WO2015116568A2 - Muscle cell-targeting nanoparticles for vaccination and nucleic acid delivery, and methods of production and use thereof - Google Patents

Abstract

Described herein are conjugates (e.g., nanoparticles), compositions (e.g., nanoparticle-based compositions), kits, methods and platforms for muscle-specific targeting of vaccines and gene therapies. The nanoparticles are composed of charged polymers or charged particles (e.g., polyamidoamide dendrimers) surface-modified with a muscle cell ligand (a targeting moiety) and an anionic enhancer (e.g. a phospholipid) resulting in a multinucleotide-carrying conjugate for introducing vaccines or genes into skeletal muscle cells of a subject (e.g., human and other mammals). The novel compositions described herein find use in muscle-targeted vaccination and gene therapy delivery (e.g., delivery of gene-based vaccines) for the prevention and treatment of disease (e.g., skeletal muscle atrophy, cancer, autoimmune disease, infection).

Description

MUSCLE CELL-TARGETING NANOPARTICLES FOR VACCINATION AND NUCLEIC ACID DELIVERY, AND METHODS OF PRODUCTION AND USE

THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional application 61/932,367, filed January 28, 2014, hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0001.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 26, 2015, is named 59077-01078(7230-184)_SL.txt and is 1,450 bytes in size.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of chemistry, immunology, and medicine. More particularly, the invention relates to compositions, kits, platforms and methods for muscle cell- specific delivery of vaccines and other gene therapies using surface-modified nanoparticles that drastically enhance nucleic acid delivery to muscle cells and enhance the expression and bioavailability of the encoded antigen/protein.

BACKGROUND

[0003] Development of deoxyribonucleic (DNA) vaccine platforms is the challenge that today's vaccine development faces, and the delivery of DNA is the challenge of DNA vaccine platforms. All approved human vaccines and the majority of gene therapies are administered into muscles. The poor efficacy of DNA-based vaccines and that of gene therapies has been attributed to the fact that muscle cells are difficult to efficiently transfect. There is a need for an effective muscle cell-specific nucleic acid delivery platform for vaccines and gene therapies.

SUMMARY [0004] Described herein are muscle cell-targeting nanoparticles for vaccination and nucleic acid delivery. The nanoparticles include a charged particle or charged polymer that is surface- modified for muscle cell-specific targeting. In a typical embodiment, the charged particle or charged polymer is a charged highly branched polymeric dendrimer such as a polyamidoamine dendrimer. Polyamidoamine dendrimer-based vaccine and gene delivery formulations disclosed herein involve a nanoparticle that includes a polyamidoamine (PAMAM) dendrimer having a nucleic acid (e.g., DNA, ribonucleic acid) cargo and conjugated to a muscle cell ligand (targeting moiety) and an anionic enhancer (e.g., a phospholipid). In these formulations, the nanoparticle (surface-modified polyamidoamine dendrimer) is targeted to muscle cells, typically skeletal muscle cells (SMCs). The primary route for administration of conventional vaccines is the intramuscular route. In a typical method described herein, skeletal muscle is the target because its microenvironment is rich in patrolling dendritic cells (Siegrist, Vaccine Immunology); a phenomenon conducive to immune response. Resurgent studies have shown that skeletal muscle cells play an active role in immunity (Marino, et. al., Gene Therapy, 2011) by acting as antigen presenting cells and expressing immunologicaly relevant chemokine and cytokine receptors and co-stimulatory molecules (Weindl, et. al., TRENDS in Immunology, 2005).

[0100] PAMAM G5 dendrimer (hereafter referred to as "G5 dendrimer" or "G5"), with a positive net charge and a 5-10 nm diameter, is a suitable nanoparticle for nucleic acid cargo delivery as it complexes with negatively charged DNA or RNA, has a defined structure, is easy to scale up, and enhances the stability of its nucleic acid cargo. To make this nanoparticle (nanocarrier) muscle-targeted, it was surface-functionalized with a peptide that is shown to have high affinity for human skeletal muscle cells, i.e., the peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 1) (hereafter referred to as "ASSLNIA" (SEQ ID NO: 1) and "the ASSLNIA peptide" (SEQ ID NO: 1) - described in Samoylova et al., Muscle Nerve 1999). Typical compositions and nanocarriers described herein include a charged (e.g., positively- charged) highly branched polymeric dendrimer conjugated to a muscle cell ligand, at least one nucleic acid, and at least one negatively charged moiety that is not a nucleic acid (e.g., a phospholipid).

[0101] Accordingly, described herein is a conjugate (e.g., a nanoparticle) having conjugated thereto (complexed with): 1) at least one muscle cell ligand, 2) at least one nucleic acid, and 3) at least one anionic enhancer. The conjugate can be further conjugated to poly(LC). The conjugate typically includes a charged highly branched polymeric dendrimer. The at least one muscle cell ligand is conjugated to the exterior surface of the conjugate such that the at least one muscle cell ligand specifically binds to muscle cells. The at least one nucleic acid (e.g., DNA, RNA, microRNA, small RNA, and siRNA) can be within an expression vector. A conjugate (e.g., charged highly branched polymeric dendrimer) is typically conjugated to the at least one nucleic acid via electrostatic forces. The at least one nucleic acid can encode an antigen, a peptide or polypeptide that promotes muscle cell growth (e.g., a growth factor), etc. The charged highly branched polymeric dendrimer can be a PAMAM dendrimer. The at least one muscle cell ligand can be, for example, ASSLNIA (SEQ ID NO: 1), TARGEHKEEELI (SEQ ID NO: 2), TGGETS GIKKAP Y AS TTRNR (SEQ ID NO: 3), S HHG V AG VDLGGG ADFKS IA (SEQ ID NO: 4), or RGD. The at least one anionic enhancer is typically a phospholipid (e.g., cardiolipin) or negatively charged molecule having a >1 negative charge in physiological pH.

[0102] Also described herein is a composition including a plurality of conjugates as described herein in a therapeutically effective amount and a pharmaceutically acceptable carrier. In a composition, the charged highly branched polymeric dendrimer is a PAMAM dendrimer. Typically, a portion of the plurality of conjugates are conjugated to the same at least one nucleic acid. The charge ratio of charged highly branched polymeric dendrimer to nucleic acid is greater than 5: 1. The at least one nucleic acid can encode a peptide or polypeptide that promotes muscle cell growth and the therapeutically effective amount is effective for promoting muscle cell growth in a subject having skeletal muscle atrophy. In another embodiment, the nucleic acid encodes an antigen and the therapeutically effective amount is effective for inducing an immune response against the antigen when administered to a subject. The composition can be a vaccine. The nucleic acid can encode an antigen and the therapeutically effective amount is effective for preventing or decreasing growth of a cancerous tumor when administered to a subject having a cancerous tumor or at risk of developing a cancerous tumor. The at least one muscle cell ligand can be, for example, ASSLNIA (SEQ ID NO: 1), TARGEHKEEELI (SEQ ID NO: 2), TGGETS GIKKAP Y AS TTRNR (SEQ ID NO: 3), S HHG V AG VDLGGG ADFKS IA (SEQ ID NO: 4), or RGD. The at least one anionic enhancer is typically a phospholipid having a >1 negative charge in physiological pH. The phospholipid can be, for example, cardiolipin. Alternatively, the anionic enhancer can be alpha-lipoic acid or heparin. Lipoic acid is a cofactor found in mitochondrial respiratory enzyme complexes such as the pyruvate dehydrogenase complex. In its reduced form, lipoic acid (or more appropriately, dihydrolipoic acid) can act as a potent antioxidant and has been shown to be able to increase glucose uptake by skeletal muscle (Eason, et. al., Diabetes, Obesity and Metabolism, 2002). Lipoic acid has been used as an enhancer of transfection in gene delivery (Zheng, et. al., Molecular Pharmaceutics, 2011).

