Mitochondria-facilitated delivery of mRNA

Abstract

Among the various aspects of the present disclosure is the provision of compositions and methods of a gene delivery system. The compositions include at least one therapeutic molecule and an isolated mitochondria, and optionally further complexed to a lipid nanoparticle. Methods of molecule delivery include the use of isolated mitochondria as a vector for the delivery of the therapeutic molecule. A method of treating a patient in need of a gene vaccine is also described, in which the mRNA of the administered composition encodes a vaccine antigen, including but not limited to at least a portion of a SARS-CoV-2 spike protein.

Description

[0001] Docket No.: 021132 / WO

[0002] TITLE OF THE INVENTION

[0003] MITOCHONDRIA-FACILITATED DELIVERY OF MRNA

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 692,560 filed on September 09, 2024, which is incorporated herein by reference in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] Not applicable.

[0008] MATERIAL INCORPORATED-BY-REFERENCE

[0009] Not applicable.

[0010] FIELD OF THE INVENTION

[0011] The present disclosure generally relates to methods of mitochondria- facilitated gene delivery.

[0012] BACKGROUND OF THE INVENTION mRNA can be rapidly synthesized and modified to develop vaccines for new emerging pathogens. mRNA can encode virtually any proteinaceous antigen and is quickly synthesized via in vitro transcription reactions without a cell line. These attributes of mRNA production, in combination with powerful nucleic acid sequencing and protein structure modeling technologies, enabled the identification and manufacturing of the spike protein-encoding mRNA sequence delivered via vaccines administered during the initial COVID-19 pandemic and subsequent outbreaks of COVID-19 variants. Several easily generated modifications to mRNA sequences were employed in the SARS-CoV- 2 mRNA vaccines to enhance the therapeutic benefits these compositions. Such modifications can improve stability, reduce mRNA recognition by Toll-like receptors, and enhance the immune system. These features, along with the ability to quickly change in vitro transcribed mRNA sequences in response to newly emerging viral variants, highlight the utility of mRNA in vaccines I the Docket No.: 021132 / WO context of new and evolving pathogens. However, the SARS-CoV-2 mRNA vaccines generated systemic immunity but did not elicit mucosal immune responses. Eliciting mucosal immunity in response to vaccines is crucial to limiting the widespread transmission of respiratory pathogens.

[0013] Existing nucleic acid vectors face challenges to safely and effectively generating mucosal immunity against the encoded antigen. Lipid nanoparticles (LNPs) are a widely-used vector for mRNA delivery of therapeutics. Ongoing research to optimize LNP delivery of mRNA to mucosal surfaces has refined LNP formulations, but LNPs remain a relatively inefficient vector for mRNA delivery to mucosal surfaces. LNPs typically accumulate primarily in the liver and spleen rather than reaching other bodily locations. A delivery vector that can stimulate mucosal immunity to prevent respiratory pathogen transmission by delivering a quickly manufactured and customizable nucleic acid to mucosal tissues is an ongoing need.

[0014] Mitochondria are the energetic hubs of nearly all eukaryotic cells and are essential for human health. An emerging body of literature has shown that many cell types can release their mitochondria and transfer them to other cell types in tissues. This mitochondria uptake process occurs in a heparan sulfatedependent endocytic process and can be used to rescue cell-intrinsic defects in mitochondrial metabolism.

[0015] In some cases, mitochondria are ejected by donor cells and are readily taken up by nearby acceptor cells. In this mitochondria transfer process, the transferred mitochondria may also deliver damage-associated molecular patterns (DAMPs) which could serve as intrinsic adjuvants. Mitochondria have been engineered to deliver tumor proteins and stimulate an anti-tumor immune response in one existing treatment method.

[0016] Mitochondria transplantation, a procedure involving isolating mitochondria from a biological source and injecting them into a recipient animal, has been used to rescue metabolically stressed cells and limit tissue damage in the setting of ischemic injury to the heart, brain, and limbs. In addition, it was recently shown that transplanting mitochondria into mice can ameliorate inherited mitochondrial diseases such as Leigh Syndrome. Docket No.: 021132 / WO

[0017] SUMMARY OF THE INVENTION

[0018] Among the various aspects of the present disclosure is the provision of compositions and methods of therapeutic compound delivery to at least one cell of a subject.

[0019] In one aspect, a composition for the delivery of a therapeutic molecule to at least one cell of a subject is disclosed. The composition comprises the therapeutic molecule and an isolated mitochondria. In some aspects, the therapeutic molecule is selected from the group consisting of a protein, a peptide, a DNA molecule, and RNA molecule, a vaccine antigen. In some aspects, the RNA molecule is a messenger RNA molecule (mRNA) configured to encode a protein. In some aspects, the mRNA molecule encodes a vaccine antigen. In some aspects, the vaccine antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some aspects, the composition further comprises a lipid nanoparticle (LNP), wherein the therapeutic molecule is complexed to the LNP. In some aspects, the composition comprises a plurality of therapeutic molecules and a plurality of isolated mitochondria, and the plurality of isolated mitochondria comprising a mitochondria concentration ranging from about 12.5 pg to about 200 pg per 200 pL of the composition. In some aspects, the mitochondria concentration is about 25 pg per 200 pL of the composition. In some aspects, the composition comprises the lipid nanoparticles at a nanoparticle concentration of about 10 pL of the lipid nanoparticles per 200 pL of the composition.

[0020] In another aspect, a method of delivering a therapeutic molecule to at least one cell of a subject is disclosed. The method includes administering a therapeutically effective amount of a composition comprising the therapeutic molecule and an isolated mitochondria to the subject. In some aspects, the therapeutic molecule is selected from the group consisting of a protein, a peptide, a DNA molecule, and RNA molecule, a vaccine antigen. In some aspects, the RNA molecule is a messenger RNA molecule (mRNA) encoding the therapeutic protein. In some aspects, the therapeutic protein encoded by the mRNA molecule is the vaccine antigen. In some aspects, the vaccine antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some aspects, Docket No.: 021132 / WO the composition further comprises a lipid nanoparticle (LNP), wherein the therapeutic molecule is complexed to the LNP. In some aspects, the composition comprises a plurality of the therapeutic molecules and a plurality of the isolated mitochondria, the plurality of isolated mitochondria comprising a mitochondria concentration ranging from about 12.5 pg to about 200 pg per 200 pL of the composition. In some aspects, the mitochondria concentration is about 25 pg per 200 pL of the composition. In some aspects, the composition comprises the lipid nanoparticles at a nanoparticle concentration of about 10 pL of the lipid nanoparticles per 200 pL of the composition. In some aspects, the composition is administered by an administration method selected from intranasal inoculation, intramuscular injection, intravenous injection, intrathecal injection, and intraperitoneal injection. In some aspects, the administration method is intranasal inoculation, and the composition is configured to impart mucosal immunity to the subject. In some aspects, the method further comprises mixing the therapeutic molecule and the isolated mitochondria prior to administering the therapeutically effective amount of the composition to the subject.

[0021] In another aspect, a kit comprising at least one lipid nanoparticle (LNP) and at least one therapeutic molecule is described. In some aspects the at least one therapeutic molecule comprises an mRNA molecule encoding a vaccine antigen. In some aspects the mRNA molecule encodes a vaccine antigen. In some aspects the vaccine antigen comprises at least a portion of SARS-CoV-2 spike protein.

