WO2019028469A1

WO2019028469A1 - Polymer-functionalized mitochondrial compositions and methods of use in cellular transplantation and for altering metabolic phenotype

Info

Publication number: WO2019028469A1
Application number: PCT/US2018/045416
Authority: WO
Inventors: Dale James HAMILTON; Anisha Anil GUPTE; Aijun Zhang; Elvin Blanco
Original assignee: The Methodist Hospital
Priority date: 2017-08-04
Filing date: 2018-08-06
Publication date: 2019-02-07

Abstract

Disclosed are methods and compositions including polymer-functionalized mammalian mitochondria, useful in a variety of therapeutic regimens for delivery of desirable genetic traits to one or more cells, tissues, and/or organs of interest. Also provided are methods for introducing mitochondria into selected host cells for altering the metabolic phenotype of such host cells using one or more mitochondrially-delivered nucleic acid molecules. The present disclosure is also directed to a method of cellular transplantation, comprising the step of administering to an individual one or more of the polymer functionalized mitochondrial compositions and formulations thereof as described herein.

Description

DESCRIPTION

POLYMER-FUNCTIONALIZED MITOCHONDRIAL COMPOSITIONS AND METHODS OF USE IN CELLULAR TRANSPLANTATION AND FOR ALTERING METABOLIC PHENOTYPE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application 62/541,647, filed August 4, 2017, the contents of which is specifically incorporated herein in its entirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] Not Applicable.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0004] The present disclosure relates to the fields of transplantation medicine, oncology, and pharmacology. In particular, the disclosure provides compositions and methods for functionahzing the surface of a mammalian mitochondrion, and populations thereof, with one or more polymers to facilitate enhanced uptake of the mitochondria by one or more selected host cells or cell types. The disclosed polymeric coatings provide a protective layer against protein absorption, and also facilitate enhanced entry into the host cells of interest. The resulting compositions find use in a variety of therapeutic and diagnostic regimens, including, for example, the in vivo transplantation of surface-functionalized mitochondria in human host cells.

DEFICIENCIES IN THE PRIOR ART

[0005] Current state-of-the-art treatment for failing organs include organ transplantation, mechanical assist devices, and/or pharmacological agents, while those for cancer include surgical resection, radiotherapy, and/or chemotherapy. The potential for autologous or homologous transfer of cellular organelles would potentially address the underlying molecular mechanism and restore physiologic function with fewer problems associated with immune reactions, device failure and adverse effects, including systemic side effects

[0006] Investigators have previously demonstrated the transfer of isolated mitochondria into homogeneic and xenogeneic cells with rescue of mitochondrial respiratory function (Kitani et al, 2014), and have reported transplantation of autologously-derived mitochondria to protect the heart from ischemia-reperfusion injury (Masuzawa et al, 2013; Kaza et al, 2016). While these studies support the concept of rescue of oxidative phosphorylation and mitochondrial dysfunction in stressed cells, isolated mitochondria were not functionalized with a polymer coating.

BRIEF SUMMARY OF THE INVENTION

[0007] The present disclosure addresses these and other unmet deficiencies inherent in the relevant medical and pharmaceutical arts, by providing novel, non-obvious, and useful compositions and pharmaceutical formulations comprising them for use in delivering one or more therapeutic and/or diagnostic agents to one or more selected cell types and/or organs in a mammal, such as a human. In particular embodiments, compositions are disclosed comprising populations of mammalian mitochondria that are surface functionalized with one or more biocompatible polymers, which are then useful for delivering therapeutics to one or more mammalian cells via cellular internalization of the polymer-coated mitochondria.

[0008] The present disclosure provides compositions and methods for functionalizing the surface of a mammalian mitochondrion, and populations thereof, with one or more biocompatible polymers to facilitate enhanced uptake (and/or to facilitate decreased blocked uptake) of the mitochondria by one or more selected host cells and/or organ or tissue types. The disclosed biocompatible polymeric coatings provide a protective layer against protein absorption, and also facilitate enhanced entry into the host cells of interest. The resulting compositions find use in a variety of therapeutic and diagnostic regimens, including, for example, the in vivo transplantation of surface-functionalized mitochondria into populations of human host cells, and or delivery of mitochondria to in vivo, as well as ex situ organs (e.g., harvested organs for transplantation).

[0009] Exemplary embodiments have shown the effectiveness of the methods for the isolation of functioning mitochondria from murine livers and hearts, using a procedure that polymer functionalizes the surfaces of the isolated mitochondira. Xenotransfer of these polymer-functionalized mitochondria has been demonstrated in a variety of established human cancer and cardiovascular cell lines, as has both autologous and homologous transfer using established in vivo murine pre-clinical models of human disease.

[0010] These compositions and their subsequent transfer into altered organs have proven useful in modifying the bioenergy profile of the altered organs.

[0011] The present disclosure also provides new and useful methods for the treatment of failing organs, such as, for example, a failing heart, or other internal organ.

[0012] In a further embodiment, the disclosure also provides compositions and methods for the metabolic reprogramming of abnormal cells, such as cancer and/or tumor cells.

[0013] Additionally, the present disclosure provides compositions and methods for the improved functional preservation of harvested organs that have been procured for transplantation into recipient individuals in need of organ replacement.

[0014] In another embodiment, the present disclosure also provides methods for the treatment and/or amelioration of one or more symptoms of cancer, and particularly for those cancers wherein the current standard of care relies primarily or solely on cheraotherapeutics having narrow therapeutic dosing windows due to significant systemic toxicities and untoward side-effects.

[0015] In a particular embodiment, the present disclosure provides methods for treating or ameliorating one or more symptoms of a disease, such as cancer, in an animal, and particularly in a mammal, such as a human in need thereof. Such methods generally include at least the step of providing or administering to the selected animal (systemically, or locally at one or more regions or sites within, or about the body of the animal) an effective amount of one or more of the disclosed functionalized mitochondrial compositions disclosed herein, for a time sufficient to treat the cancer in the animal, or to ameliorate one or more symptoms thereof.

[0016] In a further aspect, the present disclosure provides a method for silencing a gene expressed in one or more cells of a mammal in need thereof. This method, in an overall and general sense includes providing to one or more cells or to one or more tissues of the body of the mammal, an amount of one or more of the functionalized mitochondrial compositions disclosed herein in an amount and for a time effective to lessen (i.e., "knock-down") or inhibit the expression of one or more genes in the one or more cells. In certain applications, the silencing agents are interfering RNAs that inhibit the expression of one or more deleterious genes expressed in a mammalian cell (such as a human cancer cell or tumor cell).

[0017] As set forth in more detail below, the methods and compositions of the present disclosure have numerous variations. More specific and non-limiting embodiments of the present disclosure will now be described in more detail.

[0018] In addition to the functionalized mitochondria, the compositions may be formulated to include one or more additional active agents, including, without limitation, a small molecule drug or a bimolecular drug. In some embodiments, the at least one active agent is a biologically active compound selected from the group consisting of peptides, proteins, nucleic acids, antisense RNAs, interfering RNAs, therapeutic agents, diagnostic agents, non-biological materials, pharmaceuticals, chemotherapeutics, and/or any combinations thereof. The therapeutic agent may be any physiologically or pharmacologically active substance that can produce a desired biological effect. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, an anti-inflammatory compound, or a pro-drug enzyme, which may be naturally occurring, or produced by synthetic or recombinant methods, or by a combination thereof.

[0019] Drugs that are affected by classical multi-drug resistance, such as vinca alkaloids (e.g., vinblastine, vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D), and microtubule stabilizing drugs (e.g., paclitaxel) can have particular utility as the therapeutic agent. In some embodiments, the therapeutic agent may be a hydrophobic drug or a hydrophilic drug. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and to Goodman and Gilman's "Pharmacological Basis of Therapeutics" tenth edition, Eds. Hardman et al (2001).

[0020] In some embodiments, the additional therapeutic agent may be selected from the group consisting of genes, nucleic acids, shRNAs, siRNAs, microRNAs, DNA fragments, RNA fragments, plasmids, and combinations thereof. In an illustrative embodiment, the therapeutic agent is a siRNA or a microRNA present within the population of mitochondria that silences one or more genes expressed by particular mammalian cancer cells or a particular mammalian organ or tissue to which the functionalized mitochondrial formulation is administered.

[0021] The compositions of the present disclosure may be designed, formulated and processed so as to be suitable for a variety of therapeutic and diagnostic uses and modes of administration. The composition of the disclosure may be administered to a subject, such as a human, via any suitable administration method in order to treat, prevent, and/or monitor a physiological condition, such as a disease.

Embodiments of the disclosed polymer-functionalized mitochondrial compositions may be particularly useful for oncological applications, i.e., for treatment and/or monitoring cancer or a condition, such as tumor associated with cancer.

THERAPEUTIC AND PHARMACEUTICAL FORMULATIONS

[0022] The polymer-functionalized mitochondrial compositions of the present disclosure may be employed in medical arts practices and modalities as a single treatment modality, or alternatively may be combined with one or more additional therapeutic, diagnostic, and/or prophylactic agents, including, without limitation, one or more proteins, peptides, polypeptides (including, without limitation, enzymes, antibodies, antigens, antigen binding fragments etc.); RNA molecules (including, without limitation, siRNAs, microRNAs, iRNAs, mRNAs, tRNAs, or catalytic RNAs, such as ribozymes, and the like), DNA molecules (including, without limitation, oligonucleotides, polynucleotides, genes, coding sequences (CDS), introns, exons, plasmids, cosmids, phagemids, baculovirus, vectors [including, without limitation, viral vectors, virions, viral particles and such like]); peptide nucleic acids, detection agents, imaging agents, contrast agents, detectable gas, radionuclides, or such like, and one or more additional chemotherapeutic agents, surgical intervention (e.g., tumor resection), radiotherapy, and the like., or any combination thereof as part of a multifactorial, or multifocal treatment plan for the affected patient.

[0023] The polymer-functionalized mitochondrial compositions disclosed herein may also further optionally include one or more additional active ingredients, including, without limitation, one or more anti-cancer agents, one or more anti-tumorigenic agents, one or more antineoplastic or cytotoxic agents, one or more transcription factors, immunomodulating agents, immunostimulating agents, neuroactive agents, antiinflammatory agents, chemotherapeutic agents, hormones, so called "trophic factors," cytokines, chemokines, receptor agonists or antagonists, or such like, or any combination thereof.

[0024] The polymer-functionalized mitochondrial compositions may also further optionally include one or more additional components to aid, facilitate, or improve delivery of a pro-drug and/or an active metabolite contained therein, including, without limitation, one or more liposomes, lipid particles, lipid complexes, and may further optionally include one or more binding agents, cell surface active agents, surfactants, lipid complexes, niosomes, ethosomes, transferosomes, phospholipids, sphingolipids, sphingosomes, or any combination thereof, and may optionally be provided within a pharmaceutical formulation that includes one or more additional nanoparticles, microparticles, nanocapsules, microcapsules, nanospheres, microspheres, or any combination thereof.

[0025] Preferably, the polymer-functionalized mitochondrial compositions will generally be formulated for systemic and/or localized administration to an animal, or to one or more cells or tissues thereof, and in particular, will be formulated for systemic and/or localized administration to a mammal, or to one or more cancerous cells, tumor tissues, or affected organs thereof. In certain embodiments, the compounds and methods disclosed herein will find particular use in the systemic and/or localized administration of one or more antineoplastic agents to one or more cells or tissues of a human being.