[0103] Yet further described herein is a method of delivering a nucleic acid specifically to muscle cells in a subject. The method includes administering to the subject a composition as described herein. In a typical embodiment, the subject is a human, the at least one nucleic acid encodes an antigen, and the composition is a vaccine. In one such embodiment, the vaccine is an anti-cancer vaccine, the subject has a cancerous tumor or is at risk of developing a cancerous tumor, and administering the composition to the subject results in prevention of or decreased growth of the cancerous tumor. In another embodiment, the vaccine induces an immune response against an infectious agent. In the method, the subject can be a human having skeletal muscle atrophy, the at least one nucleic acid can encode a peptide or polypeptide that promotes muscle cell growth, and the therapeutically effective amount is effective for promoting muscle cell growth in the subject. The subject can have a disease or disorder such as, for example, Muscular Dystrophy, Myasthenia gravis, muscle atrophy due to aging, muscle atrophy due to prolonged bed rest, cancer, AIDS, diabetes, muscle atrophy due to spinal cord injury, muscle atrophy due to a neuromuscular disorder, and Amyotrophic Lateral Sclerosis (ALS). The composition can be administered intramuscularly or intraperitoneally.

[0104] Additionally described herein is a method of expanding muscle cells in vitro. The method includes contacting a plurality of muscle cells in vitro with a plurality of conjugates as described herein under conditions such that the muscle cells proliferate. In another embodiment, the method includes contacting a plurality of progenitor cells with a plurality of conjugates as described herein under conditions such that the progenitor cells commit to muscle cells.

[0105] Still further described herein is a kit for delivering a nucleic acid specifically to muscle cells in a subject. The kit includes: a composition as described herein; instructions for use; and packaging. The kit can further include a cell culture reagent, and the at least one nucleic acid can encode a peptide or polypeptide that promotes skeletal muscle cell growth.

[0010] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0011] As used herein, a "nucleic acid" or a "nucleic acid molecule" means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid), and chemically-modified nucleotides. A "purified" nucleic acid molecule is one that is substantially separated from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The terms include, e.g., a recombinant nucleic acid molecule incorporated into a vector (e.g., an expression vector), a plasmid, a virus, bacterial DNA, an aptamer, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced by polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A "recombinant" nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0012] "Aptamers" are oligonucleic acid (RNA, DNA or both) or peptide molecules that bind to a specific target molecule.

[0013] When referring to an amino acid residue in a peptide, oligopeptide or protein, the terms "amino acid residue", "amino acid" and "residue" are used interchangably and, as used herein, mean an amino acid or amino acid mimetic joined covalently to at least one other amino acid or amino acid mimetic through an amide bond or amide bond mimetic.

[0014] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0015] When referring to a nucleic acid molecule or polypeptide, the term "native" refers to a naturally-occurring (e.g., a wild-type (WT)) nucleic acid or polypeptide.

[0016] As used herein, the term "antigen" or "immunogen" means a molecule that is specifically recognized and bound by an antibody and/or binds to a T cell receptor.

[0017] By the term "anionic enhancer" is meant any moiety that reduces positive charges. An anionic enhancer can be, for example, a negatively-charged molecule added before, during, or after DNA-dendrimer complex formation with the purpose of enhancing transfection efficiency of said complex. Examples include phospholipids and non-phospholipids. An example of a phospholipid is cardiolipin. Examples of non-phospholipid anionic enhancers include small RNAs such as Poly (IC), Heparin and alfa lipoic acid.

[0018] The terms "specific binding" and "specifically binds" refer to that binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, etc., and which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non- covalently bound complex, the binding which occurs is typically electrostatic, hydrogen- bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.

[0019] As used herein, the term "dendrimer" means a charged (e.g., positively-charged, negatively-charged), highly branched polymeric macromolecule with roughly spherical shape. An example of a positively-charged, highly branched polymeric dendrimer is a PAMAM dendrimer. By the terms "PAMAM dendrimer" and "poly-amidoamine dendrimer" is meant a type of dendrimer in which tertiary amines are located at branching points and connections between structural layers are made by amide functional groups. PAMAM dendrimers exhibit many positive charges on their surfaces.

[0020] By the term "derivatized dendrimer" is meant a dendrimer having one or more functional groups conjugated to its surface.

[0021] A "G5-SMC dendrimer" is a nanoconstruct (e.g., conjugate, nanocarrier, nanoparticle, nanovehicle) in which one or more muscle cell ligands (e.g., the ASSLNIA peptide (SEQ ID NO: 1)) that bind SMCs are covalently attached to the functional groups on the surface of a charged (e.g., positively-charged) highly branched polymeric dendrimer (e.g., a PAMAM dendrimer).

[0022] As used herein, the term "derivatized dendrimer" means a dendrimer that has been functionalized (e.g., having one more functional groups conjugated to its surface) or surface- modified. [0023] By the term "conjugated" is meant when one molecule or agent is physically or chemically coupled or adhered to another molecule or agent. Examples of conjugation include covalent linkage and electrostatic complexation. The terms "complexed," "complexed with," and "conjugated" are used interchangeably herein. A "conjugate" as used herein is at least one molecule or agent physically or chemically coupled or adhered to another molecule or agent. The term "conjugate" is at times used interchangeably with "nanoparticle" and "dendrimer."

[0024] As used herein, the phrase "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, CA).

[0025] The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.

[0026] As used herein, the terms "nanoparticle," "nanovehicle" and "nanocarrier" are used interchangeably and mean a microscopic particle whose size is measured in nanometers. For example, a nanoparticle, nanovehicle or nanocarrier as described herein can be a charged particle or charged polymer. In a specific embodiment, a nanoparticle, nanovehicle or nanocarrier as described herein is a muscle cell ligand-nucleic acid-phospholipid-dendrimer conjugate (e.g., G5-SMC conjugated to at least one nucleic acid and an anionic enhancer such as a phospholipid) or a particle combining at least one muscle cell ligand coupled to dendrimer conjugated to nucleic acids coated with phospholipids in a range of 2-600 nm. Typically, an unloaded PDD (e.g., a PDD not formulated with a gene-based vaccine or other nucleic acid) is sized such that at least 60% of the nanoparticles in a preparation are less than 10 nm, while in a typical preparation of gene-based vaccine or other nucleic acid-loaded nanoparticles, 60% or less of the nanoparticles are less than 600 nm.

[0027] As used herein, the term "therapeutic agent" is meant to encompass any molecule, chemical entity, composition, drug, vaccine, or biological agent capable of preventing, treating or mitigating a disease. An example of a therapeutic agent is a nucleic acid encoding a vaccine. Another example of a therapeutic agent is a nucleic acid encoding a protein or peptide for treatment of a muscle-related disease (e.g., Muscular Dystrophy). The term includes nucleic acids (DNA, RNA, microRNA and siRNA reagents), aptamers, small molecule compounds, antisense reagents, antibodies, antimicrobial agents, enzymes, polypeptides, peptides, organic or inorganic molecules, natural or synthetic compounds and the like.

[0028] The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

[0029] As used herein the term "adjuvant" means any material which promotes or enhances the humoral and/or cellular immune response.

[0030] As used herein, the terms "displayed" or "surface exposed" are considered to be synonyms, and refer to molecules (moieties) that are present (e.g., accessible for muscle cell binding) at the external surface of a structure such as a nanoparticle or nanocarrier (e.g., G5- SMC dendrimer).

[0031] By the term "progenitor cells" is meant cells that can commit to muscle cells.