[0022] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0023] DESCRIPTION OF THE DRAWINGS

[0024] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0025] FIG. 1 is a schematic diagram summarizing the enhancement of mRNA vaccine delivered in isolation or within a lipid nanoparticle (LNP) composition by the inclusion of isolated mitochondria, as well as potential experimental variables Docket No.: 021132 / WO for experimental design.

[0026] FIG. 2A contains a pair of flow cytometry plots of BV2 microglial cells which were exposed to mCherry mRNA complexed to lipofectamine LNPs in the presence (right) or absence (left) of extracellular mitochondria isolated from the livers of healthy mice. mCherry expression was measured by flow cytometry one day post-exposure. Representative flow cytometry plots are shown, pre-gated on singlet live cells.

[0027] FIG. 2B is a pair of graphs of BV2 cells (left; n=4 independent experiments) and A549 cells (right; n=2 independent experiments) showing the percentage of mCherry+ cells. Student’s t-test, ****p<0.0001 .

[0028] FIG. 3A is a schematic diagram (top) and experimental timeline (bottom) of mitochondria-enhanced and mitochondria-based intramuscular vaccines that deliver mRNA encoding proteins configured to elicit an immune responses in vivo. N = 5 mice per group.

[0029] FIG. 3B is a graph of maximum luciferase intensity from various combinations of mitochondria, mRNA, and LNP. Day 2 bioluminescence imaging was performed and max luciferase intensity quantified. Student’s t-test, *P<0.05.

[0030] FIG. 3C is a graph of serum anti-Spike IgG resulting from the administration of the compositions described in FIG. 3B. Mice were euthanized and serum was collected on day 14. ELISA was performed to quantify anti- SARSCoV-2 spike IgG antibody titers.

[0031] FIG. 3D is a graph showing in vivo Luciferase expression (photons / second) on day 2 after vaccination with the compositions of FIG. 3B. D- luciferin was administered to mice and the vaccine injection site was imaged using an IVIS50 to detect activity of luciferase, the protein encoded by the luciferase mRNA contained in the indicated vaccine compositions.

[0032] FIG. 4 is a schematic diagram (top) and experimental timeline (bottom) showing different routes of vaccine administration for mitochondria-enhanced and mitochondria-based mRNA vaccines, with particular focus on intranasal inoculation and the mucosal immune response.

[0033] FIG. 5 is a schematic diagram showing the characterization of the Docket No.: 021132 / WO immune response, including humoral and cellular immunity in response to mitochondria-enhanced and mitochondria-based mRNA vaccines.

[0034] FIG. 6 is a schematic diagram summarizing the experimental setup for dose optimization of mitochondria used for mRNA delivery for each vaccine administration route to effectively generate an immune response.

[0035] FIG. 7A is a graph showing mCherry transfection efficiency in BV2 cells after adding mitochondria to the standard LNP and mRNA solution. Adding mitochondria to the standard LNP and mRNA solution reliably transfects microglial BV2 cells. Data is from 3 independent experiments.

[0036] FIG. 7B is a graph showing mitochondrial uptake in BV2 cells after adding mitochondria to the standard LNP and mRNA solutions. The fluorescent mitochondria reporter protein mtDendra2 (mtD2) was used to determine mitochondria uptake. Adding mitochondria to the standard LNP and mRNA solution reliably transfected microglial BV2 cells. Data was obtained over 3 independent experiments.

[0037] FIG. 8A is a graph of transfection efficiency of mCherry without the addition of RNase inhibitor. Data is from 3 independent experiments, n = 6 mitochondria donor mice (3 male, 3 female).

[0038] FIG. 8B is a graph of mCherry transfection efficiency with the addition of an RNase inhibitor at 40,000 U / mL. Adding RNase inhibitor improves transfection efficiency. Data was obtained from 2 independent experiments, n = 2 mitochondrial donor mice (1 male, 1 female).

[0039] FIG. 9A is a graph showing mitochondrial uptake without the addition of RNase inhibitor using the fluorescent mitochondria reporter protein mtDendra2 (mtD2). Data was obtained from 3 independent experiments, n = 6 mitochondrial donor mice (3 male, 3 female).

[0040] FIG. 9B is a graph showing mitochondrial uptake with the addition of an RNase inhibitor at 40,000 U / mL and using the fluorescent mitochondria reporter protein mtDendra2 (mtD2). Adding RNase inhibitor did not alter mitochondria uptake. Data is obtained from 2 independent experiments, n = 2 mitochondrial donor mice (1 male, 1 female). Docket No.: 021132 / WO

[0041] FIG. 10A is a graph showing the transfection efficiency of compositions incubated at room temperature and at 4 °C at different timepoints. Transfection solutions of LNPs, mCherry mRNA, and mitochondria were combined and incubated at the indicated temperature for the indicated length of time. Data was obtained from 2 independent experiments.

[0042] FIG. 10B is a graph showing mitochondrial uptake of compositions incubated at room temperature and at 4 °C at different timepoints. Transfection solutions of LNPs, mCherry mRNA, and mitochondria were combined and incubated at the indicated temperature for the indicated length of time. Data was obtained from 2 independent experiments.

[0043] FIG. 11 is a set of flow cytometry plots showing the flow cytometry gating strategy used to detect and quantify ovalbumin-specific CD8+ T cells using ovalbumin tetramers. The percentage of CD8+CD4- T cells, all CD8+ T cell, CD8+CD4- OVA tetramer+ cells, and all CD8+ OVA tetramer+ cells are indicated.

[0044] FIG. 12A is a graph showing the percentage of CD8+CD4- OVA tetramer+ cells that were positive for the ovalbumin tetramer. Mice were injected with the standard mRNA vaccine (mRNA +LNP), IVJ (InVivoJet, a commercially available LNP), or with the mitochondria-enhanced mRNA vaccine. Spleens of the injected mice were collected two weeks post-injection. Ovalbumin-specific CD8+ T cells did not develop in mRNA+Mito complex-treated mice. n=15 mice / group, injected compositions included 25 pg mito / mouse and an RNase inhibitor, incubated for 2 hours at 4 °C with the vaccine.

[0045] FIG. 12B is a graph showing the percentage of all CD8+ OVA tetramer+ cells that were positive for ovalbumin tetramer. Mice were injected with the standard mRNA vaccine (mRNA +LNP), IVJ (InVivoJet, a commercially available LNP), or the mitochondria-enhanced mRNA vaccine. Spleens collected two weeks post-injection. Ovalbumin-specific CD8+ T cells did not develop in mRNA+Mito complex-treated mice. n=15 mice / group, injected compositions included 25 pg mito / mouse, and an RNase inhibitor, incubated for 2 hours at 4 °C with the vaccine.

[0046] FIG. 13A is a graph showing the expression of the activation marker Docket No.: 021132 / WO

[0047] CD26L (mean fluorescence intensity (MFI)) on CD8+ T cells after vaccination with the standard mRNA vaccine (mRNA +LNP), IVJ (InVivoJet, a commercially available LNP), or the mitochondria-enhanced mRNA vaccine. Two weeks after injection, mice were sacrificed and their spleens were collected. Addition of mitochondria into an mRNA vaccine did not affect expression of the activation markers CD62L on CD8 T cells. n=10 mice / group, and injected compositions contained 25 pg mito / mouse and an RNase inhibitor, and were incubated for 2 hours at 4 °C with the vaccine.

