WO2019028469A1 - Compositions mitochondriales a polymère fonctionnalisé et procédés d'utilisation dans une transplantation cellulaire et pour modifier un phénotype métabolique - Google Patents

Compositions mitochondriales a polymère fonctionnalisé et procédés d'utilisation dans une transplantation cellulaire et pour modifier un phénotype métabolique Download PDF

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Publication number
WO2019028469A1
WO2019028469A1 PCT/US2018/045416 US2018045416W WO2019028469A1 WO 2019028469 A1 WO2019028469 A1 WO 2019028469A1 US 2018045416 W US2018045416 W US 2018045416W WO 2019028469 A1 WO2019028469 A1 WO 2019028469A1
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mitochondria
cells
accordance
composition
mitochondrial
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PCT/US2018/045416
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English (en)
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Dale James HAMILTON
Anisha Anil GUPTE
Aijun Zhang
Elvin Blanco
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The Methodist Hospital
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37Digestive system
    • A61K35/407Liver; Hepatocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system

Definitions

  • the present disclosure relates to the fields of transplantation medicine, oncology, and pharmacology.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • compositions and their subsequent transfer into altered organs have proven useful in modifying the bioenergy profile of the altered organs.
  • 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.
  • the disclosure also provides compositions and methods for the metabolic reprogramming of abnormal cells, such as cancer and/or tumor cells.
  • 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.
  • 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.
  • 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.
  • 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).
  • compositions may be formulated to include one or more additional active agents, including, without limitation, a small molecule drug or a bimolecular drug.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the active ingredient(s) and targeted drugs will be substantially active at physiological conditions of the animal into which they are being administered.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • delivery devices adapted to deliver the chemotherapeutic composition(s) to an animal (e.g., syringes, injectables, and the like).
  • 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.
  • the kit may also contain a second distinct container into which this second composition may be placed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, transdermal, subdermal, and/or intraperitoneal administration.
  • 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.
  • 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.
  • an isotonic solution e.g., an isotonic NaCl-based solution
  • 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.
  • 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.
  • inorganic acids such as, without limitation, hydrochloric or phosphoric acids
  • 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 inorgan
  • 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.
  • 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.
  • polymer-functionalized mitochondrial compositions 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the present disclosure are not in any way limited to use only in humans, or even to primates, or mammals.
  • the methods and compositions disclosed herein may be employed using avian, amphibian, reptilian, or other animal species.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • 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.
  • targeting moieties can also be added to "fine- tune" the delivery and/or uptake of a particular formulation of functionalized mitochondria.
  • 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.
  • fluorescence reagents including Cy5, Cy5.5, Cy7, and Bodipy
  • MRI contrast agents such as iron oxide (Fe 3 04) nanoparticles, and gadolinium (Ga)
  • CT contrast agents such as gold nanoparticles
  • PET/SPECT agents quantum dots (QDs)., and the like
  • QDs quantum dots
  • 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.
  • chemotherapeutics e.g., doxorubicin, paclitaxel, platinum-based drugs
  • drugs in other disease conditions e.g., heart failure, retinal disorders
  • 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.
  • Cardiovascular diseases including, for example, heart failure, ischemic heart disease, congenital heart disease, and related conditions.
  • 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.
  • neurodegenerative disease e.g., Parkinson's disease, frontal temporal lobe dementia
  • cerebrovascular disease e.g., acute stroke, transient ischemic attacks, etc.
  • 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.
  • donor organ e.g., heart, liver, pancreatic islets, kidneys, etc.
  • Diabetes/obesity therapy ⁇ including, for example, hepatic and muscle insulin resistance in type 2 diabetes, transplantation into adipose tissue;
  • Ophthalmologic therapies including, for example, retinopathy (e.g., diabetic retinopathy) and degenerative retinal disease (age-related macular degeneration)
  • retinopathy e.g., diabetic retinopathy
  • degenerative retinal disease age-related macular degeneration
  • Dermatologic conditions including, for example, eczema and psoriasis.
  • 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.
  • FIG. lD-1 to FIG. 1D-3 Magnification of a TPP-Dextran coated mitochondrion examined via confocal microscopy.
  • the scale bar represents 0.5 ⁇ ⁇ ;
  • FIG. 2A-1, FIG. 2A-2, FIG. 2B, and FIG. 2C show function assessment of of TPP-Dextran coated mitochondria isolated from mouse livers.
  • 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.
  • 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;
  • 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.
  • Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green).