[0026] Preferably, the polymer-functionalized mitochondrial compositions disclosed herein will be at least substantially stable at a pH from about 4.2 to about 8.2, and more preferably, will be substantially stable at a pH of from about 5 to about 7.5. Also preferably, the active ingredient(s) and targeted drugs will be substantially active at physiological conditions of the animal into which they are being administered.

[0027] As noted above, the present disclosure provides for the use of one or more of the disclosed polymer-functionalized mitochondrial compositions in the manufacture of a medicament for therapy and/or for the amelioration of one or more symptoms of a disease, disorder, dysfunction, or condition, and particularly for use in the manufacture of a medicament for treating, one or more diseases, dysfunctions, or disorders such as cancers, carcinomas, organ failure, organ transplantation, transplanted organ transport and survival, and/or other indications in a mammal, and, in a human, in particular, as may be selected by a medical practitioner in the relevant fields.

[0028] Likewise, the present disclosure also provides for the use of one or more of the disclosed polymer-functionalized mitochondrial compositions in the manufacture of a medicament for the treatment of cancer, and in particular, those cancers that can be affected by the silencing of one or more expressed gene using small interfering RNA, or microRNA to facilitate knock-down or inhibition of the expressed gene. In certain embodiments, the disclosure also includes diagnostic and/or targeting compounds that may be optionally included in or on the surface of the silicon nanoparticle carriers to facilitate improvements in the treatment or prognosis of a mammalian cancer, and a human breast tumor in particular. THERAPEUTIC METHODS AND USES THEREFOR

[0029] Other important aspects of the present disclosure concern methods for using the polymer-functionalized mitochondrial compositions to facilitate treatment or the amelioration of one or more symptoms of the disease in a mammal having, suspected of having, or at risk for developing such a condition, and in particular for those mammalian diagnosed with one or more cancers. Such methods generally involve administering to a mammal (and in particular, to a human in need thereof), one or more of the disclosed polycation-functionalized nanoporous silicon carriers formulated to contain one or more anticancer compounds, in an amount and for a time sufficient to treat (or, alternatively, to ameliorate one or more symptoms of) a cancer in a mammal to which the composition has been administered.

[0030] In certain embodiments, the polymer-functionalized mitochondrial formulations described herein may be provided to the animal as a single treatment modality, as a single administration, or alternatively provided to the patient in multiple administrations over a period of from several hours to several days, from several days to several weeks, or even over a period of several weeks to several months or longer, as needed to treat the cancer. In some aspects, it may be desirable to continue the treatment throughout the lifetime of the patient. In other embodiments, it may be desirable to provide the therapy in combination with one or more existing, or conventional, treatment regimens, including surgery, radiotherapy, brachy therapy, chemotherapy, and combinations thereof.

THERAPEUTIC KITS

[0031] Therapeutic kits that include one or more of the disclosed polymer- functionalized mitochondrial compositions (and instructions for using the kit) also represent an important aspect of the present disclosure. Such kits may further optionally include one or more of diagnostic agents, one or more additional cytotoxic agents, either alone, or in combination with one or more additional therapeutic compounds, pharmaceuticals, and such like.

[0032] The kits of the present disclosure may be packaged for commercial distribution, and may further optionally include one or more delivery devices adapted to deliver the chemotherapeutic composition(s) to an animal (e.g., syringes, injectables, and the like). Such kits typically include at least one vial, test tube, flask, bottle, syringe or other container, into which the pharmaceutical composition(s) may be placed, and preferably suitably aliquotted. Where a second pharmaceutical is also provided, the kit may also contain a second distinct container into which this second composition may be placed. Alternatively, a plurality of polymer-functionalized mitochondrial compositions disclosed herein may be prepared in a single mixture, such as a suspension or solution, and may be packaged in a single container, such as a vial, flask, syringe, catheter, cannula, bottle, or other suitable single container.

[0033] The kits of the present disclosure may also typically include a retention mechanism adapted to contain or retain the vial(s) or other container(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) or other container(s) may be retained to minimize or prevent breakage, exposure to sunlight, or other undesirable factors, or to permit ready use of the composition(s) included within the kit.

PREPARATION OF MEDICAMENTS

[0034] Another important aspect of the present disclosure concerns methods for using the disclosed polymer-functionalized mitochondrial compositions for providing therapeutic or diagnostic compounds to selected cells or tissues of a vertebrate mammal, and particularly a human in need thereof.

[0035] Such use generally involves administration to an animal in need thereof one or more of the disclosed polymer-functionalized mitochondrial compositions, in an amount and for a time sufficient to prevent, treat, lessen, or cure the disease, disorder, dysfunction, condition, or deficiency in the affected animal, and/or to ameliorate one or more symptoms thereof.

PHARMACEUTICAL FORMULATIONS

[0036] In certain embodiments, the present disclosure concerns formulation of one or more polymer-functionalized mitochondrial compositions in a pharmaceutically acceptable formulation of the disclosed polymer-functionalized mitochondrial compositions for delivering the compounds to one or more cells or tissues of an animal, either alone, or in combination with one or more other modalities of diagnosis, prophylaxis and/or therapy. The formulation of pharmaceutically-acceptable excipients and carrier solutions is well known to those of ordinary skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0037] In certain circumstances it will be desirable to deliver the polymer- functionalized mitochondrial compositions in suitably-formulated pharmaceutical vehicles by one or more standard delivery devices, including, without limitation, subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, orally, intraperitoneally, trans dermally, topically, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs within or about the body of an animal.

[0038] The methods of administration may also include those modalities as described in U.S. Patents 5,543,158; 5,641,515, and 5,399,363, each of which is specifically incorporated herein in its entirety by express reference thereto. Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in sterile water, and may be suitably mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, oils, or mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0039] For administration of an injectable aqueous solution, without limitation, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, transdermal, subdermal, and/or intraperitoneal administration. In this regard, the polymer-functionalized mitochondrial compositions of the present disclosure may be formulated in one or more pharmaceutically-acceptable vehicles, including for example sterile aqueous media, buffers, diluents, etc. For example, a given dosage of active ingredient(s) may be dissolved in a particular volume of an isotonic solution (e.g., an isotonic NaCl-based solution), and then injected at the proposed site of administration, or further diluted in a vehicle suitable for intravenous infusion {see, e.g., "Remington's Pharmaceutical Sciences" 15th Edition, pp. 1035-1038 and 1570-1580). While some variation in dosage will necessarily occur depending on the condition of the subject being treated, the extent of the treatment, and the site of administration, the person responsible for administration will nevertheless be able to determine the correct dosing regimens appropriate for the individual subject using ordinary knowledge in the medical and pharmaceutical arts.

[0040] Sterile injectable compositions may be prepared by incorporating the disclosed polymer-functionalized mitochondrial compositions in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. The polymer-functionalized mitochondrial compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein), and which are formed with inorganic acids such as, without limitation, hydrochloric or phosphoric acids, or organic acids such as, without limitation, acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, without limitation, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is effective for the intended application. The formulations are readily administered in a variety of dosage forms such as injectable solutions, topical preparations, oral formulations, including sustain-release capsules, hydrogels, colloids, viscous gels, transdermal reagents, intranasal and inhalation formulations, and the like.

[0041] The amount, dosage regimen, formulation, and administration of polymer- functionalized mitochondrial compositions disclosed herein will be within the purview of the ordinary-skilled artisan having benefit of the present teaching. It is likely, however, that the administration of a therapeutically-effective (i.e., a pharmaceutically-effective) amount of the disclosed compositions may be achieved by a single administration, such as, without limitation, a single injection of a sufficient quantity of the delivered agent to provide the desired benefit to the patient undergoing such a procedure. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the polymer-functionalized mitochondrial compositions, either over a relatively short, or even a relatively prolonged period, as may be determined by the medical practitioner overseeing the administration of such compositions to the selected individual.

[0042] Typically, formulations of one or more of the polymer-functionalized mitochondrial compositions described herein will contain at least a chemotherapeutically-effective amount of a first active agent Preferably, the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although the percentage of the active ingredient(s) may, of course, be varied, and may conveniently be present in amounts from about 0.01 to about 90 weight % or volume %, or from about 0.1 to about 80 weight % or volume %, or more preferably, from about 0.2 to about 60 weight % or volume %, based upon the total formulation. Naturally, the amount of active compound(s) in each composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological tm, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0043] Administration of the polymer-functionalized mitochondrial compositions disclosed herein may be administered by any effective method, including, without limitation, by parenteral, intravenous, intramuscular, or even intraperitoneal administration as described, for example, in U.S. Patents 5,543,158, 5,641,515 and 5,399,363 (each of which is specifically incorporated herein in its entirety by express reference thereto). Solutions of the active compounds as free-base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose, or other similar fashion. The pharmaceutical forms adapted for injectable administration include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions including without limitation those described in U.S. Patent 5,466,468 (which is specifically incorporated herein in its entirety by express reference thereto). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be at least sufficiently stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as viruses, bacteria, fungi, and such like.

[0044] The carrier(s) can be a solvent or dispersion medium including, without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like, or a combination thereof), one or more vegetable oils, or any combination thereof, although additional pharmaceutically-acceptable components may be included.

[0045] Proper fluidity of formulations of polymer-functionalized mitochondrial populations as disclosed herein may be maintained, for example, by the use of a coating, such as e.g., a lecithin, by the maintenance of the required particle size in the case of dispersion, by the use of a surfactant, or any combination of these techniques. The inhibition or prevention of the action of microorganisms can be brought about by one or more antibacterial or antifungal agents, for example, without limitation, a paraben, chlorobutanol, phenol, sorbic acid, thimerosal, or the like. In many cases, it will be preferable to include an isotonic agent, for example, without limitation, one or more sugars or sodium chloride, or any combination thereof. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example without limitation, aluminum monostearate, gelatin, or a combination thereof.

[0046] While systemic administration is contemplated to be effective in many embodiments of the disclosure, it is also contemplated that formulations of the disclosed polymer-functionalized mitochondrial compositions may be suitable for direct injection into one or more organs, tissues, or cell types in the body. Administration of the disclosed compositions may be conducted using suitable means, including those known to the one of ordinary skill in the relevant medical arts.

[0047] The pharmaceutical formulations comprising polymer-functionalized mitochondrial compositions as disclosed herein are not in any way limited to use only in humans, or even to primates, or mammals. In certain embodiments, the methods and compositions disclosed herein may be employed using avian, amphibian, reptilian, or other animal species. In preferred embodiments, however, the compositions of the present disclosure are preferably formulated for administration to a mammal, and in particular, to humans, in a variety of diagnostic, therapeutic, and/or prophylactic regimens. The compositions disclosed herein may also be provided in formulations that are acceptable for veterinary administration, including, without limitation, to selected livestock, exotic or domesticated animals, companion animals (including pets and such like), non-human primates, as well as zoological or otherwise captive specimens, and such like.

COMPOSITIONS FOR THE PREPARATION OF MEDICAMENTS

[0048] Another important aspect of the present disclosure concerns methods for using the disclosed polymer-functionalized mitochondrial compositions in the preparation of medicaments for treating and/or ameliorating one or more symptoms of one or more diseases, dysfunctions, abnormal conditions, or disorders in an animal, including, for example, vertebrate mammals. Such use generally involves administration to the mammal in need thereof one or more of the disclosed polymer-functionalized mitochondrial compositions, in an amount and for a time sufficient to treat or ameliorate one or more symptoms of a disease, defect, dysfunction, disorder, injury, trauma, or abnormal metabolic condition in an affected mammal.