[0048] FIG. 13B is a graph showing the expression of the activation marker CD44 (MFI) on CD8+ T cells after vaccination with the standard mRNA vaccine (mRNA +LNP), IVJ (InVivoJet, a commercially available LNP), or the mitochondria-enhanced mRNA vaccine. Two weeks after injection, mice were sacrificed and their spleens were collected. Addition of mitochondria into an mRNA vaccine did not affect expression of the activation markers CD44 on CD8 T cells. n=10 mice / group, and injected compositions contained 25 pg mito / mouse, included an RNase inhibitor, and were incubated for 2 hours at 4 °C with the vaccine.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure is based, at least in part, on the discovery that isolated mitochondria are exceptional vectors for the delivery of synthetic mRNA. As shown herein, gene delivery systems that make use of mitochondria isolates are described.

[0051] In brief, the present disclosure is directed to a method of gene delivery in which synthetic mRNA can be complexed with isolated mitochondria as a gene delivery vector that is more effective than delivery using standard lipid nanoparticle (LNP)-based methods of gene delivery. In one aspect, mitochondria-based vectors for delivering mRNA can be used for vaccines and therapeutic delivery of mRNA molecules. In another aspect, mitochondria-based vectors can improve the ability of delivering any mRNA composition, including, but not limited to, transgenic mRNA, to cells and subjects in vivo as a research tool and for therapeutic applications.

[0052] In various aspects of the present disclosure, the mitochondria-enhanced Docket No.: 021132 / WO or mitochondria-based vector comprises a vaccine, a therapeutic, or a transgene delivery system. In other aspects, the mitochondria-based vector delivers mRNA or circular RNA (circRNA) to a target cell, tissue, or subject.

[0053] In some aspects, the mitochondria-based vector comprises isolated mitochondria, LNPs or other drug delivery agents, and at least one mRNA. In some aspects, the mitochondria-based composition contains from about 12.5 pg to about 200 pg of isolated mitochondria. In various other aspects, the mitochondria-based composition contains about 12.5 pg, about 25 pg, about 50 pg, about 100 pg, or about 200 pg of isolated mitochondria.

[0054] In some aspect, the mitochondria-based vector comprises about 50 pg mitochondria, about 2 pL of LNPs, and about 1 pg / pL of an mRNA. In some aspect, the mitochondria-based vector comprises about 25 pg mitochondria, about 10 pL of LNPs, and about 10 picomoles of mRNA.

[0055] In various other aspects, the mitochondria-based composition may comprise an mRNA vaccine and isolated mitochondria. The isolated mitochondria may be kept on ice prior to use. The isolated mitochondria may be frozen and thawed prior to use. For a mitochondria-based vaccine, the mRNA vaccine and the isolated mitochondria are mixed immediately prior to use.

[0056] As described in the Examples herein, adding mitochondria to commercially available LNP transfection reagents enabled successful in vitro transfection of mouse BV2 and human A549 cells, neither of which can be transfected with LNPs plus mRNA alone. In addition, intramuscular immunization with a mitochondria-enhanced composition (mRNA, mitochondria, and LNPs) and a mitochondria-based (mRNA and mitochondria) SARS-CoV-2 mRNA vaccine produced antibody titers comparable to or greater than the standard LNP-mRNA vaccines in responding mice. These data established that mitochondria are a vehicle to deliver mRNA in vitro and in vivo to previously inaccessible cell types.

[0057] GENE MODULA TION AGENTS

[0058] As described herein, gene expression has been implicated in various diseases, disorders, and conditions. As such, modulation of genes (e.g., Docket No.: 021132 / WO modulation of genes through a gene delivery system) can be used for the treatment of such conditions. A gene modulation agent can modulate gene response or induce or inhibit gene expression. Gene modulation can comprise modulating the expression of genes on cells, modulating the quantity of cells that express genes, or modulating the quality of the gene-expressing cells.

[0059] In some aspects, the mRNA of the disclosed compositions may encode gene modulation agents, defined herein as composition that can modulate gene expression on cells. For example, a gene modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. In various aspects, the gene modulation can be the result of gene editing effectuated by mRNA-encoded products .

[0060] A gene modulation agent can be an antibody (e.g., a monoclonal antibody to a gene product).

[0061] A gene modulating agent can be an agent that induces or inhibits progenitor cell differentiation into gene expressing cells.

[0062] GENE INHIBITING AGENT

[0063] One aspect of the present disclosure provides for the targeting of a gene, its receptor, or its downstream signaling. The present disclosure provides methods of treating or preventing a disease based on the discovery that AAVs can be packaged into protein vaults to enhance the delivery of genes to a patient with a disease.

[0064] As described herein, inhibitors of a gene (e.g., antibodies, fusion proteins, small molecules) can reduce or prevent disease. A gene inhibiting agent encoded by mRNAs or other means may include any agent that can inhibit a gene, downregulate a gene, or knockdown a gene.

[0065] As an example, a gene inhibiting agent can inhibit gene signaling.

[0066] For example, the gene inhibiting agent can be an antibody toward a gene product associated with a disease. Furthermore, the antibody can be a murine antibody, a humanized murine antibody, or a human antibody.

[0067] As another example, the gene inhibiting agent can be an antibody, wherein the antibody prevents binding of a gene product to its receptor or Docket No.: 021132 / WO prevents activation of a gene or its downstream signaling.

[0068] As another example, the gene inhibiting agent can be a fusion protein. For example, the fusion protein can be a decoy receptor for a gene product. Furthermore, the fusion protein can comprise a mouse or human Fc antibody domain fused to the ectodomain of a decoy receptor.

[0069] As another example, a gene inhibiting agent can be an inhibitory protein that antagonizes a gene or its product. For example, the gene inhibiting agent can be a viral protein, which has been shown to antagonize a gene.

[0070] As another example, a gene inhibiting agent can be a short hairpin RNA (shRNA) or a short interfering RNA (siRNA) targeting a gene or its downstream signals.

[0071] As another example, a gene inhibiting agent can be a sgRNA targeting a gene or its downstream signaling.

[0072] Methods for preparing a gene inhibiting agent (e.g., an agent capable of inhibiting gene signaling) can comprise the construction of a protein / Ab scaffold containing the natural gene receptor as a neutralizing agent; developing inhibitors of the gene receptor “down-stream”; or developing inhibitors of the gene production “up-stream”.

[0073] Inhibiting a gene can be performed by genetically modifying at least one gene in a subject or genetically modifying a subject to reduce or prevent expression of the gene, such as through the use of CRISPR-Cas9 or analogous technologies, wherein, such modification reduces or prevents a disease.

[0074] Gene Therapy

[0075] Gene therapy is the technique that uses a gene to prevent or recover any diseases. The technique of gene therapy allows the treatment of a disorder by inserting a gene into patient’s cell instead of using drugs or surgery. Approaches to gene therapy, include: i) replacing a mutated gene that causes disease with a healthy gene; ii) ‘knocking out’ or inactivating, a mutated gene that is functioning improperly; and iii) introducing new genes into the cells to protect from any diseases.

[0076] Genetic molecules reach to the nuclei of host cells to induce gene Docket No.: 021132 / WO expression. Gene therapy has derived to provide a patient’s somatic cells with genetic information for producing specific therapeutic proteins to modulate genetic diseases. The gene delivery systems consist of the three components such as a plasmid-based gene expression system that controls the function of a gene within the targeting cell, a gene that encodes a specific therapeutic protein, and a gene delivery system that controls the delivery of the gene expression plasmid to specific location within the body.