  • 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;
  • 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.
  • 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 ⁇ ;
  • 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.
  • 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 ⁇ ;
  • 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.
  • OCR cell oxygen consumption rate
  • ECAR extracellular acidification
  • 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
  • ECAR FIG. 7A-2
  • FIG. 7A-3 show transplantation of uncoated (red) and TPP-Dextran-coated (green) mouse liver-derived mitochondria (FIG. 7A-3).
  • 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;
  • FIG. 9A, FIG. 9B, and FIG. 9C show the ⁇ -NMR spectra of TPP (FIG. 9A), dextran (FIG. 9B), and dextran-TPP (FIG. 9C);
  • FIG. 10 shows the Zeta-potential analysis of mitochondria prior to (red), and after polymer coating (green);
  • 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 ⁇ .
  • 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 ⁇ .
  • the (red) arrows denote uncoated mitochondria;
  • 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.
  • Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green).
  • FIG. 12A-1 and FIG. 12B-1 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 ⁇ .
  • 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;
  • 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.
  • Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green).
  • 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 ⁇ ;
  • 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.
  • Nuclei were stained with DAPI (blue) and mitochondria coated with TPP-Dextran/FITC (green).
  • 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 ⁇ ;
  • 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;
  • 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. 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.
  • FIG. 18 shows the L/P coupling control ratio.
  • 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.
  • Energy transfer strategies have evolved to confer a survival advantage to cells, with alteration and impairment of these underlining many prevalent chronic diseases.
  • 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).
  • OXPHOS oxidative phosphorylation
  • 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).
  • 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.
  • 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.
  • RCR reduced respiratory coupling ratio
  • LEAK state 4
  • 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.
  • 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.
  • DNAs including and not limited to genomic or extragenomic DNAs
  • genes include 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.
  • PNAs peptide nucleic acids
  • 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.
  • 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.
  • 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.
  • 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.
  • buffers or buffer solutions 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.
  • 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.
  • 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.
  • 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.
  • an effective amount refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
  • a heterologous sequence is defined in relation to a predetermined, reference sequence, such as, a polynucleotide or a polypeptide 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Link 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.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • 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.
  • laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.
  • 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).
  • nucleic acid 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.
  • 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.
  • the term "patient” refers to any host that can receive one or more of the pharmaceutical compositions disclosed herein.
  • the subject is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
  • a "patient” refers to any animal host including without limitation any mammalian host.
  • 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.
  • the mammalian patient is preferably human.
  • phrases "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.
  • salts 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, bis
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • Purified 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.
  • 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.
  • 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
  • a "recombinant virus,” e.g., a recombinant AAV virus is produced by the expression of a recombinant nucleic acid.
  • 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]
  • subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present disclosure can be provided
  • 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.
  • 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.
  • sequences may 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • subject 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.
  • synthetic shall mean that the material is not of a human or animal origin.
  • Targeting moiety is any factor that may facilitate targeting of a specific site by a particle.
  • 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.
  • 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.
  • 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.
  • terapéutica period means the period of time that is necessary for one or more active agents to be therapeutically effective.
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • 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.
  • Treating 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
  • 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.
  • Isoleucine lie AUA AUC AUU