[0049] Pharmaceutical formulations including one or more of the disclosed polymer- functionalized mitochondrial compositions also form part of the present disclosure, and particularly those compositions that further include at least a first pharmaceutically-acceptable excipient for use in the therapy and/or amelioration of one or more symptoms of disease or defect in an affected mammal.

BIOCOMPATIBLE POLYMERS SUITABLE FOR FUNCTIONALIZATION OF MITOCHONDRIA

[0050] The following polymers have been found to be useful for mitochondrial functionalization: dextran, poly-ethylene glycol (PEG), chitosan, polypeptide, Poly(amino-ester), polyphosphate, polycarbonate, hyaluronic acid, polyacrylic acid, poly ally lamine, poly(methyl-methacrylate), PAMAM, dendrimers, and amphiphilic polymers consisting of either dextran, chitosan, PEG, polypeptide, polyphosphate, hyaluronic acid )-polyester (or polypeptide, polyamino ester), which can be conjugated with TPP, cholesterol or oleic acid.

[0051] The preferred concentration range has been shown to be from about 1.4 and about 3, based on the weight ratio of polymer to mitochondrial protein, although the particular ratio may be formulated as necessary to achieve particularly desired cellular or organ uptake of a particular population of functionalized mitochondria.

EXEMPLARY TARGETING MOIETIES

[0052] As noted herein, various additional targeting moieties can also be added to "fine- tune" the delivery and/or uptake of a particular formulation of functionalized mitochondria. For example, one or more specific targeting moieties may also be added to the surface of the population of mitochondria. These targeting moieties include, but are not limited to molecules effective to facilitate TPP targeting, cholesterol targeting, folic acid targeting, transferrin targeting, LHRH targeting, biotin targeting, RGD targeting, peptide targeting, antibody targeting (EGFR, HER2, VEGF, CTLA4, CD20, CD52, CD30, CD33, etc.), and such like.

EXEMPLARY IMAGING MOIETIES

[0053] With regards to imaging moieties, various such targets may also be used in connection with the present disclosure. For example, fluorescence reagents, including Cy5, Cy5.5, Cy7, and Bodipy; MRI contrast agents such as iron oxide (Fe₃04) nanoparticles, and gadolinium (Ga); CT contrast agents; gold nanoparticles; PET/SPECT agents; quantum dots (QDs)., and the like may also be functionalized onto the surface of the populations of mitochondria, or otherwise included within the formulations that comprise them to facilitate imaging of the mitochondria either in vitro, in situ, and/or ex vivo, where indicated.

EXEMPLARY DRUG CONJUGATION TO POLYMER-FUNCTIONALIZED MITOCHONDRIA

[0054] One or more therapeutic drugs, including, without limitation, one or more chemotherapeutics (e.g., doxorubicin, paclitaxel, platinum-based drugs), as well as drugs in other disease conditions (e.g., heart failure, retinal disorders) may also be conjugated to the polymer-TPP conjugate, to act synergistically, and thereby further exploit the disclosed mitochondrial transplantation formulations and treatment modalities. EXEMPLARY THERAPEUTIC APPLICATIONS

[0055] Cancer treatments - The disclosed therapy may be used in cancers characterized by aerobic glycolysis (i.e., Warburg effect) as indicated by FDG PET imaging, for example.

[0056] Cardiovascular diseases - including, for example, heart failure, ischemic heart disease, congenital heart disease, and related conditions.

[0057] Neurologic injury and disorders - including, for example, acute spinal cord injury, neurodegenerative disease (e.g., Parkinson's disease, frontal temporal lobe dementia), cerebrovascular disease (e.g., acute stroke, transient ischemic attacks, etc.), and related disorders.

[0058] Organ transplantation - to facilitate donor organ (e.g., heart, liver, pancreatic islets, kidneys, etc.,) preservation for extended time (as well as improved organ integrity/function) between harvest and transplant.

[0059] Diabetes/obesity therapy ~ including, for example, hepatic and muscle insulin resistance in type 2 diabetes, transplantation into adipose tissue;

[0060] Ophthalmologic therapies - including, for example, retinopathy (e.g., diabetic retinopathy) and degenerative retinal disease (age-related macular degeneration)

[0061] Dermatologic conditions - including, for example, eczema and psoriasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present disclosure. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0063] For promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the disclosure as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the disclosure relates.

[0064] The disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0065] FIG. 1A, FIG. IB, FIG. lC-1. FIG. lC-2. FIG. lC-3, FIG. lD-1, FIG. 1D-2, and FIG. 1D-3 illustrate mitochondria functionalized with a TPP-Dextran polymer coating for cellular transplantation. FIG. 1A: Chemical structures of Dextran and TPP, constituent materials of the mitochondrial polymer coating. FIG. IB: Schematic of TPP-Dextran coating of mitochondria, highlighting TPP incorporation into the mitochondrion. FIG. lC-1 to FIG. lC-3: Confocal microscopy of TPP-Dextran coated mouse liver-derived mitochondria. Isolated mitochondria were stained with MitoTracker Deep Red (red) and TPP-Dextran was labeled with FITC (green). The scale bar in the images represents 50 μηι. FIG. lD-1 to FIG. 1D-3: Magnification of a TPP-Dextran coated mitochondrion examined via confocal microscopy. The scale bar represents 0.5 μι^η;

[0066] FIG. 2A-1, FIG. 2A-2, FIG. 2B, and FIG. 2C show function assessment of of TPP-Dextran coated mitochondria isolated from mouse livers. Oxygen consumption of polymer coated (FIG. 2A-2) and uncoated (FIG. 2A-1) mitochondria. The responses to the addition of pyruvate (5 mM), malate (2 mM), ADP (2 mM) and oligomycin (4 μΜ). The breaks on the ADP responses at 0 hr lasts 1.5 min. FIG. 2B: Respiratory control ratio (RCR, state 3 /state 4), with State 3 being response to ADP, and State 4 representing the response to oligomycin. FIG. 2C: Oxygen flux following the addition of oligomycin. The responses indicate the mitochondrial membrane leakage. Results represent mean ± SEM (*P < 0.05 compared with uncoated mitos group). *p < 0.05 compared with uncoated mitos group. P+M: pyruvate + malate; Oligo: oligomycin;

[0067] FIG. 3A-1, FIG. 3A-2, FIG. 3B-1, FIG. 3B-2, and FIG. 3C show uptake and intracellular localization of HeLa-derived mitochondria in H9c2 cardiac myoblast cells. Confocal microscopy images of H9c2 cardiac myoblast cells incubated for 24 hrs with uncoated (FIG. 3A-1 and FIG. 3A-2) or TPP-Dextran-coated (FIG. 3B-1 and FIG. 3B- 2)-2 HeLa-derived mitochondria. Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green). HeLa mitochondria were detected with anti-human mitochondrial antibody (MTC02) and anti-mouse IgG antibody (red). F-actin was stained with Alexa Fluor Phalloidin-647 (purple). Images represent low- (FIG. 3A-1 and FIG. 3B-1) and high- magnification 2D images (FIG. 3A-2 and FIG. 3B-2), highlighting mitochondrial internalization. The scale bar represents 50 μιη. FIG. 3C: Average number of internalized mitochondria, uncoated and TPP-Dextran coated, per field of view in H9c2 cardiac myoblast cells. Results represent mean ± SEM;

[0068] FIG. 4A-1, FIG. 4A-2, FIG. 4A-3, FIG. 4B-1, FIG. 4B-2, FIG. 4B-3, FIG. 4C- 1, FIG. 4C-2, and FIG. 4C-3 show mitochondrial transplantation into MDA-MB-231 breast cancer cells. Confocal microscopy images of MDA-MB-231 breast cancer cells incubated with TPP-Dextran coated mouse liver-derived mitochondria at timepoints of 0 5 hr (FIG. 4A-1 to FIG. 4A-3), 4 hr (FIG. 4B-1 to FIG. 4B-3) and 24 hr (FIG. 4C-1 to FIG. 4C-3). Mitochondria coated with TPP-Dextran/FITC appear as green, nuclei were stained with DAPI (blue), and F-actin was stained with Alexa Fluor Phalloidin-568 (red). Images represent low magnification (FIG. 4A-1, FIG. 4B-1, and FIG. 4C-1), enlarged view (FIG. 4A-2, FIG. 4B-2, and FIG. 4C-2), and 2D images (FIG. 4A-3, FIG. 4B-3, and FIG. 4C-3). Panels below and to the right of 2D images highlight mitochondrial internalization. The scale bar represents 20 μιτι;

[0069] FIG. 5A-1, FIG. 5A-2, FIG. 5A-3, FIG. 5B-1, FIG. 5B-2, FIG. 5B-3, FIG. 5C- 1, FIG. 5C-2, and FIG. 5C-3 show mitochondrial transplantation into SUM-159PT breast cancer cells. Confocal microscopy images of SUM-159PT breast cancer cells incubated with TPP-Dextran coated mouse liver-derived mitochondria at timepoints of 0 5 hr (FIG. 5A-1 to FIG. 5A-3), 4 hr (FIG. 5B-1 to FIG. 5B-3) and 24 hr (FIG. 5C1 to FIG. 5C-3). Mitochondria coated with TPP-Dextran/FITC appear as green, nuclei were stained with DAPI (blue), and F-actin was stained with Alexa Fluor Phalloidin-568 (red). Images represent low magnification (FIG. 5A-1, FIG. 5B-1, and FIG. 5C-1), enlarged view (FIG. 5A-2, FIG. 5B-2, and FIG. 5C-2), and 2D images (FIG. 5A-3, FIG. 5B-3, and FIG. 5C-3). Panels below and to the right of 2D images highlight mitochondrial internalization. The scale bar represents 20 μιτι;

[0070] FIG. 6A-1, FIG. 6A-2, FIG. 6A-3, FIG. 6B-1, FIG. 6B-2, and FIG. 6B-3 show transplantation of TPP-Dextran coated mitochondria into human breast cancer cells enhances mitochondrial oxygen respiration. MDA-MB-231 cell oxygen consumption rate (OCR) (FIG. 6A-1) and extracellular acidification (ECAR) (FIG. 6A-2) response 24 hrs after transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver- derived mitochondria. Cell energy phenotype changes (FIG. 6A-3) was the ratio of mitochondrial respiration (OCR) and glycolysis (ECAR). SUM-159PT cell OCR (FIG. 6B-1) and ECAR (FIG. 6B-2) response 24 hrs after transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver-derived mitochondria. Cell energy phenotype changes (FIG. 6B-3). Results represent mean ± SEM. (*P < 0.05 and †P < 0.05 compared with OCR and ECAR, respectively, in non-treated cells). O: oligomycin; F: FCCP; R+A: rotenone + antimycin A;

