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

Abstract

Compositions and drug delivery systems are provided that contain microalgae-derived extracellular vesicles (MEVs) loaded with biologically active cargo. The MEVs may be formulated and administered by a variety of routes of administration and have a variety of applications as therapeutics, including as vaccines, as anti-cancer therapeutics, as therapeutics for mental diseases, disorders and conditions, as diagnostics, and other such uses.

Description

[Technical Field]

[0001] Related Applications The benefit of priority is claimed to U.S. Provisional Application No. 63 / 418,959, filed October 24, 2022, entitled "Extracellular Vesicles from Microalgae, Their Biodistribution Upon Administration, and Uses," to inventors Lila Drittanti and Manuel Vega, and to applicant AGS Therapeutics SAS.

[0002] The benefit of priority is claimed to U.S. Provisional Application No. 63 / 368,975, filed July 20, 2022, entitled "Extracellular Vesicles from Microalgae, Their Biodistribution Upon Administration, and Uses," to inventors Lila Drittanti and Manuel Vega, and to applicant AGS Therapeutics SAS.

[0003] The benefit of priority is claimed to U.S. Provisional Application No. 63 / 305,230, filed January 31, 2022, entitled "Extracellular Vesicles from Microalgae, Their Biodistribution Upon Administration, and Uses," to inventors Lila Drittanti and Manuel Vega, and to applicant AGS Therapeutics SAS.

[0004] This application is related to International PCT Application No. PCT / EP2022 / 070371, filed July 20, 2022, entitled "Extracellular Vesicles from Microalgae, Their Preparation, and Uses," to inventors Lila Drittanti, Juan Pablo Vega, Jeremy Pruvost, and Manuel Vega, and to 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.

[0005] This application is also related to priority applications U.S. Provisional Application No. 63 / 223,828, filed July 20, 2021, and U.S. Provisional Application No. 63 / 224,656, filed July 22, 2021, respectively, entitled "Extracellular Vesicles from Microalgae, Their Preparation, and Uses," to inventors Lila Drittanti, Juan Pablo Vega, Jeremy Pruvost, and Manuel Vega, and to applicants AGS Therapeutics SAS, 10 rue Greneta, 75003 Paris, France; AGS-M SAS, 41-43 Quai de Malakoff, 44000 Nantes, France; and Universite de Nantes, 1 Quai de Tourville, 44000 Nantes, France, which was renamed Nantes Universite on January 1, 2022.

[0006] This application is also related to U.S. Provisional Application No. 63 / 349,006, filed June 3, 2022, entitled "Extracellular Vesicles from Genetically-Modified Microalgae Containing Endogenously Loaded Cargo, Their Preparation, and Uses," to inventors Lila Drittanti and Manuel Vega, and applicant AGS Therapeutics SAS.

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

[0008] Incorporation by reference of electronically submitted sequence listings 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 January 20, 2023, is 563 kilobytes in size, and is entitled 5505SEQPC01.XML.

[0009] Field Compositions containing microalgae-derived extracellular vesicles (MEVs) exogenously loaded with bioactive cargo and their use for delivery to various tissues and organs are provided. The MEVs contain bioactive cargo, such as therapeutic drugs, and the MEVs can serve to deliver the cargo to specific organs and tissues for the treatment of 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 cell membrane, and microvesicles (approximately 50-1000 nm), which are generated by the outward budding of membrane vesicles from the cell surface. Exosomes and microvesicles have similar properties and are commonly referred to as EVs.

[0011] EVs facilitate intercellular communication through the intercellular transfer of proteins and nucleic acids, such as microRNA (miRNA), long non-coding RNA (lncRNA), and mRNA. For this reason, mammalian and plant-derived EVs have been used as carriers for small interfering RNA (siRNA) delivery, microRNA (miRNA), and small molecule drugs. They are promising delivery vehicles. Conveniently produced EVs that can be easily delivered to cells and tissues are needed. Providing such EVs is the objective of this paper. Summary of the Invention

[0012] Cargo-loaded extracellular vesicles (EVs) are provided for use in administering to subjects in vivo and to cells and cell lines in vitro. The EVs are loaded with cargoes containing bioactive molecules, including biomolecules and small molecules, e.g., diagnostic and / or therapeutic molecules. The EVs are derived from microalgae. Microalgae are unicellular green algae, including those belonging to the order Chlorellales, particularly the family Chlorellaceae, and in particular those belonging to the genus Chlorella, e.g., Chlorella vulgaris. Microalgae extracellular vesicles (MEVs) can be produced on a large scale. The MEVs are exogenously loaded with bioactive molecular cargo. MEVs can be endogenously loaded (end-loaded) 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 package into MEVs.

