups, or includes substituents selected from more than one of the specified groups.

[0057] As used herein, the term "C i~j " denotes a range of carbon atoms, where i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, where j is greater than i. For example, C 1~6 indicates a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms. In some embodiments, the term "C 1~6 " represents 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms.

[0058] As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated hydrocarbon chain. The hydrocarbon chains referred to above can be straight or branched. The term "C i~j "Alkyl" means an alkyl having i to j carbon atoms. 1~6 Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc. Reference to a group such as "butyl" without further modification refers to all forms of butyl, for example, n-butyl and tert-butyl.

[0059] As used herein, the term "alkylene," whether used as part of another term or independently, refers to a saturated hydrocarbon chain. The hydrocarbon chains referred to above can be straight or branched. The term "C i~j "Alkylene" refers to an alkyl having i to j carbon atoms. 1~6 Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-), and butylene (such as -CH2-CH2-CH2-CH2- and -CH2-CH(CH3)-CH2-).

[0060] As used herein, the term "halo" refers to fluoro, chloro, bromo and iodo.

[0061] "Heterocyclyl" means, unless otherwise specified, a saturated, partially saturated or unsaturated monocyclic or bicyclic ring containing 4 to 12 atoms, at least one of which may be carbon- or nitrogen-bonded, and in which a -CH2- group may optionally be replaced by -C(O)-, and in which sulfur atoms in the ring may optionally be oxidized to form S-oxides. Examples and preferred values ​​of the term "heterocyclyl" are morpholino, piperidyl, pyridyl, pyranyl, pyrrolyl, pyrazolyl, isothiazolyl, indolyl, quinolyl, thienyl, 1,3-benzodioxolyl, thiadiazolyl, piperazinyl, thiazolidinyl, pyrrolidinyl, thiomorpholino, pyrrolinyl, homopiperazinyl, 3,5-dioxapiperidinyl, tetrahydropyranyl, imidazolyl, pyrimidyl, pyrazinyl, pyridazinyl, isoxazolyl, N-methylpyrrolyl, 4-pyridone, 1-isoquinolone, 2-pyrrolidone, and 4-thiazolidone. A particular example of the term "heterocyclyl" is pyridyl. In one embodiment, a "heterocyclyl" is a saturated, partially saturated, or unsaturated monocyclic ring containing 5 or 6 atoms, at least one of which is selected from nitrogen, sulfur, or oxygen, which, unless otherwise specified, may be a carbon or nitrogen bond; -CH2- groups may optionally be replaced with -C(O)-, and sulfur atoms in the ring may optionally be oxidized to form S-oxides.

[0062] "Carbocyclyl" refers to a saturated, partially saturated, or unsaturated monocyclic or bicyclic carbon ring containing 3 to 12 atoms; the -CH2- group can optionally be replaced by -C(O)-. In one embodiment, "carbocyclyl" refers to a monocyclic ring containing 5 or 6 atoms, or a bicyclic ring containing 9 or 10 atoms. Suitable values ​​for "carbocyclyl" include cyclopropyl, cyclobutyl, 1-oxocyclopentyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, phenyl, naphthyl, tetralinyl, indanyl, or 1-oxoindanyl. A particular example of "carbocyclyl" is phenyl.

[0063] The "compounds" of the present disclosure include all isotopes of atoms in the compound. Isotopes of an atom include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromide, or iodine in the "compounds" of the present disclosure include, but are not limited to, the following: 1 H, 2 H, 3 H, 11 C. 12 C. 13 C. 14 C. 14 N, 15 N, 16 O. 17 O. 18 O. 31 P, 32 P, 32 S, 33 S, 34 S, 36 S, 17 F, 19 F, 35 Cl, 37 Cl, 79 Br, 81 Br, 127 I and 131In some embodiments, hydrogen includes protium, deuterium, and tritium. In some embodiments, hydrogen refers to protium. In some embodiments, hydrogen refers to deuterium. In some embodiments, hydrogen refers to tritium. In some embodiments, the term "deuterium substituted" or "deuterium substituted" refers to the substitution of deuterium for other isoforms of hydrogen in a chemical group (e.g., protium). In some embodiments, carbon is 12 C and 13 Contains C.

[0064] residue When amino acids within the peptide dendrons described herein are linked together, they form a chain via a peptide bond between the α-amino and carboxy groups. Once linked into a chain, the individual amino acids are referred to as "residues." "Residue" is derived from an amino acid. "Residue" may also be used to describe terminal amino acids linked only through the α-amino or carboxy groups, optionally with the terminal amino group being a modified amino group or the terminal carboxy group being a modified carboxy group (as described herein below). Reference herein to a particular amino acid (e.g., "lysine") may refer to the amino acid itself or to a residue, depending on the context.

[0065] Modified Lysine In any embodiment where modified lysine or residues derived from modified lysine are described, these refer to modified lysine according to formula (I) and embodiments thereof.

[0066] In any embodiment where a modified lysine or modified lysine residue is described, this may refer to a modified D-lysine.

[0067] In any embodiment where a modified lysine or modified lysine residue is described, this may refer to a modified L-lysine.

[0068] Salt Form In any embodiment where a peptide dendron is described, this may refer to the peptide dendron in its salt form.

[0069] As used herein, "salt form" refers to derivatives of the peptide dendrons described herein, where the parent compound is modified by converting one or more present acidic (e.g., carboxyl, etc.) and / or basic (e.g., amine, alkali, etc.) moieties into its salt form. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto. Particular salt forms are pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" are salts that are safe and effective for use in mammals, particularly humans.

[0070] Suitable salt forms of the peptide dendrons described herein include, for example, acid addition salts that can be derived from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) or organic acids (e.g., formic acid, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, trimesic acid, citric acid, lactic acid, phenylacetic acid, benzoic acid, mandelic acid, methanesulfonic acid, napadisilic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, salicylic acid, sulfosalicylic acid, etc.). A particular acid addition salt is the hydrochloride salt.

[0071] Suitable salt forms of the peptide dendrons described herein include, for example, base addition salts of metals derived from inorganic bases (e.g., sodium, potassium, ammonium, and hydroxides, carbonates, and bicarbonates of Groups I-XII of the Periodic Table, such as calcium, magnesium, iron, silver, zinc, and copper) or organic bases (e.g., primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc.). Particular organic amines include, but are not limited to, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine. Further lists of suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences," 20th ed., Mack Publishing Company, Easton, Pa., (1985); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0072] The term peptide dendron as used herein This application describes peptide dendrons containing one or more residues derived from modified lysines of formula (I) described herein. As used herein, peptide dendrons refer to three-dimensional structures containing amino acid residues, where one side chain of the residue, e.g., the ε-amine of lysine, forms a branch point that creates subsequent generations, building the molecule into a 3D macromolecule. Peptide dendrons contain at least one branch point, forming a non-linear molecule in at least one generation (G1).

[0073] The following abbreviations are used herein: Generation 0: The sequence of amino acid residues preceding the first branch point in the peptide dendron. · Branch point: an amino acid residue whose side chain is modified by the addition of an amino acid residue that starts a new generation. Generation: the amino acid residues between branch points, each successive generation is numbered (G1, G2, G3…). Targeting group: a moiety that has affinity for a particular cell type or organ, for example, a sugar-, small molecule-, peptide-, and / or antibody-based moiety.

[0074] By convention, peptides are drawn with the N-terminal amino acid first and the C-terminal amino acid last (written from left to right).

[0075] Lysine (LYS) refers to unmodified lysine unless otherwise specified.

[0076] In any embodiment in which the amino acid may be chiral, this may refer to a D amino acid.

[0077] In any embodiment in which the amino acid may be chiral, this may refer to an L-amino acid.

[0078] In any embodiment in which the amino acids may be chiral, the amino acids may be a mixture of L- and D-amino acids.

[0079] Peptide Dendron In any embodiment where a peptide dendron is mentioned, this may refer to a polypeptide synthesized by a technique that allows precise control over its composition and purity. Suitable techniques include solid-phase peptide synthesis.

[0080] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron where the branch point, generation 0, and subsequent generations together comprise fewer than 120 amino acid residues.

[0081] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron where the branch point, generation 0, and subsequent generations together comprise less than 100 amino acid residues.

[0082] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron where the branch point, generation 0, and subsequent generations together comprise fewer than 80 amino acid residues.

[0083] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron where the branch point, generation 0, and subsequent generations together comprise fewer than 60 amino acid residues.

[0084] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes approximately 52 amino acid residues at the branch point, generation 0, and subsequent generations combined.

[0085] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes 52 amino acid residues at the branch point, generation 0, and subsequent generations combined.

[0086] In any embodiment where a peptide dendron is mentioned, the peptide dendron may be in a salt form.

[0087] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from a modified lysine as defined herein and one or more leucine residues.

[0088] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from a modified lysine as defined herein and one or more arginine residues.

[0089] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from modified lysine as defined herein and one or more lysine residues.

[0090] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more lysine residues.

[0091] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from modified lysine as defined herein, one or more leucine residues, and one or more lysine residues.

[0092] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more leucine residues.

[0093] In any embodiment where a peptide dendron is referred to, at least one generation comprises one or more residues derived from a modified lysine as defined herein and one or more leucine residues.

[0094] In any embodiment where a peptide dendron is referred to, at least one generation comprises one or more residues derived from a modified lysine as defined herein and one or more arginine residues.

[0095] In any embodiment where a peptide dendron is mentioned, at least one generation comprises one or more residues derived from a modified lysine as defined herein and one or more lysine residues.

[0096] In any embodiment where a peptide dendron is mentioned, at least one generation comprises one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more lysine residues.

[0097] In any embodiment where a peptide dendron is mentioned, at least one generation comprises one or more residues derived from a modified lysine as defined herein, one or more leucine residues, and one or more lysine residues.

[0098] In any embodiment where a peptide dendron is referred to, at least one generation comprises one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more leucine residues.

[0099] In any embodiment where a peptide dendron is referred to, a generation includes one or more residues derived from a modified lysine as defined herein and one or more leucine residues.

[0100] In any embodiment where a peptide dendron is referred to, a generation includes one or more residues derived from a modified lysine as defined herein and one or more arginine residues.

[0101] In any embodiment where a peptide dendron is mentioned, a generation includes one or more residues derived from a modified lysine as defined herein and one or more lysine residues.

[0102] In any embodiment where a peptide dendron is referred to, a generation includes one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more lysine residues.

[0103] In any embodiment where a peptide dendron is referred to, a generation includes one or more residues derived from a modified lysine as defined herein, one or more leucine residues, and one or more lysine residues.

[0104] In any embodiment where a peptide dendron is referred to, a generation includes one or more residues derived from a modified lysine as defined herein, one or more arginine residues, and one or more leucine residues.

[0105] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 25-50%.

[0106] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 25-30%.

[0107] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 30-35%.

[0108] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 35-40%.

[0109] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 40-45%.

[0110] In any embodiment where a peptide dendron is mentioned, the % of amino acids having a side chain with a pKa>7.4 is 45-50%.

[0111] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 25 to 50%.

[0112] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 25 to 30%.

[0113] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 30 to 35%.

[0114] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 35 to 40%.

[0115] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 40 to 45%.

[0116] In any embodiment in which a peptide dendron is mentioned, the percentage of amino acids having a side chain with a pKa of 4.0 to 6.5 is 45 to 50%.

[0117] In any embodiment where a peptide dendron is mentioned, the percentage of amino acid residues of subsequent generations having side chains with pKa > 7.4 is 25-50% and the percentage of side chains with pKa = 4.0-6.5 is 25-50%.

[0118] In any embodiment where a peptide dendron is mentioned, the peptide dendron has formula (II): ({X3}{X2}{X1})8({BP}{X3}{X2}{X1})4({BP}{X3}{X2}{X1})2{BP} (II) (In the formula, one of X1, X2, or X3 is a basic amino acid residue; Another X1, X2, or X3 is a hydrophobic amino acid residue; the remaining X1, X2, or X3 are residues derived from modified lysine as defined herein; and BP is the branch point amino acid residue) containing peptide dendrons.

[0119] In formula (II), one of the X groups is attached to the α-amine of the BP amino acid, and the other is attached to a reactive group on the side chain, such as an amino group, for example the ε-amine of lysine. The peptide dendron of formula (II) has the following structure: [ka]

[0120] In the peptide dendron of formula (II), the X1 amino acid residues within the same generation may be the same or different, and / or the X1 amino acid residues in different generations may be the same or different. The same applies to X2 and X3.

[0121] In one embodiment, X1 is a basic amino acid residue.

[0122] In one embodiment, X1 is a hydrophobic amino acid residue.

[0123] In one embodiment, X1 is a residue derived from a modified lysine as defined herein.

[0124] In one embodiment, X2 is a basic amino acid residue.

[0125] In one embodiment, X2 is a hydrophobic amino acid residue.

[0126] In one embodiment, X2 is a residue derived from a modified lysine as defined herein.

[0127] In one embodiment, X3 is a basic amino acid residue.

[0128] In one embodiment, X3 is a hydrophobic amino acid residue.

[0129] In one embodiment, X3 is a residue derived from a modified lysine as defined herein.

[0130] In one embodiment: X1 is a basic amino acid residue; X2 is a hydrophobic amino acid residue; and X3 is a residue derived from a modified lysine as defined herein.

[0131] In one embodiment: X3 is a basic amino acid residue; X2 is a hydrophobic amino acid residue; and X1 is a residue derived from a modified lysine as defined herein.

[0132] One of X1, X2, or X3 is a basic amino acid residue. A basic amino acid residue is an amino acid residue having a side chain capable of carrying a positive charge.

[0133] In one embodiment, the basic amino acid residues are selected from arginine, ornithine, and lysine.

[0134] In one embodiment, the basic amino acid residue is arginine.

[0135] In one embodiment, the basic amino acid residue is ornithine.

[0136] In one embodiment, the basic amino acid residue is lysine.

[0137] In one embodiment, X1 is selected from arginine, ornithine, and lysine.

[0138] In one embodiment, X1 is arginine.

[0139] In one embodiment, X1 is ornithine.

[0140] In one embodiment, X1 is lysine.

[0141] In one embodiment, X2 is selected from arginine, ornithine, and lysine.

[0142] In one embodiment, X2 is arginine.

[0143] In one embodiment, X2 is ornithine.

[0144] In one embodiment, X2 is lysine.

[0145] In one embodiment, X3 is selected from arginine, ornithine, and lysine.

[0146] In one embodiment, X3 is arginine.

[0147] In one embodiment, X3 is ornithine.

[0148] In one embodiment, X3 is lysine.

[0149] One of X1, X2, or X3 is a hydrophobic amino acid residue. A hydrophobic amino acid residue is an amino acid residue having a side chain composed mainly of carbon and hydrogen, and tends to repel water.

[0150] In one embodiment, the hydrophobic amino acid residue is selected from alanine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, methionine, and valine.

[0151] In one embodiment, the hydrophobic amino acid residue is alanine.

[0152] In one embodiment, the hydrophobic amino acid residue is isoleucine.

[0153] In one embodiment, the hydrophobic amino acid residue is leucine.

[0154] In one embodiment, the hydrophobic amino acid residue is phenylalanine.

