Microalgae-derived extracellular vesicles, their biodistribution upon intranasal administration, and their uses

Abstract

A composition containing microalgae extracellular vesicles (MEVs) formulated for intranasal delivery is provided, whereby upon intranasal administration, the MEVs travel through a specific pathway, via the olfactory nerve, through the lateral olfactory tract (LOT) to interconnected brain regions, and to specific regions within the brain. The MEVs are transported via neuronal axonal transport. The MEVs have the ability to cross synapses, including (i) synapses between olfactory nerve neurons (OSNs) and mitral / tufted neurons, (ii) synapses between mitral / tufted neurons and local neurons in various brain regions anchored by the lateral olfactory tract (LOT), and (iii) synapses between neurons in brain regions anchored by the LOT and neurons from the frontal cortex, hippocampus, thalamus, and hypothalamus. The composition contains microalgae (MEV)-derived extracellular vesicles loaded with a bioactive cargo for treating, detecting, diagnosing, or monitoring diseases, disorders, or conditions of or involving the brain, and specifically provides neuronal delivery of the cargo. The compositions and methods have a variety of applications as therapeutic and diagnostic agents for treating, diagnosing, and monitoring diseases, disorders, or conditions of or involving the brain. The compositions can be used in methods and uses for treating cancers involving the brain, for example, to deliver therapeutic agents for psychiatric diseases, disorders, and conditions, and to deliver therapeutic agents for neurodegenerative diseases, disorders, and conditions.

Description

[Technical Field]

[0001] Related Applications The benefit of priority is claimed to U.S. Provisional Application No. 63 / 517,083, filed August 1, 2023, entitled "Microalgae-derived Extracellular Vesicles, Their Biodistribution Upon Intranasal Administration, and Uses Thereof," inventors Lila Drittanti and Manuel Vega, and applicant AGS Therapeutics SAS.

[0002] The benefit of priority is claimed to U.S. Provisional Application No. 63 / 480,264, filed January 17, 2023, entitled "Microalgae-derived Extracellular Vesicles, Their Biodistribution Upon Intranasal Administration, and Uses," inventors Lila Drittanti and Manuel Vega, and applicant AGS Therapeutics SAS.

[0003] The benefit of priority is claimed to U.S. Provisional Application No. 63 / 418,959, filed October 24, 2022, entitled "Microalgae-derived Extracellular Vesicles, Their Biodistribution Upon Administration, and Uses," to inventors Lila Drittanti and Manuel Vega and applicant AGS Therapeutics SAS.

[0004] This application is related to PCT / EP2023 / 051650, filed January 24, 2023, published August 3, 2023 as International PCT Publication No. 2023 / 144127, entitled "Microalgae-derived extracellular vesicles, their biodistribution upon administration, and uses," by inventors Lila Drittanti and Manuel Vega, and applicant AGS Therapeutics SAS.

[0005] This application is related to PCT / EP2023 / 064751, filed June 01, 2023, by inventors Lila Drittanti and Manuel Vega, and applicant AGS Therapeutics SAS, entitled "Extracellular vesicles from genetically modified microalgae containing endogenously loaded cargo, their preparation, and uses."

[0006] This application is also related to International PCT Application No. PCT / EP2022 / 070371, filed July 20, 2022, and published January 26, 2023, as International PCT Publication No. WO2023 / 001894, entitled "Extracellular Vesicles from Microalgae, Their Preparation and Use," by inventors Lila Drittanti, Juan Pablo Vega, Jeremy Pruvost, and Manuel Vega, and applicants AGS Therapeutics SAS, 10 rue Greneta, 75003 Paris, France, AGS-M SAS, 41-43 Quai de Malakoff, 44000 Nantes, France, and Nantes Universite, 1 Quai de Tourville, 44000 Nantes, France.

[0007] Where permitted, the subject matter of each of these applications is incorporated by reference in its entirety.

[0008] INCORPORATION-BY-REFERENCE OF SEQUENCE LISTINGS PROVIDED ELECTRONICALLY An electronic version of the Sequence Listing is submitted herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on October 11, 2023, is 589,338 bytes in size, and is titled 5507SEQPC01.xml.

[0009] Technical Field A composition containing microalgae extracellular vesicles (MEVs) formulated for intranasal delivery is provided, whereby upon intranasal administration, the MEVs travel through a specific pathway, via the olfactory nerve, through the lateral olfactory tract (LOT) to interconnected brain regions, and to specific regions in the brain. The composition can be administered in any form suitable for intranasal administration, thereby introducing the MEVs into the olfactory nerve. The composition contains microalgae (MEV)-derived extracellular vesicles loaded with a bioactive cargo for treating a disease, disorder, or condition of or involving the brain. The compositions and methods have various applications as therapeutic and diagnostic agents for treating, diagnosing, and monitoring a disease, disorder, or condition of or involving the brain. The compositions can be used in methods and uses for treating cancer involving the brain, as well as in therapeutic agents for psychiatric diseases, disorders, and conditions. [Background technology]

[0010] Extracellular vesicles (EVs) are natural particles produced by most cells. EVs include exosomes (generally approximately 30-150 nm in size), which are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane, and microvesicles (approximately 50-1000 nm), which are produced by the outward budding of membrane vesicles from the cell surface. Exosomes and microvesicles share similar properties and are commonly referred to as EVs. EVs facilitate intercellular communication through the intercellular transport of proteins and nucleic acids, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and mRNAs. Therefore, mammalian and plant-derived EVs have been used as carriers for short interfering RNA (siRNA), microRNAs (miRNAs), and small molecule drugs. There is a need for easily produced EVs that can be easily delivered to cells and tissues. The purpose of this specification is to provide such EVs. Summary of the Invention

[0011] Cargo-loaded extracellular vesicles (EVs) are provided for administration to subjects in vivo and to cells and cell lines in vitro. In particular, compositions containing EVs formulated for intranasal delivery and their use for delivering cargo to the brain are provided. The EVs are loaded with cargo containing bioactive molecules, including biomolecules and small molecules, including diagnostic and / or therapeutic molecules. The EVs herein are derived from microalgae and are referred to as MEVs. Microalgae are unicellular green algae, including those belonging to the Chlorellales order, particularly the Chlorellaceae family, such as Chlorella vulgaris. Microalgae extracellular vesicles (MEVs) can be produced on a large scale.

[0012] MEVs can be endogenously loaded (endoloaded) by producing them in genetically modified microalgae that encode or express proteins, polypeptides, small peptides, various RNA molecules, and / or other biomolecules that the microalgae can be genetically programmed to express and thereby packaged into MEVs.

[0013] MEVs can be exogenously loaded with bioactive molecular cargo after production. MEVs can be exogenously loaded after isolation or partial purification / isolation of MEVs from microalgae by contacting the MEVs with the cargo to produce a composition in which substantially all MEVs generally have, on average, substantially the same exogenously loaded heterologous cargo. The biodistribution pattern does not depend on the mode of loading of the MEVs (see, e.g., Example 14, which delivers exogenously and endogenously loaded (as a control) biologically active cargo). The MEVs provided herein have unique biodistribution patterns that are a function of the route of administration. The biodistribution of MEVs differs from that of mammalian EVs and other EVs and / or nanoparticles.

[0014] The MEVs herein are generally formulated for intranasal administration as liquids, such as suspensions or emulsions, or as powders or other formulations that can be administered intranasally. Upon administration, MEVs are shown herein to distribute to the brain, where they migrate to specific regions of the brain. Due to this transport pattern, they can deliver cargo to such regions of the brain for the treatment, detection, diagnosis, and / or treatment of diseases, disorders, and conditions involving these target regions.

[0015] As shown and described herein, MEVs, when administered intranasally (IN), traverse a unique pathway to the brain, thereby providing a unique route for delivering bioactive molecules. Upon IN administration, MEVs are internalized by olfactory nerve neurons (OSNs) and migrate from there to the glomeruli. Reaching the glomeruli from olfactory nerve neurons (OSNs), MEVs enter mitral and tufted neurons and migrate intracellularly along distinct pathways with distinct kinetics throughout the lateral olfactory tract (LOT). The LOT is composed of long axons of mitral and tufted neurons that migrate from the olfactory bulb (OB) to various anterior-posterior brain regions directly involved in connecting the olfactory network, including the anterior olfactory nucleus, olfactory tubercle, taeniatecta, piriform cortex, amygdala, and entorhinal cortex. Lateral branches of the main long axons of mitral and tufted neurons enter and anchor in each of the following brain regions: the anterior olfactory nucleus, olfactory tubercle, taeniatecta, piriform cortex, amygdala, and entorhinal cortex. Within these regions, mitral / tufted axons connect (via synapses) with neurons from other regions (with a more secondary olfactory role), including the frontal cortex, hypothalamus, thalamus, and hippocampus.

[0016] Regions reached by MEV via IN administration include all and each of the brain regions connected to the olfactory nerve and lateral olfactory tract (LOT) in both hemispheres, the ventral, lateral, and dorsal regions, the external and internal regions, and along the anterior-posterior axis. These regions include: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus (fimbria, dentate gyrus). For example, MEV can be delivered to or intended for delivery to the limbic system, e.g., the amygdala, hippocampus, and thalamus, or the cortex, e.g., the frontal or parietal cortex.

[0017] MEVs are loaded with a variety of cargoes (also called "payloads" and described as biologically active molecules), including, but not limited to, RNAs such as interfering RNA and other RNA products, oligonucleotides, plasmids, peptides, proteins, and small molecules.

[0018] As shown herein and elsewhere (see co-owned International PCT Publication No. WO 2023 / 144127), MEVs can deliver cargo to organs, tissues, and cells, and can be targeted by delivery pathways and delivered there. MEVs, including Chlorella MEVs, are shown herein to have a remarkable ability to cross stringent natural barriers, such as the gastrointestinal tract and olfactory neurons, that is not shared by other extracellular vesicles (EVs) from other sources, including mammalian EVs.

[0019] As described herein, MEVs can be exogenously loaded (exo-loaded) with a variety of biologically active molecules, such as siRNA, mRNA, plasmids, ASOs, peptides, proteins, and / or small molecules, enabling a variety of therapeutic, diagnostic, and other uses. MEVs can also be endogenously loaded by the microalgae in which they are produced (see U.S. Provisional Application No. 63 / 349,006, filed June 3, 2022, and International PCT Application No. PCT / EP2023 / 064751). As shown herein, MEV biodistribution is determined by the route of administration. Thus, MEVs can deliver their cargo to various tissues and organs, including, for example, the lungs, intestine, GALT, spleen, liver, and brain, depending on whether they are administered intratracheally, orally, intravenously, or intranasally.

[0020] As demonstrated herein, MEVs have many applications, including therapeutic uses, including delivery of therapeutic agents for the treatment and / or prevention (including reduction of risk or severity) of diseases, disorders, and conditions. These uses include therapeutic applications including treatment of immunomodulation, immuno-oncology, genetic or metabolic disorders, neurological disorders, psychiatric disorders, respiratory disorders, among others.

[0021] Cargos (also called "payloads") include, but are not limited to, RNA, e.g., interfering RNA and other RNA products, oligonucleotides, plasmids, peptides, proteins, and small molecules. Exogenously loaded MEVs can be loaded with almost any molecule of interest, and endogenously loaded MEVs, in which the microalgae cells have been genetically engineered to express or encode the product, will produce MEVs containing cargo such as RNA, DNA, peptides, small peptides, polypeptides, and proteins that are produced and packaged in EVs by the microalgae.

[0022] Compositions containing MEVs, e.g., exogenously cargo-loaded MEVs, particularly those produced by the Chlorellales order, particularly the Chlorellaceae family, e.g., Chlorella vulgaris, are provided. The compositions include pharmaceutical compositions that can be formulated for specific delivery routes.

[0023] Methods for loading MEVs are described and provided (see also International PCT Publication Nos. WO 2023 / 001894 and WO 2023 / 144127, which detail exogenous and endogenous loading of MEVs). The cargo is a bioactive molecule or combination thereof, including biomolecules and small molecules. Cargo includes biomolecules, including, for example, biopolymers such as DNA and RNA, proteins, protein complexes, protein-nucleic acid complexes, and plasmids, and also or alternatively, small molecules, such as small molecule drugs. Bioactive molecules include therapeutic agents, such as anticancer compounds, and biomolecules, such as RNAi, oligonucleotides, and proteins, and complexes, as well as diagnostic molecules, such as detectable markers, molecules that are cosmetics, and molecules that act as anti-infectives for humans, animals, and plants. Methods for treating diseases and disorders, including pathogen infection and cancer, and the use of MEVs for the treatment of diseases and disorders, as well as diagnostic methods, are provided.

[0024] In general, cargo-loaded MEVs have applications in a variety of fields, including the diagnosis, prevention, and treatment of human and other animal diseases, industrial applications, cosmetic applications, veterinary applications, and use in the crop industry. MEVs carrying cargo appropriate for each application can be used, for example, as vaccines, as gene therapy delivery vectors, for gene silencing, for gene editing, for transfection for industry and research, for analytical methods, for cell-based assays, and for other uses and applications. Cargo-loaded MEVs can be used for the treatment of diseases, disorders, and conditions, as well as for industrial and cosmetic uses. Diseases, disorders, and conditions include, but are not limited to, genetic disorders, disorders of the gastrointestinal tract, disorders of the respiratory tract, disorders of the central nervous system (CNS), skin disorders including natural disorders and trauma-induced disorders, disorders of the genitourinary tract, disorders of the nasal-buccal cavity, disorders of the cardiovascular system, immune and immunoregulatory disorders, cancer, eye disorders, liver disorders, systemic disorders, and diseases, disorders, and conditions caused by or involving pathogens such as bacteria, viruses, or parasites.

[0025] Target tissues for treatment and / or delivery include, for example, epithelial and mucosal cells (e.g., any type of external or internal mucosa: mouth, intestine, uterus, trachea, bladder, and others), endothelial cells, sensory cells (e.g., visual, auditory), cancer cells, tumor cells, blood cells, blood cell precursors, nervous system cells (e.g., neurons, glial cells, and other CNS and peripheral nerve cells), cells of the immune system (e.g., lymphocytes, immunoregulatory cells, effector cells), germ cells, secretory cells, glandular cells, muscle cells, stem cells (e.g., embryonic or tissue-specific stem cells), liver cells, infected cells (e.g., cells infected with viruses, bacteria, fungi, or other pathogens), natural cells, and genetically engineered cells of the nervous system (NS). For purposes herein with respect to intranasally administered MEVs, the target tissue or organ is the brain. Of interest herein is delivery to the brain via intranasal administration.

[0026] A composition containing isolated microalgal extracellular vesicles (MEVs) is provided, wherein the microalgae is a species of Chlorella, and the composition is formulated for administration to a subject. The Chlorella extracellular vesicles can contain a heterologous bioactive cargo molecule introduced into the isolated extracellular vesicles, such that the vesicles in the composition containing the heterologous bioactive molecule cargo contain the same bioactive molecule cargo, where the cargo molecule is heterologous to Chlorella, and the bioactive cargo is a biomolecule or small molecule.

[0027] For all embodiments in which the MEV is derived from Chlorella, the Chlorella is any species of Chlorella, for example, but not limited to, the genus Chlorella selected from among Chlorella ellipsoidia, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. In certain embodiments, the Chlorella is Chlorella vulgaris.

[0028] A composition containing isolated microalgal extracellular vesicles (MEVs) is provided, wherein the microalgae is a species of Chlorella, and the MEVs in the composition contain a heterologous bioactive molecular cargo introduced into the isolated MEVs, such that all vesicles in the composition containing the heterologous bioactive molecular cargo contain the same cargo. The cargo is heterologous to Chlorella and is not endogenous, and the cargo is a biomolecule or a small molecule drug. Each of the cargo-containing MEVs can contain multiple different heterologous cargoes.

[0029] Cargo includes any molecule intended for delivery to or on a plant or animal, particularly to the brain herein. Due to the transport pathway following IN administration, MEVs, and therefore any cargo, cross the blood-brain barrier (BBB). Generally, cargo is bioactive in that it can be used to treat or detect a disease, disorder, or condition. Bioactive cargo includes, for example, any molecule that can have an effect on a plant or animal when administered, such as biomolecules, including biopolymers, and small molecules. Cargo includes, for example, proteins, peptides, and nucleic acids. Bioactive molecules can be synthetic, naturally occurring, and / or modified to alter their properties or activity. It includes any molecule used as a drug or therapeutic agent, or diagnostic agent, or cosmetic, or in industry. Cargo can be, but is not limited to, a therapeutic agent for treating or preventing a disease, disorder, or condition, or for treating or preventing a symptom thereof. Cargo can be a nucleic acid molecule, polypeptide, protein, plasmid, aptamer, or antisense oligonucleotide.

[0030] The cargo in the MEV in the composition can comprise a biopolymer. The biopolymer can be a naturally occurring biopolymer, a synthetic biopolymer, or a modified biopolymer. The biopolymer can be a nucleic acid or a protein containing modifications, including insertions, deletions, substitutions, and rearrangements of nucleotide or amino acid residues, and / or, if the biopolymer is a protein, the modifications can also include post-translational modifications. Post-translational modifications include, but are not limited to, glycosylation, hyperglycosylation, PEGylation, sialylation, albumination, other half-life extending moieties, and other modifications that improve or alter the pharmacological, kinetic, or kinetic properties of the protein.

[0031] Nucleic acids, such as DNA and RNA, are among the molecules that can be cargoes. When the cargo is RNA or protein, it can be provided as cargo or it can be encoded by a nucleic acid, which is then expressed in the organism to which it is administered. Examples of RNA are interfering RNA (RNAi) and mRNA, including modified mRNA. RNAi includes, for example, silencing RNA (siRNA) or short hairpin RNA (shRNA), microRNA (miRNA), small activating RNA (saRNA), and long non-coding RNA (lncRNA). RNA products also include double-stranded RNA and ribozymes. Cargoes can also be oligonucleotides, such as antisense oligonucleotides or allele-specific oligonucleotides. Cargoes can include gene editing systems, such as CRISPR-Cas systems, and modified and improved gene editing systems, such as CRISPR-related and CRISPR-like systems (see, for example, published U.S. Patent Applications Nos. 2020 / 0332273 and 2020 / 0332274, respectively, by applicant Metagenomi).

[0032] Cargos include therapeutic, diagnostic, or theragnostic proteins or peptides, protein complexes, two or more proteins or proteins and nucleic acids, or proteins and aptamers, or complexes containing a combination of proteins, nucleic acids, and other molecules. The cargo may be a protein that is, or may encode, an antibody or antigen-binding fragment thereof. Antibodies may be in any form, including single-chain forms, nanobodies, camelids, and other forms, such as scFvs, bispecific antibodies, or antigen-binding fragments thereof. Antibodies and antigen-binding fragments thereof include checkpoint inhibitor antibodies or antigen-binding fragments thereof, tumor antigen-specific antibodies or antigen-binding fragments thereof, anti-cancer gene-specific antibodies or antigen-binding fragments thereof, tumor-specific receptor, or signaling molecule antibodies or antigen-binding fragments thereof. Exemplary antibodies and antigen-binding fragments thereof specifically bind to and inhibit one or more of CTLA-4, PD-1, PD-L1, PD-L2, the PD-1 / PDL1 pathway, the PD-1 / PDL2 pathway, HER2, EGFR, TIM-3, LAG-3, BTLA-4, HHLA-2, CD28, and other checkpoint or immune suppressor factors, or tumor antigens.

[0033] The cargo in the MEV in the composition may include immunostimulatory products and antigens and may be used as a vaccine to induce an immunoprotective or immune response upon administration. The cargo may be, but is not limited to, DNA, RNA, protein, and virus. The cargo may contain a nucleic acid or protein that is a therapeutic product, or a nucleic acid encoding a protein, for the treatment of cancer, an infectious disease, a neurodegenerative disease, or other CNS disorder, or aging, or an age-related disease, or an ophthalmic disorder, or an immunological disorder. The cargo may be a cosmeceutical or cosmetic product or a cosmetically active product. The cargo may include a small molecule bioactive molecule, such as a small molecule drug. Exemplary drugs include chemotherapeutic drugs and prodrugs. The cargo in the MEV in the composition may be or include a diagnostic marker or detectable product, such as, but not limited to, luciferase or a nucleic acid encoding a luciferase, a fluorescent protein or a nucleic acid encoding a fluorescent protein, or a luciferase operon. As described herein, cargo includes anything that can be used for the treatment, detection, diagnosis, and monitoring of any disease, disorder, or condition involving the brain.

[0034] The cargo may comprise DNA. The DNA may be a plasmid, such as one encoding a product for expression in the animal or plant to which it is administered. The plasmid may encode one or more cargo products. For expression of the cargo product, the encoding nucleic acid is operably linked to a regulatory sequence recognized by the eukaryotic cell. The cargo may include RNA, proteins, peptides, small molecules, and any other molecule that can be loaded into the MEV either exogenously or endogenously by encoding it in the microalgae.

[0035] Exemplary products include, but are not limited to, therapeutic and diagnostic products. These include proteins and RNA products, including the RNA products listed above. Because MEVs are eukaryotic and intended for administration to animals, such as humans, the plasmids generally encode the products under the control of eukaryotic regulatory signals and sequences, including eukaryotic promoters and translation sequences, such as RNA polymerase II and III promoters. Exemplary promoters include RNA polymerase II promoters, such as those derived from animals, plants, and plant or animal viruses. Exemplary promoters include, but are not limited to, cytomegalovirus promoters, simian virus 40 promoters, herpes simplex promoters, Epstein-Barr virus promoters, adenovirus promoters, synthetic promoters, actin promoters, and synthetic chimeric promoters. Other eukaryotic transcriptional and translational sequences may include, but are not limited to, one or more of an enhancer, a polyA sequence, and / or an internal ribosome entry site (IRES) sequence.

[0036] Methods for preparing MEVs are described herein. The methods include introducing a cargo into isolated MEVs. The cargo includes any molecule desired for delivery into or onto an animal or plant. Generally, the cargo is, contains, or provides a bioactive molecular product, including small molecules and biopolymers. The biopolymers may be naturally occurring, synthetic, modified, or a combination thereof. The cargo may include a protein, nucleic acid, or small molecule. The cargo may be loaded into MEVs by any method known to those skilled in the art, including, for example, one or more of electroporation, sonication, extrusion, and the use of surfactants. In some embodiments, the MEVs are derived from Chlorella, for example, but not limited to, a species of Chlorella selected from Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. Any of the MEVs provided herein, including MEVs produced by the methods and compositions containing MEVs, may be used as one or more of a method of diagnosis, a vaccine, a therapy for treatment, diagnosis of disease, treatment of a disease or disorder or condition, a cosmetic, an industrial application, and / or any use known to one of skill in the art.

[0037] MEVs can be used in any such method, including methods for treating a disease, disorder, or condition. Examples of diseases, disorders, and conditions are cancer, including solid tumors or hematologic malignancies, or metastases thereof. Other diseases, disorders, and conditions include those of or involving the respiratory system, those of or involving the central nervous system or nervous system, those of or involving the skin and exposed epithelium or mucous membranes, those of or involving the gastrointestinal tract, and those of or involving infectious agents. Infectious agents include bacteria, viruses, parasites, prions, oomycetes, and fungi.

[0038] The cargo may provide a therapeutic molecule for treatment or may induce an immune response and function as a vaccine. The MEV may contain an immunostimulatory protein or antigen or a cargo that encodes an immunostimulatory protein or antigen, such that the MEV, when administered, is immunostimulatory and elicits an innate or adaptive immune response, or the MEV and / or cargo may elicit an immune protective response to prevent or treat a disease or disorder or condition.

[0039] Generally, MEVs can be used to treat diseases, disorders, or conditions resulting from trauma. Trauma includes, but is not limited to, trauma resulting from or associated with wounds, burns, surgery, cuts in the skin, fractures, hair loss, exposed dermis, exposed mucosa, fibrosis, lacerations, and ulcerations. This includes brain or CNS trauma. MEVs can be used to induce effects that treat conditions resulting from natural aging or aging induced by pathogens or diseases or in other ways. For purposes herein, diseases, disorders, and conditions are those involving the brain or CNS that are treated, detected, or monitored in the brain.

[0040] In general, compositions containing MEVs can be formulated for administration by any route of administration, including, but not limited to, local, systemic, topical, parenteral, enteral, mucosal, pulmonary inhalation or intranasal, vaginal, rectal, otic, oral, and other routes of administration. For purposes herein, MEVs are formulated for intranasal administration. They can be formulated in any form, including forms and formulations appropriate for the route of administration, including as a tablet, as a liquid, e.g., as an emulsion, as a powder, or as an aerosol, oral administration, spray, or inhalation.

[0041] The MEV compositions can be used in any of the methods and treatments described herein or known to those skilled in the art. Methods include, for example, any of those described herein, including, for example, use for one or more of gene silencing, gene interference, gene therapy, gene / protein overexpression, gene editing, inhibition or stimulation of protein activity, and pathway signaling. The compositions and MEVs can be used for prophylaxis and / or vaccination. They can also be used for industrial purposes, such as manufacturing, characterization, and calibration.

[0042] Methods are provided for treating diseases, disorders, and conditions that can be treated by delivering an active agent to the brain. Compositions are provided that are prepared for intranasal delivery. The compositions contain microalgal extracellular vesicles containing an active agent. The microalgal extracellular vesicles can be loaded by any suitable method (see the methods and MEVs described in International Patent Publication No. PCT / EP2022 / 070371 and U.S. Provisional Application No. 63 / 349,006), including exogenous loading after MEV production and endogenous loading in vivo by genetically modifying microalgae to package nucleic acids or encoded products into MEVs.

[0043] EVs are derived from microalgae, which are unicellular green algae belonging to the Chlorellales order, particularly the Chlorellaceae family, such as Chlorella vulgaris. MEVs are provided in compositions formulated for intranasal administration. MEVs can be exogenously loaded after isolation, or endogenously loaded by genetically engineered microalgae that encode and package heterologous nucleic acids and / or proteins into MEVs in vivo. The advantage of exogenously loading MEVs with cargo (exoloading) is that the amount of cargo / MEV can be controlled, and the distribution of exogenous cargo in MEVs is predictable and substantially uniform, resulting in a known average cargo molecule or amount per cargo / MEV. A wide variety of bioactive molecules, including biomolecules and small molecules such as drugs and organic compounds, can be loaded into MEVs. MEVs can also be endogenously loaded by genetically engineered microalgae to package heterologous nucleic acids and / or proteins.

[0044] The resulting MEVs, whether endo- or exo-loaded, are not toxic, and they can be administered to cells in vitro or in vivo, with a distribution pattern that depends on the route of administration.

[0045] MEVs and brain delivery For purposes herein, MEVs are intended for delivery to the brain via intranasal administration. MEVs are herein shown to be transported to the brain via unique pathways and mechanisms after intranasal (IN) administration. These pathways and mechanisms are not shared by exosomes from other sources or by nanoparticles. Following IN delivery, MEVs are herein shown to be transported via the olfactory nerve and through the lateral olfactory tract (LOT) to multiple interconnected brain regions. MEVs are transported via neuronal axonal transport. MEVs have the ability to cross synapses at least across (i) synapses between olfactory tract neurons (OSNs) and mitral / tufted neurons, (ii) synapses between mitral / tufted neurons and local neurons in various brain regions anchored by the lateral olfactory tract (LOT), and (iii) synapses between neurons in brain regions anchored by the LOT and neurons from the frontal cortex, hippocampus, thalamus, and hypothalamus.

[0046] For all embodiments of intranasal administration and / or brain or CNS related methods, uses, and compositions described and contemplated herein, the diseases, disorders, and conditions include any described herein and known to those of skill in the art that can be treated, detected, diagnosed, and / or monitored by delivery of molecules to the brain.

[0047] The biodistribution of MEVs follows pathways and connections in the neural network of olfactory nerves and mitral / tufted neurons throughout the brain. These transport pathways provide access (biodistribution) to brain regions (within 1–16 h after IN administration), including the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus (fimbria, dentate gyrus). Therefore, MEVs can deliver active agents, particularly biologically active payloads, to specific regions of the brain. Payloads include, but are not limited to, proteins, mRNA, DNA, small molecules, and any drug that can be exogenously loaded (exoloaded) into MEVs or packaged into MEVs in vivo by microalgae, particularly genetically modified microalgae that encode or produce the drug. Thus, MEVs provide effective delivery of bioactive small molecules, including lipophilic small molecules, proteins, DNA, and mRNA, to neurons, astrocytes, glial cells, and neural stem cells. In vivo MEVs offer therapeutic and diagnostic uses, as well as diagnostic and experimental uses. Delivery is exemplified in the Examples, showing effective delivery and expression of catalase, GFP, luciferase, nerve growth factor (NGF), TrkA (tropomyosin kinase A), neurotrophic factors (NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), EPO, IGF-1, bFGF (basic fibroblast growth factor), hGH), psilocybin / psilocin, harmine, temozolomide, rivastigmine, and rhodamine to neurons, astrocytes, glial cells, and / or neural stem cells in vitro and in vivo.

[0048] MEV provides a unique vehicle for, among many uses and methods: i) treatment and / or prevention of brain disorders, including but not limited to cognitive, emotional, behavioral, psychiatric, neurological, degenerative, and cancer; (ii) in vitro or in vivo study of brain disorders; (iii) diagnosis of brain disorders; and (iv) recreational and therapeutic uses.

[0049] A method for delivering bioactive molecules to the brain by intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule is provided, whereby the MEVs travel to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; the bioactive molecule is any molecule that can provide treatment for a disease, disorder, or condition, or that can be used to detect a disease, disorder, or condition, or that can be used to monitor treatment for a disease, disorder, or condition, and the bioactive molecule is heterologous to the microalgae and / or MEVs. The use of MEVs and compositions formulated for intranasal administration for such delivery is also provided.

[0050] The delivery methods include methods and uses for treating diseases, disorders, and conditions involving the brain. Methods of treating or managing a disease, disorder, or condition of the brain or a disease, disorder, or condition involving the brain (and uses of microalgal extracellular vesicles (MEVs)) are provided by intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, whereby the MEVs travel to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus, where the bioactive molecule is any molecule that can result in treatment of a disease, disorder, or condition of or involving the brain, and the bioactive molecule is heterologous to the microalgae and / or MEVs.

[0051] Also provided are methods of detecting a brain disease, disorder, or condition, or a disease, disorder, or condition involving the brain, or monitoring the treatment of a brain disease, disorder, or condition (and use of microalgal extracellular vesicles (MEVs) for such purposes) by intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive molecules, whereby the MEVs are detected in one or more of the following regions: the olfactory bulb, the anterior olfactory nucleus, the olfactory tubercle, the taenia tecta, the piriform cortex, the amygdala, the entorhinal cortex, the primary motor cortex, the frontal cortex, the agranular insular cortex, the primary somatosensory cortex, the auditory cortex, the retrosplenial granular cortex, the temporal association cortex, the basolateral amygdala, the mammillary bodies, the arcuate nucleus of the hypothalamus, the corpus callosum, the internal capsule, the thalamus, and the hippocampus (fimbria, dentate gyrus). the microalgae and / or MEVs travel via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to the brain, the bioactive molecule comprises a reporter or detectable marker, the bioactive molecule is any molecule that can be used to detect or diagnose a disease, disorder, or condition, or that can be used to monitor treatment of a disease, disorder, or condition, or that can be used to detect or diagnose or treat a disease, disorder, or condition, the disease, disorder, or condition being of or involving the brain, and the bioactive molecule is heterologous to the microalgae and / or MEVs.