[0013] MEVs can be exogenously loaded after isolation or partial purification / isolation of microalgae-derived MEVs by contacting the MEVs with a cargo to produce a composition in which substantially all of the MEVs have substantially identical exogenously loaded heterologous cargo. Biodistribution patterns do not vary depending on how the MEVs are loaded (see, e.g., Example 14, in which exogenously and endogenously loaded MEVs (as a control) deliver biologically active cargo).

[0014] The MEVs provided herein have unique biodistribution patterns that are a function of the route of administration. The biodistribution of MEVs differs from mammalian EVs and other EVs and / or nanoparticles. For example, mammalian EVs delivered systemically accumulate in the liver, kidney, and spleen. Some mammalian-derived secreted EVs have limited pharmaceutical acceptability (see, e.g., International PCT Publication No. WO2021 / 122880). While others have shown that certain photosynthetic microalgae release EVs into the growth medium, the use of such EVs as drugs or drug delivery vehicles, and their fate upon administration, has not been described or understood. Herein, MEVs are shown to distribute to organs and tissues differently from mammalian EVs upon administration via various routes. As an example, mammalian EVs, with the exception of bovine milk EVs, cannot be administered orally because they do not survive the harsh environment of the stomach, whereas MEVs can be administered orally and delivered to the intestine, from where they are transported to the spleen, including the white spleen. For example, it is shown herein that intranasally administered MEVs follow specific transport patterns and are transported to specific regions of the brain.

[0015] As shown and described herein, MEVs traverse the intrinsic pathway to the brain upon intranasal (IN) administration. Upon IN administration, MEVs are internalized by olfactory sensory neurons (OSNs) and then travel to the glomerulus. Upon reaching the glomerulus from the OSNs, MEVs enter mitral and tufted neurons and migrate intracellularly along distinct pathways with distinct kinetics through the lateral olfactory tract (LOT). The LOT is composed of long axons of mitral and tufted neurons that lead from the olfactory bulb (OB) to various anterior-posterior brain regions, which are directly involved in the connected olfactory network, including the anterior olfactory nucleus, olfactory tubercle, tectal septum, piriform cortex, amygdala, and entorhinal cortex. Collateral branches of the main long axons of mitral / tufted neurons enter and colonize each of the brain regions: the anterior olfactory nucleus, olfactory tubercle, tectal septum, piriform cortex, amygdala, and entorhinal cortex. Within these regions, the mitral / tufted axons connect (via synapses) with neurons from other regions, including the frontal cortex, hypothalamus, thalamus, and hippocampus, which have more secondary olfactory roles.

[0016] The areas reached by MEV via IN administration include all brain regions connected to the olfactory nerve and lateral olfactory tract (LOT) in both hemispheres, as well as ventral, lateral, and dorsal, external, and internal regions along the anterior-posterior axis, including the anterior olfactory nucleus, olfactory tubercle, stenosis operculum, 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 and dentate gyrus).

[0017] MEVs are loaded with a variety of cargoes (also referred to as "payloads"), including, but not limited to, RNA, e.g., inhibitory RNA and other RNA products, oligonucleotides, plasmids, peptides, proteins, and / or small molecules. As demonstrated herein, MEVs can deliver cargoes to organs, tissues, and cells, which may be targeted by a route of delivery to which they can be delivered. It is shown herein that MEVs, including Chlorella MEVs, have a remarkable ability to cross stringent natural barriers, such as the gastrointestinal tract and olfactory neurons, a capability not shared by other extracellular vesicles (EVs) derived from other sources, including mammalian EVs.