[0032] The expression "biologically compatible form suitable for administration in vivo" as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to any subject, e.g., humans.

[0033] By the phrase "immune response" is meant induction of phagocytic, cytokine, and cellular and humoral responses specific against an antigen, antigens, pathogen, pathogenic agent, cancer cells, etc. An immune response has many facets, some of which are exhibited by the cells of the immune system (e.g., B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). In the innate phase of immune system, antigen-presenting cells interact with an antigen or pathogen or other cells of the immune system, and release cytokines to direct adaptive immunity. The adaptive immune responses are generally divided into two main categories— humoral and cell- mediated. The humoral component of the immune response includes production of antibodies specific for an antigen or pathogen. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the antigen or pathogen. An immune response can include, for example, activation of a CD4 T helper response.

[0034] By the phrases "therapeutically effective amount" and "effective dosage" is meant an amount sufficient to produce a therapeutically (e.g., clinically) desirable result; the exact nature of the result will vary depending on the nature of the disorder being treated. For example, where the disorder to be treated is skeletal muscle atrophy (caused by, for example, Muscular Dystrophy, spinal cord injury, amyotrophic lateral sclerosis (ALS), etc.), the result can be growth (regeneration) of skeletal muscle cells. As another example, where the disorder to be treated is a pathogenic infection, the result can be elimination of the pathogen, a reduction in growth of the pathogen, a reduction in size or elimination of a lesion associated with the pathogen, etc. As yet another example, where the disorder to be treated is cancer (e.g., a cancerous tumor), the result can be elimination of the cancer, a reduction in size or elimination of a cancerous tumor, a reduction in growth of cancerous cells, etc. The compositions and nanocarriers described herein can be administered from one or more times per day to one or more times per week. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions and nanocarriers described herein can include a single treatment or a series of treatments.

[0035] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent described herein, or identified by a method described herein, to a patient, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.

[0036] The terms "patient" "subject" and "individual" are used interchangeably herein, and mean an organism to be treated, including invertebrates and vertebrates including, for example, humans and other mammals. In some cases, the methods of the invention find use in experimental animals, in veterinary applications (e.g., equine, bovine, ovine, canine, feline, avian, etc.), and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, as well as non-human primates.

[0037] Although compositions, kits, platforms and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, platforms and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 is a photograph of mice that were subjected to in vivo delivery of DNA and expression of protein by G5-SMC/CL. The plasmid DNA (pVAXLUC) harbors luciferase that may be detected upon intraperitoneal (ip) injection of D-Luciferin, the substrate for the bioluminescence. Mice received intramuscular (im) vaccinations of 15 ug of pVAXLUC DNA alone, complexed with G5 dendrimer, G5/CL, or G5-SMC/CL. Anesthetized mice were imaged by IVIS, 24 hours post vaccination after an ip injection of 250 μΐ_^ of the D-Luciferin (15 mg/mL). The first mouse from the left is pVACLUC DNA alone; the second is complexed with G5 dendrimer; the third is G5 complex with CL; the fourth is G5-SMC complex with CL.

[0039] FIG. 2 is a graph showing results from an experiment in which an immune response to ovalbumin was analyzed by ELISA. Serial dilutions of mice sera were prepared according to their respective groups: DNA, DNA G5-SMC and PDD (a different nanocarrier for DNA vaccine delivery). The negative control (Control) comprised of sera of untreated mice. Anti- ovalbumin IgG was detected by anti-mouse IgG conjugated to alkaline phosphatase and measured by absorbance at 405 nm.

[0040] FIG. 3 is a photograph and a plot showing in vivo delivery of DNA and expression of protein by G5-MSC. The expression of plasmid DNA (pVAXLUC) harboring firefly luciferase may be detected upon ip injection of D-Luciferin, the substrate of the bioluminescence. Mice received im. vaccinations of 10 ug of pVAXLUC DNA alone (the 2 mice in the left), complexed with G5-SMC/pVAXLUC DNA (3 mice on the right). Anesthetized mice were imaged by IVIS, 24 hours post vaccination after an ip injection of 250 ul of the D- Luciferin (15 mg/ML).

[0041] FIG. 4 Mice received sub-conjunctival injections of 2 ug of mRNA expressing firefly luciferase complexed with PDD, or G5-SMC (left mouse) or 4 ug of mRNA expressing firefly luciferase alone (right mouse both eyes), in a total volume of 5 ul in saline. After 24 hours, an IVIS was performed 10 minutes after intraperitoneal injection of 250 UL of D-Luciferin (15 mg/ML). DETAILED DESCRIPTION

[0042] Described herein are conjugates (e.g., nanoparticles or nanocarriers), compositions, kits, platforms and methods for effective delivery of a therapeutic agent (e.g., a nucleic acid (DNA, RNA) such as a gene-based vaccine or a nucleic acid encoding a polypeptide that promotes muscle cell growth) to muscle cells (e.g., SMCs) of a subject in need thereof. A muscle cell-targeted delivery platform that offers targeted nucleic acid-based vaccine and other nucleic acid delivery to muscle cells is described herein. In a typical composition, a charged (e.g., positively-charged), highly branched polymeric dendrimer is conjugated to 1) at least one muscle cell ligand, 2) at least one nucleic acid, and 3) at least one anionic enhancer (e.g., a phospholipid) for delivery of the therapeutic agent into muscle cells in the subject. The dendrimer makes a complex (conjugate) with a therapeutic agent (e.g., nucleic acid) based on the opposite charge of the dendrimer (positive) and that of the therapeutic agent (negative) or the conjugation may be a covalent chemical linkage. The novel compositions and conjugates described herein find use in muscle-targeted vaccination and gene therapy delivery (e.g., delivery of gene-based vaccines) for the prevention and treatment of disease (e.g., skeletal muscle atrophy, cancer, autoimmune disease, infection).

[0043] In the experiments described below, selective and efficient transfection of muscle cells in vitro by the conjugates (e.g., nanoparticles) described herein was demonstrated. Specifically, transfection efficiency of C2C12 cells by the nanoparticles described herein was >100 times greater than transfection by a control nanoparticle. In vivo gene transfer and protein expression using the nanoparticles described herein in a mouse model was also demonstrated. In another experiment, mice immunized with a G5-SMC dendrimer conjugated to a plasmid expressing Ovalbumin showed a significant Ovalbumin- specific antibody response after a single vaccination. Additionally, successful in vivo delivery of RNA to muscle cells in mice was demonstrated.

[0044] The below described preferred embodiments illustrate adaptations of these compositions, kits, platforms and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below. Biological Methods

[0045] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates). Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, 2006; Medical Immunology, 6^th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. Construction and use of PAMAM dendrimers is also described, for example, in Arashkia et al., Virus Genes 40 (1): 44-52, 2010; Velders et al., J Immunol. 166:5366-5373, 2001; and S. Chauhan, N. K. Jain, P. V. Diwan. (2009) Pre-clinical and behavioural toxicity profile of PAMAM dendrimers in mice. Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences (Online publication date: December 3, 2009).