[0155] In one embodiment, the hydrophobic amino acid residue is tryptophan.

[0156] In one embodiment, the hydrophobic amino acid residue is tiron.

[0157] In one embodiment, the hydrophobic amino acid residue is methionine.

[0158] In one embodiment, the hydrophobic amino acid residue is valine.

[0159] In one embodiment, X1 is selected from alanine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, methionine, and valine.

[0160] In one embodiment, X1 is alanine.

[0161] In one embodiment, X1 is isoleucine.

[0162] In one embodiment, X1 is leucine.

[0163] In one embodiment, X1 is phenylalanine.

[0164] In one embodiment, X1 is tryptophan.

[0165] In one embodiment, X1 is tyrosine.

[0166] In one embodiment, X1 is methionine.

[0167] In one embodiment, X1 is valine.

[0168] In one embodiment, X2 is selected from alanine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, methionine, and valine.

[0169] In one embodiment, X2 is alanine.

[0170] In one embodiment, X2 is isoleucine.

[0171] In one embodiment, X2 is leucine.

[0172] In one embodiment, X2 is phenylalanine.

[0173] In one embodiment, X2 is tryptophan.

[0174] In one embodiment, X2 is tyrosine.

[0175] In one embodiment, X2 is methionine.

[0176] In one embodiment, X2 is valine.

[0177] In one embodiment, X3 is selected from alanine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, methionine, and valine.

[0178] In one embodiment, X3 is alanine.

[0179] In one embodiment, X3 is isoleucine.

[0180] In one embodiment, X3 is leucine.

[0181] In one embodiment, X3 is phenylalanine.

[0182] In one embodiment, X3 is tryptophan.

[0183] In one embodiment, X3 is tyrosine.

[0184] In one embodiment, X3 is methionine.

[0185] In one embodiment, X3 is valine.

[0186] In any embodiment where a peptide dendron is referred to, the peptide dendron has formula (II) as defined herein, wherein: X1 is arginine; X2 is leucine; X3 is a residue derived from a modified lysine as defined herein, and BP is lysine) containing peptide dendrons.

[0187] In any embodiment where a peptide dendron is referred to, the peptide dendron has formula (II) as defined herein, wherein: X1 is a residue derived from a modified lysine as defined herein; X2 is leucine; X3 is arginine; and BP is lysine) containing peptide dendrons.

[0188] Generation 0 In any embodiment where a peptide dendron is referred to, the peptide dendron comprises a sequence of amino acid residues referred to as "generation 0" attached to the first branch point amino acid in the peptide dendron. The generation 0 sequence of amino acid residues can be at the C-terminus or N-terminus, particularly the C-terminus. For example, a peptide dendron having a generation 0 sequence of amino acid residues can be described as follows: [ka]

[0189] If the first branch point is a lysine, the generation 0 sequence in the diagram above can be joined as follows: [ka] (where "··" represents the remainder of the molecule).

[0190] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises a generation 0 sequence of amino acids C-terminal to the branch point amino acid preceding the first generation.

[0191] In any embodiment where a peptide dendron is referred to, the peptide dendron comprises a generation 0 sequence of amino acids N-terminal to the branch point amino acid preceding the first generation.

[0192] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes one or more glycine residues.

[0193] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises a glycine residue.

[0194] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises two glycine residues.

[0195] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises three glycine residues.

[0196] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes one or more valine residues.

[0197] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises a valine residue.

[0198] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes one or more citrulline residues.

[0199] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes a citrulline residue.

[0200] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes one or more serine residues.

[0201] In any embodiment where a generation 0 sequence of amino acids is referenced, the generation 0 sequence comprises a serine residue.

[0202] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes one or more cysteine ​​residues.

[0203] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes a cysteine ​​residue.

[0204] In any embodiment where a generation 0 sequence of amino acids is referenced, the generation 0 sequence may terminate in a cysteine ​​residue. The terminal cysteine ​​allows for further functionalization via thiol chemistry. In another embodiment, the generation 0 sequence may terminate in a reactive derivative of an amino acid, such as lysine, serine, tyrosine, and / or azidophenylalanine. Other reactive derivatives of amino acids include those containing azide, alkyne, cyclopentadiene, and tetrazine groups.

[0205] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence terminates in a cysteine ​​residue in which the terminal carboxy group has been amidated to form a C(O)NH group. Amidation renders the COOH group inactive, reducing the overall charge of the peptide and better mimicking natural peptides.

[0206] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises glycine, valine, citrulline, serine, alanine, lysine, phenylalanine and / or cysteine ​​residues.

[0207] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises glycine, valine, citrulline, serine and / or cysteine ​​residues.

[0208] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence includes glycine, valine, citrulline, serine, and cysteine ​​residues.

[0209] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence consists of GLUTAMATE- ...

[0210] In any embodiment where a generation 0 sequence of amino acids is referred to, the generation 0 sequence comprises the sequence VAL-CIT, VAL-ALA, LYS-PHE, GLY-GLY-PHE-GLY (SEQ ID NO: 1) or GLY-PHE-LEU-GLY (SEQ ID NO: 2).

[0211] Branching point A branch point is an amino acid residue whose side chain is modified by the addition of an amino acid residue that starts a new generation. In the following diagram, branch points are indicated by BP. [ka]

[0212] When the branch point is a lysine, the lysine may form the branch point in the diagram above as follows: [ka] (where "··" represents the remainder of the molecule).

[0213] In any embodiment where a peptide dendron is mentioned, the branch points within the peptide dendron can be at the same or different amino acid residues.

[0214] In any embodiment where a peptide dendron is mentioned, the branch points within the peptide dendron can be at the same amino acid residue.

[0215] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron in which lysines form one or more branch points.

[0216] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron in which lysines form all of the branch points.

[0217] generation Peptide dendrons contain generations, which can be described as follows: [ka] where G1 represents the first generation, G2 represents the second generation, and G3 represents the third generation, collectively referred to as generations. Additional generations can be added by adding an additional branch point (BP) amino acid to the terminal X3 group of the G3 generation, followed by a G4 generation sequence of amino acid residues, such as additional X3-X2-X1 groups.

[0218] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of five amino acid residues.

[0219] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of four amino acid residues.

[0220] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of three amino acid residues.

[0221] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising a generation of two amino acid residues.

[0222] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of four or more amino acid residues.

[0223] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of three or more amino acid residues.

[0224] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations of two or more amino acid residues.

[0225] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of up to five amino acid residues.

[0226] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of up to four amino acid residues.

[0227] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of up to three amino acid residues.

[0228] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations of the same peptide chain.

[0229] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations consisting of the same peptide chain in each generation.

[0230] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations of different peptide chains.

[0231] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations of different peptide chains in each generation.

[0232] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes a generation that includes a basic amino acid residue.

[0233] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations that include hydrophobic amino acid residues.

[0234] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations that include basic amino acid residues and hydrophobic amino acid residues.

[0235] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising generations that include basic amino acid residues, hydrophobic amino acid residues, and residues derived from modified lysines as defined herein.

[0236] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes one or more residues derived from modified lysine as defined herein and generations that include one or more leucine residues.

[0237] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising one or more residues derived from modified lysine as defined herein and generations comprising one or more arginine residues.

[0238] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes generations that include one or more residues derived from modified lysine, one or more arginine residues, and one or more leucine residues as defined herein.

[0239] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes one or more generations that include an ARG-LEU-LYS(modification), where LYS(modification) is a residue derived from a modified lysine as defined herein.

[0240] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising one or more generations of ARG-LEU-LYS(modification), where LYS(modification) is a residue derived from a modified lysine as defined herein.

[0241] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron including all generations containing ARG-LEU-LYS(modification), where LYS(modification) is a residue derived from a modified lysine as defined herein.

[0242] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron including all generations consisting of ARG-LEU-LYS(modification), where LYS(modification) is a residue derived from a modified lysine as defined herein.

[0243] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron that includes one or more generations that include LYS(modified)-LEU-ARG, where LYS(modified) is a residue derived from a modified lysine as defined herein.

[0244] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising one or more generations of LYS(modified)-LEU-ARG, where LYS(modified) is a residue derived from modified lysine as defined herein.

[0245] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron including all generations containing LYS(modified)-LEU-ARG, where LYS(modified) is a residue derived from modified lysine as defined herein.

[0246] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron including all generations consisting of LYS(modified)-LEU-ARG, where LYS(modified) is a residue derived from modified lysine as defined herein.

[0247] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising one generation.

[0248] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising two generations.

[0249] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising three generations.

[0250] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising four generations.

[0251] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising five generations.

[0252] In any embodiment where a peptide dendron is mentioned, this may refer to a first generation peptide dendron.

[0253] In any embodiment where a peptide dendron is mentioned, this may refer to a second generation peptide dendron.

[0254] In any embodiment where a peptide dendron is mentioned, this may refer to a third generation peptide dendron.

[0255] In any embodiment where a peptide dendron is mentioned, this may refer to a fourth generation peptide dendron.

[0256] In any embodiment where a peptide dendron is mentioned, this may refer to a fifth generation peptide dendron.

[0257] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising two or more generations.

[0258] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising three or more generations.

[0259] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising four or more generations.

[0260] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising five or more generations.

[0261] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising five or fewer generations.

[0262] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising four or fewer generations.

[0263] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising three or more generations.

[0264] In any embodiment where a peptide dendron is mentioned, this may refer to a peptide dendron comprising two or fewer generations.

[0265] Polyethylene glycol peptide dendrons In further embodiments, the peptide dendrons described herein may further comprise a biocompatible hydrophilic polymer, such as polyethylene glycol or a polysarcosine group.

[0266] Polyethylene glycol (PEG) is -(OCH2CH2) n - a polymer of repeating subunits, where n is typically >3 and <250. PEG is typically synthesized using ring-opening polymerization of ethylene oxide. PEG polymers can be linear or branched. Branched PEGs typically have 3-30 PEG chains emanating from a central core group.

[0267] In one embodiment, the peptide dendrons described herein may include polyethylene glycol groups and are referred to herein as "polyethylene glycol peptide dendrons" or "PEG peptide dendrons." The PEG groups may form a "stealth layer" that helps stabilize the nanoparticles and reduces nonspecific protein interactions through steric shielding.

[0268] The PEG group can be attached to the terminal amino acid residue in the generation 0 sequence of amino acids of the peptide dendron, optionally via a linking group, as follows: [ka]

[0269] The PEG group can be attached to the peptide dendron via a terminal -O group or via the terminal -CH2- group of the PEG, optionally via a linking group, to the amine or carboxy of the terminal amino acid residue in the generation 0 sequence of amino acids. The PEG group can also be attached to a reactive group on the side chain of the terminal amino acid residue, for example, the -SH group on the side chain of the terminal cysteine, optionally via a linking group, especially when the terminal carboxy group of the terminal cysteine ​​is also amidated.

[0270] In any embodiment in which a polyethylene glycol peptide dendron is mentioned, the terminus of the PEG group not attached to the peptide dendron may be modified or hydrogen. Suitable modifications for the terminus of the polyethylene glycol group include, for example, C 1~4 alkyl, e.g., methyl; or C 1~4 Alkoxy, for example methoxy.

[0271] In any embodiment where a polyethylene glycol peptide dendron is mentioned, there may be a reactive group at the end of the PEG group that is not attached to the peptide dendron. Suitable reactive groups include maleimide, azide, alkyne (e.g., C 2~6These reactive groups include PEG, cyclopentadiene, and cyclopentadiene. This reactive group can be used to attach species such as radiolabels, dyes, and cell-targeting ligands, either before or after PEG conjugation to the peptide dendron.

[0272] In any embodiment where a polyethylene glycol peptide dendron is mentioned, there may be a linking group between the polyethylene glycol and the peptide dendron. Suitable linking groups include C 1~4 alkylamino, such as -CH-CH-NH-PD or PEG-NH-CH-CH-PD, for example -CH-CH-NH-; or C 1~4 Alkylene, for example, to form PEG-CH2-CH2-PD, for example CH2-CH2- (where PD is a peptide dendron).

[0273] When the polyethylene glycol group is attached to a reactive group on the side chain of a terminal amino acid residue, such as the -SH group on the side chain of a terminal cysteine, various methods can be used to attach the polyethylene glycol group. Typically, this results in the polyethylene glycol group being attached to the peptide dendron via a linker group. For example, polyethylene glycol functionalized with a 3-maleimidopropanoic acid functional group linked to the polyethylene glycol via an amide bond is a thiol-reactive reagent. The other end of the polyethylene glycol molecule may be methyl terminated. The reaction using a functionalized polyethylene glycol group of this nature to provide a linker group between the PEG group and the peptide dendron may be illustrated as follows: [ka] (wherein "SH" in "PD-SH" refers to the thiol on the cysteine ​​side chain, PD is a peptide dendron, and "n" is -(OCHCH) n - the number of repeating subunits).

[0274] Further examples of functionalized PEG groups, including branched PEG groups, include the following: [ka]

[0275] Below is an example of such a peptide dendron containing branched PEG groups. [ka] where n is the number of -CHCHO- repeating subunits and Σ n (The sum of all n groups bonded together is 4 to 250.)

[0276] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of 0.5 to 30 kDa.

[0277] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of 2 to 20 kDa.

[0278] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of 4 to 11 kDa.

[0279] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of 1 to 6 kDa.

[0280] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of about 2 kDa.

[0281] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of about 5 kDa.

[0282] In any embodiment where polyethylene glycol is mentioned, this may refer to a polymer having a molecular weight range of about 10 kDa.

[0283] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>3).

[0284] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>10).

[0285] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>20).

[0286] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>30).

[0287] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>50).

[0288] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>100).

[0289] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>150).

[0290] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>200).

[0291] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n>250).

[0292] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<300).

[0293] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<250).

[0294] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<200).

[0295] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<150).

[0296] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<100).

[0297] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<50).

[0298] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<40).

[0299] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<30).

[0300] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<20).

[0301] In any embodiment where polyethylene glycol is mentioned, it is -(OCH2CH2) n - may refer to a polymer containing repeating subunits (n<10).

[0302] targeting group In further embodiments, the peptide dendrons described herein may further comprise a targeting group. A targeting group refers to a targeting moiety that binds to cells and / or promotes cellular internalization. The targeting group may be attached to the PEG group of the peptide dendron, optionally via a linking group, as follows: [ka]

[0303] In any embodiment where a targeting group is mentioned, the targeting group may be selected from a peptide, an antibody, a sugar, or a small molecule.

[0304] Suitable targeting peptides that can be attached to a PEG group, optionally via a linking group, include the following: Transfer receptor targeting peptides, such as Ac-KGGGAWSIIDCSMNYCLYIEG (SEQ ID NO: 3) (where the bold "C" indicates that the cysteines are cross-linked via a disulfide bond) (e.g., https: / / doi.org / 10.21954 / ou.ro.0000d744); Cyclic RGD peptides, such as those targeting the a3b5 integrin in tumor and inflamed vasculature, such as cyclo(-Arg-Gly-Asp-D-Tyr-Lys) (SEQ ID NO: 4): [ka] Muscle-targeting peptides, such as ASSLNIA (SEQ ID NO: 6) (TI Samoylova 1, BF Smith. Muscle Nerve. 1999 Apr;22(4):460-6;doi:10.1002 / (sici)1097-4598(199904)22:4<460::aid-mus6>3.0.co;2-I).