[0018] As described herein, MEVs can be exogenously loaded (exoloaded) with a variety of biologically active molecules, e.g., 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 the discussion herein, and also U.S. Provisional Application No. 63 / 349,006, filed June 3, 2022). As shown herein, MEV biodistribution is determined by the route of administration. Thus, depending on whether MEVs are administered intratracheally, orally, intravenously, or intranasally, they can deliver their cargo to various tissues and organs, including, for example, the lungs, intestine, GALT, spleen, liver, and brain.

[0019] As demonstrated herein, MEVs have numerous uses, including therapeutic uses including delivery of therapeutic agents for the treatment and / or prevention (including reducing the risk or severity) of diseases, disorders, and conditions. These uses include therapeutic uses including the treatment of immune modulation, immuno-oncology, genetic or metabolic disorders, neurological disorders, psychiatric disorders, and respiratory disorders, among others.

[0020] Cargo (also called "payload") includes, but is not limited to, RNA, e.g., inhibitory 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 modified to express or encode a product, produce MEVs containing cargo, e.g., RNA, DNA, peptides, small peptides, polypeptides, and proteins, that are produced and packaged in the EVs by the microalgae.

[0021] MEVs can deliver cargo to organs, tissues, and cells, potentially targeted by the route of delivery. Herein, we demonstrate that MEVs, including Chlorella MEVs, have a remarkable ability to cross stringent natural barriers, such as the gastrointestinal tract and olfactory neurons, a capability not shared by other extracellular vesicles (EVs) derived from other sources, including mammalian EVs.

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

[0023] Methods for loading MEVs are described and provided. The cargo is a bioactive molecule or a combination thereof, including biomolecules and small molecules. Cargo includes, for example, biomolecules including biopolymers such as DNA and RNA, proteins, protein complexes, protein-nucleic acid complexes, and plasmids, as well as small molecules such as small molecule drugs. Bioactive molecules include therapeutic agents such as anti-cancer compounds and biomolecules, such as RNAi, oligonucleotides, proteins, and complexes, and diagnostic molecules, such as detectable markers, cosmetic molecules, and molecules that act as anti-infective agents for humans, animals, and plants. Methods for treating diseases and disorders, including pathogen infections and cancer, and the use of MEVs for the treatment of diseases and disorders are provided, as well as methods of diagnosis.

[0024] Cargo-loaded MEVs have applications in a variety of fields, including the diagnosis, prevention, and treatment of diseases in humans and other animals, industrial use, cosmetic use, veterinary use, and use in the crop industry. MEVs carrying the appropriate cargo for each application can be used for industry and research, analytical methods, cell-based assays, and other uses and applications, such as vaccines, gene therapy delivery vectors, gene silencing, gene editing, and transfection. Cargo-loaded MEVs can be used to treat 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), disorders of the skin including natural disorders and disorders induced by trauma, disorders of the genitourinary tract, disorders of the nasal-buccal oral cavity, disorders of the cardiovascular system, immune and immune modulation 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, etc.), 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 including, e.g., embryonic or tissue-specific stem cells, liver cells, infected cells, e.g., cells infected with a virus, bacteria, fungus or other pathogen, natural cells and genetically engineered NS cells.

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

[0027] For all embodiments, the Chlorella species is any species of Chlorella, including, but not limited to, Chlorella selected from among Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. In certain embodiments, the Chlorella species is Chlorella vulgaris.

[0028] A composition containing isolated microalgal extracellular vesicles (MEVs) is provided, wherein the microalgae is a species of the genus Chlorella, and the MEVs in the composition contain heterologous bioactive molecular cargo introduced into the isolated MEVs, thereby allowing the vesicles in the composition containing the heterologous bioactive molecular cargo to contain the same cargo. The cargo is heterologous to the genus 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 in plants or animals. Generally, cargo is bioactive. Bioactive cargo includes any molecule, such as a biomolecule, including biopolymers and small molecules, that can have an effect on plants or animals when administered. Cargo includes, for example, proteins, peptides, and nucleic acids. Bioactive molecules may be synthetic, naturally occurring, and / or modified to change their properties or activity. They include any molecule used as a drug, therapeutic agent, diagnostic agent, cosmetic, or in industry. Cargo may be, but is not limited to, a therapeutic agent for treating or preventing a disease, disorder, or condition, or for treating or preventing its symptoms. Cargo may be a nucleic acid molecule, polypeptide, protein, plasmid, aptamer, or antisense oligonucleotide.