Platform for Targeting Nucleic Acid-based Vaccines and Other Nucleic Acids to

Muscle Cells In A Subject

[0046] Dendrimers have demonstrated considerable promise in shuttling negatively charged payloads (e.g., nucleic acids) into cells (Shcharbin et al., Colloids and surfaces B, Biointerfaces. 2007;58(2):286-9; Sekowski et al., Colloids and surfaces B, Biointerfaces. 2009;69(l):95-8; Medina SH, and El-Sayed MEH, Chemical reviews. 2009; 109(7):3141-57; Bracci et al., The Journal of biological chemistry. 2003; 278(47):46590-5), and are an ideal nucleic acid (including gene-based vaccine) delivery vehicle as they provide structural control over size and shape (cargo-space), are biocompatible (non-toxic and nonimmunogenic), have precise scaffolding properties, have a well-defined surface-modifiable functionality for specific targeting moieties, have the ability for cellular adhesion and endocytosis and delivery into the cytoplasm or nucleus, have acceptable biodegradation (the ability to safely degrade within the body), and are associated with easy and consistently reproducible (clinical grade) synthesis. Dendrimers are highly branched macromolecules which span from a central core and contain a series of structurally and synthetically distinct layers. Each layer added to the structure of a dendrimer is referred to as a "generation," in a clear reference to the stepwise growth of the macromolecule. PAMAM dendrimers have been developed and are now commercially available. Described herein are novel dendrimer-based compositions and platforms that when complexed with a therapeutic agent, such as a nucleic acid-based vaccine or other nucleic acid (e.g., a nucleic acid encoding a growth factor), target the therapeutic agent primarily to skeletal muscle cells in a subject via the muscle cell ligand (e.g., the muscle cell targeting peptide ASSLNIA (SEQ ID NO: 1)) and the negatively charged moiety that is not a nucleic acid which is typically a phospholipid. A nucleic acid may be complexed to the combination of dendrimer, phospholipid, and muscle cell targeting peptide by incubation in room temperature or be coupled via direct conjugation of the nucleic acid to dendrimer.

[0047] A typical composition for nucleic acid-based vaccine or other nucleic acid delivery as described herein includes a charged highly branched polymeric dendrimer complexed with (conjugated to): 1) at least one muscle cell ligand on the exterior surface of the charged highly branched polymeric dendrimer, 2) at least one nucleic acid, and 3) at least one anionic enhancer conjugated to the exterior surface of the charged highly branched polymeric dendrimer. The charged highly branched polymeric dendrimer is typically a PAMAM dendrimer, e.g., a G5 PAMAM dendrimer. The at least one muscle cell ligand is conjugated to the exterior surface of the charged highly branched polymeric dendrimer such that the at least one muscle cell ligand specifically binds to skeletal muscle cells. The at least one muscle cell ligand is any moiety that binds to muscle cells, particularly to SMCs. Examples of muscle cell ligands include ASSLNIA (SEQ ID NO: 1), TARGEHKEEELI (SEQ ID NO: 2), TGGETS GIKKAP Y AS TTRNR (SEQ ID NO: 3), SHHGVAGVDLGGGADFKSIA (SEQ ID NO: 4), and RGD. In a typical embodiment, the muscle cell targeting peptide is the ASSLNIA peptide (SEQ ID NO: 1). However, any suitable muscle cell targeting peptide can be used. Other muscle targeting moieties may include "aptamers", "darpins" (designed ankyrin repeat proteins that are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding), peptides, proteins, or non-peptide molecules that bind skeletal muscle cells or for the purpose of Daul targeting, are added to target or recruit other cells. Daul targeting may include the use of chemo-attractants, cytokines, or their receptors. The at least one nucleic acid can be any type of nucleic acid, including, for example, DNA, RNA, aptamer(s), microRNA, small RNA, and siRNA.

[0048] The at least one nucleic acid can be present within an expression vector or plasmid, and can be complexed with or conjugated to the dendrimer by any suitable means, e.g., electrostatic forces or covalently. In one embodiment, the at least one nucleic acid encodes an antigen and together with the nanoparticle is a vaccine. In this embodiment, the nanoparticle (conjugate) can be administered to a subject for inducing an immune response against the antigen in the subject. The antigen can be from a pathogen or can be a cancer cell antigen. In another embodiment, the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth, e.g., a growth factor (e.g., insulin-like growth factor 1). In this embodiment, the conjugate is particularly useful for promoting muscle cell growth in a subject having skeletal muscle atrophy. The anionic enhancer is typically a phospholipid, e.g., a phospholipid having a >1 negative charge at physiological pH. Any suitable phospholipid can be used, such as cardiolipin (CL). Other anionic enhancers include alpha-lipoic acid, heparin and small RNAs such as Poly (IC). In some embodiments, a nanoparticle as described herein may be further complexed with or conjugated to Poly I:C (also known as "poly(LC)", "poly (IC)" and "poly I:C") which is negatively charged. The anionic enhancer is typically biocompatible (non-toxic and nonimmunogenic). Poly I:C is a synthetic double- stranded RNA analog composed of one polyinosinic strand and one polycytidylic strand. This mimic serves as a ligand for the Toll-like receptor 3 (TLR3), a highly conserved receptor involved in the recognition of viral dsRNA (Matsumoto, et. al., Advanced Drug Delivery Reviews, 2008). Upon binding of dsRNA (or poly I:C), TLR3 mediates the production of cytokines required for immune response. For this reason, poly I:C is used as an adjuvant in vaccine formulations (Trumpfheller, et. al., Proceedings of the National Academy of Sciences, 2008).

[0049] Compositions including such conjugates (e.g., nanoparticles) and a therapeutically effective, pharmaceutically acceptable carrier are described herein. A typical composition includes a plurality of conjugates or nanoparticles as described herein. Generally, the at least one nucleic acid is larger than a single conjugate described herein, and in a composition, a plurality of conjugates wrap around (are conjugated to) a single nucleic acid molecule. In the experiments described below, the nucleic acid/dendrimer ratio was optimized. From these experiments, it was found that a ratio of nucleic acid to charged highly branched polymeric dendrimer greater than 1:5 is optimal for condensation and protection of the nucleic acid.

[0050] In one embodiment of a composition, the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth and the therapeutically effective amount is effective for promoting muscle cell growth in a subject having skeletal muscle atrophy. In this embodiment, the peptide or polypeptide can be, for example, a growth factor. Examples of growth factors that promote or increase muscle cell growth include insulin-like growth factor 1 and fibroblast growth factor. In another embodiment, the nucleic acid encodes an antigen and the therapeutically effective amount is effective for inducing an immune response against the antigen when administered to a subject. As mentioned above, the antigen can be derived from a pathogen for inducing an immune response against the pathogen or can be a cancer cell antigen for inducing an immune response against cancer cells. In these embodiments, the composition is a vaccine.

[0051] Examples of pathogens that can be treated and/or vaccinated for using the compositions, methods, kits and platforms described herein include but are not limited to, pathogenic parasitic, bacterial, fungal, and viral organisms. Table 1 lists several examples:

Table 1 - Pathogens

Leishmania species (e.g., L. major, L. tropica, L. aethiopica, L. mexicana, L. donovani, L. infantum syn. L. chagas),

Streptococcus species,

Candida species,

Brucella species,

Salmonella species,

Shigella species,

Pseudomonas species,

Bordetella species,

Clostridium species,

Norwalk virus,

Bacillus anthracis,

Mycobacterium tuberculosis,

HIV, Chlamydia species,

human Papillomaviruses,

Influenza virus,

Paramyxovirus species,

Herpes virus,

Cytomegalovirus ,

Varicella-Zoster virus,

Epstein-Barr virus,

Hepatitis viruses,

Plasmodium species (e.g., Plasmodium(p) falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi),

Trichomonas species,

sexually transmitted disease agents,

arthropod-borne virus including West Nile virus and dengue

viral encephalitis agents,

protozoan disease agents,

fungal disease agents,

bacterial disease agents,

or mixtures thereof.

[0052] In embodiments for treating or preventing cancer in a subject by delivering a nucleic acid-based vaccine or other nucleic acid, any type of cancer may be treated or prevented. Examples of cancers that can be prevented or treated using the methods, platforms and kits described herein include melanoma, HPV-induced cervical cancers, prostate cancer, lung cancer, breast cancer, leukemia, etc.