[0052] Compositions are provided that include microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, the compositions being for intranasal delivery to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus. The bioactive molecule is any molecule that can effect treatment of a disease, disorder, or condition, or that can be used to detect or diagnose a disease, disorder, or condition, or that can be used to monitor treatment of a disease, disorder, or condition, or that can be used to detect, diagnose, monitor, and / or treat a disease, disorder, or condition, wherein the disease, disorder, or condition is a disease, disorder, or condition of or involving the brain, and the bioactive molecule is heterologous to the microalgae and / or MEV. Compositions are provided for use in treating, diagnosing, detecting, and / or monitoring diseases, disorders, and conditions involving the brain, and for using or targeting interconnected brain regions.

[0053] Also provided are compositions comprising microalgal extracellular vesicles (MEVs) for use in delivering a cargo comprising a bioactive molecule to the brain for treating a disease, disorder, or condition of or involving the brain, or for diagnosing, detecting, or monitoring treatment of, or treating, diagnosing, detecting, and / or monitoring a disease, disorder, or condition of or involving the brain, wherein the MEVs comprise a bioactive molecule for delivery to the brain, and the composition is delivered to the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary spleen, and / or primary spleen. The bioactive molecule is formulated for intranasal delivery to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus, where the bioactive molecule is any molecule that can provide treatment for a disease, disorder, or condition, or that can be used to detect a disease, disorder, or condition, or that can be used to monitor treatment for a disease, disorder, or condition, and the bioactive molecule is heterologous to the microalgae and / or MEV. Delivery and transport are achieved by pathways and interconnected brain regions.

[0054] According to these methods and compositions, MEVs travel within the brain via intraneuronal axonal transport and transport between neurons through synapses. For example, MEVs travel within the brain by neuronal axonal transport, and MEVs are delivered to the fimbria or dentate gyrus of the hippocampus. MEVs follow pathways and connections in neural networks, including olfactory nerves and mitral / tufted neurons, throughout the brain. Upon intranasal administration, MEVs traverse one or more of the following: (i) synapses between olfactory nerve neurons (OSNs) and mitral / tufted neurons; (ii) synapses between mitral / tufted neurons and local neurons in brain regions where the LOT is established; and (iii) synapses between neurons in brain regions where the LOT is established and neurons from or to the frontal cortex, hippocampus, thalamus, and hypothalamus. For example, MEVs traverse (i), (ii), and (iii), or (i) and (ii), such as the pathways traversed by MEVs upon intranasal administration shown in FIG. 35.

[0055] The methods, compositions, and uses provided herein are for intranasal administration, after which the MEVs are transported and / or delivered to, or are for delivery to, e.g., one or more of the corpus callosum, dorsal fornix, dorsal hippocampal commissure, and fimbria of the hippocampus.

[0056] The compositions used in the compositions and methods provided herein can be formulated as suspensions or emulsions, e.g., nanoemulsions or microemulsions. They can be formulated in any form, such as powders and liquids, for mucosal uptake into the nasal cavity, thereby allowing them to be administered intranasally.

[0057] The composition may be formulated as, for example, a liquid, powder, lozenge, granule, liquid, oil, suspension, or emulsion suitable for intranasal administration or processing, such as by dilution or dissolution, for intranasal administration.

[0058] Those skilled in the art understand and are familiar with the properties of nanoemulsions and microemulsions and their formation. In the composition, the MEVs contain a biologically active cargo. For example, MEVs can be prepared so that, on average, each MEV contains a predetermined amount of biologically active molecule, e.g., 1-100, e.g., but not limited to, at least 1-10, 1-20, 1-30, 1-50, 10-20, etc., and other amounts of biologically active molecule per MEV appropriate for the indication and use. The selection of the amount of cargo per MEV is within the level of skill in the art and depends on factors known to those skilled in the art, such as the particular disease, disorder, or condition being treated or use of the MEV, the subject, the particular cargo, and other such parameters and factors. Similarly, the concentration of the MEVs depends on the particular cargo and use. For example, the concentration of the MEVs in the composition can be, for example, about 0.1-10 mg / mL or values ​​therein, as well as lower, higher, and intermediate concentrations. The compositions can be formulated for single-dose administration (direct administration without dilution) or multiple-dose administration for administration in aliquots and / or dilution to desired concentrations. Exemplary volumes of the compositions for administration are 0.1 to 100 mL, e.g., 1 to 10 mL, 1 to 5 mL, 0.1 to 1 mL, and any appropriate volume for intranasal administration. The compositions can be administered as a single dose or as a series of doses or other regimens. The compositions can be administered as part of a combination therapy protocol.

[0059] Compositions and methods include MEVs endogenously loaded with genetically modified microalgae encoding a bioactive molecule or a pathway for its production. MEVs also include purified or partially purified MEVs exogenously loaded with cargo. MEVs may contain multiple different heterologous cargoes. For purposes herein, cargo includes a therapeutic agent for treating or preventing a disease or condition of or involving the brain, or for treating or preventing a symptom thereof.

[0060] The microalgae used to produce MEVs for use in the methods can be microalgae from a microalgal phylum selected from among euglenoids (Euglena genus), goldenrods (Golden Brown Algae and Bacillariophyta), pyrophytes (Phiaeophyta), chlorophytes (Chlorophyta), red algae (Rhodophyta), brown algae (Phaeophyta), and xanthophyta (Xanthophyta). For example, the microalgae is a species of the Chlorophyceae or Trebouxiophyceae or Chlorophyceae, such as Chlorella or Chlamydomonas.

[0061] Chlorella species include, but are not limited to, Chlorella ellipsoidia, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis, e.g., Chlorella vulgaris and Chlorella variabilis. In certain embodiments, the Chlorella is Chlorella vulgaris. For example, methods and compositions include those in which the microalgae is a Chlorella species, and the MEVs in the composition contain a heterologous bioactive molecular cargo exogenously introduced into the isolated MEVs, such that, on average, vesicles in the composition containing a heterologous bioactive molecular cargo contain the same heterologous cargo, where the cargo is heterologous to the Chlorella, and the cargo is a biomolecule or small molecule drug or any cargo for delivery to the brain described herein and / or known to those of skill in the art. Also included are methods and compositions wherein the MEVs are Chlorella extracellular vesicles, the Chlorella extracellular vesicles comprising a heterologous bioactive molecular cargo endogenously introduced into the extracellular vesicles by microalgae, the cargo molecule being heterologous to Chlorella, and the bioactive cargo being a biomolecule for treating a disease, disorder, or condition of or involving the brain.

[0062] Methods and compositions are provided in which the MEVs are Chlorella extracellular vesicles, which contain a heterologous bioactive molecular cargo introduced into the isolated extracellular vesicles, such that the vesicles in the composition containing the heterologous bioactive molecular cargo contain, on average, the same bioactive molecular cargo, where the cargo molecule is heterologous to Chlorella, and the bioactive cargo is a therapeutic or detectable molecule for treating, monitoring, and / or diagnosing a disease, disorder, or condition of or involving the brain. In other embodiments, the MEVs are Chlorella extracellular vesicles, which contain a heterologous bioactive molecular cargo endogenously introduced into the extracellular vesicles by microalgae, such that the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same bioactive molecular cargo, where the cargo molecule is heterologous to Chlorella, and the bioactive cargo is a biomolecule or small molecule. In other embodiments, the MEVs in the composition contain a heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEVs, such that, on average, the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same cargo, the cargo being heterologous to the Chlorella, and the cargo being a biomolecule or small molecule. In other embodiments, the cargo is endogenously introduced into the MEVs by modifying the microalgae to express or produce the cargo, such as a nucleic acid or protein, or a biochemical pathway product. In an exemplary embodiment, the Chlorella is Chlorella vulgaris.

[0063] Cargos include, but are not limited to, biopolymers such as biomolecules, naturally occurring biopolymers, or synthetic biopolymers, or modified biopolymers such as, for example, nucleic acid molecules, polypeptides, proteins, plasmids, aptamers, or antisense oligonucleotides. Cargos include, but are not limited to, DNA or RNA, such as, for example, interfering RNA (RNAi), mRNA or modified mRNA, silencing RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), self-amplifying RNA, small activating RNA (saRNA), long non-coding RNA (lncRNA), ribozymes, or double-stranded RNA. Cargos include oligonucleotides, such as antisense oligonucleotides, allele-specific oligonucleotides, or antisense oligonucleotides (ASOs), and gene editing systems, such as CRISPR-CAS systems, CRISPR-related, or CRISPR-like systems. The cargo may comprise DNA, such as a plasmid, encoding a therapeutic and / or detectable or diagnostic product, or an RNA product, such as an RNAi and antisense oligonucleotide, or a ribozyme, or any of the RNA forms described above, including double-stranded RNA. The plasmid may encode the cargo product under the control of a eukaryotic promoter, e.g., an RNA polymerase II or III promoter, e.g., a eukaryotic viral promoter, e.g., a cytomegalovirus promoter, a simian virus 40 promoter, a herpes simplex promoter, an Epstein-Barr virus promoter, an adenovirus promoter, a synthetic promoter, or another promoter, e.g., an actin promoter, or a synthetic chimeric promoter. The plasmid may also contain other regulatory sequences for expression, such as other eukaryotic transcription and translation sequences. The MEV cargo may comprise a small molecule for providing treatment, detection, diagnosis, or monitoring of a disease, disorder, or condition of or involving the brain.

[0064] The cargo may include any molecule of interest for delivery to the brain. This includes, for example, cargo that encodes or is an immunomodulator, such as an immunomodulator for increasing or decreasing the production of one or more cytokines, up-regulating or down-regulating self-antigen presentation, masking MHC antigens, or promoting the proliferation, differentiation, migration, or activation state of one or more types of immune cells. The cargo may include or encode a hormone, cytokine, or chemokine. The cargo may include a prodrug or a vector encoding an enzyme that converts a prodrug into a drug for treating a disease, disorder, or condition of or involving the brain. The cargo may include or encode an antibiotic, antiviral, antifungal, antiparasitic, or other anti-infective agent for the treatment of a brain infection or an infection involving the brain. The cargo may include a therapeutic nucleic acid or protein, or a nucleic acid encoding a protein, that is a therapeutic product for the treatment of cancer or tumors in the brain, or an infectious disease in the brain, or a neurodegenerative disease or other central nervous system (CNS) disorder, or dementia. The cargo may comprise a chemotherapeutic agent for treating a disease, disorder or condition of or involving the brain, and / or encodes or comprises an antibody or antigen-binding fragment thereof, such as an scFv, bispecific antibody, or antigen-binding fragment thereof, etc. The cargo may comprise a nucleic acid for gene therapy.

[0065] The MEV may comprise two or more different cargo products. The cargo may comprise a therapeutic product, a diagnostic product, or a detectable product, or a combination thereof, for detecting, diagnosing, and / or monitoring a disease, disorder, or condition of or involving the brain, or a combination thereof. A diagnostic agent may comprise a luciferase or a nucleic acid encoding a luciferase, a fluorescent protein or a nucleic acid encoding a fluorescent protein, or a luciferase operon, or a combination thereof. A bioactive molecule cargo may comprise any molecule that has an effect on a cell or organism to which it is delivered, or is detectable, or functions as a detectable marker or biomarker, thereby resulting in the treatment, detection, diagnosis, or monitoring of the treatment of a disease, disorder, or condition of or involving the brain.

[0066] The cargo may comprise one or more of a bioactive small molecule, a peptide (polypeptide, protein), RNA (mRNA, siRNA, miRNA, lncRNA), DNA (antisense oligonucleotide (ASO), plasmid, DNA fragment), and a gene editing complex. The bioactive molecule may be a diagnostic or therapeutic agent or a theragnostic for treating, diagnosing, detecting, and / or monitoring treatment of a disease, disorder, or condition of or involving the brain. The cargo may comprise, for example, one or more of a hormone, growth factor, enzyme, immunomodulatory compound, receptor, receptor agonist, or receptor antagonist for treating a disease, disorder, or condition of or involving the brain.

[0067] The disease, disorder, or condition may include a tumor in the brain. The cargo may include, for example, an oncolytic virus that infects glial tumors, or may include a therapeutic agent for the treatment of glial tumors. The disease, disorder, or condition can be a neurodegenerative disease (e.g., Parkinson's disease, or Alzheimer's disease, or Huntington's disease, or Creutzfeldt-Jakob disease, or other neurodegenerative disease), or a cognitive disorder (e.g., dementia, or amnesia, or delirium, or other cognitive disorder), or a brain disorder (e.g., encephalitis, or seizures, or tumors, or other brain disorders), or a nervous system disorder (e.g., pain, or seizures, or infections, or other nervous system disorders), or a genetic disease (e.g., cystic fibrosis, thalassemia, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, Tay-Sachs disease, or other genetic disease), or a brain tumor, or Niemann-Pick disease, or a prion disease, or Parkinson's disease, or multiple sclerosis, or amyotrophic lateral sclerosis (ALS), or a muscular dystrophy, or other disease of or involving the brain. The disease, disorder, or condition of or involving the brain may be a cancer or disease, disorder, or condition treated or prevented by the vaccine, and / or may be a disease, disorder, or condition caused by or involving an infectious agent, such as one or more of bacteria, viruses, oomycetes, parasites, prions, and fungi.

[0068] Upon intranasal administration, MEVs can deliver cargo to one or more of neurons, astrocytes, glial cells, and neural stem cells for therapeutic, diagnostic, detection, or monitoring purposes. Compositions containing MEVs can be used to deliver cargo to neurons, astrocytes, glial cells, and / or neural stem cells in vivo. As discussed below, MEVs can also be used to deliver cargo to cells in vitro for cell therapy. The resulting cells can be administered.

[0069] The diseases, disorders, and conditions include one or more of cognitive, affective, behavioral, psychiatric, neurological, degenerative, genetic, malignant (cancer), and / or traumatic brain diseases, disorders, or conditions. Diseases, disorders, or conditions of or involving the brain can result from damage to the brain or central nervous system (CNS). The MEV can contain a therapeutic cargo that is psychoactive, or treats a psychiatric disorder, or is an immunomodulatory product, or is a detectable product, or treats brain injury or trauma, or treats cancer, or treats a neurological brain disorder, or treats a CNS disorder, or treats a genetic brain disorder, or treats brain tumors, or has anti-aging activity, or has brain regenerative activity.

[0070] The MEV may, for example, comprise a cargo for one or more of: (i) treating, preventing, or reducing the risk of brain diseases, disorders, and conditions; (ii) in vitro and / or in vivo research into brain diseases, disorders, and conditions; (iii) diagnosing brain diseases, disorders, and conditions; and (iv) recreational use. The diseases, disorders, and conditions include, but are not limited to, cognitive, emotional, behavioral, psychiatric, neurological, and / or neurodegenerative diseases, disorders, and conditions, or diseases, disorders, or conditions resulting from injury to the brain or central nervous system (CNS). The diseases, disorders, and conditions are selected from among cancers or tumors of the brain and / or CNS, genetic disorders, brain injury or trauma, and infectious diseases.

[0071] The cargo may be selected from antidepressants, antipsychotics, anxiolytics, analgesics, hallucinogens, hallucinogens, and memory enhancers. For example, the cargo may include carboline, lysergic acid, psilocybin, or derivatives thereof. Due to their direct route to brain regions, intranasally administered MEVs provide a vehicle for the delivery of psychoactive drugs. MEVs may deliver drugs for the treatment of psychiatric and / or mental disorders.

[0072] MEVs can be used to deliver cargoes, such as hydrophilic compounds that cannot reach the brain after hepatic first-pass metabolism or that are poorly absorbed intestinally when administered systemically or locally to a location other than the nose, and cargoes that cannot cross the blood-brain barrier. Intranasal administration in MEVs generally provides for the delivery of such compounds that cannot otherwise be administered to reach the brain.

[0073] Diseases, disorders, and conditions for treatment with intranasally administered MEV include, but are not limited to, borderline personality disorder, eating disorders, schizophrenia, attention-deficit / hyperactivity disorder (ADHD), autism, bipolar disorder, borderline personality disorder, anxiety, depression, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder (PTSD).

[0074] In some examples, the cargo comprises a bioactive molecule for the treatment of a condition such as:

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] [Table 1-3]

[0078] [Table 1-4]

[0079] [Table 1-5]

[0080] Diseases, disorders, and conditions involving or of the brain include, but are not limited to, genetic disorders, neurodegenerative diseases, and metabolic disorders that affect brain function, as well as other brain-related conditions. Examples of diseases, disorders, and conditions include, but are not limited to, human psychiatric disorders, non-human animal brain disorders, CNS disorders, anxiety disorders such as panic disorder, social anxiety, phobia-related disorders, and generalized anxiety disorder, attention deficit hyperactivity disorder such as inattentive type, hyperactive-impulsive type, and mixed type, autism spectrum disorders such as Asperger's syndrome, childhood disintegrative disorder (CDD), Kanner's syndrome, and pervasive developmental disorder (PDD-NOS), bipolar disorders such as bipolar I disorder, bipolar II disorder, bipolar with mixed features, bipolar with major depression with a seasonal pattern, cyclothymic disorder, rapid cycling bipolar disorder, eating disorders such as anorexia nervosa, bulimia nervosa, muscle dysmorphia, binge eating disorder, and other specific eating or feeding disorders. g) Disorder (OSFED), including obsessive-compulsive overeating, Prader-Willi syndrome, diabulimia, orthorexia nervosa, selective eating, drunkorexia, pregorexia, personality disorders such as, but not limited to, antisocial personality disorder, borderline personality disorder, histrionic personality disorder, narcissistic personality disorder, avoidant personality disorder, dependent personality disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD) such as acute stress disorder, simple PTSD, complex PTSD, comorbid PTSD, classic Rett syndrome, CDKL5-associated atypical Rett syndrome, schizophrenia disorders such as catatonic schizophrenia, disorganized schizophrenia, paranoid schizophrenia, residual schizophrenia, and undifferentiated schizophrenia, and other such mental and brain related conditions. Other diseases, disorders, and conditions of or involving the brain include, for example, Alzheimer's disease, prion diseases such as Creutzfeldt-Jakob disease, Niemann-Pick disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, Tay-Sachs disease, Wilson's disease, leukodystrophies, epilepsy, multiple sclerosis, encephalitis, and migraine.

[0081] MEV cargoes for delivery to the brain include, for example, cargoes comprising one or more of psychoactive agents, enzymes, growth factors, and detectable products for treating, detecting, or monitoring diseases, disorders, or conditions of or involving the brain. Such cargoes include, for example, TrkA (tropomyosin kinase A), NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocybin and / or psilocin, harmine, temozolomide, rivastigmine, GABA B1A receptor, GABA B1A receptor siRNA, PTEN Neurotrophic factors selected from siRNA (SEQ ID NOs: 136 to 138), miR-17 (miRNA, SEQ ID NOs: 139 to 141), MALAT1 (SEQ ID NO: 142), 5-hydroxytryptamine-1A (5-) and 5-hydroxytryptamine-3 (5-HT3) receptor agonists, such as azapirone, methylphenidate, dexmethylphenidate, ondansetron (e.g., products sold under the trademark Zofran®), acetylcholinesterase inhibitors, such as donepezil, galantamine, rivastigmine, alpha-1 receptor antagonists, such as prazosin, anticonvulsants, such as gabapentin, pregabalin, topiramate (e.g., products sold under the trademark Topomax®), carbamazepine, eslicarbazepine, levetiracetam, licarbazepine, oxcarbazepine, pine, valproic acid and derivatives, lamotrigine, antipsychotics such as aripiprazole, asenapine, cariprazine, chlorpromazine, clozapine, haloperidol, lumateperon tosylate (for example, products sold under the trademark Caplyta®), olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, beta-blockers such as azapirones, propranolol, drugs that modulate the cholinergic system such as biperiden, scopolamine, corticotropin-releasing factor (CRF) antagonists, drugs that modulate the GABAergic system such as benzodiazepines, brexanolone, Sage-217, glucocorticoid receptor agonists such as hydrocortisone, drugs involved in glutamatergic modulation such as AGN-241751, AV-101, AVP-786, AVP-923, AXS-05,D-cycloserine, dextromethorphan, rapastinel, glycine, and glycine reuptake inhibitors, e.g., sarcosine, drugs that modulate the hypothalamic-pituitary-adrenal (HPA) axis, e.g., fludrocortisone, metyrapone, mifepristone, and probiotics, drugs that modulate the kynurenine pathway (KP), drugs that modulate limbic and paralimbic brain regions, e.g., cannabidiol (CBD), drugs that modulate the melatonergic system, e.g., agomelatine, fatty acids, peptides, nucleic acids, and other precursor molecules, e.g., alpha-omega fatty acids, supplements Enzyme Q10, myo-inositol, methylfolate, S-adenosylmethionine, cysteamine, and oxytocin, monoamine oxidase inhibitors (MAOIs) such as isocarboxazid (Marplan®), phenelzine (Nardil®), selegiline (Emsam®), tranylcypromine (Parnate®), mood stabilizers such as lithium salts, valproate, ebselen, and divalproex, multimodal antidepressants such as vilazodone and vortioxetine, N-nitrosodimethylamine non-steroidal anti-inflammatory drugs (NDMA) receptor antagonists, such as amantadine, arketamine, ketamine, memantine, riluzole, esketamine, neurokinin-1 (NK1) receptor antagonists, neuropeptide Y (NPY) receptor agonists, drugs with neurotrophic effects, cilostazol, sildenafil, and vildagliptin, norepinephrine-dopamine reuptake inhibitors (NDRIs), bupropion (Wellbutrin®, Zyban®, Aplenzin®), drugs acting on the opiate system, such as ALKS-5461, AZD2327, BTRX-246040 (LY2940094), buprenorphine, JNJ-67953964, nalmefene, and naltrexone, protein kinase C inhibitors or antiestrogens such as endoxifen, tamoxifen, and verapamil, hallucinogens such as 3,4-methylenedioxy-methamphetamine (MDMA), ayahuasca, lysergic acid diethylamide (LSD), psilocybin, selective serotonin reuptake inhibitors (SSRIs) such as citalopram (Celexa®),stimulants, including escitalopram (Lexapro®), fluvoxamine, paroxetine (Paxil®, Pexeva®), and sertraline (Zoloft®), selective norepinephrine transporter inhibitors such as atomoxetine, serotonin-norepinephrine reuptake inhibitors (SNRIs) such as desvenlafaxine (Pristiq®), duloxetine (Cymbalta®), levomilnacipran (Fetzima®), and venlafaxine, adenosine receptor antagonists, and alpha-2-adrenergic receptor agonists such as caffeine, clonidine, guanfacine, extended-release amphetamine XR-OS, dextroamphetamine sulfate, lisdexamfetamine and one or more of benzodiazepines, methamphetamine, mixed amphetamine salts, racemic amphetamine sulfate, triple bead mixed amphetamine salts, substance P antagonists such as aprepitant (MK0869) and fosaprepitant (MK-0517), tricyclic serotonin-norepinephrine reuptake inhibitors such as amitriptyline (Elavil®), amoxapine, buspirone (Buspar™), clomipramine, desipramine (Norpramin®), doxepin, imipramine (Tofranil®), maprotiline, nortriptyline (Pamelor™), protriptyline, and trimipramine, and vasopressin 1B (V1B) receptor antagonists such as nervaptan (SSR149415). Exemplary cargoes for delivery to the brain may include, for example, neurotrophic factors, including, but not limited to, catalase, GFP, luciferase, nerve growth factor (NGF), TrkA (tropomyosin kinase A), NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocybin / psirosin, harmine, temozolomide, rivastigmine, and / or rhodamine. As noted above, compositions containing MEVs may be delivered to neurons, astrocytes, glial cells, and / or neural stem cells for administration via intranasal administration or to cells in vitro for gene therapy.

[0082] The methods, uses, and compositions can be used to treat diseases, disorders, and conditions involving the brain, such as, but not limited to, Alzheimer's disease, prion diseases, Niemann-Pick disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, Tay-Sachs disease, Wilson's disease, leukodystrophy, epilepsy, multiple sclerosis, encephalitis, and migraine. For example, the disease, disorder, or condition can be a neurodegenerative disease, the cargo is an ApoE4 inhibitor or inhibitor of its expression in neurons, or an ApoE2 and / or ApoE3 or activator of its expression in neurons, or a gene editing cassette or system for modifying one or more of the genes encoding ApoE2, ApoE3, or ApoE4 in neurons, and the composition is formulated for and administered by intranasal administration. In particular, the disease, disorder, or condition to be treated is Alzheimer's disease or a condition or pathology associated with the risk of developing Alzheimer's disease. These can be treated by administering a therapy that treats the risk-associated condition or Alzheimer's disease. For example, ApoE gene expression or expression or levels or allelic distribution can be modulated, such as by altering ApoE levels or expression by intranasal administration of cargo-loaded MEVs, thereby: a) modifying the physiological level of ApoE lipidation using MEVs loaded with either (i) a peptide or small molecule known to increase the lipid-binding capacity of ApoE, or (ii) a sequence of miRNA (miRNA-33) or an siRNA or ASO that mimics miRNA-33, to increase ABCA1 levels or decrease Aβ levels, thereby increasing the lipidation capacity of ApoE; and / or b) reducing the amount of ApoE4 in the brain using MEVs loaded with miRNA (miRNA146) or siRNA or ASO sequences to mimic miRNA-146, or other RNAi, such as siRNA or shRNA, that inhibit the expression of ApoE4, thereby inhibiting the immune response in the brain and / or reducing ApoE4 in the brain; and / or c) increasing the expression of ApoE2 isoforms in the brain using MEVs loaded with either (a) ApoE2 protein, or (b) mRNA encoding ApoE2 protein, or (c) a plasmid encoding ApoE2 sequences, to increase the protective effects of ApoE2 and compensate for the toxic effects of ApoE4; and / or d) Genome editing using MEV loaded with a gene editing complex results in editing the ApoE4 allele to produce ApoE3 and / or ApoE2.

[0083] Thus, provided herein are MEVs that contain cargo and can be used to deliver the cargo to organs, tissues, and / or cells involved in a particular disease, disorder, or condition. Of interest herein is delivery to the brain via intranasal administration. Cargos can be selected to treat, diagnose, and / or detect a disease, disorder, or condition, and / or monitor treatment. The unique trafficking of MEVs as described and demonstrated herein makes them unique delivery vehicles. [Brief explanation of the drawings]