[0030] The cargo in the MEV in the composition may comprise a biopolymer. The biopolymer may be a naturally occurring biopolymer, a synthetic biopolymer, or a modified biopolymer. The biopolymer may be a nucleic acid or a protein containing modifications, including insertions, deletions, substitutions, and rearrangements of nucleotide or amino acid residues, and / or the biopolymer may be a protein, and the modifications may 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 molecules that can be cargo.When cargo is RNA or protein, it can be provided as cargo or can be encoded by nucleic acid, and then expressed in the organism that is administered.Examples of RNA include inhibitory RNA (RNAi) and mRNA, including modified mRNA.RNAi includes, for example, silencing RNA (siRNA) or short hairpin RNA (shRNA), micro-RNA (miRNA), short activating RNA (saRNA) and long non-coding RNA (lncRNA).RNA products also include double-stranded RNA and ribozyme.Cargo can also be oligonucleotide, for example, antisense oligonucleotide or allele-specific oligonucleotide. The cargo may include gene editing systems, such as CRISPR-CAS systems, and modified and improved gene editing systems, such as CRISPR-associated and CRISPR-like systems (see, e.g., published U.S. Patent Application Nos. 20200332273 and 20200332274, respectively, to applicant Metagenomi).

[0032] Cargos include therapeutic, diagnostic, or theragnostic proteins or peptides, protein complexes, and complexes containing two or more proteins, or proteins and nucleic acids, or proteins and aptamers, or combinations of proteins, nucleic acids, and other molecules. Cargos can be proteins that are, or encode, antibodies or antigen-binding fragments thereof. Antibodies can be in any form, including single-chain, nanobody, camelid, 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, or 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 checkpoints or immunosuppressants or tumor antigens.

[0033] The cargo in the MEV in the composition may include an immunostimulatory product or antigen and can be used as a vaccine to induce an immunoprotective response upon administration. The cargo may be DNA, RNA, protein, or virus. The cargo may contain a nucleic acid, protein, or protein-encoding nucleic acid that is a therapeutic product for the treatment of cancer, an infectious disease, a neurodegenerative disease, another CNS disorder, aging, an aging-related disease, an ocular disorder, or an immunological disorder. The cargo may be a functional cosmetic, a 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 or 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.

[0034] The cargo may comprise DNA. The DNA may be a plasmid, e.g., encoding a product for expression in the animal or plant to which it is administered. Exemplary products include therapeutic products and diagnostic agents. These include proteins and RNA products, including those listed above. Because MEVs are intended for administration to animals and plants, the plasmid generally encodes the product 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, for example, RNA polymerase II promoters derived from animals, plants, and plant or animal viruses. Exemplary promoters include, but are not limited to, the cytomegalovirus promoter, the simian virus 40 promoter, the herpes simplex promoter, the Epstein-Barr virus promoter, the adenovirus promoter, synthetic promoters, the actin promoter, 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.

[0035] The plasmid may encode one or two or more cargo products. For expression of the cargo products, the encoding nucleic acid is operably linked to a regulatory sequence recognized by the eukaryotic cell.

[0036] A method for preparing MEVs is provided. The method includes 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; these methods include, for example, one or more of electroporation, sonication, extrusion, and the use of surfactants. In some embodiments, the MEVs are derived from a species of the genus Chlorella, such as, but not limited to, Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. Any of the MEVs produced by the methods and provided herein, including compositions containing MEVs, can be used as one or more of a method of diagnosis, a vaccine, a therapy for treatment, a diagnostic for disease, a treatment for 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 diseases, disorders, or conditions. Exemplary diseases, disorders, and conditions include cancer, including solid tumors or hematological malignancies or their metastases. 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 pathogens. Infectious pathogens include bacteria, viruses, parasites, prions, oomycetes, and fungi.