[0053] The compositions, conjugates, methods, platforms and kits described herein have both prophylactic and treatment applications, i.e., can be used as a prophylactic to prevent onset of a disease or condition in a subject, as well as to treat a subject having a disease or condition. A composition or targeted delivery platform as described herein can be used to deliver a therapeutic nucleic acid (e.g., a vaccine, a gene for treating skeletal muscle atrophy, etc.) to cells, reduce the growth of or eliminate any infectious pathogen or cancer cells, and mount an immune response against any infectious pathogen or cancer cells,. Synthesis of Dendrimers Conjugated to Therapeutic Agents

[0054] The surface-modified/functionalized dendrimers described herein are delivered specifically to muscle cells, typically SMCs, in a subject due to the muscle cell ligand which acts as a muscle cell targeting moiety (e.g., the ASSLNIA peptide (SEQ ID NO: 1)). Dendrimers can be prepared and conjugated to, bound to, and/or complexed with the muscle cell ligand, at least one nucleic acid, and the at least one negatively charged moiety that is not a nucleic acid using any suitable method(s). Methods of producing and using dendrimers are well known in the art and are described, for example, in Zhang J-T et. al. Macromol. Biosci. 2004, 4, 575-578, and U.S. Patent Nos. 4,216,171 and 5,795,582, both incorporated herein by reference. See also: D.A. Tomalia, A.M. Naylor, and W.A. Goddard III, "Starburst Dendrimers: Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to Macroscopic Matter", Angew. Chem. Int. Ed. Engl. 29 (1990), 138-175. In the experiments described herein, PAMAM dendrimers were used. However, any suitable positively charged, highly branched polymeric dendrimer can be used. Examples of additional positively charged, highly branched polymeric dendrimers include poly(propylene imine) (PPI) dendrimers, poly(ethylene imine) (PEI) dendrimers, or, more generally, any other dendrimers with primary amine groups on their surfaces.

[0106] A dendrimer solution is prepared by diluting dendrimer in 20 mM HEPES buffer, pH 7.4 to a final volume of 50 μί_^. A DNA solution is prepared by diluting plasmid DNA in 20 mM HEPES buffer, pH 7.4 to a final volume of 50 μΐ_^. To prepare the DNA-dendrimer complex, the dendrimer solution is added to the DNA solution and the resulting complex solution is vortexed at low speed for ten seconds and allowed to incubate at room temperature for 10 minutes. The G5 dendrimers were functionalized with an average of 2 ASSLNIA (SEQ ID NO: 1) peptides with a linker. A spacer or linker may or may not be used depending on the circumstances. As used herein, the terms "linker" or "spacer" mean the chemical groups that are interposed between the dendrimer (e.g., G5-SMC dendrimer) and the surface exposed molecule(s) such as the muscle cell ligand, the negatively charged moiety (e.g., phospholipid), and the therapeutic nucleic acid. In an embodiment in which a linker is used, the linker is conjugated to the surface molecule at one end and at its other end to the nanoparticle (e.g., G5-SMC dendrimer). Linking may be performed with either homo- or heterobifunctional agents, i.e., SPDP, DSS, SIAB. Methods for linking are disclosed in PCT/DKOO/00531 (WO 01/22995) to deJongh, et al., which is hereby incorporated by reference in its entirety. Cleavable linkers may also be used in the compositions and methods described herein.

[0107] Generally, generation-5 (G5) dendrimers are used in the compositions, kits, platforms and methods described herein. However, other generation dendrimers (see Table 2) can be used.

Table 2 - PAMAM Dendrimers

Methods of Delivering a Therapeutic Agent to Muscle Cells

[0055] Described herein are methods of delivering a therapeutic agent (nucleic acid) for treating or preventing a disease, disorder, infection, etc., specifically to muscle cells in a subject. These methods include administering to the subject a composition including a conjugate (e.g., nanoparticle) as described herein in a therapeutically effective amount and a pharmaceutically acceptable carrier and results in expression of the product encoded by the nucleic acid in muscle cells. In an embodiment in which the composition is administered as a vaccine, the nucleic acid encodes an antigen and administration of the composition induces an immune response (e.g., activation of CD4 T helper cells, production of monoclonal antibodies) against the antigen, a pathogen from which the antigen is derived, or cancer cells. In a method of delivering a nucleic acid-based cancer vaccine, the subject has a cancerous tumor or is at risk of developing a cancerous tumor, and administering the composition to the subject results in prevention of or decreased growth of the cancerous tumor. In a method of delivering a nucleic acid-based vaccine against an infectious agent (pathogen), the vaccine induces an immune response against an infectious agent and prevents, mitigates or eliminates infection by the infectious agent. In a method of delivering a nucleic acid for treating, for example, skeletal muscle atrophy in a subject, the subject is typically a human having skeletal muscle atrophy, the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth, and the therapeutically effective amount is effective for promoting muscle cell growth in the subject. In such an embodiment, the subject may have, for example, Muscular Dystrophy, Myasthenia gravis, muscle atrophy due to aging, muscle atrophy due to prolonged bed rest, cancer, AIDS, diabetes, muscle atrophy due to spinal cord injury, muscle atrophy due to a neuromuscular disorder, and ALS. As described below, the compositions may be administered by any suitable route.

[0056] Administration of the composition generally results in no local adverse reactions in the subject. The compositions, platforms, kits and methods described herein can be utilized with any suitable subject, including invertebrate and vertebrate subjects. In a typical embodiment, a subject to be treated is an animal such as a mammal (e.g., human beings, rodents, dogs, cats, goats, sheep, cows, horses, etc.). A human patient suffering from or at risk of a muscle-related disease (skeletal muscle atrophy), contracting an infectious disease, having or developing cancer, or suffering from another disease or disorder requiring treatment is a typical subject. For example, the subject is a human in need of a vaccine against an infectious pathogen or cancer.

Compositions and Methods for Tissue Engineering and Muscle Cell Expansion

[0057] The compositions, methods and platform described herein can be used for tissue engineering. For example, in vivo, in cases of muscle atrophy or muscle weakening, the platform may be used to deliver therapeutics selectively into the affected muscles. This is a significant improvement to current methods, as the delivery of DNA/RNA/VECTOR/drugs to muscles using known methodology is generally very poor. As another example, in an in vitro embodiment, the platform may be used for expansion of muscle cells or commitment of progenitor cells for tissue engineering.

Kits for Orally Delivering Drugs and Vaccines to APCs

[0058] A kit for delivering a nucleic acid or vaccine to a subject typically includes a composition including a plurality of conjugates (e.g., nanoparticles) as described herein and a pharmaceutically acceptable carrier; instructions for use; and packaging. In one example of a kit, the kit includes the therapeutic agent (e.g., a nucleic acid such as a vaccine or a nucleic acid encoding a muscle cell growth factor) and a buffer composed of physiological saline, phosphate buffer saline, or OptiMem, that has a physiological pH and is buffered. In one embodiment, the kit may be purchased and used for expanding muscle cells in vitro, and in this embodiment, the at least one nucleic acid typically encodes a growth factor for promoting muscle cell growth, and the kit may additionally include a cell culture reagent.

Administration of Compositions

[0059] The compositions described herein may be administered to invertebrates, animals, and mammals (e.g., dog, cat, pig, horse, rodent, non-human primate, human) in any suitable formulation. For example, a composition including a PADRE-dendrimer conjugated to a therapeutic agent may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice, those for delivery directly to target muscle cells are generally preferred. A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF. Other substances may be added to the compositions to stabilize and/or preserve the compositions.