[0305] Suitable targeting antibodies include: T cell-targeting antibodies, such as anti-CD3 Fab (Van Wauwe et al. J Immunol, 1980, 124(6):2708-2713); and Caveolae-targeting antibodies, such as Meca32 monomer FC (e.g., antibodies described in Gabriela M. Marchetti, et al. Commun Biol. 2019;2:92; Published online 2019 Mar 7. doi:10.1038 / s42003-019-0337-2).

[0306] Suitable targeted sugars include those that target the asialoglycoprotein receptor in the liver, such as GalNac and Tri-GalNac containing molecules, e.g. [ka] Examples include:

[0307] Suitable small molecule targeting agents include folic acid.

[0308] Some targeting groups can utilize the -SH group on the targeting group to attach to a PEG group using methods similar to those described above for attaching PEG groups. Typically, this results in the attachment of the targeting group to the polyethylene glycol group via a linker group. Advantageous reagents are bifunctional PEG reagents that can be conjugated to peptide dendrons and targeting groups via thiol chemistry. One such reagent is a PEG reagent functionalized at both ends of the molecule with maleimidopropionate groups. A simplified description of the reaction using a bifunctional polyethylene glycol group of this nature to provide linker groups between both the PEG group and the peptide dendron and the PEG group and the targeting group can be described as follows: [ka] where "SH" in "PD-SH" refers to the thiol on the cysteine ​​side chain, "SH" in "TG-SH" refers to the reactive thiol in the targeting group, PD is the peptide dendron, TG is the targeting group, and "n" is -(OCHCH) n - The number of repeating subunits.

[0309] Peptide targeting groups, or other targeting groups bearing reactive amines (e.g., amine-functionalized sugars), can be attached to PEG groups using alternatively activated PEG reagents. Typically, this results in the attachment of the targeting group to the polyethylene glycol group via a linker group. Advantageous reagents are bifunctional PEG reagents that can be conjugated to peptide dendrons via thiol chemistry and to peptide targeting groups via amide bonds. One such reagent is a PEG reagent functionalized at one end of the molecule with a maleimidopropionate group and at the other end with a tetrafluorophenyl (TFP) ester. A simplified description of reactions using bifunctional polyethylene glycol groups of this nature can be explained as follows: [ka]

[0310] The terminal amino group may be an optionally modified amino group. In one embodiment, the terminal amino group of the peptide dendron may be unmodified. By unmodified terminal amino group, we mean that it is an —NH group.

[0311] In one embodiment, the terminal amino group of the peptide dendron can be a modified amino group.

[0312] The modifications are typically chemical modifications, including, but not limited to, adding chemical groups, creating new bonds, and removing chemical groups. Modified amino groups are well known to those skilled in the art and can include, but are not limited to, acetylation, deamination, N-lower alkyl, N-di-lower alkyl, constrained alkyl (e.g., branched, cyclic, fused, adamantyl), and N-acyl modifications. Modified amino groups can also include, but are not limited to, an N-terminus (e.g., a pyroGlu-protected amino group) or an internal amide bond to attach a radiolabel, fluorescent tag, or affinity tag (e.g., biotin). Modified amino groups can also include, but are not limited to, buffering groups (2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid, 2-amino-6-[(thiomorpholin-3-carbonyl)amino]hexanoic acid, and 2-amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid), and bridging groups (i.e., cyclopentadiene).

[0313] Suitable protecting groups for amino groups are, for example, acyl groups, e.g., alkanoyl groups such as acetyl, alkoxycarbonyl groups, e.g., methoxycarbonyl, ethoxycarbonyl, or t-butoxycarbonyl groups, arylmethoxycarbonyl groups, e.g., benzyloxycarbonyl, or aroyl groups, e.g., benzoyl. A particular modified amino group is acylamino. A particular modified amino group is acetylamino.

[0314] Lower alkyl includes t-butyl, butyl, propyl, isopropyl, ethyl and methyl. 1~4 It is alkyl.

[0315] The terminal carboxy group may be an optionally modified carboxy group. In one embodiment, the terminal carboxy group of the peptide dendron is unmodified. An unmodified terminal carboxy group means that it is a —C(O)OH group.

[0316] In one embodiment, the terminal carboxy group of the peptide dendron is a modified carboxy group.

[0317] The modifications are typically chemical modifications, including, but not limited to, adding chemical groups, creating new bonds, and removing chemical groups. Modified carboxy groups are well known to those of skill in the art and include, but are not limited to, amide, lower alkyl amide, constrained alkyl (e.g., branched, cyclic, fused, adamantyl), dialkyl amide, and lower alkyl ester modifications. Modified carboxy groups can also include, but are not limited to, protecting the carboxy group or adding a radiolabel, a fluorescent tag or affinity tag (e.g., biotin), or a cell-targeting ligand. Suitable protecting groups for carboxy groups are, for example, esterifying groups, such as methylethyl, t-butyl, or benzyl groups. A particular modified carboxy group is -CONH. A particular modified carboxy group is C-terminal amidation. A particular modified carboxy group is a carboxamide group. A particular modified carboxy group is N-(C 1~4 alkyl)carbamoyl group.

[0318] Pharmaceutically Active Agents The compositions and methods described herein are suitable for the delivery of pharmaceutically active agents, which are any substances capable of exerting a pharmacological effect on the human or animal body that leads to a therapeutic result.

[0319] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from genetic material, chemically modified nucleic acids, oligonucleotides, therapeutic peptides, chemotherapeutic agents, proteins, protein conjugates, imaging agents, protein nucleic acids associated with CRISPR technology, and naturally occurring viral components such as capsids or enzymes.

[0320] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from genetic material.

[0321] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids.

[0322] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from genetic material such as DNA or RNA.

[0323] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from genetic material such as DNA and RNA.

[0324] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from genetic material such as DNA and / or RNA.

[0325] In any embodiment where DNA is mentioned, this may be a plasmid, linear DNA, short, single-stranded or double-stranded DNA, compacted vectors such as minicircles and ministrings, folded DNA including hairpin and cruciform DNA, and virus-derived DNA.

[0326] In any embodiment where RNA is mentioned, this may be mRNA or siRNA.

[0327] In any embodiment where RNA is mentioned, this may be mRNA, gRNA, and / or siRNA.

[0328] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from DNA.

[0329] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from RNA.

[0330] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from DNA and mRNA.

[0331] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from mRNA and gRNA.

[0332] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from DNA and gRNA.

[0333] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from DNA, mRNA, and gRNA.

[0334] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from oligonucleotides.

[0335] In any embodiment where an oligonucleotide is mentioned, this may be an antisense oligonucleotide (ASO), RNA interference (RNAi), and aptamer RNA.

[0336] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from chemically modified nucleic acids.

[0337] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from therapeutic peptides.

[0338] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from a chemotherapeutic agent.

[0339] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from proteins.

[0340] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from a protein conjugate.

[0341] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from an imaging agent.

[0342] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent can be selected from protein nucleic acids associated with CRISPR technology.

[0343] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from DNA, gRNA and / or mRNA associated with CRISPR technology.

[0344] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from natural viral components such as, for example, capsids or enzymes.

[0345] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent is a monoclonal antibody, e.g., abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab , rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab; enzymes; such as agalsidase beta, imiglucerase, velaglucerase alfa, taliglucerase, alglucosidase alfa, alglucosidase alfa, laronidase, idursulfase IV, and galsulfase; growth factors; and nucleic acids (i.e., plasmids and mRNA) encoding therapeutic proteins such as IL-2 and IFN-α.

[0346] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from monoclonal antibodies; enzymes; nucleic acids (i.e., plasmids and mRNA) encoding therapeutic proteins such as growth factors and cytokines.

[0347] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (i.e., plasmids and mRNA) encoding therapeutic proteins such as monoclonal antibodies; enzymes; growth factors; transcription factors and cytokines.

[0348] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (i.e., plasmids and mRNA) encoding monoclonal antibodies.

[0349] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (ie, plasmids and mRNA) encoding MEDI8852 and STK11.

[0350] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent can be selected from nucleic acids (i.e., plasmids and mRNA) encoding a monoclonal antibody selected from abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab.

[0351] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (i.e., plasmids and mRNA) encoding enzymes.

[0352] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (i.e., plasmids and mRNA) encoding an enzyme selected from agalsidase beta, imiglucerase, velaglucerase alfa, taliglucerase, alglucosidase alfa, alglucosidase alfa, laronidase, idursulfase injection, and galsulfase.

[0353] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (ie, plasmids and mRNA) encoding growth factors.

[0354] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (ie, plasmids and mRNA) encoding transcription factors.

[0355] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (ie, plasmids and mRNA) encoding the transcription factor HNF-4α.

[0356] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (ie, plasmids and mRNA) encoding cytokines.

[0357] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from nucleic acids (i.e., plasmids and mRNA) encoding cytokines selected from IL-2 and IFN-α.

[0358] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from siRNA.

[0359] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from mRNA.

[0360] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from a gRNA.

[0361] In any embodiment where a pharmaceutically active agent is mentioned, the pharmaceutically active agent may be selected from siRNAs used to reduce protein expression in applications including modulation of oncogene, growth factor, and cytokine expression.

[0362] In any embodiment in which the pharmaceutically active agent is selected from a nucleic acid, the ratio of peptide dendron basic amino acid residues to nucleic acid phosphates (N:P) is about 2:1.

[0363] In any embodiment in which the pharmaceutically active agent is selected from a nucleic acid, the ratio of peptide dendron basic amino acid residues to nucleic acid phosphates (N:P) is about 4:1.

[0364] In any embodiment in which the pharmaceutically active agent is selected from a nucleic acid, the ratio of peptide dendron basic amino acid residues to nucleic acid phosphates (N:P) is about 6:1.

[0365] In any embodiment in which the pharmaceutically active agent is selected from DNA, the ratio of peptide dendron basic amino acid residues to DNA phosphates (N:P) is about 2:1.

[0366] In any embodiment in which the pharmaceutically active agent is selected from DNA, the ratio of peptide dendron basic amino acid residues to DNA phosphates (N:P) is about 4:1.

[0367] In any embodiment in which the pharmaceutically active agent is selected from DNA, the ratio of peptide dendron basic amino acid residues to DNA phosphates (N:P) is about 6:1.

[0368] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to RNA phosphates (N:P) is about 2:1.

[0369] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to RNA phosphates (N:P) is about 4:1.

[0370] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to RNA phosphates (N:P) is about 6:1.

[0371] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to DNA and RNA phosphates (N:P) is about 2:1.

[0372] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to DNA and RNA phosphates (N:P) is about 4:1.

[0373] In any embodiment in which the pharmaceutically active agent is selected from RNA, the ratio of peptide dendron basic amino acid residues to DNA and RNA phosphates (N:P) is about 6:1.

[0374] Drug Delivery Systems In one embodiment, a pharmaceutical delivery system is provided that includes a peptide dendron that includes one or more residues derived from the modified lysine described herein, which can be used to deliver a pharmaceutically active agent to the human or animal body.

[0375] In one embodiment, there is provided the use of a peptide dendron comprising one or more residues derived from a modified lysine as described herein in a pharmaceutical delivery system.

[0376] In one embodiment, there is provided a peptide dendron comprising one or more residues derived from the modified lysines described herein for use as a pharmaceutical delivery system.

[0377] In one embodiment, a delivery system for a pharmaceutically active agent is provided that comprises a peptide dendron that includes one or more residues derived from a modified lysine as described herein.

[0378] In one embodiment, the pharmaceutical delivery systems described herein may comprise one or more different peptide dendrons, for example, two different peptide dendrons. A mixture of peptide dendrons may be used to incorporate targeting moieties, stabilize nanoparticles, and / or synergistically combine different properties.

[0379] In one embodiment, a pharmaceutical delivery system is provided that includes one or more peptide dendrons described herein.

[0380] In any embodiment where "one or more peptide dendrons" is referred to, this may refer to one peptide dendron.

[0381] In any embodiment where "one or more peptide dendrons" is referred to, this may refer to two peptide dendrons.

[0382] In any embodiment where "one or more peptide dendrons" is referred to, this may refer to two or more peptide dendrons.

[0383] In one embodiment, a delivery system for a pharmaceutically active agent is provided that comprises one or more peptide dendrons described herein.

[0384] In one embodiment, the pharmaceutical delivery systems described herein may include peptide dendrons that include a polyethylene glycol group and peptide dendrons that include both a polyethylene glycol group and a targeting group.

[0385] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:50 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0386] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:50.

[0387] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:50.

[0388] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:20 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0389] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:20.

[0390] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:20.

[0391] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:15 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0392] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:15.

[0393] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:15.

[0394] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:10 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0395] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:10.

[0396] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:10.

[0397] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:5 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0398] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:5.

[0399] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:5.

[0400] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 2:4 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0401] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 2:4.

[0402] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 2:4.

[0403] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 1:1 peptide dendron comprising a polyethylene glycol group:peptide dendron comprising both a polyethylene glycol group and a targeting group.

[0404] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 4:2 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0405] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 4:2 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0406] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 4:2.

[0407] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 5:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0408] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 5:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0409] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 5:1.

[0410] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 10:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0411] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 10:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0412] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 10:1.

[0413] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 15:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0414] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 15:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0415] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 15:1.

[0416] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 20:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0417] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 20:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0418] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 20:1.

[0419] In one embodiment, the pharmaceutical delivery system described herein may comprise a ratio of about 50:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0420] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of greater than 50:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0421] In one embodiment, the pharmaceutical delivery systems described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 50:1.

[0422] The peptide dendrons described herein and pharmaceutically active agents can be combined with gentle mixing in a physiologically isotonic buffer (e.g., 5% trehalose or sucrose, 20 mM HEPES, or phosphate-buffered saline (PBS)) to form nanoparticles. These formulations can be delivered immediately, stored at 4°C, or lyophilized for long-term storage.

[0423] The peptide dendrons described herein can be prepared in a form suitable for oral administration (e.g., as tablets or capsules), for parenteral injection (including intravenous, subcutaneous, intradermal, intramuscular, intravascular, or infusion), for topical administration (e.g., as ointments or creams), or for rectal administration (e.g., as suppositories). In particular, the peptide dendrons described herein can be prepared in a form suitable for injection (e.g., by intravenous, subcutaneous, intradermal, or intramuscular injection).

[0424] Further excipients In one embodiment, additional excipients can be added to formulations and compositions comprising one or more peptide dendrons described herein and a pharmaceutically active agent, which may enhance nanoparticle stability, enhance nucleic acid packaging, and result in improved cell delivery and transfection.

[0425] In one embodiment, a formulation is provided comprising one or more peptide dendrons described herein and a lipid.

[0426] In one embodiment, a formulation is provided comprising one or more peptide dendrons as described herein and a lipid selected from N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE) in a 1:1 (w / w) ratio.

[0427] In one embodiment, a formulation is provided comprising one or more peptide dendrons described herein and a lipid selected from a mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), distearoylphosphatidylcholine (DSPC), dimyristoylglycerol (DMG), and heptadecan-9-yl 8((2-hydroxyethyl)(6-oxo-6(undecyloxy)hexyl)amino)octanoate (SM-102), or cholesterol.