[0038] The cargo can provide therapeutic molecules for treatment or can induce an immune response and act as a vaccine. The MEV can contain a cargo that contains or encodes an immunostimulatory protein or antigen, such that upon administration, the MEV is immunostimulatory and elicits an innate or adaptive immune response, or the MEV and / or cargo can elicit an immunoprotective response to prevent or treat a disease, disorder, or condition. MEVs can be used to treat diseases, disorders, or conditions resulting from trauma. Trauma includes, but is not limited to, trauma from or involving wounds, burns, surgery, open skin, fractures, hair loss, exposed dermis, exposed mucosa, fibrosis, lacerations, and ulcers. MEVs can be used to induce effects that treat conditions resulting from natural aging or aging induced by pathogenic or diseased or otherwise. Other diseases, disorders, and conditions that can be treated by MEVs include skin or eye diseases, disorders, and conditions. These include dermatitis, wrinkles and / or other age-related changes to the skin, macular degeneration, glaucoma, diabetic retinopathy, cataracts or conditions resulting from diabetic retinopathy.

[0039] Compositions containing MEV can be formulated for administration by any route, including but not limited to, local, systemic, topical, parenteral, enteral, mucosal, pulmonary inhalation or intranasal, vaginal, rectal, otic, oral, and other routes of administration. MEV can be formulated in any form, including as a tablet, liquid, e.g., emulsion, powder, cream, gel, or aerosol, with the form and formulation being appropriate for each route of administration, including oral, nebulized, and inhaled.

[0040] The composition or MEV can be used in any of the methods and treatments described herein or known to those skilled in the art.Methods include any of those described herein, including for example, gene silencing, gene interference, gene therapy, gene / protein overexpression, gene editing, inhibition or stimulation of protein activity and pathway signal transduction.The composition and MEV can be used for prevention and / or vaccination.They can be used in agricultural and veterinary applications, such as crop science, plant pathogens and animal diseases, for dermatological applications, and for cosmetic applications.They can be used for industrial purposes, such as manufacturing, characterization, and calibration.

[0041] Provided are methods for treating diseases, disorders, and conditions, wherein treatment can be achieved by delivering active agents to the brain. Provided are compositions prepared for intranasal delivery. The compositions contain microalgal extracellular vesicles containing active agents. Microalgal extracellular vesicles can be loaded by any suitable method (see International Patent Publication No. PCT / EP2022 / 070371, U.S. Provisional Application No. 63 / 349,006 and the methods and MEVs described), including exogenous loading after MEV production and endogenous loading in vivo by microalgae genetically modified to package nucleic acids or encoded products into MEVs.

[0042] EVs are derived from microalgae, including unicellular green algae of the Chlorellales order, particularly those in the Chlorellaceae family, and in particular those in the genus Chlorella, such as Chlorella vulgaris. MEVs are provided in compositions formulated for nasal administration. MEVs can be exogenously loaded after isolation or endogenously loaded in vivo by genetically engineered microalgae that encode and package heterologous nucleic acids and / or proteins in MEVs. The advantages of exogenously loading cargo into MEVs are 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 cargo / MEV amount. 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 that package heterologous nucleic acids and / or proteins.

[0043] The resulting MEVs, whether end-loaded or exo-loaded, are not toxic, can be administered into cells in vitro, or can be administered in vivo, and have distribution patterns that vary depending on the route of administration. For purposes herein, MEVs are intended for delivery to the brain via intranasal administration.

[0044] It is shown herein that MEVs are transported to the brain via unique pathways and mechanisms after intranasal (IN) administration. These pathways and mechanisms are not shared by exosomes or nanoparticles from other sources. Following intranasal delivery, it is shown herein that MEVs are transported via the olfactory nerve through the lateral olfactory tract (LOT) to multiple interconnected brain regions. MEVs are transported via intraneuronal axonal transport and also via transsynaptic transport between neurons. MEVs have the ability to transsynapse at least: (i) synapses between olfactory sensory neurones (OSNs) and mitral / tufted neurons, (ii) synapses between mitral / tufted neurons and local neurons in various brain regions colonized by the lateral olfactory tract (LOT), and (iii) synapses between neurons in brain regions colonized by the LOT and neurons from the frontal cortex, hippocampus, thalamus, and hypothalamus.

[0045] The biodistribution of MEVs follows the pathways and connections in the olfactory neural network and mitral / tufted neurons throughout the brain. These transport pathways provide access (biodistribution) to brain regions (within 1 to 16 hours after IN administration), including the anterior olfactory nucleus, olfactory tubercle, operculum, 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 brain regions. Payloads can also include, but are not limited to, proteins, mRNA, DNA, small molecules, any agent that can be exogenously loaded (exoloaded) into MEVs or packaged in vivo into MEVs by microalgae that encode or produce the agent, particularly genetically engineered microalgae. MEVs thus 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 provide therapeutic and diagnostic uses, as well as diagnostic and experimental uses. Delivery is illustrated in the Examples, which show effective delivery and expression of various exemplary cargoes, e.g., 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 / or rhodamine, to neurons, astrocytes, glial cells, and / or neural stem cells in vitro and in vivo.