[0084] [Figure 1] 1 provides an exemplary profile of light intensity used in HECTOR PBR culture. [Figure 2]An exemplary elution profile of a highly pure MEV preparation is shown. MEVs are pre-concentrated by TFF, purified by ultracentrifugation, formulated in PBS at a concentration of 10-10 per mL, and seeded onto a pre-packed column, qEV1, from IZON. PBS solution is used to elute MEVs. 0.5 mL elution fractions are collected. MEVs are recovered in the first fraction, as shown. The most concentrated fractions (4-5) are pooled and stored at 4 °C before use. [Figure 3] 1 provides exemplary images and approximate sizes of MEVs obtained using transmission electron microscopy (TEM). [Figure 4] Electropherograms of small RNA libraries are provided. [Figure 5] Representative patterns of biodistribution as a function of administration route are provided for intravenous (IV), intratracheal (IT), and oral (PO) routes. [Figure 6] 1 shows in vivo whole body imaging of a representative animal after intravenous administration as described in Example 5. [Figure 7] 1 shows in vivo whole body imaging of per os (oral) administration of a representative animal as described in Example 5. [Figure 8] 1 shows in vivo whole body imaging of a representative animal after intranasal administration as described in Example 5. [Figure 9] 1 shows in vivo whole body imaging of a representative animal following intratracheal administration as described in Example 5. [Figure 10] 1 shows the kinetics of accumulation in the liver, lungs and spleen (average of 6 animals) after intravenous administration as described in Example 5. [Figure 11] 1 shows the accumulation kinetics in lung, spleen and intestine following oral administration (average of 6 animals) as described in Example 5. [Figure 12] 1 shows the kinetics of lung and kidney accumulation (average of 4 animals) after intranasal administration as described in Example 5. [Figure 13] 1 shows the kinetics of accumulation in the lung, spleen and intestine (average of 3 animals) after intratracheal administration as described in Example 5. [Figure 14A]Ex vivo fluorescence analysis (total radiant efficiency) in organs [A) liver, B) spleen, C) lung, and D) brain] isolated 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration is shown. [Figure 14B] Ex vivo fluorescence analysis (total radiant efficiency) in organs [A) liver, B) spleen, C) lung, and D) brain] isolated 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration is shown. [Figure 14C] Ex vivo fluorescence analysis (total radiant efficiency) in organs [A) liver, B) spleen, C) lung, and D) brain] isolated 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration is shown. [Figure 14D] Ex vivo fluorescence analysis (total radiant efficiency) in organs [A) liver, B) spleen, C) lung, and D) brain] isolated 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration is shown. [Figure 15A] A) Hematoxylin & eosin staining of the intestine (G = GALT tissue) and B) DAPI (nuclear) staining and MEV-PKH26 fluorescence are shown. [Figure 15B] A) Hematoxylin & eosin staining of the intestine (G = GALT tissue) and B) DAPI (nuclear) staining and MEV-PKH26 fluorescence are shown. [Figure 16] Splenic pulp stained with DAPI (for nuclei) and MEV-PKH26 (red fluorescence) is shown, shown as white dots. [Figure 17] A diagram showing the migration of MEVs from GALT to the spleen is shown. [Figure 18A]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18B]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18C]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18D]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18E]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18F]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18G]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18H]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 18I]For each parameter, the results of evaluation of MEV toxicity in a mouse model after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice are shown. MEV toxicity was assessed by 1) chemical parameters: ALAT, ASAT, urea, and creatine (Figures 18A–D, respectively) and 2) hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E–I, respectively). The groups were as follows: Group 1—mice administered 100 μl of PBS via PO delivery (white bars); Group 2—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with black dots); Group 3—mice administered 100 μl of 4×10 MEV / mouse via PO delivery (white bars with vertical lines); and Group 4—mice administered 100 μl of 4×10 MEV / mouse via IT delivery (square bars). Data were measured for 6 mice per group for each parameter. Figure 18A shows ALAT: alanine aminotransferase. Figure 18B shows ASAT: aspartate aminotransferase. Figure 18C shows urea. Figure 18D shows creatine. Figure 18E shows red blood cells. Figure 18F shows hemoglobin. Figure 18G shows hematocrit. Figure 18H shows MCV (mean corpuscular volume). Figure 181 shows eosinophils. PO indicates oral (oral delivery) and IT indicates intratracheal administration. [Figure 19] 1 shows in vivo delivery and expression of mRNA following local injection of MEV into rabbit eyes. [Figure 20A] 1 shows in vitro delivery of GFP protein into human monocytes. [Figure 20B] 1 shows in vitro delivery of GFP protein into human monocytes. [Figure 21A] 1 shows in vitro delivery of GFP protein into human keratinocytes. [Figure 21B] 1 shows in vitro delivery of GFP protein into human keratinocytes. [Figure 22]Confocal microscopy of Hep-G2 cells containing GFP protein expression in Hep-G2 cells after 24 h of incubation with MEV loaded with GFP protein (MEV-GFP) or MEV loaded with mRNA-eGFP (MEV-mRNA) is shown. [Figure 23] Confocal microscopy of Huh7 cells, including GFP protein expression in Hep-G2 cells. [Figure 24] 1 shows in vitro delivery of MEVs loaded with GFP mRNA and mRNA encoding GFP into human fibroblasts. [Figure 25A] 1 shows the results of flow cytometry analysis of MEV penetration and delivery studies using human fibroblasts. [Figure 25B] 1 shows the results of flow cytometry analysis of MEV penetration and delivery studies using human fibroblasts. [Figure 25C] 1 shows the results of flow cytometry analysis of MEV penetration and delivery studies using human fibroblasts. [Figure 25D] 1 shows the results of flow cytometry analysis of MEV penetration and delivery studies using human fibroblasts. [Figure 26] 1 shows the antibacterial activity of Chlorella MEV exogenously loaded with siRNA against the Pto DC3000 cfa6 and hrpL genes. [Figure 27] 1 shows delivery of bioactive flg22 peptide exogenously loaded into Chlorella MEVs. [Figure 28] FIG. 1 is a schematic diagram showing the pathway through the olfactory epithelium. [Figure 29] A drop of MEV suspension was deposited onto a brain tissue slide. Positive control DiR-MEVs are shown on a DAPI-stained brain slice. Dots are DiR-labeled MEVs. [Figure 30] Schematic diagram of the insula and its connections (reproduced from Gogolla (2017) "The insular cortex," Current Biology: 27(12): R580-R586). [Figure 31]Schematic diagram of the brain neuronal pathway from olfactory sensory neurons (OSNs), through the olfactory bulb (OB), to mitral and tufted neurons, to the olfactory tract (OT). [Figure 32] Schematic diagram showing pathways and approximate average distances from olfactory and respiratory epithelia to CNS targets (reproduced from Lochhead et al. (2019). "Perivascular and Perineural Pathways Involved in Brain Delivery and Distribution of Drugs after Intranasal Administration," Pharmaceutics 11(11):598, doi.org / 10.3390 / pharmaceutics11110598). [Figure 33] Schematic diagram of the cortical projections of mitral and tufted cells showing a ventrolateral view of the brain (reproduced from Imai (2014) "Construction of functional neuronal circuitry in the olfactory bulb," Seminars in Cell and Developmental Biology 35, DOI:10.1016 / j.semcdb.2014.07.012). [Figure 34] Transport of MEVs through the olfactory pathway. After IN administration, MEVs are transported by the olfactory epithelium to the olfactory bulb via axonal transport by olfactory sensory neurons, and then carried by mitral and tufted neurons to the primary olfactory region, where olfactory signals are processed (Reproduced from Selvaraj et al. (2018) Artificial Cells, Nanomedicine, and Biotechnology An International Journal 46:2088-2095, doi.org / 10.1080 / 21691401.2017.1420073). [Figure 35]shows the olfactory pathway used by MEV after IN administration (schematic diagram of the general pathway reproduced from "What-when-how in-Depth tutorials and information, Olfaction and Taste, Sensory system, part 1" (URL: what-when-how.com)). [Figure 36A] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36B] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36C] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36D] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36E]Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36F] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 36G] Figure 36A shows a general overview of the experimental design for the brain biodistribution study. Figure 36B shows the locations of the five brain slices tested. Figure 36C shows the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain slices tested. Figures 36D-G illustrate and identify brain regions for reference to the following figures showing MEV in the brain after IN administration. [Figure 37A] Figure 36 shows the pharmacokinetics (PK) and biodistribution of MEV in different regions of section 1. Images of labeled MEV with DiR are black dots. [Figure 37B] Figure 36 shows the pharmacokinetics (PK) and biodistribution of MEV in different regions of section 1. Images of labeled MEV with DiR are black dots. [Figure 37C] Figure 36 shows the pharmacokinetics (PK) and biodistribution of MEV in different regions of section 1. Images of labeled MEV with DiR are black dots. [Figure 37D] Figure 36 shows the pharmacokinetics (PK) and biodistribution of MEV in different regions of section 1. Images of labeled MEV with DiR are black dots. [Figure 38]Figure 1 shows the PK and biodistribution of MEVs in different regions of section 1, providing a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 1, normalized by the total analyzed area. [Figure 39A] PK and biodistribution of MEV in different regions of section 2 (images of labeled MEV with DiR are shown). [Figure 39B] PK and biodistribution of MEV in different regions of section 2 (images of labeled MEV with DiR are shown). [Figure 39C] PK and biodistribution of MEV in different regions of section 2 (images of labeled MEV with DiR are shown). [Figure 39D] PK and biodistribution of MEV in different regions of section 2 (images of labeled MEV with DiR are shown). [Figure 40A] Figure 39 shows the PK and biodistribution of MEVs in different regions of section 2 as a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 2 from Figures 39A-D, normalized by the total analyzed area. [Figure 40B] Figure 39 shows the PK and biodistribution of MEVs in different regions of section 2 as a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 2 from Figures 39A-D, normalized by the total analyzed area. [Figure 40C] Figure 39 shows the PK and biodistribution of MEVs in different regions of section 2 as a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 2 from Figures 39A-D, normalized by the total analyzed area. [Figure 40D] Figure 39 shows the PK and biodistribution of MEVs in different regions of section 2 as a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 2 from Figures 39A-D, normalized by the total analyzed area. [Figure 41A] PK and biodistribution of MEV in different regions of section 3, images of DiR-labeled MEV are shown. [Figure 41B]PK and biodistribution of MEV in different regions of section 3, images of DiR-labeled MEV are shown. [Figure 41C] PK and biodistribution of MEV in different regions of section 3, images of DiR-labeled MEV are shown. [Figure 41D] PK and biodistribution of MEV in different regions of section 3, images of DiR-labeled MEV are shown. [Figure 42A] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 42B] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 42C] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 42D] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 42E] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 42F] Figure 4 shows the PK and biodistribution of MEVs in different regions of section 3 in a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 3 from Figures 41A-D, normalized by the total analyzed area. [Figure 43A]Images of DiR-labeled MEVs are shown, along with the PK and biodistribution of MEVs in different regions of section 4. [Figure 43B] Images of DiR-labeled MEVs are shown, along with the PK and biodistribution of MEVs in different regions of section 4. [Figure 43C] Images of DiR-labeled MEVs are shown, along with the PK and biodistribution of MEVs in different regions of section 4. [Figure 43D] Images of DiR-labeled MEVs are shown, along with the PK and biodistribution of MEVs in different regions of section 4. [Figure 44A] Figures 4A-D show the PK and biodistribution of MEVs in different regions of section 4 from 3A-D, providing a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 4, normalized by the total analyzed area. [Figure 44B] Figures 4A-D show the PK and biodistribution of MEVs in different regions of section 4 from 3A-D, providing a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 4, normalized by the total analyzed area. [Figure 44C] Figures 4A-D show the PK and biodistribution of MEVs in different regions of section 4 from 3A-D, providing a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 4, normalized by the total analyzed area. [Figure 44D] Figures 4A-D show the PK and biodistribution of MEVs in different regions of section 4 from 3A-D, providing a graphical representation of the total number of labeled MEVs with DiR spots per surface of the region of section 4, normalized by the total analyzed area. [Figure 45A] PK and biodistribution of MEV in different regions of section 5, images of DiR-labeled MEV are shown. [Figure 45B] PK and biodistribution of MEV in different regions of section 5, images of DiR-labeled MEV are shown. [Figure 45C] PK and biodistribution of MEV in different regions of section 5, images of DiR-labeled MEV are shown. [Figure 45D] PK and biodistribution of MEV in different regions of section 5, images of DiR-labeled MEV are shown. [Figure 46A] The kinetics of brain penetration by MEVs from the rostral to the distal parts of the brain are shown. [Figure 46B] The kinetics of brain penetration by MEVs from the rostral to the distal parts of the brain are shown. [Figure 47] Shown is the blood-brain barrier (Reproduced from Cecchelli et al. (2007) Nat Rev Drug Discov. 6(8):650-661). [Figure 48] This shows a microscopic image of mouse intestinal epithelium 8 hours after administration of PKH26-labeled MEV. [Figure 49] Whole-body bioluminescence imaging of a representative animal treated with MEVs loaded with luciferase mRNA is shown. [Figure 50] Whole-body bioluminescence imaging of a representative animal treated with MEVs loaded with luciferase enzyme is shown. [Figure 51] 1 shows a timeline for image analysis using an Incucyte® live cell analyzer. [Figure 52] The structure of human ApoE3 is shown. The 299-residue polypeptide chain is shown in linear form, and the sites of cysteine-arginine exchanges, which distinguish ApoE2 and ApoE4 from ApoE3, are indicated at positions 112 and 158. The protein folds into two separate domains, with the N-terminal residues 1–191 containing an antiparallel four-helix bundle and the C-terminal residues 192–299 forming a separately folded domain that interacts with the helix bundle. The segment of the N-terminal domain spanning residues 135–150 (horizontal red arrow) contains a cluster of basic amino acids that forms the binding site for LDLR. The C-terminal segment spanning residues 260–299 (horizontal red arrow) contains an amphipathic alpha helix that initiates protein binding to lipid surfaces. (Phillips MC. (2014) IUBMB Life 66:616–623) DETAILED DESCRIPTION OF THE INVENTION

[0085] outline A.Definition B. Microalgae and Overview C. extracellular vesicles 1. Types of Extracellular Vesicles (EVs) a. Exosomes b. Microvesicles C apoptotic bodies 2. EV uptake 3. General Methods for Isolating EVs Ultracentrifugation b. Size-based technology c. Immunoaffinity capture-based technology d. Exosome precipitation e. Microfluidic-based isolation techniques 4. Microalgae and microalgae-derived extracellular vesicles (MEVs) 5. Green Algae – Chlorella species a. Life cycle b. Genome analysis of Chlorella species c. Commercial and biotechnological uses of Chlorella d. Chlorella MEV D. Exogenously Loaded Microalgal Extracellular Vesicles (MEVs), Cargo, and Targets 1. MEV Isolation 2. MEV Load and Cargo 3. Generation of Payload-loaded MEV a. Electroporation b. Sonication c. Extrusion d. surfactants e. Other methods 4. Exemplary Cargoes and Exemplary Uses of Exogenously Loaded MEVs a. Cargo 1) RNA cargo 2) Antibody cargo b. Diseases and treatment methods c. Agricultural veterinary applications d. Cosmetic and dermatological applications E. Pharmaceutical Compositions, Formulations, Kits, Articles of Manufacture and Combinations 1. Pharmaceutical Compositions and Formulations 2. Manufactured Products / Kits and Combinations 3. Administration and Route of Exogenously Loaded MEVs 4. Combination therapy F. Biodistribution of MEV after administration via various routes 1. Biodistribution of Mammalian EVs 2. Biodistribution of Microalgal EVs Oral administration a) Components of the lymphatic system b) Targeted GALT 3. Diseases and conditions treated by MEV G. Biodistribution and Delivery of MEV to the Brain via Intranasal (IN) Administration to Treat Diseases, Disorders, and Conditions of the Brain and CNS 1. Brain structure a. Anterior olfactory nucleus b. Taeniatecta c. Olfactory tubercle d. piriform cortex e. amygdala f. entorhinal cortex g. Frontal cortex h. Striatum: caudate nucleus and putamen i.Nucleus accumbens j. thalamus K. hypothalamus l.Substantia nigra pars compacta m.hippocampus n.Hill o.pontine raphe nucleus 2. Blood-brain barrier 3. Brain and target cells 4. Differences in biodistribution between MEVs and other delivery vehicles 5. Intranasal (IN) administration 6. Brain delivery after MEV and intranasal administration 7. Transport and Biodistribution of MEVs After Intranasal (IN) Administration 8. Primary and secondary circuits of the olfactory system and the area reached by MEV upon IN administration 9. Delivery of MEVs via IN administration to the brain - exemplary bioactive cargoes and their uses H. Formulations for Routes of Administration and Diseases and Disorders Treated Thereby I. MEV-mediated intracellular signaling J. Working Example

[0086] A.Definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. In the event of multiple definitions for terms herein, those in this section prevail. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may come and go, but that equivalent information may be found by searching the Internet. Reference thereto evidences the availability and public dissemination of such information.

[0087] As used herein, cargo refers to exogenous molecules, such as bioactive molecules and small molecules, including biomolecules, that are loaded into microalgal extracellular vesicles (MEVs) provided herein after isolation. This includes cargo that is heterologous to the MEV. As used herein, heterologous generally refers to cargo within the MEV that is not naturally present in the MEV but is exogenously loaded, as discussed above. It also refers to cargo in the MEV that is endogenously loaded into the MEV by genetically modified microalgae. MEVs with heterologous cargo include cargo that is not naturally present in the MEV. Cargo that is heterologous to the microalgae and / or MEVs is cargo that is not naturally present in the microalgae to be packaged in the MEV, and / or cargo that is not present in the MEV without modification of the microalgae or by exogenous loading into the isolated MEV.

[0088] As used herein, a bioactive molecule or bioactive agent refers to any molecule or agent that can have biological activity, such as therapeutic activity, or that can act as a detectable marker or in vivo on a subject. Bioactive agents and molecules include biomolecules such as DNA, RNA, proteins, other biopolymers, and small molecules such as small molecule drugs and pharmaceuticals, immunogens, and any molecule that can be delivered to a subject, such as a human or other animal, plant, or microorganism (bacterial or otherwise), in connection with a therapeutic, diagnostic, or other such use, such as a cosmetic. A bioactive agent or molecule can function or have activity as, for example, a therapeutic agent, immunogen, diagnostic, detectable marker, or cosmetic. A bioactive molecule for use herein is any molecule that can be loaded into microalgal extracellular vesicles (MEVs).

[0089] As used herein, a biomolecule refers to any biologically active biopolymer or molecule that is or may be present in an organism or virus, or is a modified form of such a biopolymer or molecule. Thus, a biomolecule includes modified naturally occurring biomolecules, such as proteins that contain primary sequences that have been modified, for example, by deletion, insertion, and / or substitution of amino acids to alter the primary sequence, and / or by modifications such as post-translational modifications of the protein.

[0090] As used herein, when MEVs are described as having the same or substantially the same loaded cargo or amount thereof, it is understood that this refers to the average among a population of MEVs in a composition. When MEVs are exogenously loaded, it is understood that the cargo / MEV ratio can be selected so that each MEV has, on average, a predetermined amount of cargo. As a simple example, to load an average of one molecule of cargo / MEV, one skilled in the art can calculate the amount of cargo to load into a composition of MEVs, understanding that in a composition of MEVs, some will have more than one molecule of cargo / MEV and others will not. On average, MEVs will have one molecule of cargo / MEV. One skilled in the art will understand that the amount of cargo / MEV will generally be greater than one molecule / MEV and that the amount of cargo will depend on various parameters, including the cargo, the target tissue and / or cell, the disease, disorder, or condition being treated, and the subject being treated. Generally, on average, more than one molecule of cargo is loaded per MEV, e.g., at least 10 or about 10 molecules / MEV. Substantially more cargo, 100, 500, 1000, 10 4 More than one molecule / MEV can also be loaded. The amount loaded depends on the target, disease, disorder, or condition, the subject, the cargo, and the capacity of the MEV. Selecting the amount is within the skill of one in the art.

[0091] As used herein, a subject is any organism, generally an animal or plant, into which a composition containing an MEV is introduced. Subjects include, but are not limited to, humans, plants, particularly crops, and animals, including livestock and pets, such as dogs and cats, and zoo animals.

[0092] As used herein, a disease or disorder or condition refers to a pathological or undesirable or unwanted state in an organism resulting from causes or conditions including, but not limited to, infectious, acquired, and genetic conditions, and those characterized by identifiable symptoms.

[0093] As used herein, treating a subject with a disease, disorder, or condition means that the subject's symptoms or signs of the disease or condition are partially or completely alleviated or remain static following treatment.

[0094] As used herein, treatment refers to any effect that improves the symptoms of a disease or disorder. Treatment includes prophylaxis, therapy, and / or cure. Treatment also includes any pharmaceutical use of any MEV or composition provided herein. Treatment refers to any effect that improves or prevents, or otherwise reduces or eliminates, any symptoms or signs of a disease or disorder. Treatment also includes any pharmaceutical use of any MEV or composition provided herein.

[0095] As used herein, prevention refers to the prevention of potential disease and / or the prevention of worsening symptoms or progression of disease. Prevention or prophylaxis, and grammatical equivalents, refer to methods in which the risk or probability of developing a disease or condition is reduced or eliminated, and to products that reduce or eliminate the risk or probability of developing a disease or condition.

[0096] As used herein, a disease, disorder, or condition of or involving the brain is one in which the etiology of the disease, disorder, or condition involves the brain, such that delivery of a therapeutic agent to the brain can result in treatment, including amelioration of or relief from symptoms, and / or treatment of the cause or symptomatic manifestations of the disease, disorder, or condition, or delivery of a diagnostic molecule, such as a labeled molecule, reporter, or enzyme, that can be used to aid in or make a diagnosis or that can be used to monitor the progress or effectiveness of treatment. Such diseases, disorders, and conditions can affect or involve organs or tissues other than the brain, but treatment of the brain can at least ameliorate symptoms, and administration of a detectable molecule or reporter can be used to detect or monitor the disease, disorder, or condition, or aspects thereof.

[0097] As used herein, modification, with respect to modification of the sequence of amino acids in a polypeptide or the sequence of nucleotides in a nucleic acid molecule, refers to and includes deletion, insertion, and substitution of amino acids or nucleotides, respectively. These include modifications of the primary sequence of the polypeptide or protein. Methods for modifying polypeptides and nucleic acid molecules are routine to those skilled in the art, such as by using recombinant DNA methodology. When referring to a polypeptide or protein rather than a sequence, modification refers to post-translational or post-purification changes, such as conjugation or linkage of moieties that alter the properties of the polypeptide or protein, such as half-life extending moieties, glycosylation, purification tags, detectable reporters, and other such moieties.

[0098] As used herein, genome or plasmid or gene modifications include nucleic acid deletions, substitutions, insertions, and translocations, which include any change to the native or naturally occurring nucleic acid sequence.

[0099] As used herein, RNA interference (RNAi) is a biological process in which an RNA molecule inhibits gene expression or translation by neutralizing a target mRNA molecule and inhibiting translation, thereby inhibiting expression of the target gene.

[0100] As used herein, RNA molecules that act via RNAi are said to be inhibitory because they silence the expression of a target gene. Silencing expression means that the expression of the target gene is reduced, suppressed, or inhibited.

[0101] As used herein, RNAi-mediated gene silencing refers to inhibiting, suppressing, disrupting, or silencing the expression of a target gene. The target gene contains a sequence of nucleotides corresponding to a sequence in an interfering RNA, thereby silencing mRNA expression. Small interfering RNA (siRNA) is a small piece of double-stranded (ds) RNA, typically about 21 nucleotides long, with a 3' overhang (2 nucleotides) at each end, which can be used to bind to messenger RNA (mRNA) at specific sequences and promote its degradation, thereby disrupting protein translation. In doing so, siRNAs prevent the production of specific proteins based on the nucleotide sequence of their corresponding mRNA. This process is called RNA interference (RNAi), also known as siRNA silencing or siRNA knockdown. Short hairpin RNAs or small hairpin RNAs (shRNAs) are artificial RNA molecules with tight hairpin turns that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically achieved by delivery of a plasmid or via a viral or bacterial vector.

[0102] As used herein, non-coding RNA is RNA that does not encode a protein. Classes of non-coding RNA include, but are not limited to, small interfering RNA (siRNA) and microRNA (miRNA). As used herein, inhibiting, suppressing, disrupting, or silencing a target gene refers to a process that alters the expression, e.g., translation, of a target gene, thereby reducing the activity or expression of the product encoded by the target gene. Reduction includes complete or partial knockout, thereby achieving treatment with respect to the MEVs provided herein and the administration herein.

[0103] As used herein, the tumor microenvironment (TME) is the cellular environment in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). Conditions present include, but are not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and altered metabolites or metabolism, such as higher levels of adenosine, indicative of a tumor.

[0104] As used herein, a nucleic acid or encoded RNA is said to target a gene means that it inhibits, suppresses, or silences the expression of the gene by any mechanism. Generally, such nucleic acids contain at least a portion that is complementary to the target gene, which portion is sufficient to form a hybrid with the complementary portion.

[0105] As used herein, a deletion, when referring to a nucleic acid or polypeptide sequence, refers to the deletion of one or more nucleotides or amino acids compared to a sequence such as a target polynucleotide or polypeptide or native or wild-type sequence.

[0106] As used herein, an insertion, when referring to a nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids within a target, native, wild-type, or other related sequence. Thus, a nucleic acid molecule containing one or more insertions compared to a wild-type sequence contains one or more additional nucleotides within the linear length of the sequence.

[0107] As used herein, additions to nucleic acid and amino acid sequences describe the addition of nucleotides or amino acids to either end compared to another sequence.

[0108] As used herein, substitution or replacement refers to the replacement of one or more nucleotides or amino acids in a native, target, wild-type, or other nucleic acid or polypeptide sequence with alternative nucleotides or amino acids without changing the length (described in terms of the number of residues) of the molecule. Thus, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions compared to a particular polypeptide may be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence.

[0109] As used herein, a statement that a nucleotide or amino acid position corresponds to a nucleotide or amino acid position in a disclosed sequence as set forth in the sequence listing refers to the nucleotide or amino acid position identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as a guide. Generally, to identify corresponding positions, amino acid sequences are aligned to obtain the highest order match (e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carrillo et al. (1988) SIAM J Applied Math 48:1073).

[0110] As used herein, sequence alignment refers to the use of homology to align two or more sequences of nucleotides or amino acids. Typically, two or more sequences related by 50% or more identity are aligned. A set of aligned sequences refers to two or more sequences aligned at corresponding positions and may include aligned sequences derived from RNA, such as ESTs and other cDNAs, aligned with genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules may be aligned by any method known to those of skill in the art. Such methods typically include maximizing matches, using manual alignment, and using the numerous alignment programs available (e.g., BLASTP) and others known to those of skill in the art. By aligning polypeptide or nucleic acid sequences, one of skill in the art can identify analogous portions or positions using conserved and identical amino acid residues as a guide. Furthermore, one of skill in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between human and non-human sequences. Corresponding positions may also be based on structural alignment, for example, by using computer-simulated alignment of protein structures. In other instances, corresponding regions can be identified. One skilled in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between human and non-human sequences.

[0111] As used herein, a property of a polypeptide, such as an antibody, refers to any characteristic exhibited by the polypeptide, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermotolerance, and tolerance to pH conditions. A change in the property may alter the activity of the polypeptide. For example, a change in the binding specificity of an antibody polypeptide may alter the ability to bind to an antigen and / or various binding activities, such as affinity or avidity, or the in vivo activity of the polypeptide.

[0112] As used herein, the activity or functional activity of a polypeptide, such as an antibody, refers to any activity exhibited by the polypeptide. Such activities can be empirically determined. Exemplary activities include, but are not limited to, the ability to interact with a biomolecule, for example, through antigen binding, DNA binding, ligand binding, or dimerization, or enzymatic activity, for example, kinase activity or proteolytic activity. For antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind to a particular antigen, the affinity of antigen binding (e.g., high or low affinity), the avidity of antigen binding (e.g., high or low avidity), on-rate, off-rate, effector function, for example, the ability to promote antigen neutralization or clearance, virus neutralization, and in vivo activity, for example, the ability to prevent infection or invasion by, or promote the clearance of, a pathogen, or the ability to penetrate specific tissues, fluids, or cells in the body. Activity may be assessed in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays to measure on or off rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding assays (e.g., panning assays).

[0113] As used herein, bind, bound, and grammatical variations thereof refer to the involvement of a molecule in any interaction with or between molecules that results in a stable association in which the molecules are in close proximity to one another. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules such as compounds including drugs.

[0114] As used herein, antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic, e.g., recombinantly produced, including any fragments thereof containing at least a portion of the variable heavy and light regions of an immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen. Thus, antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). For example, antibody refers to an antibody comprising two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L'), where each heavy chain can be a full-length immunoglobulin heavy chain or a sufficient portion thereof to form an antigen-binding site (e.g., heavy chains include, but are not limited to, VH, VH-CH1, and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or a sufficient portion thereof to form an antigen-binding site (e.g., light chains include, but are not limited to, VL and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody minimally comprises all or at least a portion of a variable heavy (VH) chain and / or a variable light (VL) chain. An antibody may also comprise all or a portion of a constant region.

[0115] For purposes of this specification, the term "antibody" includes full-length antibodies and portions thereof, including antibody fragments, such as anti-tumor antibodies, anti-pathogen, or gene-silencing fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies also include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intracellular antibodies. Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subsubclass (e.g., IgG2a and IgG2b).

[0116] As used herein, nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. The term nucleic acid also includes nucleic acid analogs, such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives, or combinations thereof. Nucleic acid also includes DNA and RNA derivatives containing, for example, nucleotide analogs or backbone bonds other than phosphodiester bonds, such as phosphotriester, phosphoramidate, phosphorothioate, thioester, or peptide bonds (peptide nucleic acids). The term also includes equivalents, derivatives, variants, and analogs of either RNA or DNA made from nucleotide analogs, single-stranded (sense or antisense) and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine.

[0117] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode RNAi or therapeutic proteins.

[0118] As used herein, operably linked with respect to a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, where the leader peptide results in the secretion of the fusion polypeptide. In some examples, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a second polypeptide, where the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of the two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce a fusion protein comprising the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, whereby the promoter controls or mediates transcription of the nucleic acid.

[0119] As used herein, synthetic, for example with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant and / or chemical synthesis methods.

[0120] As used herein, naturally occurring α-amino acid residues are those 20 α-amino acid residues found in nature that are incorporated into proteins in humans by specific recognition of a charged tRNA molecule bearing its cognate mRNA codon.

[0121] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms polypeptide and protein are used interchangeably herein.

[0122] As used herein, peptide refers to a polypeptide that is from 2 to about 40 amino acids in length. As used herein, reference to protein includes peptides, polypeptides, small peptides, and all forms of proteins, unless otherwise specified.

[0123] As used herein, an amino acid is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For purposes of this specification, the amino acids contained in the provided antibodies include the 20 naturally occurring amino acids (see the table below), unnatural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, the amino acids present in the various amino acid sequences of the polypeptides appearing herein are identified according to their well-known three-letter or one-letter abbreviations (see the table below). Nucleotides occurring in the various nucleic acid molecules and fragments are designated by the standard one-letter designations routinely used in the art.

[0124] As used herein, an amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide bonds. The amino acid residues described herein are generally in the L-isomer form. Residues in the D-isomer form can be substituted for any L-amino acid residue, so long as the desired functional properties are retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. In accordance with the standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and adopted in 37 CER §§ 1.821-1.822, the abbreviations for amino acid residues are set forth in the following table: [Table 2] All sequences of amino acid residues represented herein by formulas have a left-to-right orientation in the conventional direction from amino terminus to carboxyl terminus. The term amino acid residue is defined to include the amino acids listed in the Table of Correspondence above, modified amino acids, unnatural amino acids, and unusual amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, or to an amino-terminal group such as NH2, or to a carboxyl-terminal group such as COOH.

[0125] Suitable conservative substitutions of amino acids in a peptide or protein are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0126] Such substitutions may be made according to the exemplary substitutions set forth in the table below: [Table 3]

[0127] Other substitutions are permissible and may be determined empirically or in accordance with other known conservative or non-conservative substitutions. As used herein, naturally occurring amino acids refer to the 20 L-amino acids present in polypeptides.

[0128] As used herein, the term unnatural amino acid refers to an organic compound that has a structure similar to a natural amino acid, but that has been structurally modified to mimic the structure and reactivity of the natural amino acid. Thus, unnatural amino acids include, for example, amino acids or analogs of amino acids other than the 20 naturally occurring amino acids, including, but not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are known to those of skill in the art and include 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidine acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2′-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr). , N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allo-hydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), allo-isoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and orthine (Orn).

[0129] As used herein, a DNA construct is a DNA molecule, single- or double-stranded, linear or circular, that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of engineered molecules.

[0130] As used herein, a DNA segment is a portion of a larger DNA molecule having particular attributes. For example, a DNA segment encoding a particular polypeptide may be a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read in the 5' to 3' direction, encodes the sequence of amino acids of the particular polypeptide.

[0131] As used herein, the term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated as nt) or base pairs (abbreviated as bp). The term nucleotide is used for single- and double-stranded molecules where the context allows. When applied to double-stranded molecules, it is used to indicate the total length and is understood to be equivalent to the term base pairs. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends may be staggered; therefore, not all nucleotides within a double-stranded polynucleotide molecule may be paired. Such unpaired ends generally do not exceed 20 nucleotides in length.

[0132] As used herein, recombinant production refers to the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

[0133] As used herein, a heterologous nucleic acid is a nucleic acid that encodes a product (i.e., RNA and / or protein) not normally produced in vivo by the cell in which it is expressed, or a nucleic acid at a locus in which it does not normally occur, or a nucleic acid that mediates or encodes a mediator that alters the expression of an endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acids, such as DNA, are also referred to as foreign nucleic acids. Any nucleic acid, such as DNA, that one of skill in the art would recognize or consider to be heterologous or foreign to the cell in which it is expressed is encompassed herein by heterologous nucleic acid, and heterologous nucleic acids include exogenously added nucleic acids that are also expressed endogenously. Heterologous nucleic acids are generally not endogenous to the cell in which they are introduced, but are obtained from another cell or prepared synthetically, or are introduced into a genomic locus that does not naturally occur, or whose expression is under the control of regulatory sequences or sequences different from the natural regulatory sequences.

[0134] Examples of heterologous nucleic acids herein include, but are not limited to, DNA molecules, RNA molecules, plasmids, and antisense oligonucleotides. In MEVs, heterologous nucleic acids can be encoded on a plasmid. Heterologous nucleic acids, such as DNA, can include nucleic acids that can mediate in some way the expression of DNA encoding a therapeutic product, or can encode products, such as peptides or RNA, that directly or indirectly mediate in some way the expression of a therapeutic product.

[0135] As used herein, cell therapy includes the delivery of MEVs to a subject to treat a disease or condition. MEVs are exogenously loaded with a cargo so that they deliver or express a product when introduced into a subject. MEVs can also be endogenously loaded with a cargo (see, e.g., co-pending U.S. Provisional Application No. 63 / 349,006, filed June 3, 2022, which details the preparation of endogenously loaded MEVs and their production cell lines) and used as described herein. The transport of MEVs is generally independent of the method by which they are loaded with the cargo. Microalgae can be modified to alter the properties of the resulting MEVs. Endogenously loaded MEVs can be used in the methods and compositions described herein.

[0136] As used herein, gene therapy involves the transfer of heterologous nucleic acid, such as DNA, into specific cells, e.g., target cells, of a mammal, particularly a human, having a disorder or condition for which such treatment is sought. The nucleic acid, such as DNA, is introduced into the selected target cells in such a manner that the heterologous nucleic acid, e.g., DNA, is expressed and the therapeutic product encoded thereby is produced. Gene therapy can also be used to deliver nucleic acids encoding gene products that replace defective genes or supplement gene products produced by the mammal or cell into which it is introduced. The introduced nucleic acid may encode a therapeutic compound, such as a growth factor or its inhibitor, or tumor necrosis factor or its inhibitor, e.g., its receptor, that is not normally produced in the mammalian host or is not produced in therapeutically effective amounts or for a therapeutically useful time. Heterologous nucleic acid, e.g., DNA, encoding a therapeutic product can be modified prior to introduction into the cells of the affected host to enhance or otherwise alter the product or its expression. Gene therapy can also include the delivery of inhibitors or repressors of gene expression or other regulators of gene expression.