[0428] use The peptide dendrons described herein can be used to deliver pharmaceutically active agents suitable for the treatment of a wide range of conditions, including metabolic disorders, immunological disorders, hormonal disorders, cancer, hematological disorders, genetic disorders, infectious diseases, cardiac diseases, bone disorders, respiratory diseases, neurological disorders, adjunctive therapy, ocular diseases, and malabsorption disorders. Therapeutic applications can include systemic expression or targeted delivery (i.e., metastatic tumors, in vivo CAR-T) of proteins (i.e., antibodies for viral therapy).

[0429] In one embodiment, there is provided a pharmaceutical delivery system comprising one or more peptide dendrons described herein for use in therapy.

[0430] In one embodiment, there is provided a delivery system for a pharmaceutically active agent comprising one or more peptide dendrons described herein for use in therapy.

[0431] In one embodiment, a peptide dendron is provided for use in delivering a pharmaceutically active agent to a cell.

[0432] In one embodiment, there is provided a pharmaceutical delivery system comprising one or more peptide dendrons as described herein for use in the treatment of a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disease, a bone disorder, a respiratory disease, a neurological disorder, an adjunctive therapy, an ocular disease, or a malabsorption disorder.

[0433] In one embodiment, there is provided a delivery system for a pharmaceutically active agent comprising one or more peptide dendrons as described herein for use in the treatment of a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disease, a bone disorder, a respiratory disease, a neurological disorder, adjunctive therapy, an ocular disease, or a malabsorption disorder.

[0434] In one embodiment, there is provided a delivery system for pharmaceutically active agents comprising one or more peptide dendrons described herein for use in gene therapy.

[0435] As used herein, the terms "treatment" and "treating," as used herein, refer to ameliorating, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have manifested. In other embodiments, treatment may be administered even in the absence of symptoms. For example, treatment may be administered to susceptible individuals before symptoms manifest (e.g., in light of a history of symptoms and / or in light of genetic predisposition or susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0436] Pharmaceutical Composition In one embodiment, a pharmaceutical composition is provided comprising one or more peptide dendrons described herein.

[0437] In any embodiment in which the pharmaceutical composition comprises one or more peptide dendrons, the pharmaceutical composition may comprise one peptide dendron.

[0438] In any embodiment in which the pharmaceutical composition comprises "one or more peptide dendrons," the pharmaceutical composition may comprise two or more different peptide dendrons.

[0439] In any embodiment in which the pharmaceutical composition comprises "one or more peptide dendrons," the pharmaceutical composition may comprise two different peptide dendrons.

[0440] In any embodiment in which the pharmaceutical composition comprises "one or more peptide dendrons," the pharmaceutical composition may comprise at least two different peptide dendrons.

[0441] In one embodiment, a pharmaceutical composition is provided comprising one or more peptide dendrons described herein and a pharmaceutically active agent.

[0442] In one embodiment, a pharmaceutical composition is provided comprising one or more peptide dendrons described herein.

[0443] In one embodiment, a pharmaceutical composition is provided that includes two or more different peptide dendrons.

[0444] In one embodiment, a composition is provided comprising a pharmaceutically active agent and one or more peptide dendrons described herein.

[0445] In one embodiment, the pharmaceutical compositions described herein may include peptide dendrons that include a polyethylene glycol group and peptide dendrons that include both a polyethylene glycol group and a targeting group.

[0446] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:50 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0447] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:50.

[0448] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:50.

[0449] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:20 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0450] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:20.

[0451] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:20.

[0452] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:15 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0453] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:15.

[0454] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:15.

[0455] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:10 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0456] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:10.

[0457] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:10.

[0458] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:5 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0459] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 1:5.

[0460] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 1:5.

[0461] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 2:4 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0462] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 2:4.

[0463] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 2:4.

[0464] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 1:1 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0465] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 4:2 peptide dendrons comprising polyethylene glycol groups:peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0466] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 4:2.

[0467] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 4:2.

[0468] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 5:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0469] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of greater than 5:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0470] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 5:1.

[0471] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 10:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0472] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 10:1.

[0473] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 10:1.

[0474] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 15:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0475] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 15:1.

[0476] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 15:1.

[0477] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 20:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0478] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 20:1.

[0479] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 20:1.

[0480] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of about 50:1 of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups.

[0481] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of greater than 50:1.

[0482] In one embodiment, the pharmaceutical compositions described herein may comprise a ratio of peptide dendrons comprising polyethylene glycol groups to peptide dendrons comprising both polyethylene glycol groups and targeting groups of less than 50:1.

[0483] In one embodiment, there is provided a pharmaceutical composition comprising one or more peptide dendrons described herein for use in therapy.

[0484] In one embodiment, there is provided a pharmaceutical composition comprising one or more peptide dendrons described herein and a pharmaceutically active agent for use in therapy.

[0485] In one embodiment, there is provided a pharmaceutical composition comprising one or more peptide dendrons described herein for use in the treatment of a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disorder, a bone disorder, a respiratory disorder, a neurological disorder, an adjunctive therapy, an ocular disorder, or a malabsorption disorder.

[0486] In one embodiment, there is provided a pharmaceutical composition comprising one or more peptide dendrons as described herein and a pharmaceutically active agent for use in the treatment of a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disease, a bone disorder, a respiratory disease, a neurological disorder, adjunctive therapy, an ophthalmic disease, or a malabsorption disorder.

[0487] In one embodiment, there is provided a pharmaceutical composition comprising one or more peptide dendrons described herein and a pharmaceutically active agent for use in gene therapy.

[0488] In one embodiment, a pharmaceutical composition is provided comprising one or more peptide dendrons described herein for use in gene therapy.

[0489] Treatment method In one embodiment, there is provided a method of treating a metabolic disorder, immunological disorder, hormonal disorder, cancer, hematological disorder, genetic disorder, infectious disease, cardiac disease, bone disorder, respiratory disease, neurological disorder, adjunctive therapy, ophthalmic disease, or malabsorption disorder, comprising administering to said animal a pharmaceutical composition comprising an effective amount of one or more peptide dendrons described herein.

[0490] In one embodiment, there is provided a method of treating a metabolic disorder, immunological disorder, hormonal disorder, cancer, hematological disorder, genetic disorder, infectious disease, cardiac disease, bone disorder, respiratory disease, neurological disorder, adjunctive therapy, ophthalmic disease, or malabsorption disorder, comprising administering to said animal a pharmaceutical composition comprising an effective amount of one or more peptide dendrons described herein and a pharmaceutically active agent.

[0491] In one embodiment, a method of gene therapy is provided that includes administering one or more peptide dendrons described herein.

[0492] In one embodiment, a method of gene therapy is provided that includes administering one or more peptide dendrons described herein and a pharmaceutically active agent.

[0493] Use of the Pharmaceutical Composition In one embodiment, there is provided a use of a pharmaceutical composition comprising one or more peptide dendrons as described herein in the manufacture of a medicament for treating a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disease, a bone disorder, a respiratory disease, a neurological disorder, an adjunctive therapy, an ophthalmic disorder, or a malabsorption disorder.

[0494] In one embodiment, there is provided a use of a pharmaceutical composition comprising one or more peptide dendrons as described herein and a pharmaceutically active agent in the manufacture of a medicament for treating a metabolic disorder, an immunological disorder, a hormonal disorder, cancer, a hematological disorder, a genetic disorder, an infectious disease, a cardiac disease, a bone disorder, a respiratory disease, a neurological disorder, an adjunctive therapy, an ophthalmic disorder, or a malabsorption disorder.

[0495] In one embodiment, there is provided the use of a pharmaceutical composition comprising one or more peptide dendrons described herein in gene therapy.

[0496] In one embodiment, there is provided the use of a pharmaceutical composition comprising one or more peptide dendrons as described herein and a pharmaceutically active agent in gene therapy.

[0497] kit In one embodiment, a) one or more peptide dendrons as described herein within the first unit; b) a pharmaceutically active agent in a second unit; and c) container means for containing said first and second units; A kit is provided comprising:

[0498] In one embodiment, a) one or more peptide dendrons as described herein within the first unit; b) a pharmaceutical active agent in a second unit; c) container means for containing said first and second units; and d) Instructions for use A kit is provided comprising: [Brief explanation of the drawings]

[0499] [Figure 1] Representative schematic of the synthesis of peptide dendrons (PDs) as described in Example 2. [Figure 2A-2B]Results of peptidic nanoparticle (PNP) stability against anion dissociation (A) and cathepsin B degradation (B) are shown in Example 7. Nanoparticle stability is essential for efficient delivery of intact nucleic acids to target cells, but to function, nucleic acids must be released upon cellular uptake. To this end, PDs are designed to be enzymatically degraded upon cellular uptake. Stimuli-responsive release was confirmed to have improved efficacy and reduced toxicity. The results demonstrate that PD3(MN) and PD3(TM) nanoparticles exhibited significantly increased resistance to anion dissociation compared to nanoparticles formulated with PD1, unmodified (NM)PD, and PD3(His), which readily released DNA at dextran sulfate concentrations above 50 μg / mL. Furthermore, PD3(M), PD3(MN), and PD3(TM) readily released DNA in response to cathepsin B activity compared to unmodified PD3 and all PD2 formulations. [Figure 3A-3C] Results obtained from the in vitro transfection screen described in Example 8. Different cell lines were evaluated, as shown in Figure 3A) the human non-small cell lung cancer cell line H1299 and Figure 3B) the mouse myotube cell line C2C12. Luminescence was collected in quadruplicate and presented as the mean ± standard deviation. In Figure 3C, two different NP formulations, PD3(MN) and PD3(TM), were screened in various cell lines (H1299, C2C12, HEK393, and HEPG2) to identify top performers in different applications. These results show that the MN modification was superior in lung (H1299; a lymphoid-derived human non-small cell lung cancer cell line) and muscle cells (C2C12; mouse myoblasts). Alternatively, the TM modification performed best in cell lines derived from filtering organs, including the kidney (HEK393; human embryonic kidney cells) and liver (HEPG2; a human hepatoma cell line). [Figure 4]Results from luciferase mRNA mouse expression experiments described in Example 9. In vivo translation of DNA NPs was tested in mouse intramuscular expression studies. The reporter protein luciferase was expressed to allow live tracking of expression. Expression was observed and found to be consistent over a one-month monitoring period. Expression was assessed weekly using IVIS, and luminescence was collected four times and presented as the mean ± standard deviation. Results demonstrate that MeO-PEG-PD(MN) efficiently delivers DNA via IM injection in vivo, resulting in stable expression for one month. [Figure 5A-5B] Results from therapeutic expression experiments described in Example 10. Figure 5A shows results using the anti-Flu mAb MEDI8852, and Figure 5B shows results using the tumor suppressor serine / threonine kinase 11 (STK11). MEDI8852 was quantified by indirect ELISA, and expression levels were collected in triplicate and presented as the mean ± standard deviation. Mass spectrometry was used as further confirmation of expression. STK11 was quantified using Western blot. The purpose of NPs is to express therapeutic proteins using host cells. These results demonstrate that NPs have the ability to express a diverse range of therapeutics, including mAbs (i.e., MEDI8852 (anti-flu mAb)) and enzymes (i.e., tumor inhibitor serine / threonine kinase 11 (STK11)), in vivo in cell lines of interest, with the therapeutic's mode of action. [Figures 6A-6C]Results obtained from in vitro targeted DNA NP transfection, as described in Example 11. Targeted PD nanoparticles were tested in various cell lines and confirmed to enhance transfection. In H1299, TfR (Figure 6A) and cRGD (Figure 6B) enhanced overall expression levels (delivered GFP, fluorescence) and kinetics (delivered Gwiz luciferase, luminescence) compared with the commercially available transfection control polyethyleneimine (PEI). Similar improvements in transfection were observed in C2C12 and CT26 compared with non-targeted NPs (Figure 6C). Targeted DNA delivery can be used to increase transfection efficiency and / or achieve cell-specific expression. The PD platform was intentionally designed with a flexible targeting strategy that allows the incorporation of a wide range of peptide- and antibody-based targeting ligands (as shown in Examples 3 and 5), which greatly enhanced transfection. Furthermore, highly specific expression was achieved through PEGylation and targeting. These results demonstrate that targeting can enhance expression in multiple, very different cell lines. [Figure 7] Results from in vivo experiments of lung-targeted NPs described in Example 12. PV1-targeted nanoparticles were administered intravenously to BALB / C mice, and luciferase expression was monitored by IVIS for 8 days. Ex vivo imaging on days 3 and 8 showed that only nanoparticles with optimized ligand density were significantly expressed within the targeted lungs. Cell-specific delivery significantly expands the potential applications of nucleic acid-based therapeutics. These results demonstrate that antibody-based targeting moieties can be used to generate targeted PD NPs, and that PV1 targeting can be used for lung targeting. This example demonstrates that PV1-targeted NPs achieve highly specific expression within mouse lungs for a minimum of 8 days. [Figure 8]Results obtained from transfection of H1299 cells with pDNA nanoparticles versus mRNA PD3(MN) nanoparticles, as described in Example 13. H1299 cells were treated with nanoparticles formulated with either mRNA or pDNA. Transfection kinetics were monitored live using an Incucyte system, where cells were fluorescently imaged and the total red fluorescence intensity was quantified using the instrument software as a measure of transfection. mRNA has been shown to facilitate faster expression kinetics than DNA delivery, without the complex nuclear localization requirement for successful transgene expression. Our NPs successfully packaged and delivered mRNA, demonstrating the promise of achieving faster expression kinetics than DNA delivery. These results demonstrate that mRNA expression is very rapid, but reaches expression levels similar to those achieved with pDNA delivery by 48 hours. [Figure 9] Results from the luciferase mouse expression experiment described in Example 14. CD mice were treated with pDNA or mRNA complexed with MeOPEG-PD3(MN) via intramuscular injection. Expression was assessed using IVIS. Luminescence was collected in quadruplicate and presented as the mean ± standard deviation. These results demonstrate that both mRNA and pDNA can be delivered in vivo via intramuscular injection using the PD platform, with mRNA NPs exhibiting faster expression kinetics. [Figure 10] Results from anti-Flu mAb MEDI8852 expression in a mouse study described in Example 15. BALB / c mice were treated with mRNA NPs by IV injection, and MEDI8852 expression was quantified by ELISA. These results show that significant levels of mAb were detected over an 8-day period, confirming the ability of PDs to express therapeutic proteins in vivo. [Figure 11]Results obtained from transfection of non-activated primary T cells with mCherry mRNA / PD3(MN) NPs with different display of anti-CD3-targeted Fabs, as described in Example 16. These results demonstrate that while non-targeted NPs have the ability to transfect T cells, CD3 targeting significantly enhances delivery when the targeted Fab is displayed at low (25%) to moderate (50%) levels. [Figure 12] The results obtained from CTNNB1 silencing were monitored by reporter assay and Western blot as described in Example 17. The ability of NPs to deliver siRNA was demonstrated in the colon cancer cell line SW480 targeting the oncogene CTNNB1. PD3(MN) and PD3(TM) (containing 1% (w / w) DOPE:DOTMA = 1:1) were shown by Western blot to effectively silence CTNNB1 for at least 5 days. These results indicate that PD NPs can efficiently deliver siRNA to target cells. [Figures 13A-13C] Results obtained from silencing catenin-B by PD3(MN) NPs with different display targeting peptides (cRGD), as described in Example 18. CTNNB1 reporter activity SW480 TopFlash (Figure 13A), CTNNB1 protein levels SW480 (Figure 13B), and Colo205 proliferation (Figure 13C). Many applications require localized delivery. To determine whether "on / off" delivery can be achieved using the NP platform, PEGylated NPs were prepared with different amounts of cRGD display and used to transfect the colon cancer cell lines SW480 and Colo205. PEGylation was observed to abolish or reduce silencing, whereas the introduction of cRGD into the NPs restored NP activity. These results demonstrate that PEGylation can be used to reduce nonspecific cellular uptake and that targeting can be used to restore NP activity. [Figure 14]Results from tumor volume in a mouse metastatic colon cancer model, as described in Example 18. Mice bearing established colon cancer cell line Colo205 tumors were intravenously administered NPs with varying degrees of cRGD targeting on days 8, 9, 15, and 16 (indicated by black arrows). Groups included untreated (A), mice receiving untargeted NPs (B), and mice receiving targeted nanoparticles with moderate (C) to high (D) cRGD display (each data set represents one mouse). Mean values ​​over time (E) and at the end of the study (F) were also determined. In vivo translation of targeted siRNA NPs was assessed in the mouse model. High levels of cRGD display were confirmed to significantly slow tumor growth compared to untargeted NPs. These results demonstrate that cRGD-targeted NPs can enhance siRNA delivery and highlight the importance of targeting ligand density. [Figure 15] Fluorescence spectra of NPs formulated with fluorescently labeled DNA (Cy5) and mRNA (Cy5) as described in Example 19. NPs with Cy5-labeled DNA (Graph 1) are not excited by the blue LED, and therefore no fluorescence is detected at their emission wavelengths (650-700 nm). NPs with Cy3-labeled mRNA (Graph 2) fluoresce upon excitation, with their emission peak near 550-600 nm. Similarly, NPs prepared with Cy5-labeled DNA combined with NPs with Cy3-labeled mRNA (Graph 3) fluoresce at the Cy3 emission peak. Importantly, NPs prepared using a 1:1 mixture of Cy5-labeled DNA and Cy3-labeled mRNA (Graph 4) show an increase in emission at the Cy5 emission near 560-700 nm and a corresponding decrease in fluorescence intensity at the Cy3 emission peak near 550-600 nm. This indicates that the DNA and mRNA in sample 4 are in close proximity (<5 nm), allowing fluorescence resonance energy transfer (FRET) from the Cy3 fluorophore on the mRNA to excite the Cy5 fluorophore on the labeled DNA, evidence of co-encapsulation of mRNA and DNA in the peptide dendron NPs. [Figure 16]Expression kinetics of mRNA / DNA hybrid PDID NP in H1299 cells as described in Example 19. DNA expression is measured by GFP fluorescence area, and mRNA expression is measured by mCherry fluorescence area. GFP expression increases over 100 hours, while mRNA expression peaks at approximately 60 hours. DNA / mRNA co-expression, quantified by overlapping fluorescent signals, has similar kinetics to mRNA expression, peaking at approximately 65 hours. These results demonstrate the successful co-delivery of DNA and mRNA into cells. [Figure 17] Expression kinetics of mRNA / DNA hybrid PDID NP in differentiated C2C12 cells as described in Example 19. Differentiated C2C12 muscle cells are fused into myotubes and are non-dividing, making these cells difficult to transfect. DNA expression is measured by GFP fluorescence area, and mRNA expression is measured by mCherry expression area. GFP expression increases over 75 hours, after which the signal plateaus. mRNA expression peaks at approximately 60 hours and slowly declines. DNA / mRNA co-expression, quantified by overlapping fluorescent signals, has similar kinetics to mRNA expression, peaking at approximately 60 hours. These results demonstrate successful co-delivery of DNA and mRNA into cells. [Example]