[0077] Gene delivery systems include viral gene delivery systems and non-viral gene delivery systems. The viral gene delivery systems have discussed for the viral vectors based on DNA, RNA, and oncolytic viral vectors, respectively. The non-viral gene delivery systems have also treated for the physicochemical approaches such as physical method and chemical method. Several kinds of gene delivery systems utilize lipid nanoparticles and cationic biomedical polymers such as polysaccharides, polyethylenimine (PEI), and poly(L- lysine)(PLL) derivatives.

[0078] FORMULATION

[0079] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0080] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0081] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United Docket No.: 021132 / WO

[0082] States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0083] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0084] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0085] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0086] Controlled-release (or sustained-release) preparations may be formulated Docket No.: 021132 / WO to extend the activity of the agent(s) and reduce the dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0087] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.

[0088] THERAPEUTIC METHODS

[0089] Also provided is a process of treating, preventing, or reversing a disease in a subject in need of administration of a therapeutically effective amount of a gene modulation agent, so as to treat a disease.

[0090] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a disease. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject. Docket No.: 021132 / WO

[0091] Generally, a safe and effective amount of a gene modulation agent is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a gene modulation agent encoded by mRNA and other means as described herein can substantially inhibit a disease, slow the progress of a disease, or limit the development of a disease.

[0092] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0093] When used in the treatments described herein, a therapeutically effective amount of the mRNA can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat a disease.

[0094] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0095] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0096] The specific therapeutically effective dose level for any particular subject Docket No.: 021132 / WO will depend upon a variety of factors including the disorder being treated and the seventy of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0097] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician. Docket No.: 021132 / WO

[0098] Administration of an mRNA or other gene modulation agent can occur as a single event or over a time course of treatment. For example, a gene modulation agent can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0099] Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease.

[0100] A gene modulation agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a gene modulation agent can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of a gene modulation agent, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more of a gene modulation agent, an antibiotic, an anti-inflammatory, or another agent. A gene modulation agent can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a gene modulation agent can be administered before or after the administration of an antibiotic, an antiinflammatory, or another agent.

[0101] ADMINISTRATION

[0102] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body. Docket No.: 021132 / WO

[0103] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0104] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0105] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0106] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, Docket No.: 021132 / WO

[0107] CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

[0108] SCREENING

[0109] Also provided are methods for screening.

[0110] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

[0111] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0112] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, Docket No.: 021132 / WO numerous databases for commercially available compounds for screening (see e.g., ZINC database, LICSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0113] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0114] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “druglike”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0115] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to Docket No.: 021132 / WO oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0116] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and 0 atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0117] KITS

[0118] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to any one or more of an mRNA, an adeno- associated virus, a Vault protein, mitochondria, solubilizers, and any other suitable component of the mitochondria based or mitochondria-enhanced compositions including, but not limited to, the mRNA encoding various products such as vaccine antigens as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0119] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic Docket No.: 021132 / WO polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0120] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0121] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0122] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx Docket No.: 021132 / WO

[0123] (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0124] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0125] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0126] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially Docket No.: 021132 / WO in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0127] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0128] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0129] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0130] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent Docket No.: 021132 / WO application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0131] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0132] EXAMPLES

[0133] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0134] EXAMPLE 1 - GENE DELIVERY SYSTEMS

[0135] Messenger RNA (mRNA) has shown incredible therapeutic potential across a range of applications, most strikingly in the success of the SARS-CoV-2 mRNA vaccines rapidly developed and distributed in the COVID-19 pandemic. Despite the overwhelming success of these vaccines in reducing severe illness, breakthrough infections with both the original and evolved viral strains in vaccinated populations arose within months and continue to spread. As SARS- CoV-2 is a respiratory virus that enters the body through mucosal surfaces of the upper respiratory tract, the lack of mucosal immunity produced by the SARS- CoV-2 mRNA vaccines likely contributes to this continued infection and spread. These vaccines use lipid nanoparticles (LNPs) as an mRNA vector, which do not effectively deliver mRNA to mucosal surfaces. As mRNA is readily synthesized Docket No.: 021132 / WO and easily adaptable to new pathogens, and novel respiratory viruses infect immunologically naive humans through the respiratory mucosa, there is strong interest in developing an mRNA vaccine that can generate mucosal immunity.

[0136] In addition to fulfilling cellular energy demands, mitochondria are readily taken up by a variety of cell types, both as mechanisms for maintaining homeostasis and in various therapeutic applications. Murine mitochondria and GMP-grade human mitochondria are readily available and can be frozen and thawed without compromising their structural integrity or respiratory capacity, enabling long-term storage and global distribution capabilities. Shown herein is the use of mitochondria as a delivery vector to promote mRNA uptake at mucosal surfaces and induce a mucosal immune response. These studies demonstrate that adding mitochondria to in vitro transfection solutions of LNPs and mRNA allows for transfection of murine BV2 cells and human A549 cells, both of which cannot be transfected with LNPs plus mRNA alone. Furthermore, mitochondria-enhanced SARS-CoV-2 mRNA vaccines generate comparable or greater levels of anti-SARS-CoV-2 antibodies in serum than standard LNP mRNA vaccines.

[0137] These findings show that mitochondria can serve as a delivery vector for mRNA in vaccines, particularly at mucosal surfaces where LNPs are ineffective at delivering mRNA. These studies show the efficacy of intranasal mitochondria- enhanced and mitochondria-based vaccines and elucidate key characteristics of the systemic and mucosal immune responses they induce (FIG. 1 ), thus establishing powerful and versatile technology for mucosal immunization in the face of emerging respiratory viruses.

[0138] RESULTS

[0139] Isolated mitochondria are exceptional vectors for the delivery of synthetic mRNA and outperform conventional lipid nanoparticles (LNPs) for this purpose. First, we exposed BV2 microglial cells to mRNA that encodes mCherry (red fluorescent protein) complexed to either LNPs or LNPs plus isolated mitochondria. Using flow cytometry, we detected mCherry fluorescence in ~2% of BV2 cells exposed to mRNA+LNPs and ~60% of BV2 cells exposed to mRNA+LNPs+mitochondria (FIG. 2A, FIG. 2B). The degree of mCherry Docket No.: 021132 / WO expression by fluorescence intensity was also much higher using mitochondria- complexed mCherry mRNA. This observation was not limited to just BV2 cells, as we observed a similar improvement in mCherry expression in A549 cells with mitochondria-complexed mRNA (FIG. 1 B). These findings indicate that complexing synthetic mRNA with isolated mitochondria plus mitochondria is far superior at transfecting cells than mRNA complexed with LNPs alone.

[0140] Shown herein are mitochondria-based vectors that can be used to deliver mRNA vaccines. We generated 1 ) a mitochondria-enhanced LNP vaccine comprised of isolated mitochondria, LNPs, and 2 mRNAs (SARS-CoV-2 Spike mRNA and Luciferase mRNA in 1 :1 ratios). 2) A mitochondria-based vaccine comprised isolated mitochondria the 2 mRNAs, with no LNPs. 3) A conventional mRNA vaccine comprised of LNPs plus the 2 mRNAs, and 4) a negative control with mitochondria and LNPs with no mRNA.