[0046] MEVs are unique vehicles for delivering cargo to specific tissues, and delivery varies depending on the route of administration. As shown, in vivo transport of MEVs can be distinguished from MEVs from other sources. For example, in contrast to mammalian EVs, MEVs can be administered orally and transported through the GALT. Intranasally administered MEVs are transported to the brain via a unique route and can be used, for example, (i) for the treatment and / or prevention of brain disorders, including, but not limited to, cognitive, emotional, behavioral, psychiatric, neurological, degenerative, and cancer disorders; (ii) for in vitro or in vivo research of brain disorders; (iii) for the diagnosis of brain disorders; and (iv) for recreational and therapeutic uses.

[0047] MEVs and brain delivery For purposes herein, MEVs are for delivery to the brain by intranasal administration. MEVs can be used to deliver bioactive cargoes for treatment, including preventing or reducing the risk of diseases, disorders, and conditions of or involving the brain, and / or for detection or diagnosis of diseases, disorders, or conditions of or involving the brain, and / or for monitoring the treatment of such diseases, disorders, and conditions.

[0048] Generally, compositions and drug delivery systems for delivery to the brain are provided. As described above and herein, intranasal administration achieves brain delivery. It is also shown herein that MEVs are transported to the brain via unique pathways and mechanisms after intranasal (IN) administration. These pathways and mechanisms are not shared by exosomes or nanoparticles from other sources. It is also shown herein that, after intranasal delivery, MEVs are transported via the olfactory nerve through the lateral olfactory tract (LOT) to multiple interconnected brain regions. MEVs are transported via neuronal axonal transport. MEVs have the ability to transsynapse at least: (i) synapses between olfactory sensory neurons (OSNs) and mitral / tufted neurons, (ii) synapses between mitral / tufted neurons and local neurons in various brain regions colonized by the lateral olfactory tract (LOT), and (iii) synapses between neurons in brain regions colonized by the LOT and neurons from the frontal cortex, hippocampus, thalamus, and hypothalamus.

[0049] Compositions and uses thereof are provided, the compositions comprising microalgal extracellular vesicles (MEVs) that deliver bioactive molecules to the brain or to cells in vitro after intranasal administration, the MEVs comprising the bioactive molecule as cargo. Methods for delivering bioactive molecules to the brain by intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) comprising a cargo that is a bioactive molecule are provided.

[0050] Also provided are cell compositions and cell therapy compositions containing MEVs, where the cargo is for treating diseases, disorders, and conditions of specific organs, tissues, and systems. The cell compositions can be prepared in vitro by introducing MEVs containing a cargo for treating a specific disease, disorder, or condition, and then administering the cell therapy to the cells. For example, a composition containing MEVs can be administered to neurons, astrocytes, glial cells, and / or neural stem cells in vitro, and the resulting cells can be used for cell therapy, such as by administration to the brain.

[0051] For all embodiments of the methods 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 by intranasal administration.

[0052] The biodistribution of MEVs follows the pathways and connections in the olfactory neural network and mitral / tufted neurons throughout the brain. These transport pathways provide access (biodistribution) to brain regions (within 1 to 16 hours after IN administration), including the anterior olfactory nucleus, olfactory tubercle, operculum, 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 brain regions. Payloads can also include, but are not limited to, proteins, mRNA, DNA, small molecules, and any agent that can be exogenously loaded (exoloaded) into MEVs or packaged in vivo into MEVs by microalgae that encode or produce the agent, particularly genetically engineered microalgae. MEVs thus 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 provide therapeutic and diagnostic uses, as well as diagnostic and experimental uses. Delivery is illustrated in the Examples, which show 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, tomozolonide, rivastigmine, and rhodamine to neurons, astrocytes, glial cells, and / or neural stem cells in vitro and in vivo.