[0500] Abbreviations used herein ·NP: nanoparticles; ·PD: peptide dendron; ·PDID: Peptide dendron intercellular delivery Amino acid "CIT": Citrulline The following abbreviations are used herein for modified lysines: [ka]

[0501] The following polymers and ligands were incorporated into the peptide dendrons:

[0502] [Table 1]

[0503] Example 1 Preparation of modified lysine Method 1: Synthesis of 1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione [ka] (Morpholin-4-yl)acetic acid (1 g, 6.89 mmol) was dissolved in dichloromethane (DCM) (25 mL), and N-hydroxysuccinimide (NHS) (872 mg, 7.58 mmol) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (1.60 g, 8.35 mmol) were added. The reaction was stirred at room temperature for 1 hour and then filtered through a 2" x 3" pad of silica gel. The pad was washed with DCM (3 x 25 mL), and the combined filtrate and washings were concentrated to give 1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine 2,5-dione (1.3 g, 78%) as a white solid. 1 H NMR(300MHz,CDCl3)d3.75(d,J=3.6Hz,4H),3.57(s,2H),2.85(s,4H),2.67(d,J=4.2Hz,4H); MS(ESI) calculated value: 242.09, measured value: 243.3(M+1).

[0504] Method 2: Synthesis of 1-{[6-(morpholin-4-yl)pyridine-3-carbonyl]oxy}pyrrolidine-2,5-dione [ka] 6-(Morpholin-4-yl)pyridine-3-carboxylic acid (350 mg, 1.68 mmol) was dissolved in DCM (25 mL) with stirring at room temperature. NHS (213 mg, 1.85 mmol) was added, followed by EDC·HCl (418 mg, 2.18 mmol). The reaction mixture was stirred at room temperature for 1 h and then filtered through a 2" × 3" pad of silica gel. The pad was washed with DCM (3 × 25 mL) and ethyl acetate (25 mL). The combined filtrate and washings were concentrated to give 1-{[6-(morpholin-4-yl)pyridine-3-carbonyl]oxy}pyrrolidine-2,5-dione (325 mg, 63%) as a white solid. 1 H NMR(300MHz,CDCl3)d8.89(s,1H),8.07(dd,J=9.3Hz,1.5Hz,1H),6.60(d,J=9.3Hz,1H),3.8 0(d,J=4.2Hz,4H)3.72(d,J=4.2Hz,4H),2.89(s,4H).MS(ESI) calculated value: 305.1, actual value: 306.3(M+1).

[0505] Method 3: Synthesis of tert-butyl 3-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}thiomorpholine-4-carboxylate [ka] 4-(tert-Butoxycarbonyl)thiomorpholine-3-carboxylic acid (1 g, 4.04 mmol) was dissolved in DCM (25 mL) with stirring at room temperature. NHS (511 mg, 4.44 mmol) was added, followed by EDC·HCl (1.01 g, 5.25 mmol). The reaction mixture was stirred at room temperature for 1 hour and then filtered through a 2" × 3" pad of silica gel. The pad was washed with DCM (3 × 25 mL), and the combined filtrate and washings were concentrated to give tert-butyl 3-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}thiomorpholine-4-carboxylate (1.3 g, 93.4%) as a white solid. 1H NMR(300MHz,CDCl3)d5.38(br s,1H),4.43-4.22(m,1H),3.46-3.21(m,1H),3.19-3.10(m,1H),3.06-2.97(m,1H),2.8 5(s,4H),2.81-2.62(m,1H),2.61-2.42(m,1H),1.47(s,9H).MS(ESI) calculated value: 345.4(M+1).

[0506] Method 4: Synthesis of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(6-morpholinonicotinoyl)-L-lysine [ka] Fluorenylmethyloxycarbonyl chloride L-lysine (Fmoc-Lys-OH) (15.3 g, 41.53 mmol, 1.2 equiv) was dissolved in THF-water (1:1, 800 mL) with mechanical stirring at room temperature. The solution of the ester in DCM (Method 2) prepared above was added in one portion, followed by DIPEA (10.73 g, 82.99 mmol, 2.4 equiv). The reaction was further stirred at room temperature until the starting material was consumed (TLC, 2 h), and then ethyl acetate (EtOAc) (250 mL) was added. The mixture was acidified with HCl (1 M, 200 mL), poured into a separatory funnel, and the layers were separated. The aqueous layer was extracted with EtOAc (2 × 250 mL). The organic layers were combined, washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was obtained as a light brown oily residue, which was dissolved in THF, adsorbed onto silica gel, and purified by flash chromatography on a silica gel column (7" x 3"). The column was washed with 50% ethyl acetate and 100% ethyl acetate in hexanes, and the product was eluted under vacuum. Fractions containing the required product were combined and concentrated under vacuum to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(6-morpholinonicotinoyl)-L-lysine (12.6 g, 65%) as an off-white solid. 1H NMR(500MHz, CDCl3δ 9.26(br s,1H),8.62(d,J=1.5Hz,1H),7.93(dd,J=2.5,9Hz,1H),7.71(d,J=7.5Hz,2H),7.53(dd,J=4.5,7.5Hz, 2H),7.35(t,J=7.5Hz,2H),7.23(q,J=6.5Hz,2H),6.59(t,J=5Hz,1H),6.48(d,J=9Hz,1H),5.99(d,J=8H z,1H),4.41(dd,J=7.5,12.5Hz,1H),4.31(dd,J=12,18Hz,2H),4.15(d,J=7Hz,1H),3.71(t,=4.5Hz,4H ),3.56-3.32(m,6H),1.98-1.87(m,1H),1.86-1.75(m,1H),1.71-1.57(m,2H),1.56-1.39(m,2Hppm;13C NMR (125 MHz, CDCl3 δ 175.2, 166.6, 159.9, 156.6, 146.9, 144.1, 143.9, 141.4, 137.7, 127.9, 127.3, 125.3, 120.1, 119.5, 106.3, 67.2, 66.6, 53.8, 47.3, 45.3, 39.5, 32.0, 28.9, 22.4 ppm; MS (ESI) exact mass calculated for C31H34N4O6 [M+H]+: 559.26, found: 559.35.

[0507] Method 5: Synthesis of (2S)-2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid (N6-(6-morpholinonicotinoyl)-L-lysine) (LYS(MN)) Step 1 [ka] The Fmoc protecting group of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(6-morpholinonicotinoyl)-L-lysine (Method 4) can be removed by standard procedures known in the art, for example using 20% ​​piperidine in DMF.

[0508] Step 2 [ka] The modified lysine was dissolved in 700 μL of NMP (15 mg, 26.87 μmol). To this solution was then added 300 μL of piperidine (piperidine:NMP = 7:3; 1 mL) while stirring. The reaction mixture was stirred at room temperature for 30 min. The modified lysine was then precipitated by centrifugation (4000 g, 10 min, 4 °C) and washed three times with cold diethyl ether (10 mL). 1 Fmoc removal was confirmed using H NMR (500 MHz, CDCl). H NMR (500 MHz, CDCl) δ 11.89 (s, 1H), 8.85 (dd, 1H), 7.89 (dd, 1H), 7.34 (dd, 1H), 3.52-3.71 (m, 8H), 3.35 (t, 1H), 2.72-2.61 (t, 2H), 2.01-1.57 (m, 6H) ppm. MS (ESI) exact mass calculated for [M + HO]: 352.55, found: 352.04.

[0509] Method 6: Synthesis of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4-(tert-butoxycarbonyl)thiomorpholine-3-carbonyl)-L-lysine [ka] Fmoc-L-Lys-OH (8.94 g, 24.26 mmol, 1.2 equiv) was dissolved in THF-water (1:1, 800 mL) with mechanical stirring at room temperature. The solution of the activated ester (Method 3) in DCM prepared above was added in one portion, followed by DIPEA (6.27 g, 48.53 mmol, 2.4 equiv). The reaction was further stirred at room temperature until the starting material was consumed (TLC, 2 h), and then EtOAc (250 mL) was added. The mixture was acidified with HCl (1 M, 200 mL), poured into a separatory funnel, and the layers were separated. The aqueous layer was extracted with EtOAc (2 × 250 mL). The organic layers were combined, washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was obtained as a pale yellow oily residue, which was dissolved in DCM, adsorbed onto silica gel, and purified by flash chromatography on a silica gel column (7" x 3"). The column was washed with 50% to 70% ethyl acetate in hexanes, and the product was eluted under vacuum. Fractions containing the required product were combined and concentrated under vacuum to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(4-(tert-butoxycarbonyl)thiomorpholine-3-carbonyl)-L-lysine (9.1 g, 75%) as an off-white solid. 1H NMR(500MHz,CDCl3δ 7.75(d,J=7.5Hz,2H),7.63-7.51(m,2H),7.38(t,J=7.5Hz,2H),7.29(t,J=7.5Hz,2H),5.71(dd,J=7.5,23Hz,1H),4.97(br s,1H),4.57-4.23(m,4H),4.20(t,J=7Hz,1H),3.51-3.18(m,3H),3.17-2.96(br s,1H),2.77(d,J=12.5Hz,1H),2.70-2.58(m,1H),2.38(d,J=12.5Hz,1H),1.98-1.86(m,1H),1.85-1.73(m,1H),1.66-1.53(m,2H),1.46(br s,12Hppm;13C NMR(125MHz,CDCl3δ MS(ESIC 31 H 39 Calculated exact mass of N3O7S[M+Na]+: 620.24, found: 620.35.

[0510] Method 7: Synthesis of (2S)-2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid (N6-(thiomorpholine-3-carbonyl)-L-lysine) (LYS(TM)) Step 1 [ka] The Fmoc protecting group of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4-(tert-butoxycarbonyl)thiomorpholine-3-carbonyl)-L-lysine (Method 6) can be removed by standard procedures known in the art, for example, using 20% ​​piperidine in DMF. Similarly, the Boc protecting group can be removed by standard techniques known in the art using 30% TFA in DCM.

[0511] Step 2 [ka] The modified lysine was dissolved in 700 μL of NMP (15 mg, 25.12 μmol). To this solution was then added 300 μL of piperidine (piperidine:NMP=7:3; 1 mL) while stirring. The reaction mixture was stirred at room temperature for 30 min to remove the Fmoc protecting group. The modified lysine was then precipitated by centrifugation (4000 g, 10 min, 4°C) and washed three times with cold diethyl ether (10 mL). After air-drying overnight, the product was dissolved in 50% TFA:DCM (1 mL) and stirred at room temperature for 15 min. The TFA:DCM solution was removed using a rotary evaporator, and the precipitate was washed twice with cold ethyl ether. 1 Confirmed by H NMR: δ 11.56-12.04 (1H, br), 7.34 (s, 1H), 3.65-3.76 (dd, 2H), 3.42 (t, J = 7.3 Hz, 1H), 3.01-3.15 (m, 2H), 2.70-2.89 (m, 2H), 2.55-2.61 (t, J = 5.6 Hz, 2H), 2.06-2.18 (m, 2H), 1.74-1.85 (m, 2H), 1.58-1.64 (q, 2H) 1.05 (1H, s) ppm and MS (ESI exact mass calculated [M + HO]: 279.22, found: 279.62).