[0137] As used herein, expression refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, a method for determining the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantification of polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0138] As used herein, a host cell is a cell used to receive, maintain, reproduce, and / or propagate a vector. Host cells can also be used to express a polypeptide encoded by a vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acid.

[0139] As used herein, a vector is a replicable nucleic acid capable of expressing one or more heterologous proteins when the vector is transformed into an appropriate host cell. Reference to a vector includes vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to a vector also includes vectors containing a nucleic acid encoding a polypeptide or RNA. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. While vectors typically remain episomal, they can be designed to result in the integration of a gene or a portion thereof into a chromosome of the genome. Artificial chromosome vectors, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art. Vectors also include viral vectors. Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer the exogenous genes into cells (as a vehicle or shuttle).

[0140] As used herein, an expression vector includes a vector capable of expressing DNA operably linked to a regulatory sequence, e.g., a promoter region, which can effect expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, e.g., a plasmid, phage, recombinant virus, or other vector, that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, as well as those that remain episomal or that integrate into the host cell genome.

[0141] As used herein, primary sequence refers to the sequence of amino acid residues in a polypeptide or the sequence of nucleotides in a nucleic acid molecule.

[0142] As used herein, sequence identity refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test polypeptide or polynucleotide and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes of this specification, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global. Matches, mismatches, and gaps can be identified between the compared sequences. A gap is a null amino acid or nucleotide inserted between residues of aligned sequences so that identical or similar features are aligned. Generally, internal and terminal gaps can exist. When gap penalties are used, sequence identity can be determined without penalty for terminal gaps (e.g., terminal gaps are not penalized). Alternatively, sequence identity can be determined as the number of identical positions / length of all aligned sequences x 100, without considering gaps.

[0143] For purposes herein, sequence identity is determined by aligning a test polypeptide or nucleic acid molecule with a reference molecule and counting the number of differences, including gaps and insertions. The number of differences is divided by the length of the reference molecule, typically the molecule whose sequence is being sought, to determine a percentage. For example, if the desired polypeptide is 100 amino acids long and a variant contains one with 90% sequence identity, the variant may have 10 amino acid differences, including gaps and insertions.

[0144] As used herein, a global alignment is an alignment in which two sequences are aligned from beginning to end, with each character in each sequence aligned only once. Alignment is performed regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on a global alignment means that 50% of the residues are the same in the entire alignment of two compared sequences, each 100 nucleotides long. It is understood that global alignment can also be used to determine sequence identity even when the lengths of the aligned sequences are not the same. Differences at the ends of the sequences are taken into account when determining sequence identity, unless no penalty for end gaps is selected. Generally, global alignment is used for sequences that share significant similarity over most of their length. An exemplary algorithm for performing global alignment is the Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol. 48:443). Exemplary programs for performing global alignments are publicly available and include the Global Sequence Alignment Tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and the program available at deepc2.psi.iastate.edu / aat / align / align.html.

[0145] As used herein, a local alignment is an alignment in which two sequences are aligned, but only those portions of the sequences that share similarity or identity. Thus, a local alignment determines whether a subsegment of one sequence is present in another sequence. If there is no similarity, no alignment is returned. Local alignment algorithms include BLAST or the Smith-Waterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, 50% sequence identity based on a local alignment means that in a complete sequence alignment of two compared sequences of any length, a 100-nucleotide-long region of similarity or identity has 50% of the residues that are the same in the region of similarity or identity.

[0146] For purposes of this specification, sequence identity can be determined by a standard alignment algorithm program used with default gap penalties established by each supplier. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for end gaps. Whether any two nucleic acid molecules have nucleotide sequences or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, or other similar variations that recite percent identity, can be determined using known computer algorithms based on local or global alignments (see, e.g., wikipedia.org / wiki / Sequence_alignment_software, which provides links to dozens of known, publicly available alignment databases and programs).Generally, for purposes of this specification, sequence identity is determined using a computer algorithm based on global alignment, such as the Needleman-Wunsch Global Sequence Alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome), LAlign (implementing the Huang and Miller algorithm by William Pearson (Adv. Appl. Math. (1991) 12:337-357)), and the program from Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html. Typically, the full-length sequences of each of the polypeptides or nucleotides being compared are aligned over the entire length of each sequence in a global alignment. If the sequences being compared are substantially the same length, local alignments can also be used.

[0147] Thus, as used herein, the term identity refers to a comparison or alignment between a test polypeptide or polynucleotide and a reference polypeptide or polynucleotide. In one non-limiting example, at least 90% identity refers to a percent identity of 90-100% relative to the reference polypeptide or polynucleotide. For illustrative purposes, identity at a level of 90% or greater refers to the fact that, assuming a comparison of test and reference polypeptides or polynucleotides of 100 amino acids or nucleotides in length, 10% (i.e., 10 out of 100) or less of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be expressed as point mutations randomly distributed throughout the entire length of the amino acid sequence, or they can be clustered at one or more positions of varying length up to the maximum allowable, e.g., 10 / 100 amino acid difference (approximately 90% identity). Differences can also be due to deletions or truncations of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. At levels of homology or identity greater than about 85-90%, depending on the length of the sequences being compared, results can be independent of the program and gap parameters set, and such high levels of identity can often be easily assessed without relying on software.

[0148] As used herein, a pharmaceutically active agent includes any therapeutic or bioactive agent, including, but not limited to, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0149] As used herein, a therapeutic effect refers to an effect resulting from treating a subject that alters, typically improves or ameliorates, the symptoms of a disease or condition, or cures the disease or condition. As used herein, a therapeutically effective amount or therapeutically effective dose refers to the amount of a drug, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. It is thus the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0150] As used herein, therapeutic efficacy refers to the ability of a drug, compound, material, or composition containing a compound to produce a therapeutic effect in a subject to which the drug, compound, material, or composition containing the compound is administered.

[0151] As used herein, prophylactically effective amount or prophylactically effective dose refers to the amount of a drug, compound, material, or composition containing a compound that, when administered to a subject, has the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the incidence of viral infection.A complete preventive effect does not necessarily occur by administering a single dose, but may only occur after administering a series of doses.Therefore, a prophylactically effective amount can be administered in one or more administrations.

[0152] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as administration of a pharmaceutical composition or other therapeutic agent, refers to any relief, permanent or temporary, lasting or transient, of symptoms that may result from or be associated with the administration of the composition or therapeutic agent.

[0153] As used herein, an anti-cancer agent refers to any agent that is destructive or toxic to malignant cells and tissues. For example, anti-cancer agents include agents that kill cancer cells or inhibit or impair the growth of tumors or cancer cells. Exemplary anti-cancer agents are chemotherapeutic agents. As used herein, therapeutic activity refers to the in vivo activity of a therapeutic polypeptide. Generally, therapeutic activity is activity associated with the treatment of a disease or condition.

[0154] As used herein, the term subject refers to animals, including mammals such as humans.

[0155] As used herein, a patient refers to a human subject. As used herein, an animal includes any animal, including, but not limited to, primates, including humans, gorillas, and monkeys, rodents, such as mice, rats, poultry, such as chickens, ruminants, such as goats, cows, deer, and sheep, as well as pigs and other animals. Non-human animals exclude humans as intended animals.

[0156] As used herein, a composition refers to any mixture, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0157] As used herein, a combination refers to any association between two or more items. The combination can be two or more separate items, such as two compositions or two collections, a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally associated or related.

[0158] As used herein, combination therapy refers to the administration of two or more different therapeutic agents, which may be provided and administered separately, sequentially, intermittently, or in a single composition.

[0159] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements, for purposes including, but not limited to, activation, administration, diagnosis, and evaluation of biological activity or properties. As used herein, unit dosage form refers to physically discrete units suitable for human and animal subjects and packaged individually as known in the art.

[0160] As used herein, a single dose formulation refers to a formulation for direct administration.

[0161] As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and can be directly administered to provide several single doses of the therapeutic agent. The doses can be administered over minutes, hours, weeks, days, or months. A multi-dose formulation can allow for dose adjustment, dose pooling, and / or dose splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0162] As used herein, an article of manufacture is a product that is made and sold. As used throughout this application, the term is intended to encompass any of the compositions provided herein contained in a packaging article.

[0163] As used herein, fluid refers to any composition that can flow. Thus, fluid encompasses compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0164] As used herein, an isolated or purified polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof) or biologically active portion thereof (e.g., an isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. A preparation may be determined to be substantially free if it appears free of readily detectable impurities, as determined by standard analytical methods used by those of skill in the art to assess such purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), or if it is sufficiently pure such that further purification does not detectably alter the physical and chemical properties, such as the enzymatic and biological activity, of the material. Methods for purifying compounds to produce substantially chemically pure compounds are known to those of skill in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may increase the specific activity of the compound.

[0165] As used herein, a cellular extract or lysate refers to a preparation or fraction made from lysed or disrupted cells.

[0166] As used herein, a control refers to a sample that is substantially identical to the test sample except that it has not been treated with the test parameters, or, if a plasma sample, can be derived from a normal volunteer not suffering from the condition of interest. A control can also be an internal control.

[0167] As used herein, psilocin is the active form of psilocybin. Psilocin is produced by the oxidation of psilocybin in the liver. For purposes of this specification, in the context of MEV-mediated delivery, psilocybin and psilocin shall have the same meaning: reference to "psilocybin" means "psilocin," and vice versa.

[0168] As used herein, bregma is a unit of measurement for the distance between a location in the brain and the junction between the coronal and sagittal sutures of the skull.

[0169] As used herein, the tropism of a MEV refers to the cells, tissues, and / or organs in which the MEV accumulates upon administration.

[0170] As used herein, native tropism with respect to MEVs provided herein means that the MEVs have not been modified to provide a particular tropism or targeting property.

[0171] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a polypeptide comprising an immunoglobulin domain includes polypeptides having one or more immunoglobulin domains.

[0172] As used herein, the term "or" is used to mean "and / or" unless expressly stated to refer to alternatives only or unless the alternatives are mutually exclusive.

[0173] As used herein, ranges and amounts may be expressed as about a particular value or range. "About" also includes the exact amount. Thus, about 5 amino acids also means about 5 amino acids and 5 amino acids.

[0174] As used herein, "any" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur. For example, an optionally mutated moiety means that the moiety is mutant or non-mutated.

[0175] As used herein, any protecting group, amino acid, and other chemical abbreviations are consistent with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 11(9):1726-1732), unless otherwise noted. For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections:

[0176] B. Microalgae and Overview Algae are a complex, multi-planted collection of primarily photosynthetic organisms. These organisms include microscopic macroscopic forms. Macroalgae (seaweeds) are multicellular macroalgae that can be seen with the naked eye. Microalgae are microscopic, unicellular organisms and include prokaryotes (e.g., cyanobacteria) and eukaryotes such as green algae.

[0177] Compared to photosynthetic crops, microalgae have a higher growth rate and can be cultivated on uncultivable land and in bioreactors. Many species of microalgae can be grown year-round in industrial-scale photobioreactors under controlled culture conditions (Adamo et al. (2021) Journal of Extracellular Vesicles 10:e12081). Algae are generally classified into 11 major phyla: Cyanobacteria, Chlorophyta, Rhodophyta, Marine Algae, Euglenophyta, Chlorarachniophyta, Charophyta, Cryptophyta, Haptophyta, Heterocynthophyta, and Dinophyta (Barkia et al. (2019) Mar. Drugs 17(5):304). Different pigments are produced in each algal group. Cyanobacteria (or Cyanophyta) contain chlorophyll-a, -d, and -f in addition to the phycobiliproteins (proteins that capture light energy), phycocyanin, allophycocyanin, and phycoerythrin. Cypridina contains chlorophyll-a and harvests light via phycobiliproteins. Chlorophyta contain chlorophyll-a and -b, as well as carotenoids, including beta-carotene and various xanthophylls (e.g., astaxanthin, canthaxanthin, lutein, and zeaxanthin). The primary pigments in red algae (Rhodophyta) are phycoerythrin and phycocyanin, which can mask chlorophyll-a. Red algae also produce a wide range of carotene and xanthophyll light-harvesting pigments (Barkia et al. (2019) Mar. Drugs 17(5):304).

[0178] Provided herein are extracellular vesicles produced by microalgae, particularly unicellular green algae such as Chlorella species, including Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, Chlorella variabilis, Parachlorella kesslerii, Parachlorella beijerinckii, and Parachlorella hassii, for use in delivery of exogenously loaded cargo to animals and plants.

[0179] Algae are unicellular eukaryotic organisms that are typically haploid but may have a diploid stage in their life cycle. Algae can be cultured in bioreactors, and extracellular vesicles can be isolated therefrom. The resulting extracellular vesicles can be loaded with cargo, typically a cargo of heterologous bioactive molecules, by methods such as electroporation to produce compositions containing the extracellular vesicles for administration to animals and plants. The compositions can be formulated for any desired route of administration, including topical, local, systemic, parenteral, and oral. These routes include oral, intravenous, subcutaneous, inhalation, mucosal, rectal, vaginal, and other suitable routes. Cargoes include biomolecules such as DNA, RNA, proteins, protein complexes, protein-nucleic acid complexes, and plasmids, as well as small molecules such as small molecule drugs. The extracellular vesicles can be formulated as liquids, powders, including lyophilized powders, tablets, capsules, emulsions, particles, sprays, gels, ointments, creams, and other formulations. They can be used for therapeutic, diagnostic, theragnostic, cosmetic, and other applications. Extracellular vesicles can be used to treat diseases and conditions, including cancer, inflammatory diseases, and conditions in which the immune system plays a role in the pathogenesis or symptoms, nervous system disorders, and pathogen infections, including viral, bacterial, and other pathogens. They can be used to treat skin diseases and conditions, lung diseases and conditions, and gastric diseases and conditions. Extracellular vesicles can be targeted to specific organs or tissues or administered locally.

[0180] Similar to extracellular vesicles (EVs) from other sources, such as mammalian EVs, microalgal EVs (MEVs) have evolved to efficiently pass genetic material and other types of molecules from cell to cell. They integrate cell-to-cell and cell-border communication through the exchange of biologically active molecules. MEVs are natural nanoparticles. They are derived from cells and therefore free of synthetic cargo and genetic modifications. They lack synthetic components, making them safe, e.g., without the risk of endogenous viruses potentially dangerous to humans. The MEVs provided herein include MEVs from all Chlorella family members, particularly the freshwater microalga Chlorella vulgaris, which also includes Parachlorella. Also included are MEVs from other members of the Chlorellaceae family, such as Parachlorella kesslerii, Parachlorella beijerinckii, and Parachlorella hassii.

[0181] Chlorella is a unicellular haploid alga that is a natural and efficient producer of extracellular vesicles. Chlorella vulgaris has been consumed worldwide as a dietary supplement for decades; it is non-toxic and non-immunogenic and can be cultivated on a large industrial scale at low cost. The MEVs provided herein can be used to protect, transport, and deliver a wide range of innovative therapeutic molecules directly to target cells associated with specific diseases.

[0182] As shown and described herein, MEVs have several advantageous characteristics, including, for example, biodistribution patterns depending on the route of administration, low toxicity, and favorable in vivo pharmacokinetic profiles. They can be administered by a variety of routes, including oral administration, administration to the respiratory tract, intranasal administration, and intravenous administration, among others. Depending on the route of administration, they are transported to specific organs, such as the intestine, GALT, spleen, lungs, liver, and brain. Based on the data herein and comparison with data from other EVs and drug delivery systems, MEVs may have a longer clearance rate, resulting in longer persistence in target organs, tissues, and cells, than has been reported for other delivery systems, including mammalian EVs.

[0183] As shown herein, MEVs overcome natural body barriers (such as oral delivery, or specific lymphatic tissue delivery, or nose-to-brain delivery) that have not been achieved with liquid nanoparticles and EVs of mammalian origin.

[0184] The MEVs provided herein address unmet needs, including the ability to specifically carry and reliably deliver therapeutic molecules to a treatment site while avoiding premature degradation or inactivation of the therapeutic agent by the immune system or enzymes, for the treatment of diseases for which therapeutic agents already exist but cannot be adequately delivered.

[0185] As demonstrated herein, purified or partially purified MEVs can be loaded by physical methods (exogenous loading, exoloading), which is scalable and industrializable. MEVs can be exoloaded with a variety of molecules, differing in size, hydrophobicity, and properties, including siRNA, mRNA, peptides, proteins, plasmids, oligonucleotides, and small molecules. The biological activity of the exoloaded cargo is preserved while simultaneously being protected from degradation by enzymes and other agents present in vivo. MEVs can deliver their cargo to recipient cells of numerous origins, including microalgae, bacteria, higher plants, mammals, and humans. MEVs can also deliver cargo to the appropriate cellular compartments, ensuring the proper expression and biological activity of cargo molecules, including those with complex biological pathways, such as siRNA, mRNA, and receptor-binding peptides.

[0186] C. extracellular vesicles Extracellular vesicles (EVs) are biomolecular structures released from plant and animal cells and play a role in intercellular communication. Structurally, EVs are negatively charged lipid bilayer vesicles with a density of 1.13–1.19 g / mL. EVs can cross barriers such as the plasma (or cytoplasmic) membrane and the blood / brain barrier, allowing the horizontal transfer of their functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor to recipient (Kuruvinashetti et al. (2020) 20 thInternational Conference on Nanotechnology 354-357). EVs are also naturally stable in various biological fluids, immunologically inert, and can exhibit organ-specific targeting capabilities (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0187] EVs contain endogenous lipids, nucleic acids, and proteins. Although results vary depending on the isolation technique and the method used to analyze the data, EVs generally contain plasma membrane, cytosol-associated proteins, and proteins involved in lipid metabolism (see, e.g., Doyle and Wang (2019) Cells 8(7):727). Proteins involved in EV biogenesis (e.g., components of ESCRTs), EV formation and release (e.g., RAB27A, RAB11B, and ARF6), signal transduction, and antigen presentation, as well as tetraspanins, generally occur in EVs (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312). EVs are rich in cholesterol, sphingomyelin, glycosphingolipids, and phosphatidylserine (Kuruvinashetti et al. (2020) 20). thInternational Conference on Nanotechnology 354-357). Although a few studies have identified genomic and mitochondrial DNA in EVs, EVs are primarily enriched in endogenous small RNAs. Studies have identified mRNA, miRNA, rRNA, long and short non-coding RNAs, tRNA fragments, piwi-interacting RNA, vault RNA, and Y RNA in EVs. Most naturally occurring RNA in EVs is approximately 200 nucleotides long (with small segments up to 4 kb) and therefore fragmented; however, circular RNA has also been shown to be enriched and stable in EVs. RNA in EVs is protected from RNase digestion in the extracellular environment by the lipid bilayer (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312). The Exocarta, Vesiclepedia, and EVpedia databases are publicly available and provide data on the protein, nucleic acid, and lipid content of EVs (generally EVs of mammalian origin, such as human origin), as well as the isolation and purification procedures used in EV research (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312).

[0188] EVs are used by cells to mediate several physiological processes or to affect various pathological conditions related to the activation of immune responses or the spread of disease or infection, and they also constitute interspecies communication and are present in all kingdoms of life. Sources of EVs include mammalian cells, bacteria, bovine milk, and plants (Adamo et al. (2021) J. Extracell. Vesicles 10:e12081). Plants and algae release EVs, although they have a cell wall outside their plasma membrane that can be a physical barrier for the release of EVs (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0189] 1. Types of Extracellular Vesicles (EVs) a. Exosomes There are three major subtypes of EVs; they are classified based on their biogenesis, mode of release, size, content, and function: microvesicles (MVs), exosomes, and apoptotic bodies (Doyle and Wang (2019) Cells 8(7):727). Exosomes, or intraluminal vesicles (ILVs), are generally 30–150 nm in diameter and are released via multivesicular bodies (MVBs) in the endosomal pathway. In the endosomal pathway, early endosomes are formed by inward budding of the plasma membrane and can transform into late endosomes, which accumulate ILVs by inward budding of the endosomal membrane. Late endosomes containing numerous vesicles are called MVBs. MVBs either fuse with lysosomes for degradation or fuse with the plasma membrane to release ILVs into the extracellular space as exosomes. The ESCRT pathway, which regulates MVB transport and exosome formation, has been reported to be the primary driver of exosome biogenesis. However, other mechanisms of exosome biogenesis exist, including those mediated by the sphingolipid ceramide or tetraspanin family proteins, which can promote membrane invagination. The ESCRT accessory proteins Alix, TSG101, HSC70, and HSP90β are often referred to as exosome marker proteins (Doyle and Wang (2019) Cells 8(7):727).

[0190] Exosomes are released into the extracellular space by fusion of the MVB limiting membrane with the plasma membrane. Several proteins, including Rab GTPases, diacylglycerol kinase α, and SNARE proteins, are involved in exosome release (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312).

[0191] Exosomes are potential drug delivery systems: they have a long circulating half-life, are tolerated by the human body, can penetrate cell membranes, can target specific cell types, and can be loaded with genetic material, proteins, or small molecules (Doyle and Wang (2019) Cells 8(7):727).

[0192] b. Microvesicles Microvesicles (MVs, or ectosomes) are formed by the outward budding or pinching of the cell's plasma membrane and have diameters ranging from 100 nm to 1 μm. MV formation involves cytoskeletal components such as actin and microtubules, molecular motors such as kinesin and myosin, and fusion machinery such as SNAREs and tethering factors. The physiological state and microenvironment of the donor cell influence the number of MVs produced, while the physiological state and microenvironment of the recipient cell influence the number of MVs consumed. MVs also possess several marker proteins, including cytosolic and plasma membrane-associated proteins, as well as cytoskeletal proteins, heat shock proteins, integrins, and proteins containing post-translational modifications; however, no specific markers are known to distinguish MVs from exosomes. Similar to exosomes, MVs can be loaded with cargo (such as proteins, nucleic acids, and lipids) for delivery to other cells, thereby altering the function of the recipient cell (Doyle and Wang (2019) Cells 8(7):727).

[0193] C apoptotic bodies Apoptotic bodies are released into the extracellular space upon cell death and have diameters ranging from 50 nm to 5,000 nm. They form when the plasma membrane of a cell separates from the cytoskeleton due to increased hydrostatic pressure after cell shrinkage. Unlike exosomes and MVs, apoptotic bodies contain intact organelles, chromatin, and small amounts of glycosylated proteins (Doyle and Wang (2019) Cells 8(7):727).

[0194] 2. EV uptake Cells internalize EVs by fusion with the plasma membrane or, more commonly, by endocytosis (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312). Endocytic uptake can occur via several types of endocytic processes, and different processes have been described in different cell types: clathrin-dependent endocytosis and phagocytosis have been described in neurons, macropinocytosis in microglia, phagocytosis and receptor-mediated endocytosis in dendritic cells, caveolin-mediated endocytosis in epithelial cells, and cholesterol- and lipid raft-dependent endocytosis in tumor cells. Blocking heparin sulfate proteoglycans (HSPGs) on the plasma membrane with heparin reduced EV uptake in cell culture, as did blocking scavenger receptor type B1 (SR-B1) with a synthetic nanoparticle mimic of HDL, suggesting a role for HSPGs and SR-B1 in EV uptake (Abels and Breakefield (2016) Cell Mol Neurobiol 36(3):301-312). Fusion of EVs with the cell membrane is also an uptake method, which requires low pH conditions; treatment of EVs with a pH-sensitive fusogenic peptide in combination with cationic lipids resulted in increased cellular uptake of exosomes and cytoplasmic release of exosomal cargo (Nakase and Futaki (2015) Sci Rep 5:10112). Low pH conditions occur in tumors (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312), so that EVs for delivering therapeutic payloads to tumor cells can enter the cells via fusion with the plasma membrane.

[0195] Like cells, EVs have extracellular receptors and ligands on the outside and cytoplasmic proteins and nucleic acids on the inside, and therefore communicate with cells in different ways. EVs bind to the cell surface, undergo endocytosis, and / or fuse with the plasma membrane, releasing their cargo into the extracellular space. If EV cargo enters via endocytosis, it must avoid degradation pathways; since late endosomes can fuse with lysosomes or the plasma membrane, the cargo must exit before being degraded in lysosomes or re-released through MVB fusion with the plasma membrane. EVs containing cargo, including mRNA and non-coding RNA, can be transferred into recipient cells in culture and in vivo (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312; Maas et al. (2017) Trends Cell Biol. 27(3):172-188).

[0196] 3. General Methods for Isolating EVs Ultracentrifugation Ultracentrifugation is used to isolate exosomes, and alternative methods are also being developed. Due to the complex nature of the biological fluids from which exosomes are isolated, the overlap in physiochemical and biochemical properties between exosomes and other types of EVs, and the heterogeneity among exosomes, isolation methods can result in complex mixtures of EVs with other components of the extracellular space. Differential ultracentrifugation relies on the initial sedimentation of larger, denser particles from the extracellular matrix, resulting in enrichment of exosomes but not complete separation of exosomes from other components in the extracellular space. Density gradient centrifugation is another ultracentrifugation method based on size and density separation in the presence of a density gradient (typically made from sucrose or iodixanol) in a centrifuge tube. Density gradient centrifugation effectively separates EVs from protein aggregates and non-membrane particles, but recovery of exosomes is low. However, purity can be improved by combining differential ultracentrifugation with other types of density gradient centrifugation, such as rate-zonal centrifugation or isopycnic centrifugation (Doyle and Wang (2019) Cells 8(7):727).

[0197] b. Size-based technology Numerous size-based techniques exist for isolating exosomes (Doyle and Wang (2019) Cells 8(7):727). Ultrafiltration separates particles based on membrane size and molecular weight cutoff, whereby particles larger than the membrane's molecular weight cutoff are retained and particles smaller than the membrane's molecular weight cutoff are passed through the filtrate. However, low isolation efficiency can occur if the filter becomes clogged and vesicles become trapped. The ExoMir™ kit (Bioo Scientific, Austin, TX) is a commercially available kit in which two membranes (200 nm and 20 nm) are placed in a syringe, and a sample (typically pretreated with centrifugation and proteinase K) is passed through the syringe. Larger vesicles remain on top of the first 200 nm filter, the smallest vesicles pass through the syringe and are discarded, and vesicles between the 20 and 200 nm range remain between the two filters in the syringe. Serial filtration also relies on a series of filtration steps to isolate exosomes (Doyle and Wang (2019) Cells 8(7):727).

[0198] Size-exclusion chromatography (SEC) of exosomes is often used in parallel with ultracentrifugation (the exosome pellet obtained from ultracentrifugation is resuspended and further purified using SEC), similar to the use of SEC to separate proteins. In SEC, a column is packed with a porous stationary phase into which small particles permeate and therefore may elute after larger particles. Typically, SEC methods require several hours of run time; however, the qEV Exosome Isolation Kit (iZON Science, New Zealand) allows for rapid and accurate exosome isolation by SEC in less than 15 minutes (Doyle and Wang (2019) Cells 8(7):727).

[0199] In flow field flow fractionation (FFFF), a sample injected into a chamber is subjected to a parabolic flow as it is forced down the chamber, in addition to a flow perpendicular to the parabolic flow, or crossflow, which separates particles in the sample. Larger particles are more affected by the crossflow and are pushed toward the chamber walls, which have a slower parabolic flow, while smaller particles remain in the center. In FFFF, smaller particles elute faster and larger particles elute slower (Doyle and Wang (2019) Cells 8(7):727).

[0200] In hydrostatic filtration dialysis (HFD), hydrostatic pressure is used to push a sample through dialysis tubing with a membrane with a molecular weight cutoff of 1000 kDa, allowing small solutes to pass through the tubing while larger particles, including exosomes and EVs, remain within the tubing and can then be further separated using, for example, ultracentrifugation (Doyle and Wang (2019) Cells 8(7):727).

[0201] c. Immunoaffinity capture-based technology Immunoaffinity capture-based techniques can isolate exosomes based on the expression of antigens on their surface, allowing for the isolation of exosomes from specific sources. In these methods, antibodies specific for the target antigen can be attached to plates (e.g., in enzyme-linked immunosorbent assays (ELISAs)), magnetic beads (e.g., in magnetic immunoprecipitation), resins, and microfluidic devices. These surfaces are then exposed to the exosome sample, resulting in the immobilization of exosomes expressing the antigen. While this assay requires that the protein / antigen for exosome isolation be expressed on the surface of the exosomes, its specificity is limited by the specificity of the antibody used, and often results in lower yields of isolated exosomes but higher purity. These methods can also be used to separate exosomes within a mixed population of EVs. Immunoaffinity capture-based techniques are often used after ultracentrifugation or ultrafiltration (Doyle and Wang (2019) Cells 8(7):727).

[0202] d. Exosome precipitation Exosome precipitation methods include polyethylene glycol (PEG) and lectin precipitation. In PEG precipitation, PEG polymers tether water molecules, allowing other particles, including exosomes, to precipitate from solution. PEG precipitation is rapid and not limited by the starting volume of solution, but lacks selectivity, as other EVs, extracellular proteins, and protein aggregates precipitate along with EVs. Sample pretreatment using filtration and / or ultracentrifugation can improve exosome yield. Commercially available kits for isolating exosomes using precipitation include, for example, ExoQuick® (System Biosciences, Palo Alto, CA) and Invitrogen™ Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA). Alternatively, lectin precipitation can be used, typically after ultracentrifugation, whereby lectins bind to carbohydrates on the surface of exosomes, altering their solubility and resulting in their precipitation from solution (Doyle and Wang (2019) Cells 8(7):727).

[0203] e. Microfluidic-based isolation techniques Microfluidic-based techniques simultaneously isolate exosomes based on their physical and biochemical properties, and are rapid, efficient, and require small starting volumes. In acoustic nanofilters, a matrix containing EVs and other cellular components is injected into a chamber and exposed to ultrasound waves. Particles respond differently to the radiation force exerted by the waves depending on their size and density, with larger particles experiencing stronger forces and moving faster toward pressure nodes. Immuno-based microfluidic isolation techniques are similar to those used in ELISA, but unlike ELISA, do not require prior ultrafiltration or ultracentrifugation of exosomes (Doyle and Wang (2019) Cells 8(7):727). The ExoChip (Kanwar et al. (2014) Lab Chip. 14(11):1891-1900) and ExoSearch Chip (Zhao et al. (2016) Lab Chip. 16(3):489-496) have been developed to isolate exosomes using microfluidic technology.

[0204] 4. Microalgae and microalgae-derived extracellular vesicles (MEVs) The taxonomy and classification of microalgae can vary. According to some schemes, there are seven divisions of microalgae: Euglenophytes (Euglena genus), Chrysophytes (Golden Brown Algae and Bacillariophytes), Pyrrhopyta (Phoenicoptera), Chlorophyta (Chlorophyta), Rhodophyta (Rhodophyta), Phaeophyta (Phaeophyta), and Xanthophyta (Xanthophyta). Of interest herein are photosynthetic microalgae such as Chlorella species and Chlamydomonas species. The methods and uses described herein generally use MEVs derived from green algae. Examples of such algae are Chlamydomonas and Chlorella, which belong to the Chlorophyceae and Trebouxiophyceae classes, respectively.