[0141] The 4 preparations were administered to wildtype C57BL6 / J mice (FIG. 3A). On day 2, we performed in vivo bioluminescence imaging to detect luciferase expression and found that the mitochondria-enhanced LNP vaccine and the conventional vaccine produced similar degrees of luciferase expression (FIG. 3B). Strikingly, the mitochondria-based vaccine (no LNPs) produced similar luciferase expression as the conventional vaccine (FIG. 3B). This result indicates that the mitochondria-based mRNA vaccine is at least as effective at inducing expression of a synthetic mRNA in vivo as the conventional LNP-based mRNA vaccine. Next, we harvested serum on day 14 post-inoculation to measure anti- SARS-CoV-2 Spike protein antibody production by the mice. We found that the 3 vaccines produced similar degrees of anti-Spike IgG, and that the 3 highest concentrations of anti-Spike antibody were produced when mitochondria were included in the vaccine (FIG. 3C). Again, the mitochondria-based vaccine (no LNPs) was at least as effective at inducing anti-Spike antibody production as the conventional vaccine.

[0142] These data show that synthetic mRNA can be complexed with isolated mitochondria as a gene delivery vector that is more effective than standard LNP- based methods of gene delivery. This invention can be used for mitochondria- based vectors for delivering mRNA can be used for vaccines and therapeutic Docket No.: 021132 / WO delivery of mRNA molecules. This technology can also greatly improve the ability of delivering transgenic mRNA to cells and animals in vivo, which could be used widely as a research tool in biomedical research.

[0143] Example 2 - Mitochondrial vectors enable mucosal mRNA-based vaccinations mRNA lipid nanoparticle (LNP) vaccines have the potential to be used to treat rare genetic diseases and demonstrated remarkable successes in the SARS-CoV-2 pandemic by significantly reducing disease severity and death. However, they did not prevent infection and viral transmission in part due to the lack of mucosal immunity. Using mitochondria transfer biology and mRNA vaccine development, a mitochondria mRNA delivery system was developed to enable delivery of mRNA to otherwise inaccessible cell types and enhanced immune responses in mice. These studies shown that isolated mitochondria can be used as an mRNA delivery vehicle for inducing mucosal immunity via intranasal vaccination. Intranasal mitochondria-facilitated mRNA vaccination compositions are readily adaptable to combat emerging respiratory viruses and potentially other diseases.

[0144] INTRODUCTION mRNA can be rapidly synthesized and easily modified to develop vaccines for new emerging pathogens. mRNA can encode virtually any proteinaceous antigen and is quickly synthesized via in vitro transcription reactions without a cell line. These attributes of mRNA production, in combination with powerful nucleic acid sequencing and protein structure modeling technology, enabled identification and manufacturing of the crucial spike protein-encoding mRNA sequence delivered via vaccines in the COVID-19 pandemic. Several modifications to mRNA sequences are easily generated and were employed in the SARS-CoV-2 mRNA vaccines to enhance the therapeutic benefits of mRNA. Replacement of undine with N1-methyl-pseudouridine enhances stability and translation of exogenous mRNA, addressing concerns that mRNA instability would prevent meaningful protein antigen production. N1- methyl-pseudouridine substitution also significantly reduces mRNA recognition Docket No.: 021132 / WO by Toll-like receptors of the innate immune system, avoiding undesirable interferon-alpha production in response to exogenous mRNA. Alteration of the mRNA sequence to encode two proline residues that stabilize the spike protein in its prefusion conformation greatly enhanced the immune response to the desired antigen. These features, along with the ability to quickly change in vitro transcribed mRNA sequences in response to newly emerging viral variants, highlight the utility of mRNA in vaccines for new and evolving pathogens.

[0145] Although natural SARS-CoV-2 infection results in an early mucosal immune response, SARS-CoV-2 mRNA vaccines generate systemic immunity. A core component of mucosal immunity is neutralizing polymeric IgA antibodies produced by local plasma cells underlying the epithelium of mucosal sites. IgA antibodies dominate early humoral immune responses to natural SARS-CoV-2 infection, and the first wave of circulating antibody-producing plasmablasts are predominantly IgA-expressing with mucosal homing markers. IgA antibodies also are significantly more neutralizing against SARS-CoV-2 than IgG antibodies, which are found in serum and induced by the systemically administered (intramuscular) SARS-CoV-2 mRNA vaccines. Both the Pfizer-BioNTech and Moderna LNP-mRNA vaccines were incredibly successful at preventing severe lower respiratory tract disease and reducing the risk of hospitalization and death. However, months later it became clear that vaccinated individuals can still become infected and transmit SARS-CoV-2 to others. This is in part because the primary site of entry for SARS-CoV-2 is mucosal surfaces of the upper respiratory tract, and systemic and mucosal immune compartments are largely distinct: the systemically administered vaccines induced a systemic but not mucosal immune response, which in turn did not prevent upper respiratory tract infection. As IgA antibodies and mucosal homing-plasmablasts largely comprise the early immune response to natural infection, stimulating a similar mucosal immune response with an intranasal vaccine could be expected to prevent transmission.

[0146] Eliciting mucosal immunity in response to vaccines is crucial to prevent transmission of respiratory pathogens. Indeed, our team has demonstrated that a single dose intranasal SARS-CoV-2 chimp adenovirus vaccine encoding a prefusion stabilized conformation of the spike protein completely protected mice Docket No.: 021132 / WO from both upper and lower respiratory tract infection. This was not seen after intramuscular vaccination and strongly suggests that upper respiratory tract immunity could prevent transmission. A similar phenomenon has been observed with Bordetella pertussis, a highly contagious respiratory bacterial infection: intramuscular vaccination with acellular pertussis vaccines successfully prevents severe disease but not colonization or transmission, and it is hypothesized that mucosal immunity is needed to prevent colonization and transmission. Research to develop safe and effective vaccines for respiratory pathogens that stimulate mucosal immunity is of paramount importance, as they could not only protect from severe disease but also restrain transmission, potentially providing a resource to intervene before transmission of an emerging pathogen is internationally widespread.

[0147] Existing nucleic acid vectors face challenges to safely and effectively generating mucosal immunity against the encoded antigen. LNPs are the primary vector of mRNA delivery for therapeutics, and LNPs were successfully employed to deliver mRNA in the Pfizer-BioNTech and Moderna SARS-CoV-2 mRNA vaccines. Ongoing research to optimize LNP delivery of mRNA to mucosal surfaces has investigated optimized LNP formulations, using charge to stabilize LNPs against rapid degradation, and attaching mannose sugars to LNPs to enhance their delivery and uptake; despite these advances, LNPs remain an inefficient vector for mRNA delivery to mucosal surfaces. Adenovirusbased vaccines are another approach to deliver nucleic acids to mucosal surfaces; however, the risk of rare but severe blood clots with adenovirus vaccines makes this approach undesirable, as evidenced by the removal of Johnson & Johnson’s SARS-CoV-2 adenovirus vaccine from the US market. Thus, research and development of a vector that can stimulate mucosal immunity to prevent respiratory pathogen transmission by delivering a quickly manufactured and customizable nucleic acid to mucosal tissues remain urgently needed.