[0512] Method 8: Synthesis of N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine [ka] The following procedure can be used to generate N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine. Fmoc-L-Lys-OH (1.2 equiv.) can be dissolved in THF-water (1:1, 800 mL) with mechanical stirring at room temperature. A solution of the activated ester in DCM prepared above can be added in one portion, followed by DIPEA (2.4 equiv.). The reaction can be further stirred at room temperature until the starting material is consumed (TLC, 2 h), and then EtOAc (250 mL) can be added. The mixture can be acidified with HCl (1 M, 200 mL), poured into a separatory funnel, and the layers can be separated. The aqueous layer can be extracted with EtOAc (2 x 250 mL). The organic layers can be combined, washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product can be dissolved in DCM, adsorbed onto silica gel, and purified by flash chromatography on a silica gel column (7" x 3"). The column can be washed with 50% to 70% ethyl acetate in hexanes, and the product can be eluted under vacuum. Fractions containing the required product can be combined and concentrated under vacuum to give N-(N(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine.

[0513] Method 9: Synthesis of (2S)-2-amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid (N6-(2-morpholinoacetyl)-L-lysine) (LYS(M)) Step 1 [ka] The Fmoc protecting group of N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine (Method 8) can be removed by standard procedures known in the art, for example using 20% ​​piperidine in DMF.

[0514] Step 2 [ka] The modified lysine was dissolved in 700 μL of NMP (15 mg, 25.02 μmol). To this solution was then added 300 μL of piperidine (piperidine:NMP=7:3; 1 mL) while stirring. The reaction mixture was stirred at room temperature for 30 min. The modified lysine was then precipitated by centrifugation (4000 g, 10 min, 4°C) and washed three times with cold diethyl ether (10 mL). The removal of Fmoc was confirmed by H-NMR: H NMR (500 MHz, CDCl δ 12.01 (br s,1H),3.62-3.74(m,4H),3.40(t,1H),3.29(s,2H),3.10(t,1H),2.59-2.70(m,4H),1.88-2.01(m ,2H), 1.58-1.65(m,2H), and 1.46-1.56(q,2H) and MS (ESI) exact mass calculated value [M+H2O]+: 273.17, observed value: 273.33.

[0515] Example 2 Synthesis of peptide dendrons bearing modified lysines A series of peptide dendrons were synthesized (Table 2). Modified lysines were either incorporated directly during peptide synthesis using modified lysines synthesized by methods 5, 7, and 9 above (these are marked with an * in the table), or incorporated post-peptide synthesis by modifying the lysine side chain (ε-amine) in solution after resin cleavage using N-hydroxysuccinimide chemistry and compounds synthesized by methods 1–3 above. PD1–PD3 and PD3(His) were synthesized for comparison and contain either unmodified lysines (PD1–3) or a histidine instead of lysine (PD3(His)).

[0516] [Table 2]

[0517] [Table 3]

[0518] Step a) Peptide dendron synthesis by direct incorporation of modified lysines or histidines Peptide dendrons were synthesized using standard solid-phase peptide synthesis by fragment condensation on Rink amide resin. Each fragment was synthesized on 2-chlorotrityl resin (a highly acid-labile resin) using Fmoc chemistry and removed from the resin using trifluoroethanol to maintain all protecting groups on the side chains and N-terminus. Peptide dendrons were then assembled using Nα,Nε-di-Fmoc-L-lysine as the fragment and branch point, followed by cleavage from the resin with trifluoroacetic acid (TFA), resulting in C-terminal amidation. This assembly is shown schematically in Figure 1 using peptide dendrons with PD3-based sequences, e.g., PD3(M), PD3(MN), and PD3(TM). All protecting groups were removed during the cleavage step. Crude peptides were purified by high-pressure liquid chromatography (HPLC) using a water:acetonitrile gradient. Purity and mass were confirmed by HPLC and electrospray ionization (ESI) mass spectrometry. Deconvolution tools were used to interpret ESI mass spectra containing forms of the same species with different charge states. Multiply charged species were reconstituted into their singly charged forms, grouped according to m / z value and peak width, and expressed in atomic mass units (amu). PD1: MW 4540.1 [4540.2 amu]. PD2: MW 7097.1 [7097.5 amu]. PD3: MW 7097.1 [7097.5 amu]. PD3(His): MW 7223.1 [7223.1 amu]. PD3(MN)*: MW 9760.0 [9759.9 amu]. PD3(TM)*: MW 8905.5 [8905.5 amu].

[0519] Step b) Synthesis of peptide dendrons by modification of the peptide lysine ε-amine after cleavage of the dendron intermediate from the resin Two micromoles of peptide dendron (PD1, PD2, or PD3) was suspended in freshly prepared 0.1 M sodium bicarbonate (pH 8.0). The mixture was sonicated for 10 min. 40 mM of NHS-functionalized intermediate (Methods 1–3) was prepared in DMAC and added dropwise to the peptide solution (0.5 mM) with stirring. To ensure 100% conversion of the lysine ε-amide, the NHS-functionalized intermediate was added in a 1.5 molar excess relative to lysine per peptide. The reaction was stirred at room temperature for 0.5 h, followed by removal of unreacted intermediates by ultracentrifugation (MWCO 3.0 kDa). Electrospray ionization mass spectrometry was used to confirm the modification. A deconvolution tool was used to interpret ESI mass spectra containing forms of the same species with different charge states. Multiply charged species were converted to their singly charged forms, grouped according to m / z values ​​and peak widths, and expressed in atomic mass units (amu). PD2(M):MW 9904.9[9904.9amu]. PD2(MN):MW 9759.9[9808.1amu(MW+formic acid)]. PD2(TM):MW 8905.5[8905.5amu]. PD3(M):MW 9904.8[9905.0amu]. PD3(MN):MW 9759.9[9808.1amu(MW+formic acid)]. PD3(TM):MW 8905.5[8905.6amu].

[0520] Example 3 Incorporation of targeting moieties or polyethylene glycol (PEG) into peptide dendrons a) Preparation of Methoxy-PEG and Mal-PEG Peptide Dendrons Maleimide-functionalized methoxy-PEG (n=36) (m-dPEG®) 36 -MAL; Quanta BioDesign, Plain City, Ohio) and bis-maleimide PEG (n=19) (Bis-MAL-PEG 19(BroadPharm, San Diego, CA)) (compounds No. 1 and 2 in Table 1) were prepared as 1.25 mM and 5 mM solutions, respectively, in 20 mM sodium citrate buffer at pH 5.5. Equal volumes of peptide dendron solution (prepared as in Example 2, 1 mM) in the same buffer were mixed with 1.25-fold and 5-fold molar excesses of PEG solution, respectively. The reactions were stirred at room temperature for 2 hours and confirmed by mass spectrometry. Excess PEG was removed by dialysis against PBS and water, or 20 mM sodium citrate (pH 5.5) for Mal-PEG-PD conjugates. Electrospray ionization mass spectrometry was used to confirm the modification. MeO-PEG(36)-PD2(MN)MW 11528.2(11528.2amu), MeO-PEG(36)-PD3(TM):MW 10673.9(10674.1amu), MeO-PEG(36)-PD2(MN):MW 11528.2(11529.2amu), MeO-PEG(36)-PD3(TM):MW 10674.2(10673.9amu). Mal-PEG(19)-PD1:MW 8296.4(8297.2amu), Mal-PEG(19)-PD2(MN):MW 10959.3(10960.3amu), Mal-PEG(19)-PD3(MN):MW 10959.3(10960.3amu), and Mal-PEG(19)-PD2(MN):MW 10959.3(10960.3amu).

[0521] b) Preparation of targeted PEG-peptide dendron conjugates The targeting peptides (compounds (numbers 3-6 in Table 1)) were prepared by standard solid-phase peptide synthesis using Fmoc chemistry. N-terminal acetylation was performed using 10% acetic anhydride in DMF, followed by cleavage from the resin. TFP-PEG (n=36)-maleimide (Quanta BioDesign, Plain City, Ohio) (5 mM) and the acylated peptide (7.5 mM) were dissolved in freshly prepared 0.1 M sodium bicarbonate buffer (pH 8.0), and the peptide solution was added to a 1.25 molar excess of PEG solution. The reaction was stirred at room temperature for 30 min, after which the pH was lowered to 5.5 and conjugation was confirmed by mass spectrometry. The buffer was then transferred to a Vivaspin column MWCO 3.5 kDa (Sigma). The purified product was exchanged into 20 mM sodium citrate buffer (pH 5.5) using a PEG-1000 HPLC (Aldrich, St. Louis, MO). The purified product was then added to a two-fold excess of peptide dendron (prepared as in Example 2) in 20 mM sodium citrate buffer (pH 5.5) for 2 hours at room temperature. The modification was confirmed by electrospray ionization mass spectrometry. A deconvolution tool was used to interpret ESI mass spectra containing forms of the same species with different charge states. Multiply charged species were reconstituted to their singly charged forms, grouped according to m / z value and peak width, and expressed in atomic mass units (amu). TfR-PEG-PD3(MN)*: 13855.0 MW (13856.7 amu). cRGD-PEG-PD3(MN)*: 12188.5 MW (12188.2 amu). TfR-PEG-PD3(TM)*: MW 13855.0 MW(13856.7amu). cRGD-PEG-PD3(TM)*: 12188.5 MW (12188.6 amu).

[0522] c) Preparation of Sugar-PEG-Mal Conjugates N,N-Diisopropylethylamine (DIPEA) (0.0860 mmol) was dissolved in amine-functionalized mono- or triacetylgalactosamine (GalNAc) (Compound Nos. 7 and 8 in Table 1; Sussex Research, Ontario, Canada) (0.01620 mmol) and MAL-dPEG® at room temperature. 36The resulting mixture was added to a solution of 0.020261 mmol of α-TFP ester (Quanta BioDesign, Plain City, Ohio) in N,N-dimethylformamide (6 mmol). The reaction was stirred at room temperature for 1 hour, and conjugation was confirmed by HPLC-MS. The product was then purified using reverse-phase HPLC. The purified product was then added to a two-fold excess of peptide dendron (prepared as in Example 2) in 20 mM sodium citrate buffer (pH 5.5) for 2 hours at room temperature. Modification was confirmed by electrospray ionization mass spectrometry. A deconvolution tool (Agilent Mass Hunter Quantitative Analysis) was used to interpret ESI mass spectra containing forms of the same species with different charge states. Multiply charged species were reconstituted into their singly charged forms, grouped according to m / z values ​​and peak widths, and expressed in atomic mass units (amu). GalNAc-PD3™* MW 11708.6 (fragment: 11506.6) [11506.6 amu]. Tri-GalNAc-PD3™*: MW 12679.1 (fragment: 12071.2) [11507.0 amu].

[0523] Example 4 Peptide dendron / DNA nanoparticle (NP) self-assembly into monodisperse nanoparticles Cationic peptide dendrons (PDs) self-assemble with anionic nucleic acids into nanoparticles. The NPs were determined to be spherical with diameters of 50–75 nm by standard nanoparticle techniques, including transmission electron microscopy and dynamic light scattering.

[0524] a) Preparation of NPs Equal amounts of DNA (40 μg / mL; Gwiz Luciferase Plasmid (6732 bp) (Genlantis, San Diego, CA)) and peptide dendron (prepared as in Example 2) solutions were prepared in 20 mM HEPES (pH 7.0). The peptide solution was made at a concentration corresponding to a peptide dendron arginine:DNA phosphate (N:P) ratio of 2:1. The DNA solution was added dropwise to the peptide solution while gently vortexing to ensure homogeneous particles. The DNA / peptide nanoparticles were allowed to complex for 30 min at room temperature. The final concentration of DNA in the NP solution was 20 μg / mL.

[0525] b) Dynamic Light Scattering Dynamic light scattering data were collected using a Zetasizer ZS (Malvern), green laser, and ZEN2112 quartz cuvette. Hydrodynamic diameter and polydispersity index (PDI) were derived using cumulative goodness-of-fit analysis. All data points represent the average of three or more independently prepared samples, as shown in Table 3.

[0526] c) Transmission electron microscopy (TEM) Samples were applied to glow-discharged 400-mesh formva-coated copper grids, negatively stained with 1% uranyl acetate, air-dried, and examined in a transmission electron microscope (Tecnai T12, Thermo Fisher Scientific) at an operating voltage of 80 kV. Digital images were acquired using an AMT bottom-mounted CCD camera and AMT600 software. Morphology was determined as shown in Table 3.

[0527] [Table 4]

[0528] Example 5 Self-assembly of peptide dendron / RNA nanoparticles (NPs) into monodisperse nanoparticles Cationic peptide dendrons (PDs) self-assemble with anionic nucleic acids into nanoparticles. The NPs were determined to be approximately 50 nm in diameter using standard nanoparticle techniques, including dynamic light scattering.

[0529] a) Preparation of NPs Equal amounts of RNA (40 μg / mL) and peptide solution (prepared as in Example 2) were prepared in 20 mM HEPES (pH 7.0). Specifically, cleancap mRNA encoding mcherry (Trilink, San Diego, CA) and siRNA targeting CTNNB1 (Dharmacon / Horizon Discovery, Lafayette, CO) were prepared. The peptide solution was made at a concentration corresponding to a peptide dendron arginine:mRNA phosphate (N:P) ratio of 4:1. The RNA solution was added dropwise to the peptide solution with gentle vortexing to ensure homogeneous particles. The RNA / peptide nanoparticles were allowed to complex for 30 minutes at room temperature. The final concentration of RNA in the NP solution was 20 μg / mL. As an optional step, Lipofectin® (DOTMA:DOPE (% w / w) = 1) (Thermo Fischer Scientific, Waltham, MA) was added to the formulation. Lipofectin® equivalent to 1-4 w / w% of RNA was diluted in 20 mM HPES (pH 7.0). The Lipofectin® solution was added to the peptide dendron solution immediately before the addition of the RNA solution, as described above. Nanoparticles containing Lipofectin® are marked "lipid."

[0530] b) Dynamic Light Scattering Dynamic light scattering data were collected using a Zetasizer ZS (Malvern), green laser, and ZEN2112 quartz cuvette. Hydrodynamic diameter and polydispersity index (PDI) were derived using cumulative goodness-of-fit analysis. All data points represent the average of three or more independently prepared samples. Results are shown in Table 4.

[0531] [Table 5]

[0532] Example 6 Generation of targeted NP nanoparticles a) Preparation of nanoparticles containing methoxy-PEG peptide dendrons and a mixture of targeting PEG-peptide dendron conjugates with DNA or RNA Peptide solutions containing different ratios of peptide dendron (prepared as in Example 2) or methoxy-PEG dendron (prepared as in Example 3a) to targeting PEG peptide conjugate (prepared as in Example 3b) were prepared in 20 mM HEPES (pH 7.0). DNA (gwiz luciferase) (Genlantis, San Diego, CA) or RNA (cleancap mcherry) (Trilink, San Diego, CA) solution (40 μg / mL) in the same buffer was added dropwise to the PD mixture while vortexing to achieve a final peptide dendron arginine:nucleic acid phosphate (N:P) ratio of 4:1 (DNA) and 6:1 (RNA). The nucleic acid / peptide nanoparticles were allowed to complex for 30 minutes at room temperature. The final concentration of nucleic acid in the nanoparticle solution was 20 μg / mL. As an optional step, Lipofectin® (DOTMA:DOPE (% w / w) = 1) (Thermo Fischer Scientific, Waltham, MA) was added to the formulation. RNA and 1-4 w / w% equivalent of Lipofectin® were diluted in 20 mM HPES (pH 7.0). The Lipofectin® solution was added to the peptide dendron solution immediately before the addition of the RNA solution, as described above.