[0205] Microalgae are a biological resource for the production of EVs for use in nanomedicine and other fields. The mechanism of EV secretion from microalgae is known in relation to primary and motile cilia / flagella (Picciotto et al. (2021) Biomater.Sci.doi:10.1039 / d0bm01696a). Chlamydomonas flagella lack MVBs, and therefore, ciliary EVs shed from Chlamydomonas are classified as ectosomes. Studies have demonstrated the shedding of ectosomes from the flagella and ciliary tips of the green alga Chlamydomonas reinhardtii. EVs have also been observed along the length of Chlamydomonas cilia. Membrane budding and ciliary EV formation are mediated by components of the endosomal sorting complex required for transport (ESCRT), which has been found in isolated ciliary transition zones, ciliary membranes, and ciliary EVs in Chlamydomonas and may act as sensors of membrane curvature. Ciliary EV formation can also occur when ciliary membrane transport is disrupted or during ciliary resorption (Wang and Barr (2018) Essays Biochem. 62(2):205-213). Chlamydomonas-derived ciliary ectosomes contain lytic enzymes required for digesting the mother cell wall and releasing the daughter cells. Ift88 null mutants lacking flagella were unable to be released from mother cells, and addition of ciliary ectosomes from wild-type cells rescued the phenotype, suggesting a role for flagella and the intraflagellar transport (IFT) machinery in EV production (Wang and Barr (2016) Cell Mol. Neurobiol. 36(3):449-457).

[0206] EVs have been extracted from algal cells using ultracentrifugation (Kuruvinashetti et al. (2020) 20 th(International Conference on Nanotechnology 354-357). According to this method, algal cells are cultured, collected, centrifuged, and the supernatant collected (and further centrifuged). Sucrose solution is added to the supernatant, and the algal supernatant containing the sucrose solution is ultracentrifuged. Due to the sucrose solution, high-density EVs settle to the bottom of the ultracentrifuge tube and can be collected using a pipette. The extracted algal EVs can be characterized in size and concentration using nanoparticle tracking analysis (NTA). Research using this method isolated green algal EVs with a size range of 25-200 nm and a concentration of 0.89E8-0.94E8 particles / mL (Kuruvinashetti et al. (2020) 20 th International Conference on Nanotechnology 354-357).

[0207] Ultracentrifugation protocols can also be used to isolate EVs from marine microalgae grown under various conditions. NTA showed that the nanoparticles had a size distribution of 100–200 nm, and Western blotting of proteins confirmed the presence of EV markers (VES4US, Extracellular vesicles from a natural source for tailor-made nanomaterials, 2020). Subsequent studies identified microalgal small EVs (sEVs) isolated from the marine photosynthetic microalga Tetraselmis chuei, called nanoalgisomes. Nanoalgisome production is an evolutionarily conserved trait within microalgae strains, as similar results were obtained using sEVs isolated from batch cultures of two other microalgae species, the green alga Dunaliella tertiolecta and the dinoflagellate Amphidinium sp. Nanoargisomes were isolated using differential ultracentrifugation (dUC) and tangential flow filtration (TFF), as well as gradient ultracentrifugation, which were used to further purify samples enriched for small EVs by TFF or dUC. Isolated cultured nanoargisomes were shown to share characteristics of EVs from other sources. EV yields (measured by sEV protein content and sEV number) from dUC and TFF were approximately 10 times higher than the reported number of isolated EVs. 9The EV particle counts were consistent with that of EV protein per μg. Biophysical analysis of particle size using multi-angle dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), fluorescent nanoparticle tracking analysis (F-NTA), and fluorescence correlation spectroscopy (FCS) yielded a consistent size distribution, with the most frequently observed size from DLS (DLS mode) being approximately 70 nm. Compared to mammalian cell-derived exosomes, which have densities of 1.15–1.19 g / mol, nanoargisomes had a slightly lower density of 1.13 g / mol. Electron microscopy revealed that nanoargisomes were spherical, heterogeneous in size and shape, and possessed a lipid bilayer structure. Compared to microvesicles (or large EVs, lEVs) and lysates, sEVs were enriched for three of four target protein biomarkers (Alix, enolase, HSP70, and β-actin). DLS measurements indicated that nanoargisomes were resistant to pH changes and stable in human plasma. The tumorigenic MDA-MB-231 breast cancer cell line, the nontumorigenic 1-7 HB2 cell line, and the human hepatoma Hep G2 cell line showed no cytotoxic or genotoxic effects after nanoargisomes treatment. Furthermore, nanoargisomes were taken up by the MDA-MB-231 and 1-7 HB2 cell lines (Adamo et al. (2021) J. Extracell. Vesicles 10:e12081).

[0208] EVs have been isolated and studied from at least 18 microalgal strains from major microalgal lineages (Anchistrodesmus spp., Brachiomonas spp., Chlamydomonas reinhardtii, Dunaliella tertiolecta, Tetraselmis tuyi, Chloromonas spp., Rhodella violacea, Kirchnerella spp., Cranberry spp., Nannochloropsis spp., Cyanophora paradoxia, Cryptomonas pyrenoidifera, Phaeodactylum tricornutum, Phaeochromis spp., Dichronema spp., Isochrysis galbana, Sulloneis spp., and Amphidinium spp.). The strains studied include strains with a variety of characteristics, including saltwater and freshwater inhabitants, small and large cells, colonies and unicellular organisms, and species with sequenced genomes.

[0209] MEVs were isolated using a differential ultracentrifugation protocol and characterized according to the International Society for Extracellular Vesicles (ISEV) guidelines. All strains tested showed the presence of MEVs in the culture medium. EV-producing microalgae strains were established based on EV protein content, expression of EV protein markers (e.g., Alix, Hsp70, enolase, and β-actin), total scattering signal (measured by dynamic light scattering, DLS) or total particle number (measured by NTA), and sEV average size and size range. These EV-producing strains include Cyanophora paradoxia, Tetraselmis tuyi, Amphidinium species, Rodella violasia, Dichronema species, Dunaliella tertiolecta, Phaeodactylum tricornutum, Cranberry species, and Phaeochromis species (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a). Data for Cyanophora paradoxia indicate that approximately 2 × 10 EVs per mL of microalgae-conditioned medium are produced. 9 The sEV particles showed strong positive signals for EV markers and a modal size distribution of 130 ± 5 nm, consistent with data from plant-derived vesicles. Cytotoxicity and genotoxicity studies showed that sEVs isolated from the freshwater glaucophyte Cyanophora paradoxia were not toxic to tumorigenic MDA-MB-231 breast cancer or C2C12 myoblast cell lines over time or at different concentrations. MDA-MB-231 cells treated with sEVs also did not exhibit morphological nuclear changes associated with apoptotic events (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0210] EVs have also been isolated from Synechocystis sp. PCC6803 (cyanobacteria), Chlamydomonas reinhardtii (green microalgae), Euglena gracilis (euglenophyte), and Haematococcus pluvialis (green algae) in a study conducted by Zhao et al. They also performed rheological and proteomic analyses on EVs isolated from Chlamydomonas reinhardtii at different stages of cell growth and under different types of abiotic stress (Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / v1). EVs were isolated using differential ultracentrifugation and filtration, and the resuspension was shown to contain membranous structures with small clusters of particles with diameters of 110–120 nm, consistent with the reported diameters of exosomes and small MVs, although there were differences in diameter between microalgae species. Specifically, EVs derived from Chlamydomonas reinhardtii ranged in diameter from 37 to 710 nm, with an average particle diameter of 120.1 nm. EVs derived from Synechocystis ranged in diameter from 24 to 450 nm, with an average particle diameter of 94.68 nm. Despite the presence of a cell wall, Chlamydomonas cells were able to internalize EVs, as indicated by the presence of EVs labeled with fluorescent lipophilic dyes within the microalgal cells. Thus, microalgal EVs can be absorbed by recipient cells. Non-coding RNAs were detected in microalgal EVs at different growth stages and treatments (biotic stress, nitrogen depletion, and nitrogen restoration), and proteomic analysis identified many flagella-associated membrane proteins in microalgal EVs (Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / v1).

[0211] These studies have shown that microalgae produce EVs that can be isolated using traditional or standard methods; microalgae-derived EVs are similar in size and concentration and display similar markers compared to EVs isolated from other species; EVs isolated from microalgae do not display cytotoxic or genotoxic effects in vitro; and microalgae-derived EVs can be taken up by cells.

[0212] EVs of mammalian origin have been shown to be capable of delivering cargo to target cells and thus have therapeutic uses for the delivery of various cargoes for the treatment of several diseases or conditions, a feasibility not generally seen with MEVs. However, mammalian EVs, with the exception of bovine milk EVs, cannot be administered orally because they do not survive the harsh conditions of the stomach. For example, small molecules such as hydrophobic and hydrophilic drugs can be injected into exosomes, or large proteins and nucleic acids can be embedded in exosomes. Nucleic acids can include those encoding genes of interest. Specific targeting ligands, imaging probes, and covalent bonds can be attached to the exosome surface and tracked using NTA, fluorescence, or bioluminescence.

[0213] Besides the mention in the publication that microalgae EVs could potentially be used to deliver drugs of interest to targeted tissues or organs (Kuruvinashetti et al. (2020) 20 th There is no published evidence or technical description of the use of MEVs for delivery for the treatment of mammalian diseases, disorders, or conditions (International Conference on Nanotechnology 354-357). There are no publications or technical descriptions describing knowledge of the application of EV technology to microalgae-derived extracellular vesicles, or whether or how this can be done. Previous studies have not considered Chlorella species, and previous studies have not generally evaluated the biodistribution and related properties of MEVs. Therefore, methods such as the oral delivery method exemplified herein using Chlorella may use MEVs derived from other microalgae.

[0214] However, as described and demonstrated herein, microalgal EVs offer several advantages over existing drug delivery systems, such as mesenchymal stem cell-derived exosomes, gold nanoparticles, liposomes, and other plant- and animal-derived EVs. Mesenchymal stem cells are a commonly used source of exosomes, and mesenchymal stem cell-derived exosomes have enhanced passive targeting (a method of preparing a drug carrier system so that it continues to circulate in the bloodstream) and are therefore used in drug delivery, such as anti-cancer vaccines. Mesenchymal stem cell-derived EVs have the ability to passively target due to their small size, unique properties, and ability to cross biological barriers. However, mesenchymal stem cells have limited exosome secretion, making exosome production difficult to scale up due to the need to optimize purification, increase exosome homogeneity, and establish efficient transfection strategies. Nanoparticles can be toxic, and current technologies for synthesizing nanoparticles limit their ability to scale for manufacturing purposes. Nanoparticle- and liposome-based drug delivery methods can also result in the formation of teratomas (tumors composed of several different types of tissue). Liposome-based drug delivery methods have also been shown to be less efficient at internalizing specific cells, tissues, or organs compared to exosomes. Plant-derived EVs, such as those from curcumin, ginger, grapefruit, and lemon, have been used for drug delivery, but their extraction process and therapeutic use have yet to be optimized. The production of EVs from agricultural products, such as fruits and milk, is economically impractical and requires 3–4 months of growth, compared to algal EVs, which can be grown anywhere within a few days. Algal EVs avoid phagocytosis or degradation by macrophages, circulate in vivo for a long time, and have low immunogenicity. Algal EVs also have a low risk of teratoma formation. Therefore, algae provide a source from which pure, well-characterized, and high-quality EVs can be obtained (Kuruvinashetti et al. (2020) 20). thInternational Conference on Nanotechnology 354-357). Kuruvinashetti et al. do not describe the use of Chlorella species as a source of EVs, nor their advantages as a source. The prior art does not describe the biodistribution of MEVs themselves, nor the implications for administering MEVs with drugs targeted to specific organs, tissues, or systems.

[0215] 5. Green Algae – Chlorella species Previous research and discussion of EVs has not focused on or evaluated Chlorella species as a source of EVs. Chlorella and the resulting EVs offer advantages for growth, manipulation, and drug administration that other species and EVs do not offer. Green algae belong to the phylum Chlorophyta and encompass a diverse group of photosynthetic eukaryotes. Green algae include both unicellular and multicellular organisms. Algae originally included in the genus Chlorella are among the most widely distributed and frequently encountered in freshwater. These algae are found in aquatic environments and on land. They are typically small (approximately 2-10 μm in diameter), unicellular, spherical in shape, non-motile, contain a single chloroplast, and some have rigid cell walls (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Molecular analysis has classified Chlorella species into two classes of green algae: the Trebouxiophyceae, which includes true Chlorella, and the Chlorophyceae. As used herein, Chlorella species includes any that can be used or are used as a food supplement or can be consumed by humans or other animals, such as livestock. Exemplary species include, but are not limited to, Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis species.

[0216] Euphytic Chlorella species are characterized by glucosamine as the major component of their rigid cell walls. While most Chlorella species are free-living in nature, the Trebouxiophyceae contains most known green algal endosymbionts that inhabit lichens, unicellular eukaryotes, plants, and animals (e.g., mussels and hydra). For example, Chlorella variabilis NC64A is a heritable photosynthetic endosymbiont (or photobiobiont) of the unicellular protozoan Paramethium bursaria, and NC64A is also host to a family of large double-stranded DNA viruses that occur in freshwater (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0217] a. Life cycle In unicellular organisms such as microalgae, the life cycle is identical to the cell cycle. Chlorella is a haploid organism that reproduces asexually by autosporulation. The cell cycle and proliferation of Chlorella vulgaris were investigated by Rioboo et al. using flow cytometry analysis of algal cells stained with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Their results showed that, as commonly described for microalgae, proliferation of Chlorella vulgaris mother cells occurs during the light phase, while cytokinesis and release of daughter cells occur during the dark phase. Chlorella vulgaris also exhibits a distinct light / dark cycle, characterized by increases in cell size, cell complexity, and autofluorescence during the light phase, measured over a 96-hour period. Monoparametric histograms of CFSE-stained Chlorella vulgaris cells, showing only one peak for daughter cells, indicated that each mother cell undergoes only one division cycle over the 96-hour period. Furthermore, cytoplasmic division occurred during the dark phase. Therefore, the Chlorella vulgaris strain used exhibits three life cycle phases: 1) mother cell proliferation, 2) cell division, and 3) daughter cell release. Chlorella vulgaris cells proliferate during two light periods and begin to divide during the following dark period, with cell division occurring when the mother cell reaches twice the size of the daughter cells. Furthermore, Chlorella vulgaris cells exposed to the herbicide terbutryn require a longer proliferation period to reach a cell size large enough to divide. This suggests that there is a critical threshold size required for Chlorella vulgaris to complete the proliferation phase and initiate the division phase, and that this critical threshold may control progression through the G1 phase of the Chlorella vulgaris cell cycle. Finally, this study showed that the intensity of the peak CFSE fluorescence of mother cells was four times greater than that of daughter cells, indicating that four daughter cells are produced from each mother cell. Thus, Chlorella vulgaris cells undergo an initial cell division, followed by a cytoplasmic division, and then two other simultaneous mitotic divisions, which result in the liberation of four daughter cells (see, e.g., Rioboo et al. (2009) doi:10.1016 / j.aquatox.2009.07.009).

[0218] b. Genome analysis of Chlorella species Although Chlorella species have been reported to be nonmotile and lack a sexual cycle, genome analysis of Chlorella variabilis NC64A (NC64A) and Chlorella vulgaris 211 / 11P (211 / 11P) reveals the presence of genes involved in sexual reproduction and motility (Blanc et al. (2010) Plant Cell 22(9):2943-2955; Cecchin et al. (2019) Plant J. 100(6):1289-1305). The NC64A nuclear genome (GenBank accession number ADIC00000000.1) is 46.2 Mb long and consists of 12 chromosomes. NC64A encodes the meiosis-specific protein dosage suppressor of MCk1 (DMC1), its homologous counterparts HOP1 and HOP2, the meiotic recombination protein MER3, the meiotic nuclear division protein MND1, and the mutS homolog MSH4, all of which are present in most other sequenced green algae species. Nineteen homologs of the Chlamydomonas gametolysin protein, which promotes gamete cell wall degradation and enables gamete fusion, were also identified in NC64A. Furthermore, an ortholog of the Chlamydomonas GCS1 protein, which is essential for cell fusion, is present in NC64A (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Primary genes involved in meiosis are also present in the Chlorella vulgaris 211 / 11P 40 Mb genome (GenBank accession number SIDB00000000), in addition to a gene encoding gametolysin (g3347) and a gene encoding a protein containing a domain with putative GCS1 / HAP2 function (Cecchin et al. (2019) Plant J. 100(6):1289-1305). Thus, although Chlorella species have only been observed in the haploid phase, the presence of meiotic genes indicates that the Chlorella life cycle may include a diploid phase.

[0219] Similarly, although flagella were not observed in NC64A, orthologs of Chlamydomonas flagellar proteins were identified in the NC64A genome, including orthologs to the intraflagellar transport (IFT) proteins IFT52, IFT57, and IFT88, the kinesin-2 motor protein FLA8, the kinesin-associated protein KAP, and proteins involved in the axonemal outer dynein arm (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0220] Sequencing of three Chlorella sorokiniana strains, strains 1228, UTEX 1230, and DOE1412, reveals the presence of genes related to sex and flagellation (Hovde et al. (2018) Algal Research 35:449-461). The genomes of several other Chlorella species have been sequenced: Chlorella protothecoides species 0710 (Gao et al. (2014) BMC Genomics 15(1):582, GenBank accession number APJO00000000), Chlorella sorokiniana UTEX 1602 (GenBank accession number LHPG00000000) and Chlorella species strain SAG 241.80 (Micractinium conductoricus, GenBank accession number LHPF00000000) (Arriola et al. (2018) Plant J. 93(3):566-586), and Chlorella vulgaris strain UTEX 395 (Guarnieri et al. (2018) Plant J. 93(3):566-586). al.(2018)Front.Bioeng.Biotechnol.6:37;GenBank accession number LDKB00000000), UMT-M1 (Teh et al.(2019)Data Brief 27:104680, GenBank accession number VJNP00000000), UTEX 259 (GenBank accession number VATW00000000), and NJ-7 (Wang et al.(2020)Mol.Biol.Evol.37(3):849-863, GenBank accession number VATV00000000).

[0221] c. Commercial and biotechnological uses of Chlorella Commercial cultivation of microalgae for food purposes began in Japan and Taiwan in the 1960s with the production of Chlorella vulgaris. Dried biomass products from Arthrospira and Chlorella are included in dietary supplements due to their high protein content, nutritional value, and reported health benefits. For example, Chlorella extracts have been shown to lower cholesterol and have antioxidant, antibacterial, and antitumor activities. High-yield production of Chlorella is routine, and MEV can be isolated from cell culture media, as detailed herein. Regarding its use as a pharmaceutical, Chlorella consumption is known to be nontoxic and nonimmunogenic in humans.

[0222] Chlorella has been used in various biotechnological applications, including biofuel production, CO2 capture, production of molecules of high economic value, and heavy metal removal from wastewater (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Chlorella species exhibit metabolic flexibility in response to environmental perturbations and can use nutrients, such as organic carbon and minerals, directly from wastewater for growth. Among microalgae, Chlorella species have higher photosynthetic efficiency than other photosynthetic organisms. Furthermore, Chlorella vulgaris can grow under either autotrophic, heterotrophic, or mixotrophic conditions (Zuniga et al. (2016) Plant Physiol. 172(1):589-602).

[0223] Chlorella species can also be genetically modified by Agrobacterium-mediated transformation. A study by Cha et al. developed a method to genetically transform Chlorella vulgaris using Agrobacterium tumefaciens strain LBA4404. Compared to wild-type, uninfected Chlorella, the presence of a gene fragment in 30% of the transgenic lines indicates that the T-DNA had been integrated into the Chlorella genome (Cha et al. (2012) World J. Microbiol. Biotechnol. 28:1771-1779).

[0224] d. Chlorella MEV As described herein, Chlorella species, such as Chlorella vulgaris, are advantageous for the production of EVs, referred to herein as MEVs, for use in the delivery of biomolecules and small molecules for a variety of applications, including therapeutic, diagnostic, and cosmetic applications. Of particular interest herein are MEVs produced by Chlorella species. Chlorella EVs have not yet been utilized as a source of MEVs for the exogenous loading of biomolecular products or small molecule drugs or diagnostic agents. Chlorella offers numerous advantages as a source of EVs for such applications. Chlorella is a haploid organism, meaning that specific, targeted mutants can be generated through genetic engineering, and it can be easily genetically modified or loaded to produce or contain biologically active molecules and small molecules. Stable cell lines containing stable producers of the encoded product can be generated. These are defined products, and when exogenously loaded, the resulting composition contains EVs containing the same cargo.

[0225] Detailed genetic maps can be obtained, and correlations between genotype and phenotype can be established. The Chlorella genome has been completely sequenced, so the structure and function of various genes are known. Phylogenetically, Chlorella lies at the very branching point between higher plants and microalgae. Thus, Chlorella shares a significant (and useful) number of molecular biological and metabolic characteristics with higher plants, yet remains a unicellular, haploid microalga. An example of a molecular biological feature shared with eukaryotes is the intracellular machinery, including the Dicer enzyme system, for processing exogenous RNA into siRNA. Chlorella is autotrophic; therefore, unlike mammalian and other animal cells, it can be cultured and reproduced without the need for nutrients or factors of animal origin.

[0226] Regarding the therapeutic use of its EVs, Chlorella species are not toxic. For example, tablets made from Chlorella vulgaris biomass (i.e., compressed whole Chlorella cells) have been consumed regularly as a dietary supplement by people around the world for many years without any limitations related to toxicity or immunogenicity. Japan is a world leader in the consumption of Chlorella biomass. For example, in Japan, Chlorella is used in medical procedures for its immunomodulatory properties and purported anti-cancer activity, as well as for anti-aging applications such as cardiovascular disease, hypertension, and cataracts. It also reduces the risk of atherosclerosis and stimulates collagen synthesis in the skin.

[0227] Chlorella cells naturally produce extracellular vesicles (EVs) that conform to the "standard specifications" of more familiar EVs (e.g., mammalian EVs). Plant-derived EVs have many characteristics that make them more promising / convenient than synthetic nanoparticles or semi-synthetic EVs for use as drug delivery systems in humans. These include, for example, higher stability, lower toxicity, and lower immunogenicity. Because Chlorella is as close to plants as possible, it provides a source of EVs with characteristics similar to plant EVs. At the same time, large-scale mass production of Chlorella is easier and cheaper than that of higher plants. The glycosylation pattern of membrane proteins in Chlorella is similar / identical to that present in higher plants.

[0228] The size of Chlorella MEVs ranges from approximately 50 nm to 200 nm, with an average size of approximately 130 nm. Their morphology resembles plant and mammalian exosomes. For administration, the size distribution can be made more uniform by separating MEVs by size and selecting those of the desired size, which may vary depending on the intended use and route of administration.

[0229] D. Exogenously Loaded Microalgal Extracellular Vesicles (MEVs), Cargo, and Targets Targets and cargoes (see discussion below) include any known to those of skill in the art. Sections F and G below and the Examples describe the biodistribution of MEVs after administration by various routes, as well as the meaning, uses, and methods for targeting or treating specific diseases, disorders, and conditions, and for formulating and administering MEVs.

[0230] 1. MEV Isolation Isolation methods are discussed in the sections above and detailed in the Examples.

[0231] 2. MEV Load and Cargo MEVs can be loaded with any desired cargo (also referred to as payload), including, but not limited to, nucleic acid molecules including RNAi, plasmids, antisense nucleic acids, nucleic acids encoding RNAi or antisense nucleic acids, detectable marker proteins and tags, small molecule drugs, gene editing systems, and others, as well as combinations thereof. MEVs can deliver therapeutic molecules, function as vaccines, and be used in human and other animal health, agricultural applications, gene therapy applications including delivery genes, gene modification via gene editing systems, and gene silencing nucleic acids, cosmetics, dermatology, diagnostic applications, industrial applications, and the like. MEVs can deliver regulators of gene pathways to produce nutrients or beneficial products, and can be used to deliver gene editing systems such as CRISPR / Cas to achieve gene editing. MEVs can be used to deliver gene therapy vectors, including, but not limited to, adeno-associated virus (AAV) vectors, adenovirus vectors, vaccinia virus-derived vectors, and others, and products.

[0232] Diseases and conditions that can be treated include any known to those skilled in the art, including, but not limited to, cardiovascular disease, metabolic disease, infectious diseases including respiratory infections, bladder infections and other urinary tract infections, infectious diseases including viral diseases such as hepatitis, HIV, coronaviruses including SARS-Cov-2, CNS diseases, eye diseases, and liver diseases. As discussed, cargoes to be delivered include protein products such as antibodies and antigen-binding forms thereof, RNA products such as, but not limited to, siRNA, miRNA (microRNA), lncRNA (long non-coding RNA), saRNA (small activating RNA), shRNA, and mRNA, nucleic acids encoding products such as plasmids, nucleic acid products such as antisense oligonucleotides and DNA that also encode antisense oligonucleotides, and small molecule drugs.

[0233] MEVs can carry cargo including reporter genes and proteins as well as other detectable products, such as fluorescent proteins, including but not limited to enhanced green fluorescent protein (EGFP, SEQ ID NO: 10), luciferase genes (SEQ ID NO: 11), luxA (SEQ ID NO: 8), luxB (SEQ ID NO: 9), and the Lux operon (luxCDABE and luxABCDE, SEQ ID NO: 12).

[0234] Other cargoes may target genes or products involved in disease, for example, but not limited to, peptidyl-prolyl cis-trans isomerase FKBP4 or FKBP52 (SEQ ID NO: 1), gamma-aminobutyric acid type B receptor subunit 1 (GABBR1, SEQ ID NO: 3), oncogenes such as MYCN or NMYC (SEQ ID NO: 38), RAS (H-RAS, N-RAS, and K-RAS) (see SEQ ID NOs: 39, 40, and 41, respectively), BCL2 (SEQ ID NO: 43), and PLK1 (SEQ ID NO: 44). Genes and checkpoints involved in disease, such as oncogenes, can be regulated by cargos that encode products that inhibit or stimulate expression of the gene or inhibit or stimulate the gene product. Examples of such regulators are RNAi-type regulators, such as siRNA, miRNA, shRNA, antisense oligonucleotides (ASO), peptides, and / or tetratricopeptides. For example, siRNAs and ASOs targeting EGFP (SEQ ID NOs: 5 and 6), firefly luciferase (SEQ ID NO: 7), MYCN (SEQ ID NOs: 13-19), RAS (SEQ ID NOs: 20-27), BCL2 (SEQ ID NOs: 29-31), and PLK1 (SEQ ID NOs: 32-35), as well as microRNA-34A (SEQ ID NO: 28) targeting MYC and BCL2 are exemplified herein.

[0235] Gene silencing using RNA interference, including siRNA and microRNA, can be used to silence developmental genes, such as adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth / differentiation factors and their receptors, and neurotransmitters and their receptors, oncogenes, tumor suppressor genes, enzymes, genes associated with pathological conditions, genes associated with autoimmune diseases, anti-angiogenic genes, angiogenic genes, immunomodulatory genes, genes associated with alcohol metabolism and liver function, genes associated with neurological diseases, genes associated with tumorigenesis or cell transformation, and genes associated with metabolic diseases and disorders (see, e.g., WO 2009 / 082606, JP 2014-240428(A), WO 2011 / 072292(A2), WO 2010 / 141724, and WO 2020 / 097540). These types of products can be delivered or encoded in the MEV to activate genes or pathways or to provide a therapeutic effect. Certain cytokines can be used to treat diseases / disorders, such as certain cancers, in which immune suppression plays a role.

[0236] Extracellular vesicles and exosomes can also be used to deliver therapeutic agents, such as nucleic acids, such as microRNA, mRNA, tRNA, rRNA, siRNA, regulatory RNA, non-coding and coding RNA, DNA fragments, and DNA plasmids (see, e.g., CN105821081 and CN110699382), nucleotides or amino acids that contain detectable moieties or toxins or that interfere with transcription or translation, respectively, polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins) (see, U.S. Pat. No. 10,195,290). Non-limiting examples of proteins that may be encoded by nucleic acid cargo molecules include, but are not limited to, antibodies, intrabodies, single-chain variable fragments, affibodies, enzymes, transporters, tumor suppressors, viral or bacterial inhibitors, cellular component proteins, DNA and / or RNA binding proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transporters, nucleotide binding proteins, heat shock proteins, CRISPR-associated proteins, cytokines, cytokine receptors, caspases, and any combination and / or derivatives thereof (see, e.g., AU2018365299).

[0237] For example, a cocktail of three siRNA oligonucleotides (purchased from B-Bridge International Inc., Sunnyvale, CA) targeting human MYCN with two thymidine residues (dTdT) at the 3' end of the sequence can be used, as summarized in the table below. Anti-MYCN siRNA (siMYCN) and a negative control siRNA (non-targeting control pool) (siNeg) (both ON-TARGETplus siRNA, Dharmacon, Cambridge, UK) were used (see Reference 1). Exemplary target cancer genes and exemplary siRNA sequences (see also SEQ ID NOS: 13-35, respectively) are provided in the table below. [Table 4] 1 Nara et al.(2007)Int.J.Oncol.30(5):1189-1196;Silencing of MYCN by RNA interference induces growth inhibition,apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification 2 Maeshima et al.(2020)Nucleic Acid Ther.30(4):237-248;MYCN Silencing by RNAi Induces Neurogenesis and Suppresses Proliferation in Models of Neuroblastoma with Resistance to Retinoic Acid 3 Veas-Perez de Tudela et al.(2010)J.Neurochem.113(4):819-825;Human neuroblastoma cells with MYCN amplification are selectively resistant to oxidative stress by transcriptionally up-regulating glutamate cysteine ligase 4 Watson et al.(1991)Cancer Res.51(15):3996-4000;Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer 5 Yoshikawa et al.(2019)Mol.Ther.Methods Clin.Dev.13:290-302;Anti-cancer Effects of a Chemically Modified miR-143 on Bladder Cancer by Either Systemic or Intravesical Treatment 6 Tsujino et al.(2019)Cancer Sci.110(7):2189-2199;MicroRNA-143 / Musashi-2 / KRAS cascade contributes positively to carcinogenesis in human bladder cancer 7 Tirella et al.(2019)Int.J.Pharm.561:114-123;CD44 targeted delivery of siRNA by using HA-decorated nanotechnologies for KRAS silencing in cancer treatment 8 Nakada et al.(2001)Pancreatology 1(4):314-319;Antisense oligonucleotides specific to mutated K-ras genes inhibit invasiveness of human pancreatic cancer cell lines 9 Adams et al.(2015)Expert Opin.Ther.Targets 20(6):737-753;The Tumor-Suppressive and Potential Therapeutic Functions of miR-34a in Epithelial Carcinomas 10 Poeck et al.(2008)Nat.Med.14(11):1256-1263;5’-Triphosphate-siRNA:turning gene silencing and Rig-I activation against melanoma 11 Szegedi et al. (2008) Pathol.Oncol.Res.14(3):275-279;Bcl-2 Antisense Oligonucleotide Inhibits the Proliferation of Childhood Leukemia / lymphoma Cells of the B-cell Lineage 12 Ripoll et al. (2018)RSC Adv.8:20758-20763;Co-delivery of anti-PLK-1 siRNA and camptothecin by nanometric polydiacetylenic micelles results in a synergistic cell killing 13 Liu et al.(2012)BMC Cancer 12(1):519-529;MicroRNA-100 is a potential molecular marker of non-small cell cancer lung and functions as a tumor suppressor by targeting polo-like kinase 1 14 Spankuch et al.(2008)Neoplasia 10(3):223-234;Downregulation of Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles The cargo bioactive molecule may target central nervous system diseases such as neurodegenerative diseases such as Alzheimer's disease. An example of such is FKBP52 and tetratricopeptide derivatives therefrom. The complete sequence of the human peptidyl-prolyl cis-trans isomerase FKBP4 is: MTAEEMKATESGAQSAPLPMEGVDISPKQDEGVLKVIKREGTGTEMPMIGDRVFVHYTGW LLDGTKFDSSLDRKDKFSFDLGKGEVIKAWDIAIATMKVGEVCHITCKPEYAYGSAGSPP KIPPNATLVFEVELFEFKGEDLTEEEDGGIIRRIQTRGEGYAKPNEGAIVEVALEGYYKD KLFDQRELRFEIGEGENLDLPYGLERAIQRMEKGEHSIVYLKPSYAFGSVGKEKFQIPPN AELKYELHLKSFEKAKESWEMNSEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWL EYESSFSNEEAQKAQALRLASHLNLAMCHLKLQAFSAAIESCNKALELDSNNEKGLFRRG EAHLAVNDFELARADFQKVLQLYPNNKAAKTQLAVCQQRIRRQLAREKKLYANMFERLAE EENKAKAEASSGDHPTDTEMKEEQKSNTAGSQSQVETEA (SEQ ID NO: 1). Tetratricopeptide repeat (TPR) domains 260-400 are: MNSEEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWLEYESSFSNEEAQKAQALRLA SHLNLAMCHLKLQAFSAAIESCNKALELDSNNEKGLFRRGEAHLAVNDFELARADFQKVL QLYPNNKAAKTQLAVCQQRI (SEQ ID NO: 2).