[0060] The compositions described herein may be administered to a subject (e.g., mammals) by any conventional technique. Typically, such administration will be by means of intramuscular injection. In therapeutic applications, the compositions described herein are administered to an individual already suffering from a disease or disorder or infection, for example, an individual having an autoimmune disease (e.g., diabetes), an individual having a muscle-related disease (e.g., Muscular Dystrophy, ALS, any condition that results in or is associated with skeletal muscle atrophy, etc.), an individual infected with a pathogen of interest, an individual having cancer cells (e.g., a cancerous tumor). In prophylactic applications, the compositions described herein are administered to an individual at risk of developing (e.g., genetically predisposed to, or environmentally exposed to) or contracting an infectious disease, cancer, or other disease or disorder. Effective Doses

[0061] The compositions (e.g., vaccines) described herein are preferably administered to a subject (e.g., invertebrates, animals, mammals (e.g., dog, cat, pig, horse, rodent, non-human primate, human)) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., amelioration or reversal of skeletal muscle atrophy, protection against cancer, infectious disease(s), or other disease or disorder). Such a therapeutically effective amount can be determined as described below.

[0062] Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of preferred compositions lies preferably within a range that includes an ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Generally, the range of concentrations for a G5-SMC dendrimer is O.OOlmg/Kg to 200 mg/Kg per day for at least one day. Generally, the range of concentrations for the therapeutic agent is O.OOlmg/Kg to 200 mg/Kg per day for at least one day. For vaccinations an average of 2-3 injections each of a dose of 0.01/k to 1 mg fk is expected to be therapeutic.

[0063] As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.

EXAMPLES

[0064] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way. Example 1 - Establishment of the selective transfection of muscle cells by muscle-targeting particle

[0065] First, in a series of studies various peptide/dendrimer substitutions, various ratios of DNA (P) to dendrimer- ASSLNIA (SEQ ID NO: 1) (N), and various buffers were examined and compared. We performed DNA retardation assays and Dynamic Light Scattering to assess the complexation, the stability, and optimal complexation of DNA-dendrimer complexes. Charge ratios above 5: 1 (N:P) were able to effectively condense the plasmid DNA.

[0066] The C2C12 skeletal muscle cell line was employed in this study. For this study and the NP and controls at 1 ug were used. The skeletal muscle cells at 20,000 -100,000 per well in a 12- well plates were incubated with pEGFP alone, pEGFP complexed with dendrimer or with NP (skeletal muscle targeting dendrimer), for 4 hours after which the media was replaced with fresh media. Flow cytometry analysis was performed after 24 hours. In these studies various peptide/dendrimer substitutions, various ratios of DNA(P):dendrimer- ASSLNIA (SEQ ID NO: 1) (N), and various buffers were examined and compared. Ratios above 1 :5 (P:N) were able to effectively condense the plasmid DNA.

Example 2 - Platform characterization and ratio optimization of dendrimer/DNA that

condense/protects the DNA

[0067] Charge ratio is defined as the ratio of the dendrimer' s positively charged amine groups (N) to the negatively charged phosphate groups on the DNA (P). A charge ratio of 1, for example, consists of 1 amine group to 1 phosphate and is conventionally expressed as N/P = 1. Just the same, a charge ratio N/P = 5 refers to the ratio between 5 amine groups to 1 phosphate group. The charge ratio is used to calculate how much DNA and dendrimer to use when making formulations for DNA-dendrimer complexes.

[0068] To calculate how much DNA and G5 PAMAM dendrimer to use for a charge ratio N/P = 5:

1 μg DNA contains 1.71 x 10¹⁵ negative charges (Kukowska-Latallo, et. al.; Bielinska, et. al.)

So, a charge ratio N/P of 5 for a complex containing 1 μg of DNA =

N = 8.55 x 10¹⁵ positive charges

P 1.71 x 10¹⁵ negative charges

Since 1 G5 PAMAM dendrimer contains 128 positive charges (theoretical, Dendritech),

8.55 x 10¹⁵ positive x 1 unit G5 PAMAM x 1 mole G5 PAMAM = charges 128 positive charges 6.02 x 10 units = 1.10 x 10^"10 moles G5 PAMAM

Since the molecular weight of G5 PAMAM is 28,824.81 g/mol (Sigma),

1.10 x 10^"10 moles G5 PAMAM x 28,824.81 g = 3.17 x l0^"6 g =

1 mole G5 PAMAM

= 3.17 μg G5 PAMAM

[0069] So, to prepare a DNA-dendrimer complex at a charge ratio N/P = 5, use 3.17 μg G5 PAMAM for every 1 μg of DNA. Gel retention of pcOVA-G5 and pcOVA-(G5-SMC) complexes was performed. In this experiment, complexes were prepared by mixing equal volumes of pcOVA plasmid and G5- PAMAM or G5-SMC dendrimers in 20 mM HEPES, pH 7.4, at varying charge ratios (N/P). Samples were then electrophoresed on 0.7% agarose gel.

[0070] The DNA-dendrimer complex was then made with a second plasmid to demonstrate further the universality of optimized ratio in plasmids with close or similar size (4000-6000 bp). Gel retention of pcOVA-(G5-SMC) complexes was demonstrated. In this experiment, complexes were prepared by mixing equal volumes of pcOVA plasmid and G5-SMC dendrimer in 20 mM HEPES, pH 7.4, at varying charge ratios (N/P). Samples were then electrophoresed on 0.7% agarose gel.

[0071] Regarding the size and charge of the DNA-(G5-SMC) complex, various vehicle solutions and buffers (water, PBS, normal Saline, and 20 mM HEPES) were tested for the stability, size, and disparity studies of DNA-(G5-SMC) complexes. DLS diameter data and the results from the optimization of the formulation of platforms were obtained. Size disparity and stability determination of DNA-(G5-SMC) complexes were made in various vehicles.

[0072] Finally, 20 mM HEPES, pH 7.4 was selected as a more suitable buffer as DNA- dendrimer complexes made in this buffer resulted in a consistent size and Zeta potential over time. See Table 3:

Charge Ratio (N/P) Diameter (nm) Zeta Potential (mV)

5 131.5 +31.4

10 150 +32.8

Table 3: Size and Zeta Potential Characterization of pcOVA-(G5-SMC) Complexes. Complexes were prepared by mixing equal volumes of pcOVA plasmid DNA and G5-SMC dendrimer in 20 mM HEPES, pH 7.4. Diameter and zeta potential were analyzed using Zetasizer (Malvern). Values were obtained from one experiment.

[0073] Labeled G5-SMC effectively binds to the human muscle cells. One-hour post incubation of 1 ug of G5-SMC-FITC with primary human muscle cells, flow cytometry analysis was conducted. G5-SMC not only bound the muscle cells effectively (to 97% of cells), the binding did not result in cell toxicity as the viability of cells remained at the level of cells incubated with the culture media.