[0071] FIG. 7A-1, FIG. 7A-2, FIG. 7A-3, FIG. 7B-1, FIG. 7B-2, and FIG. 7B-3 show transplantation of TPP-Dextran coated mitochondria into cardiac cells enhances mitochondrial oxygen respiration. H9c2 cell OCR (FIG. 7A-1) and ECAR (FIG. 7A-2) response 24 hrs after transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver-derived mitochondria (FIG. 7A-3). Primary adult mouse cardiomyocytes (CM) OCR (FIG. 7B-1) and ECAR (FIG. 7B-2) response 24 hrs after transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver-derived mitochondria. Cell energy phenotype changes are shown in FIG. 7B-3. Results represent mean ± SEM. (*P < 0.05 and†P < 0.05 compared with OCR and ECAR, respectively, in non-treated cells). O: oligomycin; F: FCCP; R+A: rotenone + antimycin A;

[0072] FIG. 8 is a schematic representation of the mechanism of transplanted mitochondria enhances the mitochondria oxygen respiration in the recipient cells. The gray mitochondria represents the endogenous population in the cell, the green represents the mitochondria from donor population;

[0073] FIG. 9A, FIG. 9B, and FIG. 9C show the ^-NMR spectra of TPP (FIG. 9A), dextran (FIG. 9B), and dextran-TPP (FIG. 9C);

[0074] FIG. 10 shows the Zeta-potential analysis of mitochondria prior to (red), and after polymer coating (green);

[0075] FIG. llA-1, FIG. 11A-2, FIG. llB-1, FIG. 11B-2, FIG. llC-1, and FIG. 11C- 2 show confocal microscopy images of polymer coated mitochondria at various weight ratios of Dextran-TPP to mitochondria. Mitochondria coated with 1.4* (FIG. llA-1, and FIG. 11A-2); 1.9* (FIG. llB-1 and FIG. 11B-2); and 2.9x (FIG. llC-1 and FIG. llC-2) polymer by weight. The scale bar in images on the left represent 50 μηι. Images on the right, FIG. 11A-2, FIG. 11B-2, and FIG. llC-2, represent magnified regions from images in FIG. llA-1, FIG. llB-1, and FIG. llC-1, respectively. The scale bar in images on the right represents 10 μιη. The (red) arrows denote uncoated mitochondria;

[0076] FIG. 12A-1, FIG. 12A-2, FIG. 12B-1, FIG. 12B-2, and FIG. 12C show uptake and intracellular localization of HeLa-derived mitochondria in H9c2 cardiac myoblast cells. Confocal microscopy images of H9c2 cardiac myoblast cells incubated for 4 hrs with (FIG. 12A-1 and FIG. 12A-2) uncoated or (FIG. 12B-1 and FIG. 12B-2) TPP- Dextran coated HeLa-derived mitochondria. Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green). HeLa mitochondria were detected with anti-human mitochondrial antibody (MTC02) and anti-mouse IgG antibody (red). F-actin was stained with Alexa Fluor Phalloidin-647 (purple). Images represent low magnification (FIG. 12A-1 and FIG. 12B-1) and 2D images (FIG. 12A-2 and FIG. 12B-2), with panels below and to the right of 2D images highlighting mitochondrial internalization. The scale bar represents 50 μηι. FIG. 12C shows the average number of internalized mitochondria, uncoated and TPP-Dextran coated, per field of view in H9c2 cardiac myoblast cells. Results represent mean ± SEM;

[0077] FIG. 13A-1, FIG. 13A-2, FIG. 13B-1, and FIG. 13B-2 show uptake and intracellular localization of HeLa-derived mitochondria in L929 mouse fibroblast cells. Confocal microscopy images of L929 mouse fibroblast cells incubated for 24 hrs with uncoated (FIG. 13A-1 and FIG. 13A-2) or TPP-Dextran-coated (FIG. 13B-1 and FIG. 13B-2) HeLa-derived mitochondria. Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green). HeLa mitochondria were detected with anti-human mitochondrial antibody (MTC02) and anti-mouse IgG antibody (red). F-actin was stained with Alexa Fluor Phalloidin-647 (purple). Images represent low magnification (FIG. 13A-1 and FIG. 13B-1) and 2D images (FIG. 13A-2 and FIG. 13B-2), with panels below and to the right of the 2D images highlighting mitochondrial internalization. The scale bar represents 50 μιτι;

[0078] FIG. 14A-1, FIG. 14A-2, FIG. 14B-1, and FIG. 14B-2 show uptake and intracellular localization of HeLa-derived mitochondria in L929 mouse fibroblast cells. Confocal microscopy images of L929 mouse fibroblast cells incubated for 24 hrs with uncoated (FIG. 14A-1 and FIG. 14A-2) or TPP-Dextran-coated (FIG. 14B-1 and FIG. 14B-2) HeLa-derived mitochondria. Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green). HeLa mitochondria were detected with anti-human mitochondrial antibody (MTC02) and anti-mouse IgG antibody (red). F-actin was stained with Alexa Fluor Phalloidin-647 (purple). Images represent low magnification (FIG. 14A-1 and FIG. 14B-1) and 2D images (FIG. 14A-2 and FIG. 14B-2), with panels below and to the right of the 2D images highlighting mitochondrial internalization. The scale bar represents 50 μηι;

[0079] FIG. 15 shows the energy metabolism phenotype in different cells. The relative basal oxygen consumption rate and extracellular acidification in 8 different cells and cell line;

[0080] FIG. 16A-1, FIG. 16A-2, FIG. 16A-3, FIG. 16A-4, FIG. 16A-5, FIG. 16A-6, FIG. 16A-7, FIG. 16B-1, FIG. 16B-2, FIG. 16B-3, FIG. 16B-4, FIG. 16B-5, FIG. 16B-7, and FIG. 16B-7 show the dose responses for mito-transplant on human breast carcinoma cell line. Red highlighted is transplanted with uncoated mouse liver mitochondria, green highlighted with polymer coated mouse liver mitochondria. FIG. 16A-1 to FIG. 16A-7: OCR and ECAR dose responses on MDA-MB-231 cell line 24 hrs after mito-transplant. FIG. 16B-1-FIG. 16B-7: OCR and ECAR dose responses on SUM-159PT cell line 24 hrs after mito-transplant. The numbers labeled on OCR vs. ECAR, 0.1, 1, and 10, are the concentration of mitos, 0.1 μg/well, and 10 μg/well as shown. *p < 0.05 compared with OCR in cells control group. †/? < 0.05 compared with ECAR in cells control group. O: oligomycin; F: FCCP; R+A: rotenone + antimycin A;

[0081] FIG. 17A-1, FIG. 17A-2, FIG. 17A-3, FIG. 17A-4, FIG. 17A-5, FIG. 17A-6, FIG. 17A-7, FIG. 17B-1, FIG. 17B-2, FIG. 17B-3, FIG. 17B-4, FIG. 17B-5, FIG. 17B-7, and FIG. 17B-7 show the dose response for mito-transplant on cardiac cells. Oxygen consumption rate (OCR) and extracellular acidification (ECAR) dose response of H9c2 cardiac myoblast cells (FIG. 17A-1 to FIG. 17A-7) and primary adult mouse cardiomyocytes (FIG. 17B-1 to FIG. 17B-7) 24 hrs after transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver-derived mitochondria at concentrations of 0.1

as shown. Results represent mean ± SEM (*P < 0.05 and†P < 0.05 compared with OCR and ECAR, respectively, in non-treated cells. O: oligomycin; F: FCCP; R+A: rotenone + antimycin A; and

[0082] FIG. 18 shows the L/P coupling control ratio. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0083] Illustrative embodiments of the disclosure are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

MITOCHONDRIA

[0084] Mitochondria are the primary source of cellular energy. These organelles can be isolated and then transferred to cells in organs with energy disorders to improve the energy status in conditions like heart failure, or disrupt the energy transfer strategy (i.e. , Warburg effect) in neoplastic cells.

[0085] Energy transfer strategies have evolved to confer a survival advantage to cells, with alteration and impairment of these underlining many prevalent chronic diseases. As an example, high-energy demand tumor cells exhibit a transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis (Vander Heiden et al., 2009), providing a growth advantage over surrounding differentiated cells (Zong et al., 2016). In another example, the failing heart has been documented as energy depleted as evidenced by reduced phosphocreatine-to-ATP ratio (Vander Heiden et al., 2009; Zong et al., 2016; Neubauer et al., 1997; Neubauer, 2007). Despite this, the mitochondria in failing hearts and tumors continue to be active (Senft and Ronai, 2016). Altering these pathological cellular energy strategies should disrupt alternate energy procuring mechanisms of neoplastic cells and enable replenishment of energy depleted organs such as the failing heart.

[0086] Recent reports have documented endogenous mitochondrial intercellular transfer through extracellular spaces into stressed cells, providing a rationale for the potential benefits of mitochondrial transplantation. This has been shown in neurological tissue (Falchi et al, 2013), and from astrocytes to neurons in mice following transient ischemia (Hayakawa et al, 2016). F actin-based tunneling nanotubes facilitate the transfer of functional mitochondria into cultured pheochromocytoma cells, resulting in apoptotic reversal (Wang and Gerdes, 2015). Moreover, mitochondrial transfer from bone marrow-derived stromal cells to pulmonary alveoli led to a reduction of inflammatory injury (Islam et al, 2012). Physiological mitochondrial transfer in pathological states thus provides a rationale for the orthotopic or autologous transplantation of isolated mitochondria from healthy differentiated cells into failing or energy procuring tissues.

[0087] While an innovative therapeutic option to manage devastating chronic conditions, transplantation of isolated mitochondria into malignant or energy -deprived cells in vivo proves challenging. Re-introduction of isolated mitochondria into local or systemic vasculature can potentially render the organelle as a foreign material susceptible to macrophage engulfment and degradation (Murray and Wynn, 2011).

[0088] An objective of this disclosure was to enable in vivo mitochondrial transplantation by functionalizing the surface of mitochondria with the biocompatible polymer dextran (FIG. 1A). Incorporation of hydrophilic polymers such as poly (ethylene glycol) (PEG) onto the exterior of nanoparticle platforms has resulted in prolonged circulation times (Blanco et al, 2015), mostly through prevention of adsorption of opsonins, and recognition by resident macrophages of the mononuclear phagocyte system. Natural polysaccharides such as dextran have previously been used as coatings for nanoparticles, providing several advantages for in vivo delivery of therapeutics. These include reduced opsonization and lessened macrophage uptake (i.e., enhanced "stealthness") and stabilization that prevented aggregation (Silva et al, 2014). Moreover, the versatility of dextran allows for functionalization with moieties that enable molecular imaging, active targeting to specific diseased cells, and incorporation of additional therapeutics for synergy. Findings from this study highlight the feasibility of grafting dextran to the surface of isolated mitochondria, with coated mitochondria entering into a dormant state characterized by reduced respiratory coupling ratio (RCR) and a reduced LEAK state (state 4), a phenomenon not observed in uncoated mitochondria. The present disclosure demonstrates that dextran-functionalization facilitated cellular internalization in a time-dependent fashion, after which transplanted mitochondria induced a metabolic shift from glycolysis to OXPHOS with an increase in the OCR/ECAR ratio in breast cancer and cardiac cells. EXEMPLARY DEFINITIONS

[0089] In accordance with the present disclosure, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Dictionary of Biochemistry and Molecular Biology, (2ⁿ Ed.) J. Stenesh (Ed ), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3^rd Ed ), P. Singleton and D. Sainsbury (Eds ), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2^nd Ed ), P. Walker (Ed.), Chambers (2007); Glossary of Genetics (5^th Ed.), R. Rieger et al. (Eds.), Springer-Verlag (1991); and The HarperCollins Dictionary of Biology, W.G. Hale and J.P. Margham, (Eds.), HarperCollins (1991).