[0238] An in vitro model was developed and described by a group from the Institut National de la Santé et de la Recherche Medicale, Universite Paris XI (see Chambraud et al. (2007) FASEB J. 21(11):2787-97 and Chambraud et al. (2010) Proc. Natl. Acad. Sci. USA 107(6):2658-63). The effect of FKBP52 depletion in cultured PC12 cells was tested by introducing two different small interfering RNA (siRNA) duplexes specific for rat FKBP52, designated RNAi 1 and RNAi 2. An oligonucleotide duplex with the sense sequence of the siRNA and a scrambled sequence corresponding to RNAi 1 was used as a negative control. In these experiments, FKBP52 levels, analyzed by Western blot, were substantially reduced 48 hours after transfection and remained low 72 hours after transfection. Tubulin and FKBP52 staining was performed 72 hours after transfection. In cells transfected with RNAi 1 or 2, FKBP52 staining was significantly lower than that observed in control cells. Tubulin staining revealed changes in PC12 cell phenotype. In particular, loss of FKBP52 in PC12 cells leads to these cells forming outgrowths. Thus, these cells acquired a differentiated phenotype comparable to that of PC12 cells treated with NGF. No significant alterations were observed in control-transfected cells. In another study, Chambraud et al. (Proc. Natl. Acad. Sci. USA, cited above) reported that FKBP52 prevents tau accumulation and neurite outgrowth in PC12 cells. An inducible FKBP52 expression system based on a tetracycline-responsive element was used. This system allows the generation of stably transformed PC12 cell lines to determine the cellular role of FKBP52.Among the clones that tested positive, one clone, designated H7C2, was selected and used to study the effects of FKBP52 overexpression on PC12 cells and to further investigate the relationship between FKBP52 and tau. Under basal conditions, H7C2 cells expressed endogenous FKBP52, and treatment with doxycycline (Dox) resulted in a significant increase in recombinant FKBP52 protein expression. FKBP52 induction in H7C2 cells was approximately fourfold after 5 days of Dox treatment. Next, we examined the effect of FKBP52 on tau accumulation. The amount of tau protein was determined by Western blotting of extracts from cultures of PC12 or H7C2 cells treated or not with nerve growth factor (NGF) (50 nM) for 5 days in the presence or absence of Dox. In PC12 cells, FKBP52 expression did not change after treatment with NGF. As expected, an increase in tau was observed in both PC12 and H7C2 cells after NGF treatment. When H7C2 cells were exposed to Dox in addition to NGF, resulting in overexpression of FKBP52, no further accumulation of tau protein occurred. Increased tau protein was still observed in PC12 cells treated with NGF and Dox, ruling out the possibility that Dox was responsible for the lack of tau reduction. This report concludes that FKBP52 prevents NGF-induced tau accumulation in PC12 cells.

[0239] Because one role of tau is to stimulate neurite outgrowth, we also investigated the effects of FKBP52 overexpression on neurite length in PC12 and H7C2 cells. In the absence of NGF, no neurite outgrowth was observed in H7C2 cells, regardless of whether they were treated with Dox for 1 week. A 40% (±7%) decrease in neurite length was observed in H7C2 cells treated with 50 nM NGF and Dox compared to the control (H7C2 cells not treated with Dox). The same effect of Dox on neurite length was observed in H7C2 cells treated with 10 or 20 nM NGF. Since no difference in neurite length was observed between Dox-treated and untreated PC12 cells, Dox itself was not involved in the neurite growth process. The inhibition of neurite outgrowth resulting from FKBP52 overexpression is consistent with a previous report from Chambraud et al. showing that loss of FKBP52 in PC12 cells results in the formation of neurite outgrowth. The effect of FKBP52 on neurite length can be explained by tau binding to FKBP52 and its removal from microtubules. Prevention of tau accumulation by overexpression of FKBP52 coincides with a reduction in neurite length, suggesting a potential role for this immunophilin in tau function. Therefore, the above targets and sequences can be delivered or encoded in MEVs to treat Alzheimer's disease by preventing tau accumulation.

[0240] Pathogens involved in AD pathology Microbial, viral, and fungal infections have been reported to increase the risk of, induce, or contribute to the pathology of AD (Catumbela et al. (2023) Transl. Neurodegener. 37, doi:10.1186 / s40035-023-00369-7). For example, oral bacteria have been identified in the brains of patients with AD. The oral bacterium Porphyromonas gingivalis has been identified in the brains of patients with Alzheimer's disease (AD). Porphyromonas gingivalis has been identified as a risk factor for AD, and its components, gingipain and lipopolysaccharide, have been shown to cause AD-like neurodegeneration in infected neurons derived from induced pluripotent stem cells in an in vitro culture system with sustained expression of active gingipain. Porphyromonas gingivalis has been detected in brain tissue from patients with AD and associated with pathological changes. The MEVs provided herein can be loaded with agents that inhibit Porphyromonas gingivalis and / or gingipains to prevent or treat AD. The amyloid pathway has been proposed to be an inflammatory response to infection and toxic products, including gingipains. The resulting amyloid plaques and abnormal tau protein can contribute to neuroinflammation and neurodegeneration (see, e.g., Seymour et al. (2022) J. Exploratory Res. in Pharmacology 7:45-53). The MEVs provided herein can be loaded with bacterial inhibitors and / or gingipain inhibitors, such as atzagstat, and delivered to the brain, including neurons, via intranasal administration. Treatment of infections with antiviral, antibacterial, and antifungal therapeutics, such as acyclovir, famciclovir, ganciclovir, idoxuridine, penciclovir, tromantadine, valacyclovir, and valganciclovir, has been shown to reduce the risk of developing AD. Some antibiotic regimens have resulted in cognitive improvement. For example, H. pylori -positive AD patients were treated with an H. pylori eradication regimen of omeprazole (a proton pump inhibitor), clarithromycin (a macrolide antibiotic), and amoxicillin (a penicillin-type antibiotic).At the study endpoint, AD patients successfully treated with a Helicobacter pylori eradication regimen showed significantly improved cognitive scores on the Cambridge Cognitive Examination for the Elderly and Functional Rating Scale for Symptoms of Dementia compared with patients who did not show a decrease in Helicobacter pylori levels after similar treatment. Therefore, Helicobacter pylori infection may contribute to AD pathology, and disease progression may be ameliorated upon eradication of this pathogen. Therefore, various therapeutic agents for pathogens whose infection is associated with AD can be administered via intranasal administration of MEV, and the cargo may include therapeutic agents, such as antivirals or antibiotics, or other antibacterial agents.

[0241] ApoE and Alzheimer's Disease As described in Example 33, the ApoE gene, particularly the ApoE4 allele, has been implicated as a risk factor for late-onset AD. The ApoE gene and allele are therapeutic targets for treating AD. As described herein, orally administered MEVs can traverse neurons in the brain and be loaded with cargo to alter ApoE expression or levels in neurons to treat, prevent, or reduce the risk of AD. For example, as detailed in the Examples, MEVs can be loaded with RNAi that inhibits ApoE4 expression for intranasal administration, thereby delivering the RNAi directly to neurons. Other approaches include administering gene editing systems in MEVs to convert the ApoE4 allele to ApoE3 or ApoE2, as well as other approaches. Intranasally administered MEVs provide direct entry into neurons, whereas other therapeutic regimens do not provide such access.

[0242] The reporter gene, reporter protein, and / or its regulator can be delivered in the MEV. Reporter proteins Target sequences in the form of siRNA, miRNA, antisense oligonucleotides (ASO), peptides and / or tetratricopeptides for regulating (repressing or stimulating) marker genes, such as GFP protein, eukaryotic luciferase, or prokaryotic luciferase, such as the Lux operon (luxCDABE) and the lux operon (luxABCDE), respectively, can be used, for example, for diagnostics and gene expression assessment (SEQ ID NOs: 5-6, 7, and 62-65, respectively): [Table 5]

[0243] Other exemplary cargoes may include chemotherapeutic agents, including, but not limited to, alkylating agents such as thiotepa and cyclophosphamide (available under the trademark CYTOXAN®), sulfonate alkyl esters such as busulfan, improsulfan, piposulfan, androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, antiadrenal agents such as aminoglutethimide, mitotane, and trilostane, antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin, antibiotics such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibitors such as 4(5)-imidazole, 4-hydroxytamoxifen, 4-hydroxybenzoates ...117018, onapristone, and toremifene (sold under the trademark Fareston®), antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate, aziridines such as benzodepa, carboquone, meturedepa, and uredepa, altretamine, ethyleneimines and methylmelamines including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, folic acid supplements such as furoic acid, nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobe citabine, phenesterine, prednimustine, trofosfamide, and uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, proteins such as arginine deiminase, asparaginase, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, taxanes such as paclitaxel (e.g., paclitaxel sold under the trademark TAXOL®, Bristol-Myers Squibb Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), a topoisomerase inhibitor RFS2000, thymidylate synthase inhibitors (e.g., Tomudex™), additional chemotherapy including aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, Bestravcil, bisantrene, edatrexate, defosfamide, demecolcine, diaziquone, difluoromethylornithine (DFMO), eflornithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamt, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK®, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziconazole, 2,2',2''-trichlorotriethylamine, urethane , vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (Ara-C), cyclophosphamide, thiotepa, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine (e.g., vinorelbine tartrate sold under the trademark Navelbine®), Novantrone™, teniposide, daunomycin, aminopterin, capecitabine (e.g., sold as Xeloda®), ibandronate, CPT-11, retinoic acid, esperamycin, capecitabine, and topoisomerase inhibitors such as irinotecan. Pharmaceutically acceptable salts, acids or derivatives of any of the above may also be used.

[0244] Chemotherapeutic agents include, but are not limited to, prodrugs including phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active cytotoxic free drugs.

[0245] Other cargoes include, for example, anti-angiogenic agents. Anti-angiogenic agents can be small molecules or proteins, such as antibodies, Fc fusions, and cytokines, that bind to growth factors or growth factor receptors involved in promoting angiogenesis. Examples of anti-angiogenic agents include antibodies that bind to vascular endothelial growth factor (VEGF) or VEGF-R, RNA-based therapeutics that reduce the level of VEGF or VEGF-R expression, VEGF-toxin fusions, Regeneron's VEGF trap, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, BeneFin, bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyltransferase inhibitor, fibronectin fragment, GRO-beta, halofuginone, heparinase, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon-inducible protein 10 (IP-10), interleukin-12, kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (e.g., TIMPs), 2-methoxyestradiol, MMI 270 (CGS 27023A), plasminogen activator inhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDa fragment, proliferin-related protein (PRP), PTK 787 / ZK 222594, retinoids, solimatat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP470, transforming growth factor beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

[0246] Other cargoes include tyrosine kinase inhibitors, including but not limited to quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706, pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo(2,3-d)pyrimidine, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostins containing a nitrothiophene moiety, PD-0183805 (Warner-Lambert), antisense molecules (e.g., those that bind to nucleic acids encoding ErbB), quinoxalines (U.S. Pat. No. 5,804,396), tyrphostins (U.S. Pat. No. 5,804,396), PTK-787 (Novartis / Schering AG), pan-ErbB inhibitors such as CI-1033 (Pfizer), Affinitac (ISIS 3521, Isis / Lilly), imatinib mesylate (STI571, Gleevec®, Novartis), PKI 166 (Novartis), GW2016 (GlaxoSmithKline), CI-1033 (Pfizer), EKB-569 (Wyeth), semaxinib (Sugen), ZD6474 (AstraZeneca), IMC-1C11 (ImClone), or those described in any of the following patent publications: U.S. Pat. No. 5,804,396, PCT Publication No. WO 99 / 09016 (American Cyanamid), PCT Publication No. WO 98 / 43960 (American Cyanamid), PCT Publication No. WO 97 / 38983 (Warner-Lambert), PCT Publication No. WO 99 / 06378 (Warner-Lambert), PCT Publication No. WO 99 / 06396 (Warner-Lambert), PCT Publication No. WO 96 / 30347 (Pfizer, Inc.), PCT Publication No. WO 96 / 33978 (AstraZeneca), PCT Publication No. WO 96 / 33979 (AstraZeneca), PCT Publication No. WO 96 / 33980 (AstraZeneca), gefitinib (Iressa®, ZD1839, AstraZeneca), and OSI-774 (Tarceva®, OSI Pharmaceuticals / Genentech).

[0247] Other cargoes include immunomodulatory agents that increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells. Examples of immunomodulatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketorolac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g., glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisolone); steroids, such as anthralin, prednisolone, triamcinolone, azulfidine eicosanoids, e.g., prostaglandins, thromboxanes, and leukotrienes, and topical steroids, e.g., anthralin, calcipotriene, clobetasol, and tazarotene), cytokines, e.g., TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-10, cytokines, chemokines, or receptor antagonists, including antibodies, soluble receptors, and receptor-Fc fusions, B7, CCR2, CC R5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, C D147, CD152, complement factors (C5, D), CTLA4, eotaxin, Fas, ICAM, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL- 9, IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, T cell receptors including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab), heterologous antilymphocyte globulins, other immunomodulatory molecules such as 2-amino-6-aryl-5-substituted pyrimidines,Anti-idiotypic antibodies against MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitrilamide (e.g., leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasalazine.

[0248] Other cargoes include cytokines, including, but not limited to, lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-alpha and -beta, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factor, such as NGF-beta, platelet growth factor, transforming growth factor (TGF), such as TGF-α. and TGF-beta, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-alpha, -beta, and -gamma, colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta, and other polypeptide factors including LIF and Kit ligand (KL).

[0249] Other exemplary cargoes include cytokines and other agents that stimulate cells of the immune system and enhance desired effector functions. For example, agents that stimulate NK cells include IL-2, and agents that stimulate macrophages include, but are not limited to, C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine. Cargoes include agents that stimulate neutrophils, such as G-CSF and GM-CSF. Additional agents include, but are not limited to, interferon gamma, IL-3, and IL-7.

[0250] The cargo includes antibiotics for the treatment of infections, particularly difficult-to-treat bacterial infections, including urinary tract infections, respiratory infections, particularly Pseudomonas aeruginosa or Staphylococcus aureus infections in subjects with cystic fibrosis, and sinus infections, which can be treated by topical administration, such as inhalation of an aerosol containing MEV. Antibiotic treatment of lung infections in subjects with cystic fibrosis can be combined with gene therapy using the same or different MEVs containing nucleic acid, DNA, or RNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, or providing a gene editing system to correct defects in the CFTR protein.

[0251] Antibiotics that can be loaded as cargo into MEVs include, but are not limited to, aminoglycosides (e.g., apramycin, arbekacin, bambermycin, butirodin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), aminocyclitols (e.g., spectinomycin), amphenicol antibiotics (e.g., azidamphenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., thiamphenicol ... e.g., rifamide and rifampin), carbapenems (e.g., imipenem, meropenem, and panipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxime, cefixime, cephalexin, and cephradine), cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan), lincosamides (e.g., clindamycin and lincomycin) , macrolides (e.g., azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, tobramycin), monobactams (e.g., aztreonam, carumonam, tigemonam), mupirocin, oxacephems (e.g., flomoxef, latamoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillin acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamecillin, penetacillin, matehydriodide, penicillin o-benethamine, penicillin O, penicillin V, penicillin V benzoate, penicillin V hydrabamine, penimepicycline, and phenethicillin potassium), polypeptides (e.g., bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin), quinolones (amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, losoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, and trovafloxacin), rifampin, streptogramins (e.g., quinupristin and dalfopristin), sulfonamides (sulfanilamide, sulfamethoxazole), tetracyclines (chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, Duramycin®, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

[0252] Cargos also include antifungal agents, including, but not limited to, amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, terconazole, and tioconazole. In some examples, the cargo-loaded MEVs described herein are administered with one or more antiviral agents, including, but not limited to, protease inhibitors, reverse transcriptase inhibitors, and others, including type I interferons, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, amprenavir, clevudine, enfuvirtide, entecavir, foscarnet, ganciclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine.

[0253] In all cases, the cargo form includes proteins and also nucleic acids encoding proteins, such as plasmids, and also mRNA. The nucleic acid may be operably linked to regulatory elements recognized in a particular subject, such as a mammal, to which the nucleic acid is delivered. [Table 6]

[0254] 3. Generation of Payload-loaded MEV As demonstrated herein, isolated Chlorella can be loaded with a cargo for delivery to humans by any suitable route, including, but not limited to, intravenous, oral, topical, mucosal, inhalation, and any other route known to those skilled in the art for delivery of vehicles such as lipid nanoparticles, vectors, therapeutic bacteria, and therapeutic viruses. Upon administration, the MEVs are taken up by cells. Any cargo currently delivered in vectors, bacteria, exosomes, nanoparticles, and other such delivery vehicles can be loaded into the MEVs provided herein. The loaded cargo can be selected to be expressed or produced only in the target cells, for example, when the cargo is a plasmid encoding a therapeutic product. Transcriptional regulatory signals can be selected so that the encoded product is expressed in the target cells. For example, for expression in the liver, the encoded product can be expressed under the control of a liver-specific promoter, or the product can be targeted to a receptor or target expressed in the targeted cells, e.g., the tumor or tumor microenvironment. Loading methods described above and in the examples below include, but are not limited to, the following: a. Electroporation b. Sonication c. Extrusion d. surfactants e. Other methods known to those skilled in the art for introducing exosomes into cells.

[0255] 4. Exemplary Cargoes and Exemplary Uses of Exogenously Loaded MEVs a. Cargo As described above, MEVs can be loaded with cargo that can be used for any purpose of interest, including any purpose for which other delivery vehicles are used. These uses include delivery of mRNA, such as mRNA encoding coronavirus spike proteins and modified spike proteins to improve immune responses to viruses, RNAi, such as siRNA, and antisense RNA or antisense DNA (ASO), to silence genes, such as bacterial and viral pathogen virulence genes, antibiotic resistance genes, antimicrobial resistance genes, genes that suppress the immune system, tumor genes, such as oncogenes, and host factors for viral infection, such as targeted angiotensin-converting enzyme-2 (ACE2), transmembrane protein serine 2 (TMPRSS2), and other such genes. The cargo can also include any therapeutic antibody. Therapeutic antibodies include, but are not limited to, anti-cancer antibodies, antibodies for treating autoimmune or inflammatory diseases, antibodies for treating transplant rejection, antibodies for treating graft-versus-host disease (GVHD), and antibodies for treating infectious diseases.

[0256] 1) RNA cargo The mechanism of RNA interference, or RNAi, was first described as a process of sequence-specific silencing of gene expression in the nematode Caenorhabditis elegans (Fire et al. (1998) Nature 391(6669):806-11; Fire and Mello, 2006 Nobel Prize in Medicine awarded to Andrew Fire and Craig Mello). The process of small RNAs targeting (and silencing) messenger RNA involves specific RNAi machinery (including silencing factors such as DICER and ARGONAUTE).

[0257] In the plant kingdom, RNAi is involved in antiviral defense mechanisms and defense mechanisms against plant pathogenic fungi and oomycetes. Small regulatory RNAs can be active in silencing genes in bacterial cells that lack the aforementioned RNAi mechanisms. The silencing activity of siRNA has been demonstrated to be interkingdom (see, for example, Singla, Navarro., 2019a, PCT / EP2019 / 072169; Singla, Navarro., 2019b, PCT / EP2019 / 072170; Singla et al. (2019c) bioRxiv, doi:doi.org / 10.1101 / 863902).

[0258] RNAi-mediated modulation of gene expression has been exploited in biotechnology for several years to confer resistance to viruses (Baulcombe (2015) Current Opinion in Plant Biology 26:141-146). Interkingdom RNAi has been used to characterize the function of genes in eukaryotic pathogens and / or eukaryotic parasites, as well as to induce defenses against these organisms.

[0259] In Drosophila and Caenorhabditis, RNAi plays an important role in antiviral defense by directly targeting viral RNAs via small RNAs produced by the host in response to viruses. Recent studies have shown that plant EVs derived from human food plants, naturally loaded with small RNAs (loaded by the EV-producing plant cells), can alter the composition of the human gut and oral microbiota by silencing the expression of specific genes in certain symbiotic bacteria (Teng et al. (2018) Cell Host & Microbes 24:637-652; Sundaram et al. (2019) iScience 21:308-327).

[0260] Small interfering RNA (siRNA) and microRNA (miRNA) are non-coding RNAs that play important roles in gene regulation. They have recently been investigated as a novel class of therapeutic agents for the treatment of a wide range of disorders, including cancer and infectious diseases. Clinical trials of siRNA- and miRNA-based drugs have already begun. Although siRNA and miRNA share many similarities—both are short, double-stranded RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA)—their mechanisms of action and clinical applications differ. The main difference between siRNA and miRNA is that the former is highly specific for only one mRNA target, whereas the latter has multiple targets. siRNA and miRNA play roles in gene regulation and serve as targets for drug discovery and development. Compared to traditional therapeutic small molecules, siRNA and miRNA are highly potent and offer the potential to act on “undruggable” targets (e.g., proteins lacking enzymatic function). Furthermore, RNAi can be designed to target and / or affect the expression of any gene of interest.

[0261] 2) Antibody cargo Examples of anti-cancer antibodies and other antibodies include, but are not limited to, anti-17-1A cell surface antigen antibodies, such as the antibody sold or offered under the trademark Panorex® (edrecolomab), anti-4-1BB antibodies, anti-4Dc antibodies, anti-A33 antibodies, such as A33 and CDP-833, anti-α1 integrin antibodies, such as natalizumab, anti-α4β7 integrin antibodies, such as LDP-02, anti-αVβ1 integrin antibodies, such as F-200, M-200, SJ-749, anti-αVβ3 integrin antibodies, such as abciximab, CNTO-95, Mab-17E6, and Vitaxin®, anti-complement factor antibodies, CD5 (C5) antibodies, e.g., 5G1.1; anti-CA125 antibodies, e.g., those sold or offered under the trademark OvaRex® (oregovomab); anti-CD3 antibodies, e.g., those sold or offered under the trademarks Nuvion® (vigilizumab) and Rexomab™; anti-CD4 antibodies, e.g., IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies, e.g., oncolicin B and oncolicin CD6; anti-CD7 antibodies, e.g., HB2; anti-CD19 antibodies, e.g., B43, MT-103, oncolicin B; anti-CD20 antibodies, e.g., 2H7, 2H7.v16, 2H7.v114, 2H7.v115, products sold or offered under the trademark Bexxar® (tositumomab), antibodies sold or offered under the trademark Rituxan® (rituximab), and antibodies sold or offered under the trademark Zevalin® (ibritumomab tiuxetan), anti-CD22 antibodies, such as those sold or offered under the following generic names, trade names, or trademarks: Lymphocide® (epratuzumab), anti-CD23 antibodies, such as IDEC-152, anti-CD25 antibodies, such as basiliximab and Zenapax® (daclizumab), anti-CD30 antibodies, such as AC10, MDX-060, and SGN-30, anti-CD33 antibodies, such as gemtuzumab ozogamicin (sold under the trademark Mylotarg®), oncolycin M, and Smart M195, anti-CD38 antibodies, anti-CD40 antibodies such as SGN-40 and toralizumab, anti-CD40L antibodies such as 5c8, Antova®, and IDEC-131, anti-CD44 antibodies such as bivatuzumab, anti-CD46 antibodies, anti-CD52 antibodies such as alemtuzumab (sold under the trademark Campath®), anti-CD55 antibodies such as SC-1, anti-CD56 antibodies such as huN90 1-DM1, anti-CD64 antibodies, e.g., MDX-33, anti-CD66e antibodies, e.g., XR-303, anti-CD74 antibodies, e.g., IMMU-110, anti-CD80 antibodies, e.g., galiximab and IDEC-114, anti-CD89 antibodies, e.g., MDX-214, anti-CD123 antibodies, anti-CD138 antibodies, e.g., B-B4-DM1, anti-CD146 antibodies, e.g., AA-98, anti-CD148 antibodies, anti-CEA antibodies, e.g., cT84.66, labetuzumab, and Pentacea, anti-CTLA-4 antibodies such as MDX-101, anti-CXCR4 antibodies, anti-EGFR antibodies such as ABX-EGF, cetuximab (e.g., products sold under the trademark Erbitux®), IMC-C225, and Merck Mab 425, anti-EpCAM antibodies such as Crucell's anti-EpCAM, ING-1, and KS-IL-2, anti-ephrin B2 / EphB4 antibodies, anti-Her2 antibodies such as trastuzumab (trade name Herceptin®), MDX-210, anti-FAP (fibroblast activation protein) antibodies such as sibrotuzumab, anti-ferritin antibodies such as NXT-211, anti-FGF-1 antibodies, anti-FGF-3 antibodies, anti-FGF-8 antibodies, anti-FGFR antibodies, anti-fibrin antibodies, anti-G250 antibodies such as WX-G250 and Girenz Ximab (sold under the trademark Rencarex®), anti-GD2 ganglioside antibodies such as EMD273063 and TriGem®, anti-GD3 ganglioside antibodies such as BEC2, KW-2871, and mitumomab, anti-gpIIb / IIIa antibodies such as ReoPro®, anti-heparinase antibodies, anti-Her2 / ErbB2 antibodies such as trastuzumab, MDX-210, pertuzumab, anti-HLA antibodies (e.g., products sold under the trademark Oncolym®), Smart 1D10, anti-HM1.24 antibodies, anti-ICAM antibodies such as ICM3, anti-IgA receptor antibodies, anti-IGF-1 antibodies such as CP-751871, EM-164, anti-IGF-1R antibodies such as IMC-A12, anti-IL-6 antibodies such as CNTO-328 and elcilimomab, anti-IL-15 antibodies (e.g., products sold under the trademark HuMax®-IL15), anti-KDR antibodies, anti-laminin 5 antibodies, anti-Lewis Y antigen antibodies such as Hu3S193 and IGN-311, anti-MCAM antibodies, anti-Muc1 antibodies such as BravaRex and TriAb™, anti-NCAM antibodies such as ERIC-1 and ICRT, anti-PEM antigen antibodies such as Theragyn and Therex, anti-PSA antibodies, anti-PSCA antibodies such as IG8, anti-Ptk antibodies, anti-PTN antibodies, anti-RANKL antibodies such as AMG-162, anti-RLIP76 antibodies, anti-SK-1 antigen antibodies such as Monopharm Antibodies include anti-STEAP antibodies, anti-TAG72 antibodies such as CC49-SCA and MDX-220, anti-TGF-β antibodies such as CAT-152, anti-TNF-α antibodies such as CDP571, CDP870, D2E7, adalimumab (e.g., products sold under the trademark Humira®), and infliximab (e.g., products sold under the trademark Remicade®), anti-TRAIL-R1 and TRAIL-R2 antibodies, anti-VE-cadherin-2 antibodies, and anti-VLA-4 antibodies (e.g., products sold under the trademark Antegren®). Anti-idiotypic antibodies, including but not limited to the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7, may also be used. Bispecific antibodies, including but not limited to the anti-CD3 / CD20 antibody Bi20, may also be used.

[0262] Further exemplary cargoes, uses and treatments that can be achieved with cargo-loaded MEVs are described below by way of example.

[0263] b. Diseases and treatment methods As described above, MEVs can be loaded with any desired cargo, including, but not limited to, nucleic acid molecules, detectable marker proteins and tags, small molecule drugs, gene editing systems, and the like, and combinations thereof, for delivering therapeutic molecules, functioning as vaccines, and for use in human and other animal health, agriculture, cosmetics, dermatology and diagnostic applications, industrial applications, and other uses. MEVs can deliver regulators of gene pathways to produce nutrients or beneficial products, gene editing systems such as CRISPR / cas to effect gene editing, and gene therapy vectors and products.

[0264] MEVs can carry cargo, for example, to treat diseases characterized by genetic defects resulting in the lack of functional proteins or to treat diseases characterized by the overexpression of polypeptides. Non-limiting examples of diseases that can be treated by silencing target genes, e.g., using siRNA or microRNA (see, e.g., WO 2013 / 048734), include cancer (e.g., lung cancer, leukocyte and lymphoid cancer, pancreatic cancer, colon cancer, prostate cancer, glioblastoma, ovarian cancer, breast cancer, head and neck cancer, liver cancer, skin cancer, and uterine cancer), cardiovascular disease, eye diseases (e.g., age-related macular degeneration, herpes stromal keratitis, glaucoma, dry eye syndrome, diabetic retinopathy, and ocular neovascularization and hypervascularization), and other diseases (e.g., ocular hypervascularization and hyperplasia). conditions related to intraocular pressure), neurological diseases (e.g., amyotrophic lateral sclerosis, Alzheimer's disease, myasthenic disorders, Huntington's disease, spinocerebellar ataxia, frontotemporal dementia, Parkinson's disease, prion diseases, and Lafora's disease, and those resulting from ischemic or hypoxic conditions), renal disorders, inflammatory or autoimmune diseases (e.g., ischemia or reperfusion injury, restenosis, rheumatoid arthritis, inflammatory bowel disease, e.g., Crohn's disease or ulcerative colitis, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, and diabetic conditions, arthritis, These include conditions related to: rheumatism or psoriasis), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, acute respiratory distress syndrome (ARDS), emphysema, and acute lung injury), hearing impairment, epilepsy, spinal cord injury, oral mucositis, male infertility, uterine disorders, endometrial disorders or conditions, and conditions related to metabolism (e.g., obesity), ischemia, stroke, alcohol metabolism, and liver function (see, e.g., WO 2006 / 029161, WO 2007 / 022470, WO 2007 / 130604). No. 2008 / 021157, WO 2009 / 104051, WO 2009 / 142822, WO 2019 / 217459, WO 2020 / 123083, EP 2504435, and U.S. Patent Publication Nos. 2011 / 0223665, U.S. 2012 / 0116360, U.S. 2012 / 0071540, U.S. 2016 / 0257956, U.S. 2015 / 0196648, and U.S. 2017 / 0304459).RNAi molecules can be targeted to genes encoding, for example, oncogenes, transcription factors, receptors, enzymes, structural proteins, cytokines, cytokine receptors, lectins, selectins, immunoglobulins, kinases, and phosphatases.