[0533] b) Preparation of nanoparticles containing a mixture of methoxy-PEG peptide dendrons and targeting PEG-antibody conjugates with DNA or RNA For antibody-based targeting ligands, mal-PEG PD nanoparticles were prepared and then conjugated to antibodies (compounds Nos. 5 and 6 in Table 1, modified as follows). Peptide solutions containing different ratios of methoxy-PEG dendron (Example 3a) and mal-PEG dendron (Example 3a) were prepared in 20 mM HEPES (pH 7.0). DNA (gwiz luciferase) (Genlantis, San Diego, CA) or RNA (cleancap mcherry) (Trilink, San Diego, CA) solutions (40 μg / mL) in the same buffer were added dropwise to the PD mixture while vortexing to achieve a peptide dendron arginine:nucleic acid phosphate (N:P) ratio of 4:1 (DNA) and 6:1 (RNA). The nucleic acid / peptide nanoparticles were allowed to complex for 30 minutes at room temperature. The final concentration of nucleic acid in the nanoparticle solution was 20 μg / mL.

[0534] Fabs and / or halfmers (one light and one heavy chain) were engineered with the following unnatural amino acid substitutions in the heavy chain: [ka]

[0535] The unnatural amino acid (NnaaCP1) contains a cyclopentadiene group in its side chain, which can serve as a reactive handle for the Diel-Alder reaction. NnaaCP1 was genetically encoded into an antibody sequence that delivers NnaaCP1 in response to an amber stop (TAG) codon using Chinese hamster ovary (CHO) cells expressing the Methanosarcina mazei pyrrolysine-tRNA synthetase / tRNA(PylRS) / tRNA(Pyl) pair. (Amant et al. Angew Chem, 2019, 58(25):8489-93)

[0536] The engineered antibody-based targeting moiety was added to the nucleic acid / peptide nanoparticle solution at a 1.25 molar excess relative to maleimide for 2 hours at room temperature, as described in Example 6. The mixture was then quenched by the addition of N-acetylcysteine ​​(10-fold molar excess). Excess antibody was removed using ultracentrifugation (MWCO 100 kDa), and the reaction was monitored using the A260 / A280 ratio and dynamic light scattering.

[0537] c) Dynamic Light Scattering Dynamic light scattering data were collected using a Zetasizer ZS (Malvern), green laser, and ZEN2112 quartz cuvette. Hydrodynamic diameter and polydispersity index (PDI) were derived using cumulative goodness-of-fit analysis. All data points represent the average of three or more independently prepared samples.

[0538] [Table 6]

[0539] [Table 7]

[0540] Example 7 NP generated stable nanoparticles that released DNA in response to stimuli a) Anion stability test A dextran sulfate (DS) solution was prepared in 20 mM HEPES and added to the NP solution (prepared as in Example 4) to a final DS concentration of 100 mg / mL to 1000 mg / mL and a DNA concentration of 10 μg / mL. After a 15-minute decomplexation period, 15 μL of each formulation was added to a 2% E-gel containing ethidium bromide and run for 10 minutes. pDNA release was calculated using ImageJ (NIH, Bethesda, Maryland), and the results are shown in Figure 2A (percent intact nanoparticles plotted as a function of DS concentration).

[0541] b) Enzymatic release of DNA NPs were prepared as in Example 4, but in 8 mM L-cysteine ​​HCl buffer (pH 6) at a final DNA concentration of 0.1 μg / μL. Cathepsin-B stock solution (60 units / mL) was then added. The Cat-B NP solution was incubated at 37°C for up to 4 hours. Aliquots were removed at appropriate time points and analyzed by agarose gel electrophoresis to visualize the released DNA. DNA dilutions were prepared at different concentrations as controls and were also loaded onto the same agarose gel for subsequent quantification by bio-rad intensity software analysis. The results are shown in Figure 2B.

[0542] [Table 8]

[0543] Example 8 NPs exhibit cell specificity in in vitro transfection a) In vitro transfection H1299, C2C12, HEK393, and HEPG2 cell lines were seeded into 96-well plates and targeted for 80% confluency within 24 hours. C2C12 cells were then incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% horse serum for 7 days to differentiate from myoblasts to myotubes. After a 24-hour recovery period, NPs (prepared as in Example 2) were added to the cells at 0.2 μg per well in OPTI-MEM (1:10 dilution). After 16 hours, the NP-supplemented medium was removed and fresh culture medium was added. GFP was imaged using an Incucyte (EssenBio) and luciferase / viability was quantified using the standard protocol for the ONE-Glo™ + Tox luciferase reporter (Promega). The results are shown in Figures 3A, 3B, and 3C.

[0544] Example 9 Intramuscular DNA NPs for in vivo transfection a) Intramuscular delivery BALB / C mice were treated with 5 μg per limb of pDNA(Gwiz luciferase) (Genlantis, San Diego, CA) complexed with MeO-PEG-PD(MN) (prepared as in Example 5). Expression was assessed weekly using IVIS (Perkin Elmer). Luminescence was collected in quadruplicate and presented as the mean ± standard deviation. The results are shown in Figure 4.

[0545] Example 10 Therapeutic expression of DNA NPs a) Preparation of MEDI8852 and STK11-encoded PD nanoparticles Nanoparticles were prepared using PD3(MN) and plasmids encoding the therapeutics MEDI8852 and STK11, as described in Example 4. For MED8852 expression, NPs were also prepared with TfR-PEG-PD3(MN) and MeO-PD3(MN) (peptide dendron arginine:nucleic acid phosphate (N:P 4:1), where 25% of the peptide was TfR-PEG-PD3(MN). Equal amounts of DNA (40 μg / mL; MEDI8852 plasmid (prepared in a similar manner as the Minimal CMV expression plasmid from Sigma-Aldrich, St. Louis, MO) or STK11 (Origene, Rockville, MD) plasmid) and peptide dendron solutions were diluted to 20 mM The DNA solution was prepared in HEPES (pH 7.0). The peptide dendron solution was made at a concentration equivalent to a peptide dendron arginine:nucleic acid phosphate (N:P) ratio of 4:1. The DNA solution was added dropwise to the peptide solution while gently vortexing to ensure homogeneous particles. The DNA / peptide nanoparticles were allowed to complex for 30 minutes at room temperature. The final concentration of DNA in the NP solution was 20 μg / mL. As a control, the MEDI8852 plasmid was also prepared using Lipofectamine-2000 (ThermoFisher, Waltham, MA) according to the manufacturer's protocol.

[0546] b) In vitro transfection studies using therapeutic coded nanoparticles H1299 (6,600 cells / well), HEPG2 (50,000 cells / well), and C2C12 (12,000 cells / well) were seeded in 96-well plates in RPMI medium or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S), respectively. After a 24-hour recovery period, cells were washed twice with 100 μL of PBS and once with culture medium. Nanoparticles were then added to the cells at 0.2 μg (H1299) or 0.5 μg (HEPG2 and C2C12) per well in culture medium. After 16 hours, the nanoparticle-containing medium was removed and fresh culture medium was added. MEDI8852 expression was quantified using ELISA as described in (c) below, and STK11 expression was detected using Western blot as described in (d) below. The results are shown in Figures 5A and 5B, respectively.

[0547] c) MEDI8852 ELISA NUNC™ MaxiSorp™ 96-well microplates were coated with 100 μL / well of anti-MEDI8852 antibody (Ali, S et al. Antimicrob Agents Chemother. 2018 Nov;62(11):e00694-18.) at 1 μg / mL in bicarbonate buffer. The plates were incubated overnight at 4°C and washed three times the next morning with 300 μL of PBS-T (phosphate-buffered saline (PBS) + 0.1% Tween 20). All standards, quality control samples, and test samples were diluted 1:250 in PBS + 0.1% Tween 20 containing 0.5% bovine serum albumin (BSA). 100 μL of diluted sample was loaded into each well and incubated for 2 hours. After incubation, the plates were washed three times with 300 μL of PBS-T. The secondary antibody, biotinylated anti-MEDI8852 (Ali, S et al. Antimicrob Agents Chemother. 2018 Nov;62(11):e00694-18), was diluted to 2 μg / mL in PBS-T, and 100 μL was added to each well and incubated at 37°C for 1 hour. The plate was then washed four times with PBS-T, and 100 μL of streptavidin-HRP conjugate (Jackson ImmunoResearch Laboratories, Inc., code 016-030-084) was added at a 1:40 dilution. The plate was then washed four times with PBS-T, and 100 μL of room-temperature equilibrated SureBlue TMB substrate (Thermo Fischer Scientific) was added to all wells. After 15 minutes in the dark, the reaction was quenched with 100 μL of TMB stop solution (Thermo Fischer Scientific). The optical density (OD) of each well was read at 450 nm using a BMG Labtech PHERAstar FSX microplate reader, and the results are shown in Figure 5A.

[0548] e) STK11 Western blot After transfection, cells were lysed using RIPA lysis buffer (Teknova Catalog No. R3792) supplemented with protease and phosphatase inhibitors (Pierce Catalog No. 78442) and centrifuged at 12,000 x g for 5 minutes. The supernatant was collected and quantified using a BCA assay (Thermo Product No. 23227). Ten micrograms of extracted protein per sample was then separated using SDS-polyacrylamide gel electrophoresis at 200 mA for 90 minutes and then transferred to a PVDF membrane using the iBlot system. The membrane was then blocked with TBS-T (20 mM Tris-HCl, pH 7.6) supplemented with 5% BSA. The cells were hybridized overnight with rabbit anti-STK11 (Cell Signaling Catalog No. D60C5) and anti-GAPDH (Cell Signaling Catalog No. 14C10) primary antibodies at a 1:10,000 dilution. The membrane was washed for 30 minutes and then hybridized with HRP-conjugated goat anti-rabbit IgG for 30 minutes. After incubation, the membrane was washed to remove any nonspecific binding and incubated with Pierce SuperSignal West Pico chemiluminescence reagent (Pierce catalog number 34579). Bands were detected using an Image Quant LAS 4000. The results are shown in Figure 5B.

[0549] Example 11 Targeted DNA NP transfection in vitro a) In vitro transfection H1299 (6,600 cells / well), CT26 (10,000 cells / well), and C2C12 (12,000 cells / well) cells were seeded in 96-well plates in RPMI medium or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S), respectively. Before transfection of H1299 and CT26 cells, a 24-hour recovery period was allowed, during which C2C12 myoblasts were incubated in DMEM supplemented with 2% horse serum for at least 7 days to differentiate into myotubes. Subsequently, cells were washed twice with 100 μL of PBS and once with culture medium. Targeted and non-targeted nanoparticles were prepared using the Gwiz luciferase plasmid (Genlantis, San Diego, CA) as described in Example 6. TfR-targeted NPs and cRGD-targeted NPs were prepared using a mixture of MeO-PEG-PD(MN):TfR-PEG-PD(MN) or cRGD-PEG-PD(MN) (materials prepared in Examples 3a and b). 0, 10, and 50% TfR-PEG-PD(MN) and 0, 5, 10, 20, and 30% cRGD-PEG-PD(MN) were used. NPs were added to cells at 0.2 μg per well in culture medium (1:10 dilution). After 16 hours, the nanoparticle-containing medium was removed and fresh culture medium was added. GFP expression was imaged using an Incucyte (Essen BioScience, Ann Arbor, MI), and luciferase / viability was quantified using the ONE-Glo™ + Tox luciferase reporter (Promega, Madison, WI) and standard protocols on a PHERAstar FSX instrument (BMG Lab Tech, Cary, NC). The results are shown in Figures 6A to 6C.

[0550] Example 12 a) Preparation of nanoparticles PV1-targeted MECA32 was engineered and expressed as a halfmer (one light chain and one heavy chain) with an unnatural amino acid substitution in the heavy chain as described in Example 6. Using the protocol described in Example 6, NPs were prepared using PD3(MN)PEG conjugate (prepared as in Example 6) and Gwiz luciferase plasmid (Genlantis, San Diego, CA) in a 5% trehalose buffer at a peptide dendron arginine:nucleoside phosphate (N:P) ratio of 4:1 (75% MeO-PEG-PD3(MN):25% Mal-PEG-PD3(MN)). Nucleic acid phosphate (N:M:Ratio of 4:1 (75% MeO-PEG-PD3(MN):25% Mal-PEG-PD3(MN)) using the protocol described in Example 6. CP1-MECA32 (Gabriela M. Marchetti, et al. Commun Biol. 2019;2:92; published online March 7, 2019, doi:10.1038 / s42003-019-0337-2) was added in a 1.25-fold molar excess based on the moles of maleimide and incubated overnight at room temperature. The reaction was quenched by the addition of N-acetylcysteine ​​(10-fold molar excess), and excess antibody was removed by ultracentrifugation (MWCO 100 kDa).

[0551] b) Lung-targeted delivery in mice BALB / C mice were treated intravenously with 20 μg of Gwiz luciferase pDNA complexed with PD(MN). 10% of the pDNA was labeled with Cy-5 (12 labels / plasmid). Expression and biodistribution were assessed on days 1, 3, and 8 using IVIS (Perkin Elmer). Each treatment was performed in quadruplicate, and quantitative data are presented as the mean ± standard deviation. Results are shown in Figure 7.

[0552] Example 13 In vitro mRNA NP transfection a) Preparation of DNA and mRNA NPs A peptide dendron solution was prepared with PD3(MN) (prepared as in Examples 4 and 5) in 20 mM HEPES (pH 7.0). DNA (Origene, Rockville, MD) or mRNA (Trilink, San Diego, CA) encoding mCherry was diluted in the same buffer (40 μg / mL) and added dropwise with vortexing to a final peptide dendron-arginine-nucleic acid-phosphate (N:P) ratio of 2:1 (DNA) or 4:1 (mRNA). The nucleic acid / peptide nanoparticles were allowed to complex for 15 min at room temperature. The final concentration of nucleic acid in the nanoparticle solution was 20 μg / mL. NPs were characterized using DLS as described in Examples 4 (DNA) and 5 (mRNA).

[0553] b) In vitro transfection H1299 cells were seeded into 96-well plates and allowed to reach 80% confluency within 24 hours. After a 24-hour recovery period, NPs were added to the cells at 0.2 μg per well in culture medium (1:10 dilution). After 4 hours, the medium containing NPs was removed and fresh medium was added. mCherry was imaged using an Incucyte (EssenBio) microscope. The results are shown in Figure 8.

[0554] Example 14 In vivo transfection of pDNA and mRNA Intramuscular delivery NPs were prepared using MeOPEG-PD3(MN) as described in Examples 4 and 5. BALB / C mice were treated with 5 μg per limb of luciferase-encoding conjugated cleancap mRNA (Trilink, San Diego, CA) or Gwiz DNA (Genlantis, San Diego, CA). Expression was assessed weekly using IVIS (Perkin Elmer). Luminescence was collected in quadruplicate and presented as the mean ± standard deviation. The results are shown in Figure 9.