[0148] Mitochondria present a novel vehicle with potential to deliver mRNA to mucosal surfaces and induce a mucosal immune response. Mitochondria are readily taken up by cells, and our preliminary data show that mitochondria combined with LNP transfection reagents can transfect cell lines that cannot be Docket No.: 021132 / WO transfected by LNPs alone. We also show that intramuscular immunization with a mitochondria-enhanced SARS-CoV-2 mRNA vaccine (mRNA, mitochondria, and LNPs) and a mitochondria-based SARS-CoV-2 mRNA vaccine (mRNA and mitochondria) produced comparable or higher anti-SARS-CoV-2 antibody titers than the standard LNP-mRNA vaccine in responding mice. As such, we hypothesize that mitochondria may be able to deliver mRNA to mucosal surfaces where LNPs are inefficient and adenovirus-based vaccines have presented safety concerns. LUCA Science has developed a novel method to isolate human mitochondria that are intact, functional, and storable. These mitochondria are stable, can be frozen and thawed without compromising respiration, and ready to use in various applications as the shelf-stable biopharmaceutical product MRC- Q. If this mitochondria vaccine technology could be employed to deliver mRNA to mucosal surfaces, the combination of rapid, versatile, and efficient generation of mRNA that encodes a protein antigen and an effective mRNA delivery vehicle to mucosal tissues would constitute a significant improvement in pandemic preparedness.

[0149] RESULTS

[0150] Established herein is a vaccine composition that comprises adding mitochondria to an LNP plus mRNA transfection solution. This composition successfully transfects multiple cell lines that are naturally resistant to LNP-only transfection, and mitochondria-enhanced and mitochondria-based vaccines can immunize mice in vivo to produce antibodies to the antigen encoded by the delivered mRNA.

[0151] Mitochondria can transfect BV2 and A549 cells in vitro.

[0152] Cationic lipid-based transfection reagents are considered the gold standard for delivery of exogenous DNA or mRNA to cells. These reagents are LNP formulations. Lipofectamine MessengerMAX reagent is specifically designed for mRNA transfection. BV2 cells (mouse microglial cell line) and A549 cells (human alveolar epithelial cell line) are both extremely difficult to transfect with commercially available transfection reagents. As mitochondria are readily taken up by various murine and human cell types, including BV2 cells as we have previously shown, we show that adding mitochondria to the transfection Docket No.: 021132 / WO solution could increase mRNA uptake and therefore protein expression. Strikingly, we observed a transfection efficiency >60% for BV2 cells and ~35% for A549 cells with the addition of mitochondria to LNP reagent and mRNA transfection solution (FIG. 2A, FIG. 2B).

[0153] Mitochondria-enhanced and mitochondria-based vaccines can immunize mice in vivo.

[0154] Based on the findings that mitochondria facilitate gene delivery in vitro, we next investigated whether mitochondria could serve as an in vivo delivery vehicle for mRNA. We injected mice intramuscularly with a mitochondria-enhanced SARS-CoV-2 mRNA vaccine (mRNA, mitochondria, and LNPs) and a mitochondria-based SARS-CoV-2 mRNA vaccine (mRNA and mitochondria), delivering both SARS-CoV-2 spike mRNA (GenScript, SpikeSA-RBD-STMD mRNA) and Firefly Luciferase mRNA (TriLink, CleanCap FLuc mRNA) (FIG. 3A). We measured Luciferase protein expression two days after vaccination via bioluminescence imaging and measured anti-SARS-CoV-2 serum antibody titers and found that both the mitochondria-enhanced and the mitochondria-based mRNA vaccines generated luciferase protein expression and anti-SARS-CoV-2 antibody titers comparable to or greater than the standard LNP-mRNA vaccine in responding mice (FIG. 3B, FIG. 3C).

[0155] Routes of administration for mitochondria-enhanced and mitochondria-based mRNA vaccines

[0156] Epithelial cells line mucosal surfaces of the respiratory, gastrointestinal, and urogenital tracts, and play a crucial role in sensing and promoting a host immune response to foreign organisms or particles. Mucosal surfaces are often the point of entry into the body for pathogens and as such generating mucosal immunity to prevent infection and subsequent transmission is a critical goal of vaccination. Lung alveolar epithelial cells have been shown to take up mitochondria, resulting in functional improvement, in models of cigarette smoke exposure, acute lung injury, and asthma. The uptake of mitochondria by epithelial cells can be employed as an mRNA delivery vehicle to mucosal surfaces.

[0157] Conversion of successful mitochondria-enhanced and mitochondria- Docket No.: 021132 / WO based intramuscular vaccines to intranasal vaccines. We have demonstrated that mitochondria can deliver mRNA to generate a systemic immune response via an intramuscular vaccine (FIG. 3B, FIG. 30, FIG. 3D). Here we administer mitochondria-enhanced and mitochondria-based SARS-CoV-2 mRNA vaccines intranasally and evaluate the mucosal immune response (FIG.4). The vaccines are prepared by isolating mitochondria from the liver of healthy wild-type mice, combining LNP reagent in v / vo-jetRNA+ (Polyplus) and mRNAs encoding FLuciferase and SARS-CoV-2 spike protein receptor binding domain (RBD), and then adding mitochondria to the LNP and mRNA solution and administering intranasally to wild-type mice.

[0158] The vaccine design may include circular RNA (circRNA) to encode an antigen of interest and incorporate circRNA rather than mRNA in the mitochondria-enhanced and mitochondria-based vaccine. mRNA delivery and translation to protein is evaluated. Two days after vaccination by intranasal vaccination, FLuciferase mRNA delivery is assessed and translation to protein using bioluminescence imaging is evaluated. We quantify FLuciferase expression in the upper and lower respiratory tracts as separate anatomical regions in order to understand spatial distribution of mRNA delivery and translation to protein via the mitochondria-enhanced and mitochondria-based vaccines.

[0159] After two weeks after vaccination by intranasal vaccination, the mucosal immune response to the SARS-CoV-2 spike RBD is assessed by mitochondria- enhanced and mitochondria-based vaccines. The mucosal immune response is stimulated by the mitochondria-enhanced and mitochondria-based mRNA vaccines. The production of anti-SARS-CoV-2 RBD antibodies and antigenspecific CD8+ T cells are evaluated. Two weeks after intranasal vaccination, we will harvest blood, bronchoalveolar lavage (BAL) fluid, spleen, and lung tissue from mice. We will isolate serum from blood and perform ELISA assays to quantify IgG and IgA anti-spike RBD antibodies on both serum and BAL fluid. The lung tissue is digested and spleen tissue is homogenized to obtain single cells, then tetramer staining is performed using MHC class I tetramers to identify and quantify antigen-specific CD8+ T-cells (FIG. 12A, FIG. 12B). Understanding Docket No.: 021132 / WO the site distribution of both antibodies and antigen-specific T cells will allow us to determine the mucosal immunity in the upper and / or lower respiratory tracts and / or systemic immunity is induced in response to intranasal mitochondria- enhanced and mitochondria-based vaccines. Additionally, mice expressing the human angiotensin-converting enzyme receptor 2 will be vaccinated with the intranasal vaccines and subsequently challenge with SARS-CoV-2 infection.

[0160] Characterization of the immune response: humoral and cellular immunity.

[0161] Mice intramuscularly vaccinated with the mitochondria-enhanced and mitochondria-based mRNA vaccines containing SARS-CoV-2 spike RBD mRNA and FLuciferase mRNA resulted in FLuciferase expression 40-48 hours after vaccination and generation of anti-SARS-CoV-2 spike RBD IgG antibodies in serum two weeks after vaccination (FIG. 3B, FIG, 3C, FIG. 3D). The longevity of the humoral immune response will be assessed and cellular characteristics of the immune response to our mitochondria vaccines is compared to standard LNP mRNA vaccines administered intramuscularly and intranasally (FIG. 5).