[0265] Other cargoes and uses are also contemplated. For example, the MEVs may carry cargo for treating conditions resulting from trauma, such as wounds, burns, cuts in the skin, fractures, hair loss, exposed dermis, exposed mucous membranes, fibrosis, lacerations, and ulcers. The MEVs may carry cargo for treating conditions resulting from natural or induced aging, particularly on the skin, or visual conditions, for example.

[0266] MEVs can be used to deliver cargoes to treat infectious diseases, for example, by gene silencing, or to prevent infectious diseases, for example, through vaccination. For example, MEVs derived from antigen-pulsed macrophages or dendritic cells have been shown to elicit immune responses when introduced into naive animals (Gyorgy et al. (2015) Annu. Rev. Pharmacol. Toxicol. 55:439-464). Gene silencing can also be used to target pathogen-associated proteins, such as viral proteins involved in host immunosuppression, pathogen replication, pathogen transmission, or maintenance of infection, or host proteins that facilitate pathogen invasion into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen genome, establishment or spread of infection in the host, or assembly of subsequent generations of pathogens.Pathogens include, for example, RNA and DNA viruses, such as arenaviruses, coronaviruses, influenza viruses, paramyxoviruses, flaviviruses (e.g., West Nile virus), picornaviruses (e.g., coxsackieviruses, polioviruses, and rhinoviruses), rhabdoviruses, filoviruses, retroviruses (e.g., lentiviruses and Rous sarcoma virus), adenoviruses, poxviruses, herpesviruses, human papillomaviruses, cytomegaloviruses, hepadnaviruses (e.g., hepatitis B and C), rotaviruses, respiratory syncytial viruses, polyomaviruses, and others, as well as bacteria, fungi, helminths, cystosomes, trypanosomes, and malaria parasites (e.g., Plasmodium malariae). Parasites, including mammalian transposable elements (e.g., WO 2010 / 141724, WO 2011 / 071860, WO 2011 / 072292, WO 2013 / 126803, WO 2020 / 035620, and WO 2020 / 097540, Australian Publication No. AU2004257373(A1), Australian Publication No. AU201 3203219(B2), and AU2016225873(A1), European Publication No. 2395012, and European Patent No. 2888240, U.S. Patent Publication No. 2011 / 0223665, U.S. Publication No. 2014 / 0256785, and U.S. Publication No. 2019 / 0032051, Japanese Publication No. 2018-197239(A), Taiwan Publication No. 201204351(A) may be mentioned.

[0267] MEVs can also be used to deliver DNA or mRNA sequences encoding therapeutically useful polypeptides. For example, if a subject lacks a particular gene product, the gene can be encoded in a nucleic acid molecule, such as a DNA or RNA molecule. The nucleic acid molecule encoding the gene product can be loaded into a MEV and delivered to the subject lacking the gene product.For example, diseases resulting from the absence or deficiency of a gene product include, but are not limited to, lysosomal storage disorders, metabolic disorders of the urea cycle, SLC3A1-related disorders including SMN1-associated spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), GALT-associated galactosemia, cystic fibrosis (CF), cystinuria, COL4A5-related disorders including Alport syndrome, galactocerebrosidase deficiency, X-linked adrenoleukodystrophy and adrenomyeloneuropathy, Friedreich's ataxia, Pelizaeus-Merzbacher disease, TSC1- and TSC2-associated tuberous sclerosis, Sanfilippo B syndrome (MPS), and others. IIIB), FMR1-related disorders including CTNS-associated cystinosis, fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X premature ovarian insufficiency syndrome; Prader-Willi syndrome, hereditary hemorrhagic telangiectasia, Niemann-Pick disease type C1, juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Hartzia-Santa Vori disease, Jansky-Bielskowski disease, and neuronal ceroid lipofuscinosis-related disorders including PTT-1 and TPP1 deficiency; EIF2B1-, EIF2B2-, EIF2B3-, EIF2B4-, and EIF2B5-associated childhood ataxia with central nervous system hypomyelination / white matter loss; CACNA1A- and CACNB4-associated episodic ataxia type 2; classic Rett syndrome; MECP2-associated severe neonatal encephalopathy and PPM Examples of such disorders include MECP2-related disorders including cerebral thrombosis-X syndrome, CDKL5-associated atypical Rett syndrome, Kennedy disease (SBMA), Notch-3-associated cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), SCN1A- and SCN1B-associated seizure disorders, Alpers-Huttenlocher syndrome, POLG-associated sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, and polymerase G-related disorders including autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions, X-linked adrenal hypoplasia, X-linked agammaglobulinemia, and Wilson's disease (see, e.g., WO 2011 / 068810, WO 2019 / 243574, WO 2019 / 092287, and WO 2020 / 099682).

[0268] MEVs can be loaded with CRISPR / Cas systems to effect gene editing. Clustered regularly interspaced short palindromic repeats (CRISPR) technology enables genome modification in living organisms and is based on the bacterial CRISPR / Cas9 antiviral defense system. This system enables DNA cleavage at target sites. Type II CRISPR systems incorporate sequences from invading foreign nucleic acids, such as DNA from viruses or plasmids, between CRISPR repeats encoded within the host genome. Transcripts from the CRISPR repeats are processed into CRISPR RNAs (crRNAs). Each crRNA contains a variable sequence transcribed from the foreign DNA and a portion of the CRISPR repeats. Each crRNA hybridizes with a second trans-activating CRISPR RNA (tracrRNA), and these two RNAs form a complex with Cas9 nuclease, directing it to cleave the target DNA sequence. By delivering Cas nucleases complexed with synthetic guide RNAs (gRNAs) consisting of a fusion of crRNA and tracrRNA into cells, the cell's genome can be cleaved at desired locations, allowing existing genes to be removed and / or new genes to be added in vivo (Sander and Joung (2014) Nat. Biotechnol. 32(4):347-355). CRISPR technology can be used with Cas polypeptides or the single RNA-guided endonuclease Cpf1 to effect genome modification and can be delivered in lipid nanoparticles, Evs, and other vesicles (see, e.g., WO 2017 / 161010, WO 2019 / 238626, and WO 2020 / 097540).

[0269] Examples of gene therapy approaches include gene replacement, e.g., to replace a gene encoding a defective product. An example of a disease that can be treated by gene replacement is Rett syndrome. Rett syndrome symptoms are caused by mutations in a single gene, MECP2, which in turn produces a mutant protein. As demonstrated in animal models, restoring MECP2 protein levels reverses symptoms, which, when delivered to the brain, could provide a cure. Gene replacement adds a healthier MECP2 gene to the brain, resulting in more unmutated MECP2 protein. MECP2 is encoded on the X chromosome. The goal is to replace as many defective genes in the brain as possible. MEVs provide a method for achieving gene replacement in the brain. This can be achieved by gene editing in the brain through the delivery of gene editing machinery, such as the CRISPR-Cas system, to MEVs. By editing existing genes to correct mutations, the regulatory mechanisms controlling expression are not affected.

[0270] RNA editing is an alternative to DNA editing. Unused RNA molecules are rapidly degraded, so errors introduced by therapeutic drugs are not permanent. RNA can be introduced by RNA trans-splicing. RNA trans-splicing is a technique that hijacks this naturally occurring phenomenon to remove the mutated portion of MECP2 protein RNA and replace it with a healthy version. A single RNA trans-splicing therapeutic could treat 97% of all Rett patients, avoiding any possibility of producing excess MECP2 protein.

[0271] Another alternative treatment that could be delivered by MEV is the administration of MECP2 protein to replace the mutated one. Protein replacement is a well-established method used to treat cancer, diabetes, autoimmune disorders, blood disorders, and many other disorders. Protein replacement for Rett syndrome allows for titration of the dose administered to each individual, allowing just the right amount to improve their symptoms.

[0272] These methods for treating genetic disorders described for Rett Syndrome can be applied to any disease, disorder, or condition of or involving the brain that involves a defective protein product. These include the diseases, disorders, and conditions listed above, as well as any such disease, disorder, and condition in which replacement or editing of a defective gene or gene product can result in therapeutic delivery to the brain. Nucleic acids and / or proteins can be delivered via intranasal administration of MEVs carrying the nucleic acid and / or protein as cargo.

[0273] MEVs can also be used to treat diseases, disorders, and conditions, including but not limited to those listed above, by introducing a payload in the form of a therapeutic protein, polypeptide, or small organic molecule or compound into target cells. Non-limiting examples of such therapeutically effective agents or drugs include oncology drugs (e.g., chemotherapeutic agents, hormonal therapy agents, immunotherapy agents, and radiotherapy agents), lipid-lowering agents for treating lipid disorders, antiviral agents, antifungal agents, anticholinergic agents, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, birth control drugs, antipyretics, vasodilators, antiangiogenic agents, cytovascular agents, antifibrotic agents, antihypertensive agents, aromatase or esterase inhibitors, signal transduction inhibitors, synthase inhibitors, cardiovascular drugs, such as antiarrhythmic drugs, phosphatase inhibitors, and the like. These include steroids, ... Therapeutic agents also include allergens, adjuvants, antigens, immunogens, antibodies (e.g., whole, polyclonal, monoclonal, and recombinant antibodies), fragments thereof, as well as single chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab)2, Fv, dAb and Fd fragments, diabodies, and antibody-related polypeptides. The therapeutic agent may be a biological therapeutic agent selected from a group consisting of a cytokine, hormone, factor, cofactor, cellular component protein, metabolic enzyme, immunoregulatory enzyme, interferon, interleukin, gastrointestinal enzyme, enzyme or factor involved in hemostasis, growth regulatory enzyme, vaccine, antithrombotic agent, toxin, antitoxin, diagnostic agent, or imaging biological agent (see, e.g., WO 2017 / 203260, WO 2018 / 102397, WO 2019 / 081474, WO 2019 / 155060,See WO 2020 / 041720, Australian Patent Publication No. 2018365299(A1), Singapore Patent Publication No. 11201811149, and U.S. Patent Publication No. 2019 / 0202892). For example, MEV therapy has been shown to be effective in treating Crohn's disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist (DIRA) deficiency, endometritis, autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, meningitis, encephalitis, liver failure, and nonalcoholic steatohepatitis (NASH). ASH), non-alcoholic fatty liver disease (NAFLD), renal failure, heart failure or any acute or chronic organ failure and associated underlying etiology, graft-versus-host disease, Duchenne muscular dystrophy and other muscular dystrophies, lysosomal storage diseases, neurodegenerative diseases, cancer-induced cachexia, anorexia nervosa, type 2 diabetes mellitus, and cancers (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, cerebellum or brain, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain stem glioma, brain cancer, brain tumor, tumor) (cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor (childhood, gastrointestinal), cancer of unknown primary, central nervous system lymphoma, cerebellar astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibroid small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (extracranial, extragonadal, or ovarian), gestational trophoblastic tumor, glioma (brain stem glioma, cerebral astrocytoma, visual pathway and hypothalamic glioma), gastric carcinoid, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) carcinoma,Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, follicular cell leukemia), lip and oral cancer, intracavitary carcinoma, liposarcoma, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, medulloblastoma, Merkel cell carcinoma, mesothelioma, primary non-small cell lung cancer Clear metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, Mycosis fungoides, myelodysplastic / myeloproliferative disorders, myeloid leukemia, chronic myeloid leukemia, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian epithelial cancer (with surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell Alveolar carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and primitive neuroectodermal tumor of the upper cerebral hemisphere, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (Ewing's sarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer (non-melanoma, melanoma), small intestine cancer, squamous cell carcinoma, squamous cell carcinoma of the cerebral plexus, gastric cancer, cerebral hemisphere It may be used to treat supraborbal primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, ureteropelvic transitional cell carcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and / or Wilms' tumor (see, e.g., WO 2017 / 203260 and WO 2019 / 155060, and U.S. Patent Publication No. 2019 / 0388347).

[0274] c. Agricultural veterinary applications MEVs carrying biomolecular cargoes can be used in agricultural veterinary applications. For example, immune ribonucleic acids can be used to treat and prevent poultry diseases, and resistance to virulent pathogens can be enhanced in plants and animals by selectively modulating miRNA pathways (see WO 2008 / 087562). Thus, cargo-loaded MEVs can be used to treat diseases in animals, including livestock.

[0275] Cargo-loaded MEVs can be used to treat plant diseases. Illustratively, MEVs can be loaded with therapeutic molecules, such as siRNAs, that target virulence genes in plant pathogens, including bacteria and viruses, and delivered to plants, such as by application or spraying onto leaves and / or other surfaces, to target the gene and eliminate or control the pathogen.

[0276] d. Cosmetic and dermatological applications MEVs carrying a pharmacological payload can also be used for cosmetic and dermatological applications. For example, skin care products, such as creams, lotions, gels, emulsions, ointments, pastes, powders, liniments, sunscreens, and shampoos, specifically containing stem cell-derived EVs, can be used to improve and / or alleviate symptoms and problems such as dry skin, elasticity, wrinkles, folds, ridges, and / or skin creases (see, for example, Singapore Patent Publication No. 11201811149). Stem cell EVs inherently possess cytokines, as well as growth and transcription factors, among their cargo, and have been shown to control inflammation, accelerate skin cell migration and proliferation, control wound scarring, improve angiogenesis, and ameliorate signs of skin aging. While the exact mechanism is still being elucidated, the effect of stem cell EVs on wound healing may depend on the vertical transfer of microRNAs or proteins to skin cells. Angiogenesis, a part of wound healing, can be induced by stem cell EVs. Stem cell EVs have also been shown to have beneficial effects on cell matrix maintenance and collagen production, and are involved in skin cell rejuvenation (da Fonseca Ferreira, A. and Gomes, D. (2019) Bioengineering (Basel) 6(1):4). MEVs loaded with desired cargo can thus be used in cosmetic and dermatological applications.

[0277] E. Pharmaceutical Compositions, Formulations, Kits, Articles of Manufacture and Combinations 1. Pharmaceutical Compositions and Formulations Compositions containing MEVs and loaded MEVs provided herein can be formulated as pharmaceutical compositions provided for administration by any desired route, such as oral, mucosal, or intravenous. Pharmaceutically acceptable compositions are prepared in consideration of regulatory or other agency approval and in accordance with generally recognized pharmacopoeias for use in animals and humans, and for agricultural use in plants. Typically, compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).

[0278] Pharmaceutical compositions can be used for therapeutic, prophylactic, cosmetic, and / or diagnostic applications. The MEVs and cargo-loaded MEVs provided herein can be formulated with a pharmaceutically acceptable carrier or diluent. Generally, such pharmaceutical compositions contain components that do not significantly impair the biological or other properties of the cargo. Each component is pharmaceutically and physiologically acceptable so as to be compatible with the other components and not harmful to the subject to which it is administered. Formulations can be provided in unit dosage form and can be prepared by methods well known in the art of pharmacy, including, but not limited to, tablets, pills, powders, liquid solutions or suspensions (e.g., injectable, ingestible, and topical formulations, including eye drops, gels, pastes, creams, or ointments), aerosols (e.g., nasal sprays and inhalers), liposomes, suppositories, pessaries, injectable and infusible solutions, and sustained-release forms. See, for example, Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8 th Ed.,Pergamon Press;and Remington's Pharmaceutical Sciences,17 thed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets, Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems, Dekker, NY. When administered systemically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenterally acceptable solution having regard to pH, isotonicity, and stability. These conditions are known to those skilled in the art. Methods for preparing parenterally administrable compositions are well known or apparent to those skilled in the art, and are described, for example, in "Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences)," 1999. th ed., Mack Publishing Company, Easton, Pa. (1995).

[0279] The pharmaceutical compositions provided herein can be in various forms, for example, solid, semi-solid, liquid, powder, aqueous, and lyophilized form.Examples of suitable pharmaceutical carriers are known in the art, and include, but are not limited to, water, buffer, saline solution, phosphate buffered saline solution, various types of wetting agents, sterilized solution, alcohol, gum arabic, vegetable oil, benzyl alcohol, gelatin, glycerin, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oil, fatty acid monoglyceride and diglyceride, pentaerythritol fatty acid ester, hydroxymethylcellulose, and powder. The pharmaceutical compositions provided herein may contain, for example, antioxidants, preservatives, antibacterial agents, analgesics, binders, disintegrants, colorants, diluents, excipients, bulking agents, glidants, solubilizers, stabilizers, isotonicity agents, vehicles, viscosity agents, flavoring agents, emulsions, e.g., oil / water emulsions, emulsifiers and suspending agents, e.g., acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl ... Other additives may be included, including propyl methylcellulose, methylcellulose, octoxynol-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and crystalline cellulose, e.g., microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, and starch (see generally Alfonso R. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20 th(See, e.g., "Patent Citation 100 of the American Journal of Clinical Oncology," vol. 1, pp. 111-114, 1999, Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and / or additives can be formulated by conventional methods and administered to a subject at an appropriate dosage. Stabilizers, such as lipids, nuclease inhibitors, polymers, and chelating agents, can protect the composition from degradation in the body.

[0280] The route of administration will be in accordance with known methods, such as intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical, rectal, mucosal injection or infusion, and by sustained release systems. The MEV or cargo-loaded MEV may be administered continuously by infusion or bolus injection. The MEV or cargo-loaded MEV may be administered in a local or systemic manner.

[0281] The MEV or cargo-loaded MEV can be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulating and administering compounds are known to those of skill in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.). The therapeutic composition can be administered intravenously, or through the nose or lungs, such as by liquid or powder aerosol (lyophilized). The composition can also be administered parenterally or subcutaneously, as desired. When administered systemically, the therapeutic composition should be sterile, pyrogen-free, and in a parenterally acceptable solution having regard to pH, isotonicity, and stability. These conditions are known to those of skill in the art.

[0282] Pharmaceutical compositions suitable for use include compositions containing MEVs or cargo-loaded MEVs in an amount effective to achieve their intended purpose. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. Therapeutically effective dosages can be determined by using in vitro and in vivo methods and / or by those skilled in the art.

[0283] Therapeutic formulations can be administered in many conventional dosage formulations. Dosage formulations of the MEVs and cargo-loaded MEVs provided herein are prepared for storage or administration by mixing the compounds having the desired degree of purity with physiologically acceptable carriers, excipients, or stabilizers. Such substances are non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as Tris HCl, phosphate, citrate, acetate and other organic acid salts, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) peptides such as polyarginine, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamic acid, aspartic acid, arginine, monosaccharides, disaccharides, and other carbohydrates, including cellulose or its derivatives, glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol, sorbitol, counterions such as sodium, and / or non-ionic surfactants such as polysorbate (TWEEN®), Pluronic®, polyethylene glycol, and the like.

[0284] In certain examples herein, a pharmaceutical composition containing a stabilizer is provided. The stabilizer can be an amino acid, an amino acid derivative, an amine, a sugar, a polyol, a salt, or a surfactant. In some examples, the stable co-formulation contains a single stabilizer. In other examples, the stable co-formulation contains two, three, four, five, or six different stabilizers. For example, the stabilizer can be a sugar or polyol, such as glycerol, sorbitol, mannitol, inositol, sucrose, or trehalose. In certain examples, the stabilizer is sucrose. In other examples, the stabilizer is trehalose. The sugar or polyol concentration is between or approximately between 100 mM to 500 mM, 100 mM to 400 mM, 100 mM to 300 mM, 100 mM to 200 mM, 200 mM to 500 mM, 200 mM to 400 mM, 200 mM to 300 mM, 250 mM to 500 mM, 250 mM to 400 mM, 250 mM to 300 mM, 300 mM to 500 mM, 300 mM to 400 mM, or 400 mM to 500 mM, inclusive.

[0285] In examples, the stabilizer can be a surfactant that is polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer, and polysorbate. For example, the surfactant can be polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer, and polysorbate, such as poloxamer 188, polysorbate 20, and polysorbate 80. In particular examples, the stabilizer is polysorbate 80. The concentration of the surfactant as a % of mass concentration (w / v) in the formulation is between or about 0.005% to 1.0%, 0.01% to 0.5%, 0.01% to 0.1%, 0.01% to 0.05%, or 0.01% to 0.02%, inclusive.

[0286] For in vivo administration, the formulation should be sterile and may be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, before or after lyophilization and reconstitution. The MEV or cargo-loaded MEV may be stored in lyophilized form or in solution, which may be frozen or refrigerated. Other vehicles may be included, such as naturally occurring vegetable oils such as sesame oil, peanut oil, or cottonseed oil, or synthetic fatty vehicles such as ethyl oleate. Buffers, preservatives, and antioxidants may be incorporated according to accepted pharmaceutical practice.

[0287] The MEVs or cargo-loaded MEVs provided herein may be provided in a concentration in the composition of 0.1 to 10 mg / mL or higher or lower amounts, such as at least or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 mg / mL or higher, depending on the application and target. The volume of the solution can be at or about 1 to 100 mL, e.g., at least or about at least or 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mL or more. In some examples, the MEVs or cargo-loaded MEVs are provided in phosphate buffered saline.

[0288] The MEVs or cargo-loaded MEVs provided herein can be provided as controlled- or sustained-release compositions. Polymeric materials are known in the art for formulating pills and capsules that can achieve controlled- or sustained-release of the MEVs and cargo-loaded MEVs provided herein (e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer and Peppas (1983) J. Macromol. Sci. 23:61; also, Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105; U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326, and International Patent Publication Nos. WO 99 / 15154 and WO 99 / 20253.) Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. Generally, polymers used in sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Any technique known in the art for producing sustained release formulations can be used to produce sustained release formulations containing the MEVs or cargo-loaded MEVs provided herein.

[0289] In some examples, the pharmaceutical composition contains a MEV or cargo-loaded MEV provided herein and one or more additional agents, such as an antibody or other therapeutic agent for combination therapy.

[0290] 2. Manufactured Products / Kits and Combinations Pharmaceutical compositions of MEVs or cargo-loaded MEVs can be packaged as articles of manufacture that include packaging materials, a pharmaceutical composition effective for treating a disease or condition that can be treated by administration of the particular MEV or cargo-loaded MEV (e.g., diseases and conditions described herein or known in the art), and a label indicating that the cargo, such as an antibody or nucleic acid molecule, is to be used to treat the infection, disease, or disorder. Pharmaceutical compositions can be packaged in unit dosage forms containing an amount of the pharmaceutical composition for a single dose or multiple doses. Packaged compositions can contain lyophilized powders of pharmaceutical compositions containing cargo-loaded MEVs that can be reconstituted (e.g., with water or saline) prior to administration.

[0291] The articles of manufacture provided herein include packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art (see, for example, U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252). Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers (e.g., pressurized metered dose inhalers (MDIs), dry powder inhalers (DPIs), nebulizers (e.g., jet or ultrasonic nebulizers) and other single-breath liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material appropriate for the selected formulation and intended mode of administration and treatment.

[0292] MEVs or cargo-loaded MEVs can be provided in combinations and as kits. Kits can optionally include one or more components such as instructions for use, devices, and additional reagents (e.g., sterile water or saline for diluting compositions and / or reconstituting lyophilized proteins), as well as components such as tubes, containers, and syringes for carrying out the methods. Exemplary kits can include MEVs or cargo-loaded MEVs provided herein and, optionally, instructions for use, devices for administering the MEVs or cargo-loaded MEVs to a subject, devices for detecting the MEVs or cargo-loaded MEVs in a sample obtained from the subject, and devices for administering additional therapeutic agents to the subject.

[0293] The kit may optionally include instructions. The instructions typically include a tangible representation describing the MEV or cargo-loaded MEV, and optionally other components included in the kit, as well as methods for administration, including methods for determining the appropriate condition of the subject, the appropriate dosage, dosing regimen, and the appropriate method of administration for administering the MEV or cargo-loaded MEV. The instructions may also include guidance for monitoring the subject over the duration of the treatment period.

[0294] Kits can also include pharmaceutical compositions and items for diagnosis as described herein, for example, such kits can include items for measuring the concentration, amount, or activity of MEVs and cargo-loaded MEVs in a subject.

[0295] In some examples, the MEV or cargo-loaded MEV is provided in a diagnostic kit for detection of the MEV or cargo-loaded MEV or cargo in an isolated biological sample (e.g., tumor cells, e.g., circulating tumor cells obtained from a subject or tumor cells excised from a subject).

[0296] The kits provided herein may also include a device for administering MEV to a subject. Any of a variety of devices known in the art for administering drugs to a subject may be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, nebulizers, and inhalers. Typically, the device for administering the composition will be compatible with the desired method of administration of the composition.

[0297] 3. Administration and Route of Exogenously Loaded MEVs The cargo-loaded MEVs provided herein can be administered to a subject by any method known in the art for administering polypeptides, including, for example, systemic or local administration. The cargo-loaded MEVs can be administered by routes such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intracavity routes), topical, epidural, or mucosal (e.g., topical, intranasal, oral, intravaginal, vulvovaginal, esophageal, bronchial, rectal, and pulmonary routes). The cargo-loaded MEVs can be administered externally to a subject at the site of disease to exert a local or transdermal effect. Compositions containing cargo-loaded MEVs can be administered by any convenient route, for example, by injection, inhalation, bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., topical, oral, vaginal, rectal, and intestinal mucosa). Compositions containing cargo-loaded MEVs can be administered together with or sequentially with other biologically active agents. For example, cargo-loaded MEVs may be administered by infusion delivery, eg, by infusion pump or syringe pump, and may be administered in combination with another therapeutic agent or as a monotherapy.

[0298] The method and / or route of administration may be modified to mitigate adverse side effects associated with the administration provided herein. For example, if a patient experiences a mild or moderate (i.e., Grade 1 or 2) infusion reaction, the infusion rate may be reduced (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). If a patient experiences a severe (i.e., Grade 3 or 4) infusion reaction, the infusion may be temporarily or permanently discontinued.

[0299] In some cases, if a subject experiences adverse side effects, such as severe skin toxicity, e.g., a severe acne-like rash, treatment adjustments may be made. For example, after the onset of adverse side effects, administration may be delayed, for example, for 1-2 weeks or until the adverse side effects have improved. In some cases, after further adverse side effects have occurred, the dosage may be reduced. Specific regimens and treatment protocols may be established by a skilled physician or other medical practitioner.

[0300] An appropriate method for delivery can be selected by one of skill in the art based on the properties of the dosage of the cargo-loaded MEV or pharmaceutical composition comprising the cargo-loaded MEV, including but not limited to, solubility, hygroscopicity, crystallization properties, melting point, density, viscosity, flowability, stability, and degradation profile.

[0301] 4. Combination therapy The cargo-loaded MEVs provided herein can be administered before, after, or simultaneously with one or more other therapeutic regimens or agents. A skilled physician can determine the appropriate dosage of each therapeutic regimen or agent, as well as the appropriate timing and method of administration, empirically or by considering the pharmacokinetics and mode of action of the agent. The additional therapeutic regimens or agents may improve the efficacy, safety, or other properties of the cargo-loaded MEV. In some examples, the additional therapeutic regimens or agents may treat the same disease or coexisting disease. In some examples, the additional therapeutic regimens or agents may improve, reduce, or eliminate one or more side effects known in the art or described herein associated with administration of the cargo-loaded MEV or cargo.

[0302] For example, the cargo-loaded MEVs described herein can be administered in conjunction with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy, or for antiviral or antibacterial or other pathogen therapy, the cargo-loaded MEVs can be administered in conjunction with other anti-pathogen therapeutics and treatments. The cargo-loaded MEVs can be administered in combination with one or more other prophylactic or therapeutic agents, including, but not limited to, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, antihormones, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressants, agents that promote the proliferation of blood cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, FcγRIIb or other Fc receptor inhibitors, or other therapeutic agents.

[0303] The one or more additional agents may be administered simultaneously, sequentially, or intermittently with the cargo-loaded MEV. The agents may be co-administered, for example, as part of the same pharmaceutical composition or the same delivery method. In some examples, the agents may be co-administered simultaneously with the cargo-loaded MEV but by a different delivery means. The agents may also be administered at a different time from the administration of the cargo-loaded MEV, but close enough in time to have a combined prophylactic or therapeutic effect. In some examples, the one or more additional agents are administered after or before administration of the cargo-loaded MEV separated by a selected period of time. In some examples, the period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. In some examples, the one or more additional agents are administered multiple times, and / or the cargo-loaded MEVs provided herein are administered multiple times.

[0304] F. Biodistribution of MEV after administration via various routes 1. Biodistribution of Mammalian EVs The pharmacokinetics and biodistribution of mammalian EVs in organs and tissues have been extensively studied (Vader et al. (2016) Advanced drug delivery reviews 106(Pt A):148-156, doi.org / 10.1016 / j.addr.2016.02.006; Morishita et al. (2017) Journal of pharmaceutical sciences 106(9):2265-2269, hdoi.org / 10.1016 / j.xphs.2017.02.030). Treatment with mammalian cell-derived EVs is generally based on intravenous or intraperitoneal administration routes. The primary target organs for systemic administration of mammalian EVs are the liver, spleen, and lungs. A comprehensive study of the tissue distribution of fluorescently labeled mammalian EVs from various cell sources (see Wiklander et al. (2015) J. Extracellular Vesicles 4:26316) demonstrated that 24 h after intravenous (i.v.) injection in mice, the highest fluorescent signal was in the liver, followed by the spleen, gastrointestinal tract, and lungs. Furthermore, the cell source, EV dose, and administration route were shown to affect EV distribution; for example, injection of higher EV doses resulted in relatively lower liver accumulation compared with lower doses, likely caused by saturation of the mononuclear phagocyte system (MPS). A comparison between intraperitoneal ...

Claims

1. 1. A composition comprising microalgal extracellular vesicles (MEVs) for use in delivering bioactive molecules to the brain to treat, detect, or monitor a disease, disorder, or condition of the brain or a disease, disorder, or condition involving the brain, comprising: the MEV comprises the biologically active molecule, and the composition is formulated for intranasal administration; the MEV, when administered intranasally, travels to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; the biologically active molecule is any molecule that can effect treatment of the brain disease, disorder, or condition, or that can be used to detect the brain disease, disorder, or condition, or that can be used to monitor treatment of the brain disease, disorder, or condition, or that involves the brain; A composition wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

2. 1. A method of delivering a bioactive molecule to the brain, comprising intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising the bioactive molecule, whereby the MEVs travel to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; The biologically active molecule is any molecule that can provide treatment for a disease, disorder, or condition, or that can be used to detect a disease, disorder, or condition, or that can be used to monitor treatment for a disease, disorder, or condition; the disease, disorder, or condition can be treated, detected, or monitored by delivery of the bioactive molecule to one or more of the interconnected regions; The method, wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

3. 3. The composition or method of claim 1 or claim 2, wherein the disease, disorder, or condition can be treated, detected, or monitored by delivery of the bioactive molecule to neurons and / or other brain cells.

4. 4. The composition or method of any one of claims 1 to 3, wherein the bioactive molecule provides treatment for a neurodegenerative disease, condition, or disorder.