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DCC N,N'-dicyclo- hexylcarbodiimide
  • DMAP 4-(dimethylamino) pyridine
  • TPP-COOH (5-carboxypentyl) triphenylphosphonium bromide
  • 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-LyzerTM 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.
  • FITC fluorescein 5(6)-isothiocyanate
  • 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% C0 2 .
  • DMEM-Dulbecco's Modified Eagle Medium ThermoFisher Scientific
  • 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 CaK 2 EGTA, 7.2 mM K 2 EGTA, 5.7 mM Na 2 ATP, 6.6 mM MgCl 2 » 6H 2 0, 20 mM taurine, 15 mM Na 2 Phospho-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.
  • BIOPS buffer 2.8 mM CaK 2 EGTA, 7.2 mM K 2 EGTA, 5.7 mM Na 2 ATP, 6.6 mM MgCl 2 » 6H 2 0, 20 mM taurine, 15 mM Na 2 Phospho-creatine, 20 mM, imi
  • 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).
  • mice 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.
  • 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.
  • 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 MgS0 4 , 1 mM EDTA, 1 mM ATP, 0.05% BSA, pH 7.4]), mixed well, and allowed to settle for 3 min.
  • ATP medium 100 mM KCl, 50 mM Tris, 5 mM MgS0 4 , 1 mM EDTA, 1 mM ATP, 0.05% BSA, pH 7.4
  • 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 MgS0 4 , 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.
  • 1 KCl medium 100 mM KCl, 50 mM Tris, 5 mM MgS0 4 , 1 mM EDTA; pH 7.4
  • 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 LiberaseTM (Roche LifeScience, Indianapolis, IN, USA).
  • 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.
  • Mitochondrial uptake was also examined in 4T1 mouse breast cancer cells.
  • Cells were cultured in 8-chamber slides (2 ⁇ 10 4 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.
  • 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 MgCl 2 » 6H 2 0, 60 mM K-lactobionate, 2 mM taurine, lO mM KH 2 PO 4 , 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.
  • MiR05 medium 0.5 mM EGTA, 3 mM MgCl 2 » 6H 2 0, 60 mM K-lactobionate, 2 mM taurine, lO mM KH 2 PO 4 , 20 mM HEPES, 110 mM sucrose, 1 g/L fatty acid free bovine serum albumin (BSA), pH 7.1
  • BSA bovine serum albumin
  • the substrates pyruvate-malate (PM, 5 mM each), ADP (4 mM) and oligomycin (5 ⁇ ) 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.
  • 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.
  • 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.
  • TPP triphenylphosphonium
  • FIG. IB a lipophilic, cationic ligand with mitochondriotropic properties 16
  • Dextran-TPP conjugation proved successful, as demonstrated by ⁇ -NMR (FIG.
  • 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.
  • RCR respiratory control ratio
  • 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).
  • uncoated mitochondria underwent increased internalization within H9c2 cells compared to the 4-hr time-point (FIG. 3A).
  • mitochondrial uptake into cells after 24 hrs was substantially greater following functionalization with Dextran-TPP (FIG. 3B).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • CMs isolated adult cardiomyocytes
  • 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).
  • the triple-negative breast cancer cell lines MDA-MB-231 and SUM-159PT exhibited a baseline glycolytic profile.
  • 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.
  • 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 CO 2 and carbonic acid release.
  • 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.
  • 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.
  • 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.
  • 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.
  • the isolated murine liver mitochondria were transplanted into human (a xeno-transplant) tumor cell cultures.
  • 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.
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  • HAYAKAWA K et al, "Transfer of mitochondria from astrocytes to neurons after stroke," Nature, 535:551-555, doi: 10.1038/naturel8928 (2016).
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  • MASUZAWA A et al, "Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury," Am. J. Physiol. - Heart Circulatory Physiol, 304:H966-H982, doi: 10.1152/ajpheart.00883.2012 (2013).
  • NEUBAUER S et al, "Myocardial phosphocreatine-to-ATP ratio is a predictor of mortality in patients with dilated cardiomyopathy," Circulation, 96:2190- 2196 (1997).
  • SIEGEL MP et al, Aging Cell 12:763 (2013).
  • SILVA AK et al, "Polysaccharide nanosy stems for future progress in cardiovascular pathologies," Theranostics, 4:579-591, doi: 10.7150/thno.7688 (2014).
  • TASSA C et al, "Dextran-coated iron oxide nanoparticles: a versatile platform for targeted molecular imaging, molecular diagnostics, and therapy," Accts. Chem. Res., 44:842-852, doi: 10.1021/ar200084x (2011).
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  • 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.

Abstract

L'invention concerne des procédés et des compositions comprenant des mitochondries mammaliennes à polymère fonctionnalisé, utiles dans une variété de régimes thérapeutiques pour l'administration de traits génétiques souhaitables à une ou à plusieurs cellules, tissus et/ou organes d'intérêt. L'invention concerne également des procédés d'introduction de mitochondries dans des cellules hôtes sélectionnées pour modifier le phénotype métabolique de telles cellules hôtes à l'aide d'une ou de plusieurs molécules d'acide nucléique délivrées de manière mitochondriale. La présente invention concerne également un procédé de transplantation cellulaire, comprenant l'étape consistant à administrer à un individu une ou plusieurs des compositions mitochondriales à polymère fonctionnalisé et des formulations de celles-ci telles que décrites dans la description.
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WO2021132735A3 (fr) * 2019-12-27 2021-09-02 Luca Science Inc. Mitochondries isolées ayant une taille plus petite et vésicules à base de membrane lipidique encapsulant les mitochondries isolées
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