[0091] Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, and compositions are described herein. For purposes of the present disclosure, the following terms are defined below for sake of clarity and ease of reference:

[0092] In accordance with long standing patent law convention, the words "a" and "an," when used throughout this application and in the claims, denote "one or more."

[0093] The terms "about" and "approximately" as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

[0094] Biocompatible" refers to a material that, when exposed to living cells, will support an appropriate cellular activity of the cells without causing an undesirable effect in the cells, such as a change in a living cycle of the cells, a change in a proliferation rate of the cells, or a cytotoxic effect.

[0095] The term "biologically-functional equivalent" is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally-equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the methods and compositions set forth in the instant application.

[0096] As used herein, "biomimetic" shall mean a resemblance of a synthesized material to a substance that occurs naturally in a human body and which is not rejected by (e.g., does not cause an adverse reaction in) the human body.

[0097] As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition.

[0098] Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.

[0099] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) or such like, or a combination thereof, that is pharmaceutically acceptable for administration to the relevant animal. The use of one or more delivery vehicles for chemical compounds in general, and chemotherapeutics in particular, is well known to those of ordinary skill in the pharmaceutical arts. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the diagnostic, prophylactic, and therapeutic compositions is contemplated. One or more supplementary active ingredient(s) may also be incorporated into, or administered in association with, one or more of the disclosed chemotherapeutic compositions.

[0100] As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term "DNA segment," are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

[0101] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

[0102] The term "for example" or "e.g.," as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.

[0103] As used herein, a "heterologous" sequence is defined in relation to a predetermined, reference sequence, such as, a polynucleotide or a polypeptide sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.

[0104] As used herein, "homologous" means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an "analogous" polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).

[0105] As used herein, the term "homology" refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences. The word "identity" may substitute for the word "homology" when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.

[0106] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.

[0107] As used herein, "implantable" or "suitable for implantation" means surgically appropriate for insertion into the body of a host, e.g., biocompatible, or having the desired design and physical properties.

[0108] As used herein, the phrase "in need of treatment" refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein. [0109] The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.

[0110] As used herein, the term "kit" may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the assay methods of the present disclosure. Optionally, such kit may include one or more sets of instructions for use of the enclosed reagents, such as, for example, in a laboratory or clinical application.

[0111] "Link" or "join" refers to any method known in the art for functionally connecting one or more proteins, peptides, polysaccharides, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.

[0112] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.

[0113] As used herein, the term "nucleic acid" includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term "nucleic acid," as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. "Nucleic acids" include single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.

[0114] As used herein, the term "patient" (also interchangeably referred to as "host" or "subject") refers to any host that can receive one or more of the pharmaceutical compositions disclosed herein. Preferably, the subject is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a "patient" refers to any animal host including without limitation any mammalian host. Preferably, the term refers to any mammalian host, the latter including but not limited to, human and non-human primates, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, ranines, racines, vulpines, and the like, including livestock, zoological specimens, exotics, as well as companion animals, pets, and any animal under the care of a veterinary practitioner. A patient can be of any age at which the patient is able to respond to inoculation with the present vaccine by generating an immune response. In particular embodiments, the mammalian patient is preferably human.

[0115] The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and in particular, when administered to a human.

[0116] As used herein, "pharmaceutically-acceptable salt" refers to a salt that preferably retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, without limitation, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like); and salts formed with organic acids including, without limitation, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from NN'-dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0117] As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of compositions.

[0118] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post- translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post- translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures. [0119] For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue provided the desired properties of the polypeptide are retained.

[0120] As used herein, the terms "prevent," "preventing," "prevention," "suppress," "suppressing," and "suppression" as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.

[0121] "Protein" is used herein interchangeably with "peptide" and "polypeptide," and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including those of short peptides from about 2 to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including from about 100 amino acid residues or more in length. Furthermore, the term is also intended to include enzymes, i.e., functional biomolecules including at least one amino acid polymer. Polypeptides and proteins of the present disclosure also include polypeptides and proteins that are or have been post-translationally modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.

[0122] "Purified," as used herein, means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.

[0123] The term "recombinant" indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant" when it is produced by the expression of a nucleic acid segment engineered by the hand of man. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus," e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid.

[0124] The term "RNA segment" refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term "RNA segment," are RNA segments and smaller fragments of such segments. [0125] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure can be provided

[0126] The term "substantially complementary," when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence. As such, typically the sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e., be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.

[0127] Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or "% exact-match") to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the disclosure, and in such instances, the nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds.

[0128] Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0129] As used herein, the term "substantially free" or "essentially free" in connection with the amount of a component preferably refers to a composition that contains less than about 10 weight percent, preferably less than about 5 weight percent, and more preferably less than about 1 weight percent of a compound. In preferred embodiments, these terms refer to less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent.

[0130] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule. [0131] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[0132] As used herein, "synthetic" shall mean that the material is not of a human or animal origin.

[0133] "Targeting moiety" is any factor that may facilitate targeting of a specific site by a particle. For example, the targeting moiety may be a chemical targeting moiety, a physical targeting moiety, a geometrical targeting moiety, or a combination thereof. The chemical targeting moiety may be a chemical group or molecule on a surface of the particle; the physical targeting moiety may be a specific physical property of the particle, such as a surface such or hydrophobicity; the geometrical targeting moiety includes a size and a shape of the particle. Further, the chemical targeting moiety may be a dendrimer, an antibody, an aptamer, which may be a thioaptamer, a ligand, an antibody, or a biomolecule that binds a particular receptor on the targeted site. A physical targeting moiety may be a surface charge. The charge may be introduced during the fabrication of the particle by using a chemical treatment such as a specific wash. For example, immersion of porous silica or oxidized silicon surface into water may lead to an acquisition of a negative charge on the surface. The surface charge may be also provided by an additional layer or by chemical chains, such as polymer chains, on the surface of the particle. For example, polyethylene glycol chains may be a source of a negative charge on the surface. Polyethylene glycol chains may be coated or covalently coupled to the surface using methods known to those of ordinary skill in the art.

[0134] The term "therapeutically-practical period" means the period of time that is necessary for one or more active agents to be therapeutically effective. The term "therapeutically-effective" refers to reduction in severity and/or frequency of one or more symptoms, elimination of one or more symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and the improvement or a remediation of damage.

[0135] A "therapeutic agent" may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally occurring, produced by synthetic or recombinant methods, or a combination thereof. Drugs that are affected by classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician 's Desk Reference and Hardman and Limbird (2001 ).

[0136] As used herein, a "transcription factor recognition site" and a "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.

[0137] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.

[0138] "Transcriptional unit" refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first cw-acting promoter sequence and optionally linked operably to one or more other czs-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis- sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.

[0139] "Treating" or "treatment of as used herein, refers to providing any type of medical or surgical management to a subject. Treating can include, but is not limited to, administering a composition comprising a therapeutic agent to a subject. "Treating" includes any administration or application of a compound or composition of the disclosure to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present disclosure may also be administered prophylactically, i.e., before development of any symptom or manifestation of the condition, where such prophylaxis is warranted. Typically, in such cases, the subject will be one that has been diagnosed for being "at risk" of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.

BIOLOGICAL FUNCTIONAL EQUIVALENTS

[0140] Modification and changes may be made in the structure of the nucleic acids, or to the vectors comprising them, as well as to mRNAs, polypeptides, or therapeutic agents encoded by them and still obtain functional systems that contain one or more therapeutic agents with desirable characteristics. As mentioned above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide.

[0141] When it is desirable to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, second-generation molecule, the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.

[0142] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

TABLE 1

AMINO ACIDS CODONS

Alanine Ala GCA GCC GCG GCU

Cysteine Cys UGC UGU

Aspartic acid Asp GAC GAU

Glutamic acid Glu GAA GAG

Phenylalanine Phe UUC uuu

Glycine Gly GGA GGC GGG GGU

Histidine His CAC CAU

Isoleucine lie AUA AUC AUU

Lysine Lys AAA AAG

Leucine Leu UUA UUG CUA cue CUG CUU

Methionine Met AUG

Asparagine Asn AAC AAU

Proline Pro CCA ccc CCG ecu

Glutamine Gin CAA CAG

Arginine Arg AGA AGG CGA CGC CGG CGU

Serine Ser AGC AGU UCA ucc UCG UCU

Threonine Thr ACA ACC ACG ACU

Valine Val GUA GUC GUG GUU

Tryptophan Trp UGG

Tyrosine Tyr UAC UAU

[0143] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0144] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively based on hydrophilicity. U.S. Patent No. 4,554,101 (specifically incorporated herein in its entirety by express reference thereto), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0145] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0146] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of ordinary skill in the art, and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0147] The section headings used throughout are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application (including, but not limited to, patents, patent applications, articles, books, and treatises) are expressly incorporated herein in their entirety by express reference thereto. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

EXAMPLES

[0148] The following examples are included to demonstrate illustrative embodiments of the disclosure. It should be appreciated by those of ordinary skill in the art that the techniques disclosed in these examples represent techniques discovered to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of ordinary skill in the art should, in light of the present disclosure appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. EXAMPLE 1 - POLYMER FUNCTIONALIZATION OF ISOLATED MITOCHONDRIA FOR CELLULAR TRANSPLANTATION AND METABOLIC PHENOTYPE ALTERATION

[0149] Disruption in mitochondrial energy transfer is associated with prevalent chronic health conditions. Examples include aerobic glycolysis in cancers and energy depletion in the failing left ventricle. Strategies that alter cellular energy handling can affect growth of rapidly dividing cells and replenish energy depleted organs. The present example describes the functionalization of isolated mitochondria with a dextran polymeric outer layer to facilitate transplantation into cancer and energy-depleted cells for restoring cellular mitochondrial metabolism.

[0150] Herein, it is shown that grafting of a polymer conjugate composed of dextran with triphenylphosphonium (TPP) onto isolated mitochondria protected the organelles and facilitated cellular internalization compared to uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells had profound effects on respiration, mediating a shift towards improved oxidative phosphorylation and reduced glycolysis. These data represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.

MATERIALS AND METHODS

[0151] Dextran Polymer Conjugation and Characterization. N,N'-dicyclo- hexylcarbodiimide (DCC) (Sigma-Aldrich, Corp., St. Louis, USA), 4-(dimethylamino) pyridine (DMAP) (Sigma-Aldrich, Corp.), and (5-carboxypentyl) triphenylphosphonium bromide (TPP-COOH) (Alfa Aesar, Lancashire, UNITED KINGDOM) were dissolved in anhydrous dimethyl sulfoxide (DMSO) and stirred for 5 min. The resulting mixture was added to DMSO solution containing Dextran derived from Leuconostoc mesenteroides (Mw 150 kDa, Sigma-Aldrich, Corp.) and left stirring overnight. The solution underwent purification dialysis against water for 3 days using Slide-A-Lyzer™ MINI Dialysis Devices (3.5K MWCO, ThermoFisher Scientific, Waltham, MA, USA), after which functional Dextran-TPP was obtained through lyophilization. Conjugation of TPP to dextran was confirmed by ^-NMR using a Varian 400 MHz NMR spectrometer (Santa Clara, CA, USA). Deuterated-DMSO was used as solvent. Grafted TPP amount on each dextran chain was calculated from the ratio of the characteristic peak area between TPP and dextran.