5. 5. The composition or method of any one of claims 1 to 4, wherein the disease, disorder, or condition is Alzheimer's disease or a psychiatric disease, disorder, or condition.

6. 6. A composition or method according to any one of claims 1 to 5 for use in treating a disease, disorder or condition of the brain or a disease, disorder or condition involving the brain.

7. 1. A method of treating a disease, disorder, or condition of the brain or a disease, disorder, or condition involving the brain, comprising intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a biologically active molecule, whereby the MEVs travel to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; the bioactive molecule is any molecule that can provide treatment for a disease, disorder, or condition of or involving the brain; The method, wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

8. 1. A method of detecting a brain disease, disorder, or condition, or a disease, disorder, or condition involving the brain, or monitoring the treatment of a brain disease, disorder, or condition, comprising intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive molecules, whereby the MEVs travel to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus, wherein the bioactive molecule comprises a reporter or detectable marker; the biologically active molecule is any molecule that can be used to detect or diagnose the disease, disorder, or condition, or that can be used to monitor the treatment of the disease, disorder, or condition, or that can be used to detect or diagnose the disease, disorder, or condition, or that can be used to treat the disease, disorder, or condition; the disease, disorder, or condition is a disease, disorder, or condition involving one or more of the brain or the interconnected brain regions; The method, wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

9. 1. A composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, the composition is formulated for intranasal delivery to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; The biologically active molecule is any molecule that can provide treatment for a disease, disorder, or condition, or that can be used to detect or diagnose a disease, disorder, or condition, or that can be used to monitor treatment for a disease, disorder, or condition; the disease, disorder, or condition is a disease, disorder, or condition of the brain or involving one or more of the interconnected brain regions; A composition wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

10. for use in delivering a cargo comprising a biologically active molecule to the brain for treating a disease, disorder or condition of or involving the brain, or for diagnosing or detecting or monitoring the treatment of a disease, disorder or condition of or involving the brain, or for treating, diagnosing, detecting and / or monitoring a disease, disorder or condition of or involving the brain, the MEV comprises the bioactive molecule for delivery to the brain, and the composition is formulated for intranasal delivery to the brain via the olfactory nerve through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, taenia tecta, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus; the biologically active molecule is any molecule that can provide treatment for the disease, disorder, or condition, or that can be used to detect the disease, disorder, or condition, or that can be used to monitor the treatment of the disease, disorder, or condition; 10. The composition of claim 9, wherein the bioactive molecule is heterologous to the microalgae and / or the MEV.

11. 11. The method or composition of any one of claims 1 to 10, wherein the MEVs travel within the brain via intraneuronal axonal transport.

12. 12. The method or composition of any one of claims 1 to 11, wherein the MEV is delivered to or is for delivery to the limbic system, e.g., the amygdala, hippocampus, and thalamus, or the cortex, e.g., the frontal cortex or parietal cortex.

13. 13. The method or composition of any one of claims 1 to 12, wherein the MEV after intranasal administration follows or is intended to follow the pathways and connections of neural networks including olfactory nerves and mitral / tufted neurons throughout the brain.

14. 14. The method or composition of any one of claims 1 to 13, wherein upon intranasal administration, the MEV traverses one or more of: (i) the synapse between the olfactory sensory neuron (OSN) and the mitral / tufted neuron; (ii) the synapse between the mitral / tufted neuron and the local neuron in the brain region where LOT is established; and (iii) the synapse between the neuron in the brain region where LOT is established and neurons from or to the frontal cortex, the hippocampus, the thalamus, and the hypothalamus.

15. 15. The method or composition of claim 14, wherein the MEV traverses (i), (ii), and (iii), or (i) and (ii).

16. 36. The method or composition of any one of claims 1 to 15, wherein the pathway traversed by the MEV upon intranasal administration is shown in Figure 35.

17. 17. The method or composition of any one of claims 1 to 16, wherein after intranasal administration, the MEV is delivered to or is for delivery to one or more of the corpus callosum, the dorsal fornix, the dorsal hippocampal commissure, and the fimbria of the hippocampus.

18. 18. The method or composition of any one of claims 1 to 17, wherein the disease, disorder, or condition involves neurons.

19. The method or composition of any one of claims 1 to 18, wherein the composition is formulated as a suspension or as an emulsion.

20. 20. The method or composition of claim 19, wherein the composition is formulated as an emulsion that is a nanoemulsion or a microemulsion.

21. 21. The method or composition of any one of claims 1 to 20, wherein each MEV in the composition contains, on average, 1 to 100 of the biologically active molecules.

22. 22. The method or composition of any one of claims 1 to 21, wherein the amount of MEVs in the composition is about 10e10 to 10e14 or 10e10 to 10e14 MEV particles.

23. the composition is formulated for single dose administration or multiple dose administration, the volume of the composition is 0.1 to 100 mL; 23. The method or composition of any one of claims 1 to 22, wherein a single dose contains about 10e10 to 10e14 or 10e10 to 10e14 MEV particles.

24. 24. The method or composition of any one of claims 1 to 18 and 21 to 23, wherein the composition is formulated as a powder, lozenge, granule, liquid, oil, suspension, or emulsion for nasal administration.

25. The MEVs are extracellular vesicles derived from microalgae from the Chlorellaceae family, The Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo introduced into the isolated extracellular vesicles, whereby the vesicles in the composition containing the heterologous bioactive molecular cargo contain, on average, the same bioactive molecular cargo; the cargo molecule is heterologous to the Chlorellaceae family, 25. The method or composition of any one of claims 1 to 24, wherein the bioactive cargo is a therapeutic or detectable molecule for treating, monitoring and / or diagnosing a disease, disorder or condition involving the brain.

26. The MEV is a Chlorella extracellular vesicle, The Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo that is endogenously introduced into the extracellular vesicles by the microalgae, whereby the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same bioactive molecular cargo; the cargo molecule is heterologous to Chlorella; 25. The method or composition of any one of claims 1 to 24, wherein the bioactive cargo is a therapeutic or detectable molecule for treating, monitoring and / or diagnosing a disease, disorder or condition involving the brain.

27. the microalgae is a species of the Chlorellaceae family of microalgae; the MEVs in the composition contain a heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEVs, whereby the vesicles in the composition that contain the heterologous bioactive molecular cargo contain the same cargo; the cargo is heterologous to Chlorella; 25. The method or composition of any one of claims 1 to 24, wherein the cargo is a biomolecule or a small molecule.

28. The MEV is a Chlorella extracellular vesicle, The Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo that was endogenously introduced into the extracellular vesicles by the microalgae, whereby the vesicles in the composition containing a heterologous bioactive molecular cargo contain the same bioactive molecular cargo; the cargo molecule is heterologous to Chlorella; 25. The method or composition of any one of claims 1 to 24, wherein the biologically active cargo is a biomolecule.

29. 25. The method or composition of any one of claims 1 to 24, wherein the MEVs are derived from a phylum of microalgae selected from among Euglenophyta (Euglena algae), Chrysophyta (golden brown algae and diatoms), Pyrrhopphyta (fire algae), Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae), and Xanthophyta (yellow-green algae).

30. The method or composition according to any one of claims 25 to 28, wherein the chlorella is a species of the family Chlorellaceae selected from among Chlorella ellipsoidia, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis, or a species of the genus Parachlorella selected from among Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hassii.

31. 31. The method or composition of claim 30, wherein the chlorella is Chlorella vulgaris or Parachlorella kesslerii.

32. 25. The method or composition or drug delivery of any one of claims 1 to 24, wherein the MEVs are derived from microalgae that are species of the Chlorophyceae or Trebouxiophyceae.

33. 25. The method or composition of any one of claims 1 to 24, wherein the MEV is from the Chlorophyta.

34. 34. The method or composition of claim 33, wherein the MEV is derived from a Chlorellaceae species, such as Chlorella species or Parachlorella species, or Chlamydomonas.

35. 35. The method or composition of any one of claims 1 to 34, wherein the cargo is a biomolecule.

36. 36. The method or composition of any one of claims 1 to 35, wherein the cargo comprises a biopolymer.

37. 37. The method or composition of claim 36, wherein the biopolymer is a naturally occurring biopolymer, a synthetic biopolymer, or an engineered biopolymer.

38. 38. The method or composition of any one of claims 1 to 37, wherein each of the cargo-containing MEVs comprises a plurality of different heterologous cargoes.

39. 39. The method or composition of any one of claims 1 to 38, wherein the cargo is a therapeutic agent for treating or preventing a disease or condition of or involving the brain, or for treating or preventing a symptom thereof.

40. 40. The method or composition of any one of claims 1 to 39, wherein the cargo is a nucleic acid molecule, a polypeptide, a protein, a plasmid, an aptamer, or an antisense oligonucleotide.

41. 41. The method or composition of claim 40, wherein the cargo is a nucleic acid molecule that is DNA or RNA.

42. 42. The method or composition of claim 40 or claim 41, wherein the cargo is an interfering RNA (RNAi).

43. 42. The method or composition of claim 40 or claim 41, wherein the cargo is mRNA or modified mRNA.

44. 43. The method or composition of claim 42, wherein the RNAi is a silencing RNA (siRNA), or a short hairpin RNA (shRNA), or a microRNA (miRNA).

45. 42. The method or composition of claim 41 , wherein the RNA molecule is a small activating RNA (saRNA), or a long non-coding RNA (lncRNA), or a double-stranded RNA (dsRNA).

46. 43. The method or composition of claim 41 or claim 42, wherein the cargo is an oligonucleotide.

47. 43. The method or composition of claim 41 or claim 42, wherein the cargo is an antisense oligonucleotide (ASO) or an allele-specific oligonucleotide.

48. 43. The method or composition of claim 41 or claim 42, wherein the cargo comprises a gene editing system.

49. 49. The method or composition of Claim 48, wherein the gene editing system comprises a CRISPR-CAS system.

50. 49. The method or composition of Claim 48, wherein the gene editing system comprises a CRISPR-associated system or a CRISPR-like system.

51. 51. The method or composition of any one of claims 1 to 41 and 46 to 50, wherein the cargo comprises DNA.

52. 52. The method or composition of claim 51 , wherein the cargo comprises a plasmid.

53. 53. The method or composition of claim 52, wherein the plasmid encodes a therapeutic or diagnostic product.

54. 53. The method or composition of claim 52, wherein the plasmid encodes a therapeutic RNA product.

55. 55. The method or composition of claim 54, wherein the RNA product is an interfering RNA (RNAi).

56. 56. The method or composition of claim 55, wherein the product is an siRNA, shRNA, or miRNA, or a small activating RNA (saRNA).

57. 53. The method or composition of claim 52, wherein the plasmid encodes an antisense oligonucleotide or a ribozyme or a double-stranded RNA.

58. 58. The method or composition of any one of claims 52 to 57, wherein the plasmid encodes the cargo product under the control of a eukaryotic promoter.

59. 59. The method or composition of claim 58, wherein the promoter is recognized by RNA polymerase II or III.

60. 60. The method or composition of claim 59, wherein the promoter is recognized by RNA polymerase II and is a eukaryotic viral promoter.

61. 61. The method or composition of claim 59 or claim 60, wherein the promoter is selected from a cytomegalovirus promoter, a simian virus 40 promoter, a herpes simplex promoter, an Epstein-Barr virus promoter, an adenovirus promoter, a synthetic promoter, an actin promoter, and a synthetic chimeric promoter.

62. 62. The method or composition of any one of claims 52 to 61, wherein the plasmid further comprises other eukaryotic transcription and translation sequences.

63. 63. The method or composition of any one of claims 1 to 62, wherein the cargo encodes or is an immunomodulatory factor.

64. 64. The method or composition of any one of claims 1 to 63, wherein the cargo comprises or encodes an immunomodulatory agent for increasing or decreasing production of one or more cytokines, upregulating or downregulating self-antigen presentation, masking MHC antigens, or promoting proliferation, differentiation, migration, or activation state of one or more types of immune cells.

65. 65. The method or composition of any one of claims 1 to 64, wherein the cargo comprises or encodes a hormone, or cytokine, or chemokine.

66. 66. The method or composition of any one of claims 1 to 65, wherein the cargo comprises a prodrug or a vector encoding an enzyme that converts a prodrug into a drug for treating a disease, disorder, or condition of or involving the brain.

67. 67. The method or composition of any one of claims 1 to 66, wherein the cargo comprises or encodes an antibiotic for the treatment of an infection in or involving the brain.

68. 68. The method or composition of any one of claims 1 to 67, wherein the cargo comprises an antifungal agent.

69. 69. The method or composition of any one of claims 1 to 68, wherein the cargo comprises a therapeutic nucleic acid or protein or a nucleic acid encoding a protein that is a therapeutic product for the treatment of a cancer or tumor in the brain, or for the treatment of an infectious disease in the brain, or for the treatment of a neurodegenerative disease or other central nervous system (CNS) disorder, or for the treatment of dementia.

70. 70. The method or composition of any one of claims 1 to 69, wherein the cargo comprises a chemotherapeutic agent for treating a disease, disorder, or condition of or involving the brain.

71. 70. The method or composition of any one of claims 1 to 69, wherein the cargo comprises or encodes a protein that is an antibody or an antigen-binding fragment thereof.

72. 72. The method or composition of claim 71, wherein the antibody is an scFv, a bispecific antibody, or an antigen-binding fragment thereof.

73. 73. The method or composition of any one of claims 1 to 72, wherein the cargo comprises a nucleic acid for gene therapy.

74. 74. The method or composition of any one of claims 1 to 73, wherein the disease, disorder, or condition comprises a tumor.

75. 75. The method or composition of any one of claims 1 to 74, wherein the cargo comprises an oncolytic virus that infects glial tumors or comprises a therapeutic agent for the treatment of glial tumors.

76. 76. The method or composition of any one of claims 1 to 75, wherein the MEV comprises two or more cargo products.

77. 77. The method or composition of any one of claims 1 to 76, wherein the cargo comprises a diagnostic or detectable product for detecting, diagnosing, and / or monitoring a disease, disorder, or condition of or involving the brain.

78. 78. The method or composition of claim 77, wherein the diagnostic or detectable product comprises a luciferase or a nucleic acid encoding a luciferase, a fluorescent protein or a nucleic acid encoding a fluorescent protein, or a luciferase operon, or a combination thereof.

79. 79. The method or composition of any one of claims 1 to 78, wherein the bioactive molecular cargo comprises any molecule that has an effect on a cell or organism to which it is delivered, or that is detectable, or that acts as a detectable marker or biomarker, thereby resulting in the treatment, detection, diagnosis, or monitoring of treatment of a disease, disorder, or condition of or involving the brain.

80. 80. The method or composition of any one of claims 1 to 79, wherein the biologically active molecular cargo comprises one or more of a biologically active small molecule, a peptide (polypeptide, protein), RNA (mRNA, siRNA, dsRNA, miRNA, lncRNA), DNA (antisense oligonucleotide (ASO), plasmid, DNA fragment), and a gene editing complex.

81. 81. The method or composition of any one of claims 1 to 80, wherein the bioactive molecule is a diagnostic or therapeutic agent or theragnostic for treating, diagnosing, detecting, and / or monitoring the treatment of a disease, disorder, or condition of or involving the brain.

82. 82. The method or composition of any one of claims 1 to 81, wherein the disease, disorder, or condition is a neurodegenerative disease (e.g., Parkinson's disease, or Alzheimer's disease, or Huntington's disease, or Creutzfeldt-Jakob disease, or other neurodegenerative disease), or a cognitive disorder (e.g., dementia, or amnesia, or delirium, or other cognitive disorder), or a brain disorder (e.g., encephalitis, stroke, or tumor, or other brain disorder), or a nervous system disorder (e.g., pain, or stroke, infection, or other nervous system disorder), or a genetic disease (e.g., cystic fibrosis, thalassemia, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, Tay-Sachs disease, Rett syndrome, or other genetic disease), or a brain tumor, or Niemann-Pick disease, or a prion disease, or Parkinson's disease, or multiple sclerosis, or amyotrophic lateral sclerosis (ALS), or muscular dystrophy, or other disease of or involving the brain.

83. 82. The method or composition of any one of claims 1 to 81, wherein the disease, disorder, or condition of or involving the brain is cancer or a disease, disorder, or condition that is treated or prevented by a vaccine.

84. 84. The method or composition of any one of claims 1 to 83, wherein the disease, disorder, or condition is caused by or involves an infectious agent.

85. 85. The method or composition of claim 84, wherein the infectious agent is one or more of a bacterium, a virus, an oomycete, and a fungus.

86. 77. The method or composition of any one of claims 1-76, wherein the MEVs, upon intranasal administration, deliver the cargo to one or more of neurons, astrocytes, oligodendrocytes, microglial cells, ependymal cells, and / or neural stem cells.

87. 87. The method or composition of any one of claims 1 to 86, wherein the cargo is delivered in vivo to neurons, astrocytes, oligodendrocytes, microglial cells, ependymal cells, and / or neural stem cells.

88. 88. The method or composition of any one of claims 1 to 87, wherein the disease, disorder, or condition is one or more of cognitive, affective, behavioral, psychiatric, neurological, degenerative, genetic, malignant (cancer), and / or traumatic brain diseases, disorders, or conditions.

89. 89. The method or composition of claim 88, wherein the disease, disorder, or condition of or involving the brain results from damage to the brain or central nervous system (CNS).

90. 90. The method or composition of any one of claims 1 to 89, wherein the MEV comprises a therapeutic cargo that is psychoactive, or treats a psychiatric disorder, or is an immunomodulatory product, or is a detectable product, or treats brain injury or trauma, or treats cancer, or treats a neurological brain disorder, or treats a CNS disorder, or treats a genetic brain disorder, or treats brain tumor, or has anti-aging activity, or has brain regenerative activity.

91. 91. The method or composition of any one of claims 1-90, wherein the MEV comprises a cargo comprising one or more of a hormone, a growth factor, an enzyme, an immunomodulatory compound, a receptor, a receptor agonist, or a receptor antagonist.

92. 92. The method or composition of any one of claims 1-91, wherein the MEV comprises a cargo for one or more of: (i) treating or preventing or reducing the risk of brain diseases, disorders, and conditions; (ii) studying brain diseases, disorders, and conditions in vitro and / or in vivo; (iii) diagnosing brain diseases, disorders, and conditions; and (iv) recreational use.

93. 93. The method or composition of claim 92, wherein the diseases, disorders, and conditions are selected from among cognitive, emotional, behavioral, psychiatric, neurological, and / or neurodegenerative diseases, disorders, and conditions, or diseases, disorders, or conditions resulting from injury to the brain or CNS.

94. 93. The method or composition of claim 92, wherein the disease, disorder, or condition is selected from among cancer or tumor of the brain and / or CNS, genetic disorder, brain injury or trauma, and infectious disease.

95. 95. The method or composition of any one of claims 1 to 94, wherein the MEV cargo comprises a small molecule.

96. 96. The method or composition of any one of claims 1 to 95, wherein the cargo is selected from among antidepressants, antipsychotics, anxiolytics, analgesics, hallucinogens, hallucinogens, and memory enhancers.

97. 97. The method or composition of any one of claims 1 to 96, wherein the cargo is a carboline, or lysergic acid, or psilocybin, or a derivative thereof.

98. 98. The method or composition of any one of claims 1 to 97, wherein the cargo is incapable of crossing the blood-brain barrier when not within a MEV (naked).

99. 99. The method or composition of any one of claims 1 to 98, wherein the cargo comprises a hydrophilic compound.

100. The cargo, as a naked molecule or in the MEV, is one that, when administered systemically or locally by a route other than nasal, is unable to reach the brain after hepatic first-pass metabolism or is poorly absorbed in the intestine, but 100. The method or composition of any one of claims 1 to 99, wherein upon intranasal administration in MEV, the cargo reaches the brain.

101. 101. The method or composition of any one of claims 1 to 100, wherein the disease, disorder, or condition is a psychiatric disorder, or a mental disorder, or a neurological disorder.

102. 102. The method or composition of claim 101, wherein the disease, disorder, or condition is selected from among borderline personality disorder, eating disorders, schizophrenia, attention-deficit / hyperactivity disorder (ADHD), autism, bipolar disorder, anxiety, depression, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder (PTSD).

103. 103. The method or composition of any one of claims 1 to 102, wherein the cargo comprises a bioactive molecule for the treatment of the following conditions: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

104. Diseases, disorders, and conditions involving the brain include human psychiatric or neurological disorders, non-human animal brain disorders, CNS disorders, anxiety disorders such as panic disorder, social anxiety, phobia-related disorders, and generalized anxiety disorder, attention deficit hyperactivity disorder such as inattentive type, hyperactive-impulsive type, and mixed type, autism spectrum disorders such as Asperger's syndrome, childhood disintegrative disorder (CDD), Kanner's syndrome, pervasive developmental disorder (PDD-NOS), epilepsy, drug-resistant epilepsy, bipolar disorders such as bipolar I disorder, bipolar II disorder, bipolar with mixed features, bipolar with seasonal pattern of major depression, cyclothymic disorder, rapid cycling bipolar disorder, eating disorders such as anorexia nervosa, bulimia nervosa, muscle dysmorphia, binge eating disorder, other specific eating or feeding disorders (OSD).

104. The method or composition of any one of claims 1 to 103, wherein the subject is a disorder selected from among FED), compulsive overeating, Prader-Willi syndrome, diabulimia, orthorexia nervosa, selective eating, drunkorexia, pregorexia, personality disorders including, but not limited to, antisocial personality disorder, borderline personality disorder, histrionic personality disorder, narcissistic personality disorder, avoidant personality disorder, dependent personality disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorders including PTSD, acute stress disorder, simple PTSD, complex PTSD, comorbid PTSD, classic Rett syndrome, CDKL5-associated atypical Rett syndrome, schizophrenia, catatonic schizophrenia, disorganized schizophrenia, paranoid schizophrenia, residual schizophrenia, and undifferentiated schizophrenia, and other such mental and brain related conditions.

105. 104. The method or composition of any one of claims 1 to 103, wherein the diseases, disorders, and conditions involving the brain are selected from among genetic disorders, neurodegenerative diseases, neurological disorders, and metabolic disorders that affect brain function, and other brain-related metabolic diseases, disorders, or conditions.

106. 106. The method or composition of claim 105, wherein the diseases, disorders, and conditions involving the brain are selected from Alzheimer's disease, prion diseases, Niemann-Pick disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Huntington's disease, disease with Lewy bodies, Parkinson's disease, spinal muscular atrophy, Tay-Sachs disease, Wilson's disease, leukodystrophy, epilepsy, drug-resistant epilepsy, multiple sclerosis, encephalitis, and migraine.

107. the disease, disorder, or condition is a neurodegenerative disease, and the cargo is an Apo E4 inhibitor or an inhibitor of its expression in neurons, or an activator of Apo E2 and / or Apo E3 or their expression in neurons, or a gene editing cassette or system for modifying one or more of the genes encoding Apo E2, Apo E3, or Apo E4 in neurons; 106. The method or composition of claim 105, wherein the composition is formulated for and administered by intranasal administration.

108. 108. The method or composition of any one of claims 105 to 107, wherein the disease, disorder, or condition is Alzheimer's disease.

109. Modification of Apo levels or expression is achieved by intranasal administration of cargo-loaded MEVs, thereby: a) modifying the physiological level of ApoE lipidation using MEVs loaded with either i) peptides or small molecules known to increase the lipid-binding capacity of ApoE, or (ii) miRNA (miRNA-33) or siRNA or ASO sequences that mimic miRNA-33, to increase ABCA1 levels or decrease Aβ levels, thereby increasing the lipidation capacity of ApoE; and / or b) reducing the amount of ApoE4 in the brain using MEVs loaded with miRNA (miRNA146) or siRNA or ASO sequences to mimic miRNA-146, or other RNAi, such as siRNA or shRNA, that inhibit the expression of ApoE4, thereby inhibiting the immune response in the brain and / or reducing ApoE4 in the brain; and / or c) increasing the expression of ApoE2 isoforms in the brain using MEVs loaded with either (a) ApoE2 protein, or (b) mRNA encoding ApoE2 protein, or (c) a plasmid encoding ApoE2 sequences, to increase the protective effects of ApoE2 and compensate for the toxic effects of ApoE4; and / or d) The method or composition of any one of claims 105 to 108, wherein genome editing using MEV loaded with a gene editing complex results in editing of the ApoE4 allele to produce ApoE3 and / or ApoE2.

110. 110. The method or composition of any one of claims 1 to 109, wherein the MEV cargo for delivery to the brain is selected from cargo comprising one or more of psychoactive agents, enzymes, growth factors, and detectable products for treating or detecting or monitoring a disease, disorder, or condition of or involving the brain.

111. The cargo in the MEV may be a neurotrophic factor selected from among TrkA (tropomyosin kinase A), NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocybin and / or psilocin, harmine, temozolomide, rivastigmine, GABAB1A receptor, GABAB1A receptor siRNA, PTEN siRNA (SEQ ID NOs: 136-138), miR-17 (miRNA, SEQ ID NOs: 139-141), MALAT1 (SEQ ID NO: 142), 5-hydroxytryptamine-1A (5-) and 5-hydroxytryptamine-3 (5-HT3) receptor agonists, such as azapirone, methylphenidate, dexmethylphenidate, ondansetron (e.g., products sold under the trademark Zofran®), acetylcholinesterase inhibitors, or the like. anticonvulsants such as gabapentin, pregabalin, topiramate (e.g., products sold as Topomax®), carbamazepine, eslicarbazepine, levetiracetam, licarbazepine, oxcarbazepine, valproic acid and derivatives, lamotrigine, antipsychotics such as aripiprazole, asenapine, cariprazine , chlorpromazine, clozapine, haloperidol, lumateperone tosylate (e.g., products sold as Caplyta®), olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, beta-blockers such as azapirone, propranolol, drugs that modulate the cholinergic system such as biperiden, scopolamine, corticotropin-releasing factor (CRF) antagonists, drugs that modulate the GABAergic system such as benzodiazepines, brexanolone, Sage-217, glucocorticoid receptor agonists such as hydrocortisone, drugs involved in glutamatergic regulation such as AGN-241751, AV-101, AVP-786, AVP-923, AXS-05, D-cycloserine, dextromethorphan, rapastinel, glycine, and glycine reuptake inhibitors such as sarcosine, drugs that modulate the hypothalamic-pituitary-adrenal (HPA) axis such as fludrocortisone, metyrapone,Mifepristone, and probiotics, drugs that modulate the kynurenine pathway (KP), drugs that modulate limbic and paralimbic brain regions, such as cannabidiol (CBD), drugs that modulate the melatonergic system, such as agomelatine, fatty acids, peptides, nucleic acids and other precursor molecules, such as alpha-omega fatty acids, coenzyme Q10, myo-inositol, methylfolate, S-adenosylmethionine, cysteamine, and oxytocin, monoamine oxidase inhibitors (MAOIs), such as isocarboxazid (Marplan), phenelzine (Nardil), selegiline (Emsam), tranylcypromine (Parnate), mood stabilizers such as lithium salts, valproate, ebselen, and divalproex, multimodal antidepressants such as vilazodone and vortioxetine, N-nitrosodimethylamine (NDMA) receptor antagonists such as amantadine, arketamine, ketamine, memantine, riluzole, esketamine, neurokinin-1 (NK1) receptor antagonists, neuropeptide Y (NPY) receptor agonists, drugs with neurotrophic effects, silos acetaminophen, sildenafil, and vildagliptin, norepinephrine-dopamine reuptake inhibitors (NDRIs), bupropion (Wellbutrin®, Zyban®, Aplenzin®), drugs acting on the opiate system, such as ALKS-5461, AZD2327, BTRX-246040 (LY2940094), buprenorphine, JNJ-67953964, nalmefene, and naltrexone, protein kinase C inhibitors or antiestrogens, such as endoxifen, tamoxifen lanthanum, and verapamil, hallucinogens such as 3,4-methylenedioxymethamphetamine (MDMA), ayahuasca, lysergic acid diethylamide (LSD), psilocybin, selective serotonin reuptake inhibitors (SSRIs) such as citalopram (Celexa®), escitalopram (Lexapro®), fluvoxamine, paroxetine (Paxil®, Pexeva®), and sertraline (Zoloft®), selective norepinephrine transporter inhibitors such as atomoxetine,Serotonin-norepinephrine reuptake inhibitors (SNRIs) such as desvenlafaxine (Pristiq®), duloxetine (Cymbalta®), levomilnacipran (Fetzima®), and venlafaxine, adenosine receptor antagonists, and stimulants including alpha-2-adrenergic receptor agonists such as caffeine, clonidine, guanfacine, extended-release amphetamine XR-OS, dextroamphetamine sulfate, lisdexamphetamine, methamphetamine, mixed amphetamine salts, racemic amphetamine sulfate, triple bead mixed amphetamine salts, substance P antagonists such as Apraxol 111. The method or composition of any one of claims 1 to 110, comprising one or more of: amitriptyline (Elavil®), amoxapine, buspirone (Buspar™), clomipramine, desipramine (Norpramin®), doxepin, imipramine (Tofranil®), maprotiline, nortriptyline (Pamelor™), protriptyline, and trimipramine; and a vasopressin 1B (V1B) receptor antagonist, such as nervaptan (SSR149415).

112. the cargo in the MEV comprises a neurotrophic factor, including, but not limited to, catalase, GFP, luciferase, nerve growth factor (NGF), TrkA (tropomyosin kinase A), NT-3, NT-4, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), psilocybin / psilocin, harmine, temozolomide, rivastigmine, and / or rhodamine; 112. The method or composition of any one of claims 1-111, optionally wherein the composition is formulated for neurons, astrocytes, oligodendrocytes, microglial cells, ependymal cells, and / or neural stem cells for administration via intranasal administration.

113. 113. The method or composition of any one of claims 1 to 26 and 28 to 112, wherein the cargo in the MEV is endogenously loaded by genetically modified microalgae.

114. 113. The method or composition of any one of claims 1 to 25 and 27 to 112, wherein the cargo in the MEVs is exogenously loaded into purified or partially purified MEVs.

115. The microalgae is a species of Chlorella; the MEVs in the composition contain a heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEVs, such that, on average, the vesicles in the composition that contain the heterologous bioactive molecular cargo contain the same heterologous cargo; the cargo is heterologous to Chlorella; 115. The method or composition of any one of claims 1 to 114, wherein the cargo is a biomolecule or a small molecule drug.

116. The MEV is a Chlorella extracellular vesicle, The Chlorella extracellular vesicles comprise a heterologous bioactive molecular cargo endogenously introduced into the extracellular vesicles by the microalgae; the cargo molecule is heterologous to Chlorella; 115. The method or composition of any one of claims 1 to 114, wherein said bioactive cargo is a biomolecule for treating a disease, disorder or condition of or involving the brain.

117. 117. The method or composition of any one of claims 1-116, wherein the microalgae is a species of Chlorella selected from among Chlorella ellipsoidia, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis.

118. 117. The method or composition of any one of claims 1 to 116, wherein the microalgae is a member of the Chlorellaceae family.

119. 117. The method or composition of any one of claims 1 to 116, wherein the Chlorella species is Parachlorella species.

120. 119. The method or composition of claim 118, wherein the Chlorella species is Chlorella vulgaris or a parachlorella selected from among Parachlorella kesslerii, Parachlorella beijerinckii, and Parachlorella hassii.

121. 119. The method or composition of any one of claims 1 to 118, wherein the biologically active molecule is any molecule that can be used to detect or diagnose a disease, disorder, or condition according to any one of claims 103 to 112.

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