[0074] In vitro transfection of muscle cells was performed. For the first experiment, -75,000 C2C12 myoblast cells per well were seeded on a 12 well plate 48 hours prior to transfection. Two wells were seeded with cells for each condition. For the conditions receiving DNA- dendrimer complex, each complex was prepared using 5 micrograms pEGFP and 17.25 micrograms G5-SMC in a total volume of 100 microliters. Cells were analyzed by flow cytometry 24 hours post-transfection. pEGFP-(G5-SMC)-CL enhanced the transfection of C2C12 cells >100 times over pEGFP-G5-CL and ten times over pEGFP-(G5-SMC). CL at various molar ratios was added to G5-SMC or G5. The reagents were then complexed with DNA (pEGFP) at a 5: 1 charge ratio and co-cultured with C2C12 cells. The flow cytometry analysis was performed 24 or 48 post-transfection. It was also demonstrated that transfection of C2C12 muscle cells using pEGFP-(G5-SMC)-CL enhanced the transfection of C2C12 cells >100 times over background or control DNA vaccines including that of pEGFP-G5-CL or pEGFP-(G5- SMC). CL at various molar ratios was added to G5-SMC or G5. The reagents were then complexed with DNA, pEGFP at a 5: 1 molar ratio and co-cultured with C2C12 cells. The flow cytometry analysis was performed 24 or 48 post-transfection. [0075] For other experiments, -25,000 C2C12 myoblast cells per well were seeded on a 24 well plate 48 hours prior to transfection. One well was seeded with cells for each condition. For the conditions receiving DNA-dendrimer complex, each complex was prepared using 1 microgram pEGFP and 3.45 micrograms G5-SMC in a total volume of 100 microliters. Cells were analyzed by flow cytometry 24 hours post-transfections. The results of the flow cytometry analysis of a series of representative experiments using various controls showed the significant >100 fold increase in transfection efficacy elicited by the invention (G5-SMC-CL).

[0076] Nuclease protection assays were performed and it was shown that the G5-SMC platform protects nucleic acids from degradation. Dendriplexes comprised of pcOVA and G5 PAMAM or G5-SMC dendrimer were incubated in complete cell medium containing 10% FBS for four hours at 37 °C. The nuclease reaction was stopped by addition of EDTA (5 mM final concentration) and dendriplexes were dissociated by addition of 1% SDS. Samples were purified by ethanol precipitation and electrophoresed in 0.7% agarose at 80 V. The platform complexation with DNA was optimized to protect nucleic acid cargo from degradation by nucleases. Dendriplexes comprised of DNA (pcOVA) and G5-SMC PAMAM dendrimer at charge ratios (N/P) of 1: 1, 5: 1, 10: 1, and 20: 1 (N:P) were co-cultured with complete cell medium containing 10% FBS for four hours at 37°C. Subsequently, the serum nuclease activity was stopped by addition of EDTA (5 mM final concentration) and dendriplexes were dissociated by addition of 1% SDS. Samples were purified by ethanol precipitation and electrophoresed in 0.7% agarose at 80 V. G5-SMC protected the DNA from degradation by nucleases.

[0077] A dendrimer solution can be prepared by diluting dendrimer in 20 mM HEPES buffer, pH 7.4 to a final volume of 50 μί_^. A DNA solution is prepared by diluting plasmid DNA in 20 mM HEPES buffer, pH 7.4 to a final volume of 50 μL·. To prepare the DNA-dendrimer complex, the dendrimer solution is added to the DNA solution and the resulting complex solution is vortexed at low speed for ten seconds and allowed to incubate at room temperature for 10 minutes. UV-visible spectroscopic analysis can be performed to analyze dendriplex formation. Complexes containing pEGFP and G5 or G5-SMC were analyzed by means of UV- visible spectroscopy.

Example 3 - In vivo gene transfer and protein expression in the muscle of live mice. [0078] The following vaccines were constructed and tested:

[1] plasmid DNA encoding for luciferase (15 ug) alone (FIG. 1, DNA)

[2] plasmid DNA encoding for luciferase (15 ug) complexed with G5 (FIG. 1, DNA-G5)

[3] plasmid DNA encoding for luciferase (15 ug) complexed with G5/CL (FIG. 1, DNA-G5-CL) [4] plasmid DNA encoding for luciferase (15 ug) complexed with complexed and G5-SMC, a phospholipid, cardiolipin (5%), which is the NP platform (FIG. 1, DNA-(G5-SMC)-CL).

[0079] Cardiolipin is a phospholipid mainly found in the mitochondrial inner membrane. Unlike most other phospholipids, cardiolipin contains three glycerol backbones and four acyl chains. This phospholipid plays a role in mitochondrial membrane enzyme complex formation and metabolism (Houtkooper, et. al., Cellular and Molecular Life Sciences, 2008). Studies have elucidated the use of cardiolipin as a potential gene delivery vehicle (Zhang, et. al., Die

Pharmazie, 2006). In these experiments, Cardiolipin was added after the formation of the DNA- (G5-SMC) complexes at various charge ratios of up to 200% of the G5-SMC. Cardiolipin at charge ratios greater than 5% (-200%). In a typical flow cytometry experiment, C2C12 myoblast cells were seeded at a density of 50,000 cells per well in a 24 well plate and allowed to adhere for 24 hours prior to the experiment. In some experiments cells were then treated with 2.2 μΜ of either G5 or G5-SMC dendrimer labeled with VivoTag 680 and incubated for 15 minutes (A) or 30 minutes (B). For comparison, cells were treated with DNA-dendrimer complexes comprising 1 μg pEGFP and 2.2 μΜ of either G5 or G5-SMC dendrimer labeled with VivoTag 680 and incubated for 30 minutes (C). Control cells were treated with 20 mM HEPES buffer, pH 7.4. The in vivo expression of the payload DNA was enhanced with the addition of optimized phospholipid, cardiolipin (at least 5% charge ratio).

Example 4 - Elicitation of humoral immune responses.

[0080] Mice were immunized (once) with 10 ug of plasmid DNA expressing Ovalbumin (pcOVA) alone or complexed with G5-SMC or dendrimer controls and the anti-OVA serum titers were measured using an ELISA. FIG. 2 shows that DNA-(G5-SMC) complex (G5-SMC) elicits significant antigen specific antibody responses upon a single vaccination.

Example 5 - In vivo transgene induction for vaccination or gene therapy [0108] Referring to FIG. 3, mice received intramuscular injections of the mentioned amount of plasmids expressing firefly luciferase alone, complexed with G5-SMC or G5-SMC/CL (10%). The morning after, mice were anesthetized and were subjected to in vivo imaging (IVIS) of the luciferase 10 minutes after they received 250 ug of D-Luciferin intra-peritoneally.

Example 6 - Delivery of RNA

[0109] Flow cytometry plots and a confocal microscopy micrograph showed targeting of and shuttling cargo into skeletal muscle cells. Cells co-cultured with fluorescently labeled (Vivo Tag, NIR 680) G5 dendrimer or G5-SMC for 15 or minutes were analyzed by flow cytometry for cell binding, Gated on viable and single cells only. Fluorescently labeled (FITC) G5-SMC complexed with DNA (pcOVA-RITC labeled) were cultured with C2C12 muscle cells and analyzed by flow cytometry or confocal microscopy.

[0110] An experiment was performed demonstrating that the platform described herein can be used for mRNA-based delivery. In this experiment, mice received sub-conjuctival injections of 1 ug of mRNA (either 4 micrograms of mRNA alone or two micrograms of mRNA complexed with G5-SMC or PDD) expressing luciferase alone or complexed with G5-SMC, in a total volume of 5 ul in saline. After 24 hours, an IVIS was performed 10 minutes after intraperitoneal injection of 250 UL of D-Luciferin. An IVIS image of mice post sub-conjuctival injection of RNA alone, RNA complexed with G5-SMC (muscle binding) and PDD (which is a control nanocarrier) showed the effective expression of luciferase was achieved in the eyes of mice that received G5-SMC only using a small amount of the mRNA, which was superior to that all control treatments.