[0152] To visualize polymer coating, fluorescein 5(6)-isothiocyanate (FITC, Sigma- Aldrich) with λ_εχ = 495 nm and λ_επι = 525 nm was conjugated onto Dextran-TPP polymer by mixing Dextran-TPP and FITC (molar ratio of 1 :3 Dextran-TPP: FITC) in DMSO for 4 hrs in the dark, followed by dialysis of the mixture against water to remove residual solvent and unconjugated FITC, after which, Dextran-TPP: FITC was lyophilized.

[0153] Cell Culture. The human triple-negative breast adenocarcinoma cell line MDA-MB-231 (American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured with Leibovitz's L-15 medium (ATCC) supplemented with 10% (vol/vol.) fetal bovine serum (ThermoFisher Scientific) and 1% (vol. /vol.) penicillin/streptomycin (ThermoFisher Scientific) in a humidified incubator with atmospheric air at 37°C. Triple-negative breast cancer cells (SUM-159PT) (Asterand, Detroit, MI, USA), mouse breast cancer 4T1 cells (ATCC), BD1X rat myoblast H9c2 cells (ATCC) and human cervical carcinoma HeLa cells (ATCC) were all maintained in DMEM-Dulbecco's Modified Eagle Medium (ThermoFisher Scientific) supplemented with 10% (vol. /vol.) fetal bovine serum and 1% (vol/vol.) penicillin/streptomycin in a humidified incubator at 37°C with 5% C0₂.

[0154] Mitochondrial Isolation from Cells. All chemicals used in mitochondrial isolation were obtained from Sigma-Aldrich, Corp. HeLa cells (ATCC) were washed with ice-cold BIOPS buffer (2.8 mM CaK₂EGTA, 7.2 mM K₂EGTA, 5.7 mM Na₂ATP, 6.6 mM MgCl₂ ^»6H₂0, 20 mM taurine, 15 mM Na₂Phospho-creatine, 20 mM, imidazole, 0.5 mM dithiothreitol, 50 mM MES, pH 7.1), detached with a cell scraper, and transferred to 1.5-mL Eppendorf tubes. Following gentle homogenization of cells in Buffer A (220 mM mannitol, 70 mM sucrose, 5 mM MOPS, pH 7.4), Buffer B (2 mM EGTA, 0.2% free fatty acid-free-BSA in Buffer A) was added to a volume of 1.5 mL, and then centrifuged at 800 rpm for 10 min at 4°C. The supernatant was transferred to another tube filled with cold Buffer B and centrifuged at 12,000 rpm for 5 min at 4°C. The pellet was washed twice in ice-cold Buffer B and one time in cold Buffer A, suspended and centrifuged at 12,000 rpm for 5 min at 4°C. Pellets were then suspended in 30 of cold Buffer E (0.5 mM of EGTA in Buffer A). Mitochondrial number was normalized with protein concentration measured using a BCA protein assay (Bio-Rad, Hercules, CA, USA).

[0155] Mitochondrial Isolation from Mouse Tissues. All animal studies were conducted using Institutionally-approved protocols. Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA), aged 7-9 wks, were used for the isolation of mitochondria from liver and skeletal muscle. For liver mitochondrial isolation, mice were sacrificed, livers quickly excised, and washed with ice-cold BIOPS buffer. Tissues were then minced in a petri dish with minimal ice-cold Buffer A. Minced tissues were transferred to 1.5-mL Eppendorf tubes, homogenized with a hand-held pestle grinder in Buffer A, and then centrifuged at 800 rpm for 10 min at 4°C. The supernatant was transferred to another tube and centrifuged at 12,000 rpm for 5 min. The pellet was suspended in cold Buffer B and centrifuged at 12,000 rpm for 5 min. The resulting pellet was rinsed with Buffer A and centrifuged at 12,000 rpm for 5 min, after which, the pellet was suspended in 30 of cold Buffer E.

[0156] For mitochondrial isolation from muscle, skeletal muscle tissue (250-500 mg) was minced into small pieces in minimal ice cold BIOPS buffer, and incubated on ice for 10 min. It was then transferred to a dounce homogenizer vessel and incubated with 1 mL of ice cold, fresh proteinase medium (2 mg/mL proteinase Subtilisin A in ATP medium [100 mM KCl, 50 mM Tris, 5 mM MgS0₄, 1 mM EDTA, 1 mM ATP, 0.05% BSA, pH 7.4]), mixed well, and allowed to settle for 3 min. The supernatant was removed, and the pellet was then suspended in 6 mL ice-cold ATP medium, followed by tissue homogenization using a dounce homogenizer on ice. The homogenate was then transferred to a 15-mL centrifuge tube, and centrifuged at 1,700 rpm for 5 min at 4°C. The supematant was decanted into another 15-mL centrifuge tube, and centrifuged at 3,700 rpm for 20 min at 4°C. The pellet was then suspended in 1 mL KCl medium (100 mM KCl, 50 mM Tris, 5 mM MgS0₄, 1 mM EDTA; pH 7.4), transferred to a 1.5-mL Eppendorf tube, and centrifuged at 6,800 rpm for 10 min at 4°C. The residual pellet was resuspended in KCl medium and centrifuged. The final pellet was suspended in 30 Buffer E.

[0157] Cardiomyocyte Isolation from Adult Mice. Cardiomyocytes were isolated from 8-10 week old male C57BL/6J mice (Jackson Laboratory) by enzymatic digestion with a Langendorff perfusion system (Hugo Sachs Elektronik, GERMANY) followed by calcium reintroduction. The collagenase cocktail isolation perfusion buffer contained 0.15 mg/mL Liberase™ (Roche LifeScience, Indianapolis, IN, USA).

[0158] Mitochondrial Coating with Dextran-TPP. Dextran-TPP in Buffer E was mixed with the pellet of isolated mitochondria at concentrations ranging from 3- to 6- mg/mL, and left shaking for 20 min at 4°C. After incubation for another 20 min at 4°C, coated mitochondria were centrifuged and washed 2X with Buffer E to remove excess Dextran-TPP. Uncoated mitochondria (serving as a control) underwent the same process. Mitochondria coated with fluorescent polymer were obtained using the same process with FITC -labeled Dextran-TPP.

[0159] Cellular Uptake of Mitochondria, MDA-MB-231 cells and SUM 159 cells were seeded in 8-well chamber slides (EMD Millipore, Billerica, MA, USA) for overnight at a density of 2 ^χ 10⁴ cells and 1.2 ^χ 10⁴ cells/well, respectively. Cells were then incubated with fluorescently-coated and uncoated mitochondria at various weight ratios of mitochondria to cell (1.4: 1, 1.9: 1, or 2.9: 1) and different doses of fluorescent polymer. At predetermined time-points (0.5 hr, 4 hrs, and 24 hrs) cells were washed 2X with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and permeabilized with 0.1% Triton X-100 for 5 min. F-actin was stained with Alexa Fluor® 568 Phalloidin (Fisher Scientific. Cells were mounted with DAPI medium before imaging using a confocal imaging system (Al Nikon, Inc., Melville, NY, USA.)

[0160] Mitochondrial uptake was also examined in 4T1 mouse breast cancer cells. Cells were cultured in 8-chamber slides (2 χ 10⁴ cells/well) and treated with fluorescently-coated or uncoated mitochondria obtained from HeLa cells. After 4 and 24 hrs, immunofluorescent detection of HeLa-derived mitochondria in 4T1 cells was performed by incubation with anti-human mitochondria monoclonal antibody MTC02 (Abeam, Cambridge, MA, USA) in 1% BSA solution overnight at 4°C after fixing, permeabilizing, and blocking with 2% BSA in PBS. Anti-mouse IgG H&L (Cy3®) preadsorbed (Abeam) was applied as a secondary antibody to visualize HeLa-derived mitochondria in 4T1 cells. F-actin was stained with Alexa Fluor® 647 Phalloidin. Slides were mounted with DAPI medium onto a coverslip and examined using confocal microscopy. [0161] Mitochondrial Functional Analysis. Mitochondrial respiratory function was assessed with Oroboros high-resolution respirometry (Innsbruck, AUSTRIA) using coated and uncoated mitochondria. Mitochondria were suspended in MiR05 medium [0.5 mM EGTA, 3 mM MgCl₂ ^»6H₂0, 60 mM K-lactobionate, 2 mM taurine, lO mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose, 1 g/L fatty acid free bovine serum albumin (BSA), pH 7.1] in Oroboros chambers with a final concentration of -0.1 mg/mL mitochondrial protein. The substrates pyruvate-malate (PM, 5 mM each), ADP (4 mM) and oligomycin (5 μΜ) were sequentially added to measure oxidative phosphorylation LEAK and oxidative phosphorylation (OXPHOS) capacity. Respiratory control ratios (RCR), indices of coupling between respiration and OXPHOS, were calculated as the ratio of state 3 (ADP -supported respiration) to oligomycin state 4 (ATP-synthase- independent respiration after oligomycin addition). All readings were normalized for mitochondrial protein content as determined by BCA protein assay.

[0162] Extracellular Flux Analysis. Cells were plated (1.5 ^χ 10⁴ cells/well) in 24- well Seahorse XF24 cell culture microplates 6 hrs' prior to incubation with mitochondria. Isolated cardiomyocytes were cultured onto laminin-coated Seahorse plates with 5% fetal bovine serum (FBS) overnight prior to incubation with mitochondria. At a time-point of 24 hr after incubation, plates were washed twice with Seahorse Assay Medium (Seahorse base medium containing 25 mM D-glucose, 1 mM sodium pyruvate and 1 mM L-glutamine), the assay medium changed and maintained at 37°C in a non-C0₂ incubator for 30 min. After baseline measurements, the medium was injected sequentially with: 1) oligomycin (1 μΜ); 2) FCCP (1 μΜ); and 3) rotenone (0.5 μΜ) plus antimycin A (0.5 μΜ). OCR and ECAR were measured using the Seahorse XF24 Analyzer (Agilent, Santa Clara, CA, USA), as recommended by the manufacturer for Mito Stress Test Kit (Agilent Technologies, 13015-100). OCR and ECAR measurements were normalized to cell number in each well. [0163] Statistical Analyses. Prism software (version 7.00 for Microsoft Windows®) (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis unless otherwise stated. Results are expressed as mean ± SEM. Student's /-test was used to assess differences between means of two independent data sets. One-way ANOVA followed by Tukey's multiple comparison tests were used for differences in oxygen flux and extracellular acidification rate. A / value < 0.05 was considered significant.