[0162] Serum antibody titers decrease over time after SARS-CoV-2 vaccinations. Both natural infection with SARS-CoV-2 and vaccination with SARS-CoV-2 mRNA vaccines results in virus-specific antibody production, with natural infection producing more diverse antibodies against multiple viral antigens. Neutralizing antibodies induced by vaccination exhibit a time-dependent reduction, with significantly lower titers by 4-6 months post-vaccination, and consequently breakthrough infections with SARS-CoV-2 are common. The absence of protective mucosal IgA antibodies after intramuscular vaccination may also contribute to the high prevalence of breakthrough infections.

[0163] Temporal assessment of humoral immunity following mitochondria- enhanced and mitochondria-based mRNA vaccination is assessed, including the duration of antibody responses produced by intramuscularly and intranasally administered mitochondria-enhanced and mitochondria-based mRNA vaccines. The levels of IgG and IgA anti-SARS-CoV-2 RBD antibodies present in serum and BAL fluid are quantified at different post-vaccination time points via ELISA. Serum is obtained at 4 weeks, 3 months, 6 months, and 9 months post Docket No.: 021132 / WO vaccination and BAL fluid at 4 weeks, 3 months, and 9 months post-vaccination using non-terminal collection techniques in order to understand temporal dynamics of systemic and mucosal antibody titers following our mitochondrial vaccines. With different mouse cohorts, we also quantify spike-specific antibodysecreting B cells in the spleen over time after intramuscular and intranasal mitochondrial vaccination using ELISpot assays.

[0164] Vaccine-induced resident memory T cells play an important role in longterm immunity Breakthrough infections are associated with milder disease and substantially lower risk of long COVID symptoms than primary infection in unvaccinated individuals. Protection against severe disease in breakthrough infections is in part attributed to vaccine-induced spike-specific T cell responses, which are effective in preventing severe disease caused by viral variants against which neutralizing antibodies do not prevent infection.

[0165] Temporal assessment of cellular immunity following mitochondria- enhanced and mitochondria-based mRNA vaccination is assessed. The longevity of spike-specific T cell responses after vaccination with our mitochondrial vaccines is compared to standard LNP-mRNA vaccines. Splenocytes and lung tissue are harvested from cohorts of mice vaccinated which each of the mitochondria and standard LNP-mRNA vaccines at 4 weeks, 3 months, 6 months, and 9 months post-vaccination. The splenocytes will be restimulated ex vivo with a pool of spike protein peptides and quantify intracellular interferon-g (IFNg) and granzyme B expression of CD4+and CD8+T cells using flow cytometry (FIG. 11 ). We will similarly restimulate cells isolated from lung tissue with spike protein peptides and use flow cytometry to quantify IFNg and granzyme B expression after restimulation. We will also stain for surface markers CD103 and CD69 to detect CD103+CD69+CD8+resident memory T cells in the lung. Additionally, bone marrow may be harvested and antigen-specific antibody-secreting cells quantified to understand the cellular origin of persistent antibodies.

[0166] Dosage of mitochondria for mRNA delivery of each vaccine and administration route.

[0167] The intramuscular vaccination with mitochondria-enhanced and Docket No.: 021132 / WO mitochondria-based mRNA vaccines containing SARS-CoV-2 spike RBD mRNA, FLuciferase mRNA, and 25ug of mitochondria induced anti-SARS-CoV-2 spike RBD IgG antibodies in serum two weeks after vaccination (FIG. 3A).

[0168] Dosage of mitochondria affects outcomes in mitochondrial transplantation for ischemia-reperfusion injury (IRI). Mitochondrial transplantation is a promising and rapidly expanding field with interesting implications for our mitochondria vaccine studies. In studies of mitochondrial transplantation for IRI to the heart, delivery of respiration-competent mitochondria improved left ventricular function and decreased infarct size compared to vehicle, in part by significantly preserving cell viability. Mitochondrial dosage affects outcome: doses of 200,000 to 2 million mitochondria per gram of heart tissue demonstrated cardioprotection in IRI, with mitochondria doses below this range demonstrating decreased cardioprotection and doses above this range failing to increase the cardioprotective benefit. This dose of mitochondria is also effective for mitochondria transplantation in lung and kidney IRI, but mitochondrial transplantation for skeletal muscle IRI requires a higher mitochondria concentration to be optimally beneficial. As optimal mitochondria dose for mitochondrial transplantation in IRI varies between tissues, optimal dose of mitochondria as an mRNA vector may vary based on vaccine administration route and may impact magnitude and / or qualities of the induced immune response (FIG. 6).

[0169] Evaluation of mitochondria dose on immune response induced by mitochondria-enhanced and mitochondria-based vaccines. Mitochondria- enhanced and mitochondria-based SARS-CoV-2 mRNA vaccines are prepared with 12.5ug, 25ug (previously used dose), 50ug, 100ug, and 200ug mitochondria and administer to cohorts of mice via intramuscular injection or intranasal inoculation. We will quantify anti-SARS-CoV-2 RBD antibodies and antigenspecific CD8+ T cells two weeks after vaccination with titrated mitochondria dose vaccines. On post-vaccination day 14, we will harvest blood, bronchoalveolar lavage (BAL) fluid, spleen, and lung tissue from mice. For vaccines administered intramuscularly, we will perform ELISA assays on serum isolated from blood to quantify IgG and IgA anti-spike RBD antibodies and use MHC class I tetramer staining to quantify antigen-specific CD8+ T cells in the spleen and lung (FIG. Docket No.: 021132 / WO

[0170] 11 ). For vaccines administered intranasally, we will perform ELISA assays on BAL fluid to quantify IgG and IgA anti-spike RBD antibodies and use MHC class I tetramer staining to quantify antigen-specific CD8+ T cells in the spleen and lung (FIG. 12A, FIG. 12B, FIG. 13A, FIG. 13B). Understanding the relationship between mitochondria dose in the vaccine preparation and magnitude and / or changes in characteristics of the humoral and cellular immune response will allow us to optimize the mitochondrial vaccines for diverse applications. As mouse mitochondria are readily isolated and human mitochondria are commercially available (LUCA Science Inc.), dose titration is feasible and could inform production of an effective, easily adaptable mitochondria mRNA vaccine platform for future pandemics.

[0171] METHODS

[0172] Bioluminescence imaging: mice intraperitoneally injected with 150mg / kg of D-luciferin and imaged 10 minutes later with an IVIS50 bioluminescence system, which provides sensitive and quantitative in vivo imaging of bioluminescent reporters.

[0173] ELISA: IgG and IgA antibody titers are assessed using a standard enzyme-linked immunosorbent assay with antigen-coated plates and absorbance values will be interpolated using a standard curve.

[0174] Tetramer staining: cells are isolated and labeled with MHC class I antigen-specific tetramers conjugated to a fluorescent reporter and antibodies to key surface markers. High-dimensional spectral flow cytometry is performed to quantify CD8+ antigen-specific T cells.

[0175] High-dimensional spectral flow cytometry: The Cytek Aurora system is used to perform 31 -color spectral flow cytometry to assess surface markers and qualitatively phenotype T cells in spleen and lung of vaccinated mice.