[0555] Example 15 Therapeutic expression of mRNA in vivo Intravenous delivery NPs were prepared using mRNA encoding MeO-PD3(MN) and MEDI8852 as described in Example 5. NPs were administered intravenously to BALB / C mice via tail vein injection (20 μg per mouse). Expression was assessed weekly using an IVIS (Perkin Elmer). Luminescence was collected in quadruplicate and presented as the mean ± standard deviation. The results are shown in Figure 10.

[0556] Example 16 mRNA-targeted transfection of primary T cells Preparation and characterization of T cell-targeted NPs In vitro transfection Fresh primary T cells (similar to those obtained from Cellero, Lowell, MA, but fresh and not frozen) were seeded in 24-well plates (3e6 / well) in RPMI 1640 medium (Sigma). After a 24-hour recovery period, NPs were added to the cells at 100 μM / well in OPTI-MEM. NPs were prepared as described in Example 6 using cleancap mRNA encoding mcherry (Trilink, San Diego, CA) and a mixture of PD3(MN) and Mal-PEG-PD3(MN). NPs were prepared using 0, 25, 50, and 100% Mal-PEG-PD3(MN) and then conjugated with anti-CD3 Fab as described in Example 6. After 4 hours, the medium containing NPs was removed and fresh culture medium was added. RFP was imaged using an Incucyte (EssenBio) and quantified using flow cytometry. The results are shown in Figure 11, where A) mean mcherry intensity, B) percent transfected cells, and C) surface expression of CD3.

[0557] Example 17 In vitro testing of siRNA transfection a) Cell culture Colo205 and SW480 cell lines were obtained from ATTC (Manassas, Virginia). Cultures were maintained in Roswell Park Memorial Institute (RPMI) growth medium (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Colo205 and SW480 CTNNB1 shRNA cell lines were generated by lentiviral transduction of doxycycline-inducible human CTNNB1 shRNA (Dharmacon / Horizon Discovery, Lafayette, CO).

[0558] b) TopFlash CTNNB1 reporter assay and Western blot SW480 tumor cells (ATCC, Manassas, VA) were stably transfected with the TCF1 TopFlash luciferase reporter (EMD Millipore, Burlington, MA). SW480 TopFlash cells were seeded at a density of 2.0 × 10 cells per well in 6-well dishes and treated with cholesterol-conjugated siRNA (Accell siRNA) or siRNA NPs for 48–72 hours as described above. Nanoparticles were prepared as described in Example 5 using a mixture of PD3(MN) or MeO-PEG-PD(MN) with cRGD-PEG-PD(MN) (containing 0, 33, 66, or 100% cRGD-targeting). Cells were lysed, treated with BrightGlo reagent (Promega, Madison, WI), transferred to a 96-well plate, and luciferase reporter activity was measured using an Envision X (Perkin Elmer, Waltham, MA). TopFlash results for PD3(MN):cRGD-PEG-PD3(MN) are shown in Figure 12. TopFlash results for MeO-PEG-PD3(MN):cRGD-PEG-PD3(MN) are shown in Figure 13A. Cell lysates were also evaluated after 5 days of CTNNB1 expression using a standard Western blot protocol, as shown in Figure 13B.

[0559] c) CTNNB1 Western blot and cell proliferation assay Colo205 cells were plated in a 6-well plate at a density of 2.0 x 10 cells per well. As a control, cells were treated with cholesterol-conjugated siRNA. Specifically, 500 nM of Accell control non-targeting siRNA and Accell CTNNB1 siRNA were added to cells in 1 ml per well of Accell transfection medium purchased from Dharmacon / Horizon Discovery (Lafayette, CO). Cholesterol conjugation promotes siRNA delivery at high concentrations. 48 hours after transfection, 2 ml of RPMI + 10% FBS was added per well. For the experiment, Colo205 cells were treated with CTNNB1 siRNA-encapsulated NPs and seeded in a 6-well plate at a density of 2.0 x 10 cells per well. Nanoparticles were prepared as described in Example 5 using a mixture of MeO-PEG-PD(MN) and cRGD-PEG-PD(MN) (containing 0 or 66% cRGD-PD and 1% (w / w siRNA) lipofectin). The growth medium was replaced with 900 μl of optimal minimal essential medium (OPTIMEM), and 100 μl of NP formulation was added per well (100 μM mRNA per well). 16 hours after transfection, 2 ml of RPMI + 10% FBS was added per well. For cell proliferation assays, cells were harvested and counted 72–96 hours after transfection using a ViCell XR cell counter, as shown in Figure 13C.

[0560] Example 18 Targeted siRNA transfection in vivo study a) In vivo tumor growth inhibition assay Seven-week-old female nude mice were injected subcutaneously with 5.0 × 106 Colo205 cells in 200 μl of PBS. The average tumor volume was approximately 100 mm. 3When tumors reached 2000 mm, mice were randomized into treatment groups and injected with NPs (0, 33, and 66% cRGD PD(MN):PEG-PD(MN) prepared as in Example 6) for three consecutive days, followed one week later by two consecutive days. Tumor measurements and mouse weights were recorded two or three times a week for the duration of the experiment. 3 Mice were euthanized when tumors reached a size of 0.05 mm or showed ulceration encompassing more than 50% of the tumor surface. The results are shown in Figure 14.

[0561] Example 19 Hybrid RNA / DNA peptide dendron nanoparticle (NP) self-assembly a) Preparation of NPs Cleancap mRNA (Trilink, San Diego, CA) encoding mCherry and gWiz™ DNA (Aldevron, Fargo, ND) encoding GFP were prepared in a 1:1 mass ratio in 20 mM HEPES (pH 7.0) to a final concentration of 40 μg / mL. A solution of PD3(MN) (prepared as in Example 2) was prepared in 20 mM HEPES (pH 7.0) at a concentration corresponding to a final peptide dendron arginine:nucleic acid phosphate (N:P) ratio of 4:1. The nucleic acid solution was added to the PD3(MN) solution in a 1:1 volume ratio and mixed thoroughly by pipetting. The NPs were allowed to complex for 30 minutes at room temperature. The final concentration of nucleic acid in the NP solution was 20 μg / mL (10 μg / mL mRNA and 10 μg / mL DNA). Control NPs were prepared using only DNA and mRNA at a final nucleic acid concentration of 20 μg / mL.

[0562] b) Dynamic Light Scattering Dynamic light scattering (DLS) data were collected using a Zetasizer ZS (Malvern), green laser, and ZEN2112 quartz cuvette. Hydrodynamic diameter and polydispersity index (PDI) were derived using cumulative goodness-of-fit analysis. All data points represent the average of three or more independently prepared samples. Results are shown in Table 8.

[0563] [Table 9]

[0564] c) Evaluation of co-encapsulation by fluorescence resonance energy transfer (FRET) Using the Label IT® Nucleic Acid Labeling Kit (Mirus Bio, Madison, WI), gWiz™ DNA encoding GFP (Aldevron, Fargo, ND) was labeled with Cy5, and Cleancap mRNA encoding mCherry (Trilink, San Diego, CA) was labeled with Cy3. The following nanoparticles were formulated with PD3(MN) as described above at a final nucleic acid concentration of 20 μg / mL: 1. Cy5 DNA + unlabeled DNA (1:1) co-encapsulated NPs 2. Cy3 mRNA + unlabeled mRNA (1:1) co-encapsulated NPs 3. Cy5 DNA NP + Cy3 mRNA NP (1:1) separately encapsulated 4. Cy5 DNA + Cy3 mRNA (1:1) co-encapsulated NPs The NPs were allowed to complex for 10 minutes before fluorescence analysis. The three NPs listed above were formulated separately, complexed for 10 minutes, and then combined in a 1:1 ratio immediately prior to fluorescence analysis. Fluorescence analysis was performed using a NanoDrop™ 3300 fluorescence spectrophotometer (ThermoFisher Scientific, Waltham, MA). A 2 μL NP sample was placed on the stage, and the sample was excited using a blue LED. Fluorescence emission (relative fluorescence units, RFU) was measured at wavelengths between 450 and 750 nm. The fluorescence spectra of these NP samples are shown in Figure 15.

[0565] d) mRNA and DNA expression in H1299 cells NPs were formulated with PD3(MN) as described in a) using a 1:1 weight ratio of GFP DNA and mCherry mRNA for a final nucleic acid concentration of 20 μg / mL. H1299 cells were plated at 10,000 cells / well in tissue-culture-treated 96-well plates 24 hours prior to treatment. Immediately prior to treatment, growth medium was removed and replaced with 100 μL of Opti-MEM™ I reduced serum medium (ThermoFisher, Waltham, MA). Ten microliters of nanoparticle solution was added to each well (200 ng / well nucleic acid dose). The nanoparticles were incubated with the cells for 4 hours, then the Opti-MEM and nanoparticles were removed and replaced with growth medium. GFP and mCherry protein expression was monitored using an Incucyte live cell analysis system (Sartorius, Goettingen, Germany), capturing fluorescent images of the cells every 4 hours. Images were analyzed using Incucyte software to determine the fluorescent areas in the green (GFP) and red (mCherry) channels, as well as the overlapping area of ​​the red and green signals. Signals were normalized to the peak signal area to monitor expression kinetics. The results are shown in Figure 16.

[0566] e) Expression of mRNA and DNA in C2C12 cells C2C12 cells were plated at 20,000 cells / well in tissue culture-treated 96-well plates. The growth medium was replaced with low-serum differentiation medium (DMEM + 2% horse serum) and cultured for 7 days until confluent, elongated myotubes formed. Nanoparticles were formulated with PD3(MN) as described in a) using GFP DNA and mCherry mRNA at a 1:1 weight ratio for a final nucleic acid concentration of 50 μg / mL. Lipofectin® (DOTMA:DOPE (% w / w) = 1) (Thermo Fischer Scientific, Waltham, MA) was added to the formulation. Lipofectin® equivalent to 1% w / w of the nucleic acid was diluted in 20 mM HPES (pH 7.0). The Lipofectin® solution was added to the PD3(MN) solution immediately before adding the RNA solution, as described above. Immediately prior to treatment, the medium was removed and replaced with 100 μL of Opti-MEM™ I Reduced Serum Medium (ThermoFisher, Waltham, MA). 10 μL of nanoparticle solution was added to each well (200 ng / well) for a nucleic acid dose. The nanoparticles were incubated with the cells for 4 hours, after which the Opti-MEM and nanoparticles were removed and replaced with growth medium. GFP and mCherry protein expression was monitored using an Incucyte sperm cell assay, as described. The results are shown in Figure 17.

Claims

1. Formula (I): 【Chemical 1】 (In the formula, A is a bond, C is １～６ alkylene, carbocyclyl, or heterocyclyl; said carbocyclyl or heterocyclyl optionally has one or more R ２ When said heterocyclyl contains an —NH— moiety, the nitrogen may be substituted with R ２ and optionally substituted with a group selected from Q is a bond, carbocyclyl, or heterocyclyl; said carbocyclyl or heterocyclyl optionally has one or more R ３ When said heterocyclyl contains an —NH— moiety, the nitrogen may be substituted with R Ａ and optionally substituted with a group selected from Ring B is morpholinyl or thiomorpholinyl; where said morpholinyl or thiomorpholinyl contains an —NH— moiety, the nitrogen is optionally substituted with R Ｃ may be substituted with a group selected from R １ , R ２ and R ３ are each independently selected from halo, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl and N-methyl-N-ethylsulfamoyl; n is 0 to 4; R Ａ , R Ｂ and R Ｃ are independently selected from methyl, ethyl, propyl, isopropyl, acetyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, carbamoyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, and N-methyl-N-ethylcarbamoyl. A peptide dendron comprising one or more residues derived from modified lysines of

2. A is a bond, C is １～６ The peptide dendron of claim 1 , which is alkylene or heterocyclyl.

3. The peptide dendron of claim 1 , wherein Q is a bond.

4. The peptide dendron of claim 1 , wherein n is 0.

5. The peptide dendron of claim 1 , wherein ring B is morpholinyl.

6. The peptide dendron of claim 1 , wherein ring B is thiomorpholinyl.

7. A is a bond, methylene, or pyridyl; Q is a bond; Ring B is morpholinyl or thiomorpholinyl; and n is 0; The peptide dendron of claim 1 .

8. The modified lysine-derived residue has formula (IB): 【Chemistry 2】 The peptide dendron according to claim 1 , represented by:

9. The modified lysine-derived residue is (S)-2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid; (S)-2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid; and (S)-2-amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid The peptide dendron of claim 1 ,

10. The peptide dendron of claim 1 comprising less than six generations.

11. 2. The peptide dendron of claim 1, comprising a branch point, generation 0, and a subsequent generation, wherein the branch point, generation 0, and subsequent generations collectively comprise less than 100 amino acid residues.

12. Formula (II): ({X ３ }{X ２ }{X １ }) ８ ({BP}{X ３ }{X ２ }{X １ }) ４ ({BP}{X ３ }{X ２ }{X １ }) ２ {BP} (II) (In the formula, X １ , X ２ , or X ３ is a basic amino acid residue; Another X １ , X ２ , or X ３ is a hydrophobic amino acid residue; The remaining X １ , X ２ , or X ３ is a residue derived from a modified lysine as defined in claim 1; and BP is the branch point amino acid residue.

2. The peptide dendron of claim 1, comprising a peptide dendron of the formula:

13. The peptide dendron of claim 12, wherein the basic amino acid residue is selected from arginine.

14. The peptide dendron of claim 12, wherein the hydrophobic amino acid residue is selected from leucine.

15. 13. The peptide dendron of claim 12, wherein BP is lysine.

16. The peptide dendron of claim 1 , further comprising a generation 0 sequence of amino acid residues attached to the first branch point amino acid of the peptide dendron.

17. The generation 0 sequence consists of GLY-VAL-CIT-GLY-GLY-SER-CYS (SEQ ID NO: 5), with the terminal CYS carboxy group amidated to C(O)NH ２ 17. The peptide dendron of claim 16, which is a group-forming peptide dendron.

18. -(OCH ２ CH ２ ) ｎ 2. The peptide dendron of claim 1, further comprising a polyethylene glycol group consisting of repeating subunits, where n>3.

19. 10. The peptide dendron of claim 1, further comprising a targeting group selected from a peptide, an antibody, a sugar, or a small molecule targeting group.

20. 20. An agent for use in the delivery of a pharmaceutically active agent to a cell, the agent comprising one or more of the peptide dendrons according to any one of claims 1 to 19.

21. A pharmaceutical composition comprising one or more peptide dendrons according to any one of claims 1 to 19 and a pharmaceutically active agent.

22. 22. The pharmaceutical composition of claim 21, wherein the pharmaceutically active agent is genetic material.

23. 23. The pharmaceutical composition of claim 22, wherein the genetic material is DNA.

24. 23. The pharmaceutical composition of claim 22, wherein the genetic material is RNA.

25. 23. The pharmaceutical composition of claim 22, wherein the genetic material is DNA and RNA.

26. 22. The pharmaceutical composition of claim 21, further comprising a lipid.

27. ​​A drug for gene therapy comprising the pharmaceutical composition described in claim 21.

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