RESULTS AND DISCUSSION

[0164] Dextran-TPP polymer comprehensively coated isolated mitochondria. The objective of this example was to polymerically functionalize the exterior of isolated mitochondria to enable transplantation of these organelles within diseased cells. Dextran was selected based on its wide use in a number of biomedical applications, with advantages that include biocompatibility and the potential for further functionalization with targeting ligands and therapeutic and imaging moieties. To obtain a stable association with mitochondrial membranes, Dextran was conjugated to triphenylphosphonium (TPP) (FIG. 1A), a lipophilic, cationic ligand with mitochondriotropic properties¹⁶ (FIG. IB). Dextran-TPP conjugation proved successful, as demonstrated by ^-NMR (FIG. 9A-FIG. 9C). Upon functionalization of mitochondria with the Dextran-TPP polymer, coating of mitochondria was shown to be dependent on Dextran-TPP: mitochondria weight ratio, with higher ratios resulting in a more complete and comprehensive coating of isolated mitochondria (FIG. lC-1 to FIG. lC-3). Mitochondrial coating was further confirmed by zeta potential analysis, (FIG. 10) with findings demonstrating that the negative surface charge of the organelles became positive after functionalization with Dextran-TPP. Confocal microscopy examination of coated mitochondria indeed demonstrates successful coating of mitochondria with polymer. As can be seen in FIG. llA-1 to FIG. llC-2, a comprehensive coating of mitochondria was achieved, as demonstrated by co- localization of mitochondria stained with MitoTracker Red and FITC (green) fluorescently-labeled Dextran-TPP, with successful polymer functionalization highly evident in the magnified image of a single coated mitochondria (FIG. lD-1 to FIG. 1D- 3).

[0165] Polymer surface functionalization of mitochondria resulted in a dormant respiratory state. Respiratory function of uncoated and Dextran-TPP coated mitochondria was assessed with Oroboros high resolution respirometry. The 0 hr oxygen flux response to ADP after substrate (State 3) in uncoated controls (680 ± 45 pmol/[s*mg]) compared to the pre-coated group (685 ± 76 pmol/[s*mg]) showed no difference (FIG. 12A-1 to FIG. 12C). Counting began at the 0 hr time-point, immediately before the Dextran-TPP functionalization process; the respiratory function at this time point can be regarded as basal level. At the 2-hr time-point, the ADP response in coated mitochondria dropped to 153 ± 25 pmol/(s*mg), a 78% decline compared to the uncoated group, which remained unchanged at 2 hrs (683 ± 51 pmol/[s*mg]) and 8 hrs (656 ± 38 pmol/[s*mg]). Oxygen flux in uncoated controls at each time point had no significant difference compared with the 0 hr time point. In contrast, coated mitochondria at 8 hrs (63 ± 12 pmol/[s*mg]) remained significantly suppressed compared with the 0 hr time-point but without a significant change from the 2-hr time-point (FIG. 2A-1 and FIG. 2A-2).

[0166] To assess the OXPHOS coupling efficiency, the respiratory control ratio (RCR), defined as the ratio of State 3 (substrate and ADP) to State 4 respiration after oligomycin addition, was determined (FIG. 2B). The RCR index decreased after Dextran-TPP from 5.26 ± 0.20 at time 0 h to 2.44 ± 0.17 at 2 hrs. The RCR of uncoated mitochondria had no significant drop after 2 hrs (5.85 ± 0.42 compared to 5.57 ± 0.41 at the 0 hr time-point). [0167] To determine if the RCR decline was due to loss of ADP-activated state 3 respiration or increased LEAK respiration, oxygen flux rates were assessed after addition of oligomycin, a mitochondria complex V inhibitor that enhances membrane LEAK respiration (FIG. 2C). At 4 hrs, there was no difference in LEAK respiration rates between coated and uncoated mitochondria. After 6 hrs, the LEAK rate in the Dextran- TPP coated group significantly decreased from 106 ± 9 pmol/(s*mg) at time O hr to 58 ± 29 pmol/(s*mg) after 6 hrs. By comparison, a slight LEAK increase was observed in the uncoated group between time O hr (105 ± 19 pmol/[s*mg]) and 6 hrs (121 ± 16 pmol/[s*mg]). The reduced RCR in the coated group was the result of reduced ADP State 3 respiration rather than increased LEAK, given that a reduced LEAK rate was observed in Dextran-TPP coated mitochondria compared to uncoated controls.

[0168] Dextran Functionalization of Isolated Mitochondria Facilitates Cellular Internalization. The ability of Dextran-TPP coated mitochondria to undergo cellular entry was examined in H9c2 cardiac myoblast cells via confocal microscopy (FIG. 3A-1 to FIG. 3C). H9c2 cells isolated from mice were chosen for this experiment for ease of identification of human mitochondria derived from HeLa cells via fluorescence-based immunohistochemistry, enabling differentiation of native mouse mitochondria from transplanted human mitochondria. A small degree of internalization of uncoated mitochondria occurred after 4 hrs' incubation with H9c2 cells, with mitochondria mostly confined to extracellular spaces and cellular membranes. Mitochondria functionalized with Dextran-TPP polymer underwent a greater degree of uptake in H9c2 cells after 4 hrs compared to the uncoated group, albeit, with a large extent of mitochondria also found outside of the cell at this time-point. An approximate 3 -fold difference in internalization was observed between coated and uncoated mitochondria 4 hrs after incubation (FIG. 10). After 24-hrs' incubation, uncoated mitochondria underwent increased internalization within H9c2 cells compared to the 4-hr time-point (FIG. 3A). However, mitochondrial uptake into cells after 24 hrs was substantially greater following functionalization with Dextran-TPP (FIG. 3B). Once again, an approximate 3-fold difference in accumulation compared to the uncoated mitochondria group was observed at 24 hrs (FIG. 3C). Similar findings were observed in a separate study performed in L929 mouse fibroblast 6 cells, wherein HeLa-derived mitochondria coated with Dextran- TPP remained confined to extracellular spaces and cellular membranes at earlier time points (FIG. llA-1 to FIG. 11C-), but underwent substantial uptake by the 24-hr timepoint. It is important to note that an increase in uptake of coated mitochondria by L929 cells was observed compared to uncoated mitochondria at both time points.

[0169] Having successfully demonstrated increased cellular internalization of Dextran-TPP functionalized mitochondria compared to uncoated mitochondria, the kinetics of cellular association and internalization were examined in human triple negative breast cancer cell lines. As can be observed in FIG. 4A-1 to FIG. 4C-3, mitochondrial internalization in MDA-MB-231 triple negative breast cancer cells was time dependent. At early time points of 0.5 hrs, polymer-coated mitochondria were found associated with cell membranes. However, at time points of 4 hrs after cellular incubation, mitochondria were found internalized within cells. By 24 hrs, there was a substantial increase in mitochondrial uptake within cells (FIG. 4A-1 to FIG. 4C-3). As is evident in the confocal micrographs, internalized mitochondria were found in perinuclear regions of the cell at time-points as early as 4 hrs after incubation, indicating trafficking of transplanted mitochondria to the nucleus. A similar pattern of time- dependent internalization of polymer coated mitochondria was also observed in SUM159PT triple negative breast cancer cells (FIG. 5A-1 to FIG. 5C-3).

[0170] Transplantation of Dextran-TPP Functionalized Mitochondria into Breast Cancer Cell Lines Result in Respiratory Changes. To determine whether internalization of transplanted mitochondria were capable of triggering a bioenergetic switch, simultaneous oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were assessed by a Seahorse XF24 extracellular flux analyzer. MDA-MB- 231 cells were examined alone, and after the addition of either uncoated or coated mitochondria. As can be seen in FIG. 6A-1 to FIG. 6B-3, MDA-MB-231 is a glycolysis dominated human breast carcinoma cell line. All cells regardless of the treatment respired effectively with substrate and following sequential addition of inhibitors and uncouplers. Interestingly, the basal OCR was significantly increased in the Dextran-TPP coated group (210.5 ± 30.7 pMoles/min) compared with non-treated MDA-MB-231 cells (158.7 ± 5.2 pMoles/min). The maximum OCR capacity (FCCP addition) also increased significantly in the coated group (265.4 ± 38.7 pMoles/min) compared with non-treated MDA-MB-231 cells (192.3 ± 6.75 pMoles/min). These findings indicate that addition of Dextran-TPP coated mitochondria accelerated the electron transfer rate to oxygen in the basal and uncoupled state. LEAK respiration (oligomycin addition) and non- mitochondrial oxygen consumption (rotenone and antimycin A addition) did not change. The average basal OCR and ECAR was assessed prior to addition of oligomycin (FIG. 6A-1) and a significant shift was found in both the coated and the uncoated groups from ECAR to OCR, indicating a switch from glycolytic to oxidative phenotype. However, the Dextran-TPP coated group shifted to a greater extent than the uncoated group, with both OCR and ECAR changing significantly (p < 0.05). The OCR of the non-treated MDA-MB-231 group was 180.2 ± 4.7 pMoles/min, while that of the uncoated group and coated group increased to 203.6 ± 3.0 pMoles/min and 249.7 ± 4.4 pMoles/min, respectively. The ECAR of the non-treated MDA-MB-231 group was 17.6 ± 0.7 mpH/min, while that of the uncoated group and coated group decreased to 15.9 ± 0.9 mpH/min and 13.8 ± 0.5 mpH/min, respectively.

[0171] To confirm the bioenergetic switch observed in MDA-MB-231, OCR and ECAR were examined in the SUM-159PT triple negative breast cancer cell line. The OCR and ECAR changes after coated and uncoated mitochondrial transplantation were consistent with those observed in the MDA-MB-231 cell line. The basal SUM-159PT OCR significantly increased in the coated group (126 ± 2.8 pMoles/min) compared with non-treated MDA-MB-231 cells (71 ± 4.6 pMoles/min). The maximum OCR capacity (FCCP addition) increased in the coated group (110 ± 11.8 pMoles/min) compared with non-treated MDA-MB-231 cells (86.4 ± 2.8 pMoles/min). Upon averaging of the OCR and ECAR, a significant shift from ECAR to OCR in both the coated and uncoated groups was indicative of a cell metabolism switch from a glycolytic to oxidative state. The coated group shifted much more than the uncoated group, with both OCR and ECAR changing significantly (p < 0.05) compared to the uncoated group. The OCR of the non-treated SUM-159PT control group was 80 ± 3.9 pMoles/min, after uncoated 104 ± 7.2 pMoles/min, while that of the coated group increased to 127 ± 2.8 pMoles/min. The ECAR of the non-treated SUM-159PT group was 26.8 ± 3.2 mpH/min, while that of the uncoated group and coated group decreased to 23 ± 3.2 mpH/min and 19.2 ± 0.8 mpH/min, respectively.

[0172] Transplantation of Dextran-TPP functionalized mitochondria into cardiac myoblast cells and primary cardiomyocyte isolates result in respiratory changes. To determine the effect of transplantation of Dextran-TPP functionalized mitochondria on cardiac cell bioenergetics, OCR and ECAR were examined in H9c2 cell line and isolated adult mouse cardiomyocytes (FIG. 7A-1 to FIG. 7B-3). Basal OCR significantly increased in the coated group (202.9 ± 13.3 pMoles/min) compared with non-treated H9c2 cells (102.1 ± 4.2 pMoles/min). The maximum OCR capacity (FCCP addition) also increased in the coated group (322.4 ± 22.6 pMoles/min) compared with non-treated H9c2 cells (229.3 ± 4.7 pMoles/min). A significant shift from ECAR to OCR occurred in the coated group (OCR 209.1 ± 8.0 pMoles/min, ECAR 9.1 ± 0.7 mpH/min) compared to non-treated H9c2 cells (OCR 126.2 ± 14.8 pMoles/min, ECAR 11.0 ± 0.7 mpH/min). The ECAR did not change significantly in the uncoated group, but the OCR significantly increased to 150.4 ± 5.5 pMoles/min. Minimal LEAK respiration occurred after the addition of oligomycin, and differences in mitochondrial oxygen consumption following addition of rotenone and antimycin A were not significant.