Example 7 - Targeting of and Shuttling Cargo Into Skeletal Muscle Cells

[0111] Experimental results demonstrated targeting of and shuttling Cargo into SMCs using the nanoparticles described herein. G5 PAMAM (control) and G5-SMC were labeled with VivoTag 680, co-cultured with muscle cells and analyzed by flow cytometry. G5-SMC bound significantly to more cells and with higher intensity at the same time points as G5 (A and B). The fluorescently labeled G5 and G5-SMC also were complexed with DNA and the same experiment as above was performed; again increased binding and intensity in cells co-cultured with G5-SMC was observed (C). Cell binding and intracellular shuttling of the complexes were confirmed by confocal microscopy performed using pcOVA plasmid labeled with rhodamine isothiocyanate (RrrC) and G5-SMC labeled with FITC (D).

Example 8 - Cells transfected with pEGFP-G5-SMC complexes and cardiolipin

[0112] An experiment was performed in which C2C12 cells were seeded on 24 well plates at a density of 25,000 cells per well and allowed to adhere for two days. Cells were then transfected with pEGFP-G5-SMC complexes at a charge ratio (N/P) of 5 with varying percentages of cardiolipin. Complexes were prepared with 1 microgram of DNA. Flow cytometry was performed 24 hours post transfection to measure the percentage of EGFP-positive cells.

Example 9 - Cells transfected with pEGFP-G5-SMC complexes and lipoic acid

[0113] An experiment was performed in which C2C12 cells were seeded on 12 well plates at a density of 75,000 cells per well and allowed to adhere for two days. Cells were then transfected with pEGFP-G5-SMC complexes at a charge ratio (N/P) of 5 with varying percentages of lipoic acid. Complexes were prepared with 5 microgram of DNA. Flow cytometry was performed 24 hours post transfection to measure the percentage of EGFP-positive cells.

Example 10 - Specific binding of SMC to Human Skeletal Muscle cells

[0081] A flow cytometry analysis was performed which demonstrated 99.9% specific binding of the SMC to Human Skeletal Muscle cells. The SSC/FSC indicated that the cells have complete viability as was shown in trypan blue staining (97% live cells).

Other Embodiments

[0114] Any improvement may be made in part or all of the compositions, vaccines, kits, platforms, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. For example, although the experiments describe construction and use of charged highly branched polymeric dendrimers, non-branched charged particles and polymers are also encompassed by the invention. Any statement herein as to the nature or benefits of the invention or of preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is

1. A conjugate comprising at least one charged highly branched polymeric dendrimer having conjugated thereto: 1) at least one muscle cell ligand, 2) at least one nucleic acid, and 3) at least one anionic enhancer.

2. The conjugate of claim 1, wherein the at least one charged highly branched polymeric dendrimer is conjugated to poly(LC).

3. The conjugate of claim 1, wherein the at least one muscle cell ligand is conjugated to the exterior surface of the at least one charged highly branched polymeric dendrimer such that the at least one muscle cell ligand specifically binds to muscle cells.

4. The conjugate of claim 1, wherein the at least one nucleic acid is comprised within an expression vector.

5. The conjugate of claim 1, wherein the at least one nucleic acid is selected from the group consisting of: DNA, RNA, microRNA, small RNA, and siRNA.

6. The conjugate of claim 1, wherein the at least one charged highly branched polymeric dendrimer is conjugated to the at least one nucleic acid via electrostatic forces.

7. The conjugate of claim 1, wherein the at least one nucleic acid encodes an antigen.

8. The conjugate of claim 1 , wherein the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth.

9. The conjugate of claim 8, wherein the nucleic acid encodes a growth factor.

10. The conjugate of claim 1, wherein the at least one charged highly branched polymeric dendrimer is a polyamidoamine (PAMAM) dendrimer.

11. The conjugate of claim 1 , wherein the at least one muscle cell ligand is selected from the group consisting of: ASSLNIA (SEQ ID NO: 1), TARGEHKEEELI (SEQ ID NO: 2),

TGGETSGIKKAPYASTTRNR (SEQ ID NO: 3), SHHGVAGVDLGGGADFKSIA (SEQ ID NO: 4), and RGD.

12. The conjugate of claim 1, wherein the at least one anionic enhancer is a phospholipid or negatively charged molecule having a >1 negative charge in physiological pH.

13. The conjugate of claim 12, wherein the phospholipid is cardiolipin.

14. A composition comprising a plurality of conjugates as set forth in claim 1 in a therapeutically effective amount and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein the at least one charged highly branched polymeric dendrimer is a PAMAM dendrimer.

16. The composition of claim 14, wherein a portion of the plurality of conjugates are conjugated to the same at least one nucleic acid.

17. The composition of claim 16, wherein the charge ratio of charged highly branched polymeric dendrimer to nucleic acid is greater than 5: 1.

18. The composition of claim 14, wherein the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth and the therapeutically effective amount is effective for promoting muscle cell growth in a subject having skeletal muscle atrophy.

19. The composition of claim 14, wherein the nucleic acid encodes an antigen and the therapeutically effective amount is effective for inducing an immune response against the antigen when administered to a subject.

20. The composition of claim 19, wherein the composition is a vaccine.

21. The composition of claim 14, wherein the nucleic acid encodes an antigen and the therapeutically effective amount is effective for preventing or decreasing growth of a cancerous tumor when administered to a subject having a cancerous tumor or at risk of developing a cancerous tumor.

22. The composition of claim 21, wherein the composition is a vaccine.

23. The composition of claim 11, wherein the at least one muscle cell ligand is selected from the group consisting of: ASSLNIA (SEQ ID NO: 1), TARGEHKEEELI (SEQ ID NO: 2), TGGETSGIKKAPYASTTRNR (SEQ ID NO: 3), SHHGVAGVDLGGGADFKSIA (SEQ ID NO: 4), and RGD.

24. The composition of claim 14, wherein the at least one anionic enhancer is a phospholipid having a >1 negative charge in physiological pH.

25. The composition of claim 24, wherein the phospholipid is cardiolipin.

26. A method of delivering a nucleic acid specifically to muscle cells in a subject, the method comprising administering to the subject the composition of claim 14.

27. The method of claim 26, wherein the subject is a human, the at least one nucleic acid encodes an antigen, and the composition is a vaccine.

28. The method of claim 27, wherein the vaccine is an anti-cancer vaccine, the subject has a cancerous tumor or is at risk of developing a cancerous tumor, and administering the composition to the subject results in prevention of or decreased growth of the cancerous tumor.

29. The method of claim 27, wherein the vaccine induces an immune response against an infectious agent.

30. The method of claim 26, wherein the subject is a human having skeletal muscle atrophy, the at least one nucleic acid encodes a peptide or polypeptide that promotes muscle cell growth, and the therapeutically effective amount is effective for promoting muscle cell growth in the subject.

31. The method of claim 30, wherein the subject has a disease or disorder selected from the group consisting of: Muscular Dystrophy, Myasthenia gravis, muscle atrophy due to aging, muscle atrophy due to prolonged bed rest, cancer, AIDS, diabetes, muscle atrophy due to spinal cord injury, muscle atrophy due to a neuromuscular disorder, and Amyotrophic Lateral Sclerosis (ALS).

32. The method of claim 26, wherein the composition is administered intramuscularly or intraperitoneally.

33. A method of expanding muscle cells in vitro comprising contacting a plurality of muscle cells in vitro with a plurality of conjugates as set forth in claim 1 under conditions such that the muscle cells proliferate.

34. A method of expanding muscle cells in vitro comprising contacting a plurality of progenitor cells with a plurality of conjugates as set forth in claim 1 under conditions such that the progenitor cells commit to muscle cells.

35. A kit for delivering a nucleic acid specifically to muscle cells in a subject, the kit comprising:

a) the composition of claim 14;

b) instructions for use; and

c) packaging.

36. The kit of claim 35, wherein the kit further comprises a cell culture reagent, and the at least one nucleic acid encodes a peptide or polypeptide that promotes skeletal muscle cell growth.