[0176] ELISpot assay: Splenocytes are applied to spike protein-coated plates, incubate with biotinylated anti-IgG or anti-lgA antibodies, and subsequently add streptavidin-conjugated horseradish peroxidase. Spots are developed with 3- amino-9-ethylcarbazole substrate and count of spike-specific antibody-secreting B cells. Docket No.: 021132 / WO

[0177] Transfection Efficiency: BV2 cells were plated at 50k cells per well in 24 well plates. Different combinations of LNPs, mRNA, and mitochondria isolated from mouse liver and containing a fluorescent reporter protein were added to cells. In the 1 step condition, LNPs, mRNA, and mitochondria were combined and immediately added to cells. In the 1 step wash condition, LNPs, mRNA, and mitochondria were combined, and solution was centrifuged to pellet mitochondria then resuspended in PBS 4 times before it was added to the cells.

[0178] In the 2 step condition, LNPs, mRNA, and mitochondria were combined and incubated for 2 hours on ice, then added to cells. Transfection solutions of LNPs, mCherry mRNA, and mitochondria were combined and incubated at the indicated temperature for the indicated length of time. Then the transfection solutions were added directly to the cells. After 24 hours, cells were detached from the plate using trypsin, stained with Zombie NIR to label dead cells, and characterized using an Aurora Cytek spectral flow cytometer.

[0179] In the 2 step wash condition, LNPs, mRNA, and mitochondria were combined and incubated for 2 hours on ice, then the solution was centrifuged to pellet mitochondria then resuspended in PBS 4 times before it was added to the cells. After 24 hours, cells were detached from the plate using trypsin, stained with Zombie NIR to label dead cells, and characterized using an Aurora Cytek spectral flow cytometer (FIG. 7A, FIG. 7B, FIG. 8A, FIG. 8B, FIG. 9A, FIG. 9B, FIG. 10A, FIG. 10B).

[0180] Vaccine efficacy: Vaccines were prepared and administered under general anesthesia via intramuscular injection into the thigh of mice. The following vaccines were tested: a negative control vaccine (mitochondria and lipid nanoparticles (LNP) without mRNA), an mRNA only vaccine, a standard mRNA vaccine containing mRNA and LNPs, a mitochondria-enhanced vaccine containing mRNA, LNPs, and mitochondria, and a mitochondria-based vaccine containing mRNA and mitochondria. Two mRNAs were included in each vaccine preparation, ovalbumin mRNA and Luciferase mRNA. All vaccine preparations also contained RNase inhibitor at the manufacturer’s recommended concentration of 40,000U / mL. Mitochondria were isolated from the liver of wildtype mice and combined with the mRNA and LNP. Vaccines were incubated on Docket No.: 021132 / WO ice for 2 hours to standardize the length of time the vaccine components were together in solution before injection. Vaccines were mixed thoroughly immediately before injection.

[0181] Antigen-specific T cell response: Ovalbumin-specific CD8+ T cells were quantified using ovalbumin tetramers. Two weeks after injection, mice were sacrificed and the spleens were collected. Each spleen was processed to a single cell suspension and cells were stained with antibodies to identify T cells and ovalbumin tetramers (FIG. 12A, FIG. 12B).

[0182] Expression of activation markers CD62L and CD44 on CD8+ T cells was assessed after vaccination with the standard mRNA vaccine (mRNA + LNP; IVJ is InVivoJet, a commercially available LNP for in vivo experiments) or the mitochondria-enhanced mRNA vaccine. Two weeks after injection, mice were sacrificed and each spleen was collected. Spleen was processed to a single cell suspension and cells were stained with antibodies to identify T cells, activation markers CD62L and CD44, and ovalbumin tetramers (FIG. 13A, FIG. 13B).

Claims

Docket No.: 021132 / WOCLAIMSWhat is claimed is:1 . A composition for the delivery of a therapeutic molecule to at least one cell of a subject, the composition comprising the therapeutic molecule and an isolated mitochondria.

2. The composition of claim 1 , wherein the therapeutic molecule is selected from the group consisting of a protein, a peptide, a DNA molecule, an RNA molecule, and a vaccine antigen.

3. The composition of claim 2, wherein the RNA molecule is a messenger RNA molecule (mRNA) encoding a protein.

4. The composition of claim 3, wherein the protein encoded by the mRNA molecule is the vaccine antigen.

5. The composition of claim 4, wherein the vaccine antigen comprises at least a portion of a SARS-CoV-2 spike protein.

6. The composition of claim 1 , wherein the composition further comprises a lipid nanoparticle (LNP), wherein the therapeutic molecule is complexed to the LNP.

7. The composition of claim 1 , comprising a plurality of therapeutic molecules and a plurality of isolated mitochondria, the plurality of isolated mitochondria comprising a mitochondria concentration ranging from about 12.5 pg to about 200 pg per 200 pL of the composition.

8. The composition of claim 7, wherein the mitochondria concentration is about 25 pg per 200 pL of the composition.

9. The composition of claim 6, wherein the composition comprises the lipid nanoparticles at a nanoparticle concentration of about 10 pL of the lipid nanoparticles per 200 pL of the composition.

10. A method of delivering a therapeutic molecule to at least one cell of a subject, the method comprising administering a therapeutically effectiveDocket No.: 021132 / WO amount of a composition comprising the therapeutic molecule and an isolated mitochondria to the subject.11 . The method of claim 10, wherein the therapeutic molecule is selected from the group consisting of a protein, a peptide, a DNA molecule, and RNA molecule, a vaccine antigen.

12. The method of claim 11 , wherein the RNA molecule is a messenger RNA molecule (mRNA) configured to encode a protein.

13. The method of claim 12, wherein the mRNA molecule encodes a vaccine antigen.

14. The method of claim 13, wherein the vaccine antigen comprises at least a portion of a SARS-CoV-2 spike protein.

15. The method of claim 10, wherein the composition further comprises a lipid nanoparticle (LNP), wherein the therapeutic molecule is complexed to the LNP.

16. The method of claim 10, wherein the composition comprises a plurality of the therapeutic molecules and a plurality of the isolated mitochondria, the plurality of isolated mitochondria comprising a mitochondria concentration ranging from about 12.5 pg to about 200 pg per 200 pL of the composition.

17. The method of claim 16, wherein the mitochondria concentration is about 25 pg per 200 pL of the composition.

18. The method of claim 15, wherein the composition comprises a plurality of lipid nanoparticles at a nanoparticle concentration of about 10 pL of the lipid nanoparticles per 200 pL of the composition.

19. The method of claim 13, wherein the composition is administered by an administration method selected from intranasal inoculation, intramuscular injection, intravenous injection, intrathecal injection, and intraperitoneal injection.Docket No.: 021132 / WO20. The method of claim 19, wherein the administration method is intranasal inoculation and the composition is configured to impart mucosal immunity to the subject.21 .The method of claim 10, further comprising mixing the therapeutic molecule and the isolated mitochondria prior to administering the therapeutically effective amount of the composition.

22. A kit comprising at least one lipid nanoparticle (LNP) and at least one therapeutic molecule.

23. The kit of claim 22, wherein the at least one therapeutic molecule comprises an mRNA molecule encoding a vaccine antigen.

24. The kit of claim 24, wherein the vaccine antigen comprises at least a portion of SARS-CoV-2 spike protein.

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