[0173] In isolated adult cardiomyocytes (CMs), the basal OCR significantly increased in the coated group (505.3 ± 27.6 pMoles/min) compared with non-treated CMs (273.5 ± 75.0 pMoles/min) (FIG. 7A-1 to FIG. 7B-3). The maximum OCR capacity (FCCP addition) was lowest in the coated group (805.2 ± 23.7 pMoles/min) compared with non-treated CMs (1068.1 ± 67.9 pMoles/min). A significant shift from ECAR to OCR was observed in the coated group (OCR 592.2 ± 49.5 pMoles/min, ECAR 4.4 ± 1.3 mpH/min) compared with non-treated CMs (OCR 382.0 ± 14.8 pMoles/min, ECAR 11.6 ± 1.0 mpH/min). While the ECAR decreased in the uncoated group (6.0 ± 1.0 mpH/min), the OCR did not change significantly.

[0174] The triple-negative breast cancer cell lines MDA-MB-231 and SUM-159PT exhibited a baseline glycolytic profile. In contrast, high energy -demanding cardiomyocytes have an abundance of mitochondria, constituting 30-40% of the cellular volume. Consequently, 95% of the ATP is produced by OXPHOS and the remaining 5% by glycolysis. Thus, cardiomyocytes are committed to OXPHOS and cannot efficiently balance between oxidative phosphorylation and glycolysis as efficiently as other cells. The acidification observed may result not only from glycolytic lactate release, but also from increased Kreb's cycle flux releasing CO₂ and carbonic acid release. Therefore, the inverse relationship of ECAR to OXPHOS is not as closely linked in cardiomyocytes as in MDA-MB-231, SUM-159PT, or H9c2 cells, the latter of which demonstrate an ECAR increase in a compensatory response to OCR decrease.

[0175] The considerable shift in bioenergetic phenotype following transplantation of polymer-functionalized mitochondria is most likely the result of increased number of cellular mitochondria. Mitochondria functionalized with dextran showed a much higher accumulation than uncoated mitochondria, and thus, a greater shift towards increased OXPHOS. It is now well known that mitochondrial number and function change in response to cell stress and changes in phenotype. Increased mitochondrial biogenesis and maintenance of mitochondrial DNA has been observed in conditions of oxidative stress. Upon stem cell differentiation into motor neurons, an increase in mitobiogenesis was also observed that correlated with a transition from glycolysis to OXPHOS. Another potential explanation for the shift observed following mitochondrial transplantation may be a result of compensatory fusion of mitochondria that would alter the number of mitochondria impacting metabolic functions. As an example, in heart failure, despite the low energy state, a sub-population of mitochondria in failing hearts remains active.

[0176] In conclusion, this example illustrates the development of a new strategy to polymerically functionalize isolated mitochondria for the purposes of transplantation into cells and tissues to alter dynamics of energy handling such as substrate selection and efficiency of electron transport. A Dextran-TPP polymer conjugate comprehensively coated isolated mitochondria, with the coating shown to protect mitochondrial respiratory function. These results highlighted the efficient internalization of dextran- coated mitochondria into breast cancer and cardiac cell lines, with an approximate 3-fold increase compared to uncoated mitochondria. Similarly, mitochondrial transplantation into both cancer and cardiac cell lines resulted in a significant shift in the bioenergetic phenotype from a glycolytic to oxidative state. These findings represent the first demonstration of polymer functionalization of mitochondria for purposes of cellular transplantation, opening potential avenues for translation of this approach for treatment of diseases characterized by metabolic impairment. In light of these results, in vivo approaches to cancer treatment, prevention of heart failure, and modulation of neurodegenerative disease have now been made possible.

EXAMPLE 2 - POLYMER-FUNCTIONALIZED MITOCHONDRIA FOR TREATMENT OF HEART FAILURE

[0177] The present example describes a strategy wherein mitochondria are polymerically functionalized for purposes of mitochondrial transplantation in diseases such as cancer and heart failure. The polymer serves to enhance transmembrane delivery into cells and protect mitochondria from compliment opsonization in vivo. Moreover, the polymeric coating can in turn be functionalized with targeting moieties for enhanced retention at target sites, as well as internalization into cells. Energy depletion due to inefficient mitochondrial metabolism is a hallmark of heart failure.

[0178] The concept of reenergizing the heart through mitochondrial transplantation, rather than refueling, reprogramming, or replacing it, represents a highly innovative approach for the treatment of heart failure. In cancer, neoplastic cells become reliant on glycolysis, termed the "Warburg Effect." Mitochondrial transplantation to cancer cells could alter their dependence on glucose for fuel, changing the highly aggressive phenotype of cancer cells.

[0179] Mitochondria from liver were isolated from mice and a polymeric coating for isolated mitochondria was designed and optimized. The polymer consists of a dextran conjugate that offers a hydrating layer or shell, to the mitochondria Encapsulation conditions were optimized to yield enhanced viability and resp ratory status of mitochondrial isolates, as well as optimal amounts that ensured uniform coating of all mitochondria. After establishing the nano-coating, the isolated murine liver mitochondria were transplanted into human (a xeno-transplant) tumor cell cultures. [0180] Confocal microscopy studies confirmed entry into cells. In a separate study, it was demonstrated (via immunohistochemistry) that mitochondria functionalized with an outer polymeric shell did indeed undergo more internalization into breast cancer cells than uncoated mitochondria. Detailed functional studies comparing the energy state of control cells without mite-transfer to those co-cuitured with both uncoated and coated mitochondria were quantitatively compared. The ability of transplanted polymeric- coated mitochondria to reverse the dependence of triple negative breast cancer cells (MDA-MB-231 cells) on glycolysis has been demonstrated. This was especially striking considering it was accomplished with a xenotransplant from mouse liver into human tumor cells.

[0181] These findings confirmed feasibility of the process in cardiomyocytes, with results demonstrating that internalization of mitochondria within cardiomyocytes permitted the modification of the metabolic activity of the cells by increasing respiratory capacity, and by decreasing glycolytic activity.

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[0233] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0234] The description herein of any aspect or embodiment of the disclosure using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the disclosure that "consists of, "consists essentially of, or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0235] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents that are chemically and/or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.

[0236] All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS:

A composition comprising a population of mammalian mitochondria that are surface-functionalized with at least one biocompatible polymer.

The composition in accordance with claim 1 , wherein the biocompatible polymer comprises poly-arginine, poly-lysine, polyethyleneimine, dextran, poly-ethylene glycol (PEG), chitosan, polypeptide, poly(amino-ester), polyphosphate, polycarbonate, hyaluronic acid, polyacrylic acid, polyallylamine, poly(methyl- methacrylate), PAMAM, dendrimers, or one or more amphiphilic polymers comprising dextran, chitosan, PEG, polypeptide, polyphosphate, (hyaluronic acid)-poly ester (or polypeptide, polyamino ester), or any combination thereof.

The composition in accordance with claim 1 or claim 2, wherein the biocompatible polymer is operably linked to at least a first portion of the outer surface of at least a majority of the population of mammalian mitochondria by conjugation, by grafting, or by functionalization with one or more biocompatible chemical linking agents.

The composition in accordance with any preceding claim, wherein the one or more biocompatible chemical linking agents comprises triphenylphosphonium (TPP), cholesterol, oleic acid, or any combination thereof.

The composition in accordance with any preceding claim, wherein surface functionalization of the population of mammalian mitochondria increases cellular uptake of the functionalized organelles. The composition in accordance with any preceding claim, wherein surface functionalization overcomes at least one biological barrier selected from the group consisting of a hemo-rheology barrier, a reticulo-endothelial barrier, a blood brain barrier, a tumor associated osmotic interstitial pressure barrier, an ionic and molecular pump barrier, a cell membrane barrier, an enzymatic degradation barrier, a nuclear membrane barrier, and combinations thereof.

The composition in accordance with any preceding claim, wherein the population of surface-functionalized mitochondria further includes the presence of at least one targeting moiety on the surface of the mitochondria.

The composition in accordance with any preceding claim, wherein the at least one targeting moiety is selected from the group consisting of folate, biotin, cholesterol, LHRH peptide, RGD peptide, transferrin, an antibody, an antibody fragment, a peptide, an aptamer, a small molecule, and combinations thereof.

The composition in accordance with any preceding claim, wherein the targeting moiety is specific for a particular population of mammalian cells.

The composition in accordance with any preceding claim, wherein the targeting moiety is specific for mammalian cancer cells, or for one or more cell types that are found in a particular mammalian organ, such as a mammalian heart, or a mammalian tumor, or a mammalian liver, for example. The composition in accordance with any preceding claim, where the population of functionalized mitochondria further comprise a therapeutic agent that is selected from the group consisting of a gene, a nucleic acid, an shRNA, an siRNA, a microRNA, a DNA fragment, an RNA fragment, a plasmid, and combinations thereof.

The composition in accordance with any preceding claim, formulated for human administration.

A composition in accordance with any preceding claim, for use in therapy.

A therapeutic kit comprising a composition in accordance with any one of claims 1 to 13, and a set of instructions for using the composition in the treatment or amelioration of one or more symptoms of a mammalian disease, dysfunction, impairment, injury, trauma, or abnormal metabolic condition.

The therapeutic kit in accordance with claim 14, further comprising a second distinct therapeutic molecule, such as a chemotherapeutic agent, an antiinflammatory agent, a therapeutic agent, or any combination thereof.

Use of a composition in accordance with any one of claims 1 to 13, in the manufacture of a medicament for treating a disease, dysfunction, disorder, injury, trauma, or abnormal condition in a mammal, or ameliorating at least one or more symptoms thereof.

17. Use in accordance with claim 16, in the manufacture of a medicament for diagnosis, imaging, treating, alleviating, delaying, reducing the severity of, or ameliorating at least one or more symptoms of cancer, and particularly metastatic or therapy-resistant breast cancer, in a mammal, and preferably, in a human.

18. A method of selective gene silencing in a mammalian cancer cell, comprising administering to a mammal, an effective amount of the composition in accordance with any one of claims 1 to 13.

A method of treating a diseased or damaged heart in vivo, the method comprising administering to a mammal having such a diseased or damaged heart, a composition in accordance with any one of claims 1 to 13 in an amount, and for a time effective to treat the disease or the damage in the heart.

The method in accordance with claim 19, further comprising administering to the mammal an effective amount of a second, distinct therapeutic agent.

A method of modulating the bioenergetics phenotype of a plurality of mammalian cells, the method comprising administering to the plurality of cells, an amount of the composition in accordance with any one of claims 1 to 13, and for a time effective to modulate the bioenergetics phenotype of the plurality of mammalian cells.

The method of claim 21, wherein the plurality of mammalian cells is located within an organ to be implanted within the body of a human transplanted organ recipient.

The method of claim 22, wherein the transplant organ is a heart, a liver, a kidney, a pancreas, or a population of islet cells.

A method of treating a bioenergetic deficiency or a metabolic impairment in one or more selected cells, tissues or an organ of a mammal, and particularly a human, the method comprising administering to the mammal an amount of the composition in accordance with any one of claims 1 to 13, and for a time effective to treat the bioenergetics deficiency or the metabolic impairment in the one or more selected cells, tissue or the organ of the mammal.

25. The method of claim 24, wherein the bioenergetics deficiency is a REDOX state alteration, or the metabolic impairment is a deficiency in one or more oxidoreductive molecules.