[0053] 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) for in vitro or in vivo research of brain disorders; (iii) for diagnosis of brain disorders; and (iv) recreational and therapeutic uses.

[0054] A method of delivering a bioactive molecule to the brain is provided by intranasally administering a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, whereby the MEVs travel 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, stenosis tectum, 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 nucleus of the amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus, where the bioactive molecule is any molecule that can achieve treatment of 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 of a disease, disorder, or condition, and the bioactive molecule is heterologous to the microalgae and / or MEVs.

[0055] Methods of delivery include methods of treatment, including intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, whereby the MEVs travel via the olfactory nerve to the brain and to interconnected brain regions through the lateral olfactory tract (LOT) for delivery to one or more of the following: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, stenosis tectum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association area, basolateral nucleus of the amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus, where the bioactive molecule is any molecule capable of treating a disease, disorder, or condition of or involving the brain, and the bioactive molecule is heterologous to the microalgae and / or MEVs, thereby providing a method of treating a disease, disorder, or condition of the brain or involving the brain.

[0056] Also, a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive molecules is administered intranasally, whereby the MEVs travel via the olfactory nerve to the brain and to interconnected brain regions through the lateral olfactory tract (LOT) for delivery to one or more of the following: the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, stenosis tectum, 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 nucleus of the amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus (fimbria, dentate gyrus), wherein the bioactive molecules comprise a reporter or detectable marker, and the bioactive molecules are useful for treating diseases, Methods for detecting or monitoring the treatment of a disease, disorder, or condition of the brain or a disease, disorder, or condition involving the brain are provided, where the bioactive molecule is any molecule that can be used to detect or diagnose a disorder or condition, or that can be used to monitor the treatment of a disease, disorder, or condition, or that can be used to detect or diagnose and treat a disease, disorder, or condition, wherein the disease, disorder, or condition is of or involving the brain, and the bioactive molecule is heterologous to the microalgae and / or MEV.

[0057] Compositions are provided that include microalgal extracellular vesicles (MEVs) containing a cargo comprising a bioactive molecule, the compositions being suitable for intranasal delivery to interconnected brain regions via the lateral olfactory tract (LOT) for delivery via the olfactory nerve to the brain, to one or more of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, stenosis tectum, 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 nucleus of the amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, 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 detects, diagnoses, monitors and / or treats a disease, disorder or condition, and 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.

[0058] Also provided are compositions comprising microalgal extracellular vesicles (MEVs) for use in delivering 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 the treatment of a disease, disorder, or condition of or involving the brain, wherein the MEVs comprise the bioactive molecule for delivery to the brain, and the composition is delivered to the brain via the olfactory nerve through the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, tectal strip, piriform cortex, amygdala, entorhinal cortex, primary olfactory cortex, or olfactory septum. and formulated for intranasal delivery to brain regions interconnected through the lateral olfactory tract (LOT) for delivery to one or more of the motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, retrosplenial granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus, wherein the bioactive molecule is any molecule that can effect treatment of 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 of a disease, disorder, or condition, and the bioactive molecule is heterologous to the microalgae and / or MEVs.

[0059] Consistent with these methods and compositions, MEVs are transported via intraneuronal axonal transport and interneuronal transport across synapses. For example, MEVs are transported in 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 neurons and mitral / tufted neurons throughout the brain. Upon intranasal administration, MEVs pass through one or more of the following: (i) synapses between olfactory sensory neurons (OSNs) and mitral / tufted neurons; (ii) synapses between mitral / tufted neurons and local neurons in brain regions colonized by LOT; and (iii) synapses between neurons in brain regions colonized by LOT and neurons from or to the frontal cortex, hippocampus, thalamus, and hypothalamus. For example, MEVs pass through (i), (ii), and (iii), or (i) and (ii), e.g., the pathways traveled by MEVs upon intranasal administration depicted in Figure 35.

[0060] The methods and compositions provided herein are for intranasal administration.After intranasal administration, MEV is delivered to, for example, one or more of the corpus callosum, dorsal fornix, dorsal hippocampal commissure and fimbria of the hippocampus, or is for delivery to one or more of the corpus callosum, dorsal fornix, dorsal hippocampal commissure and fimbria of the hippocampus.

[0061] The compositions provided herein and used in the methods can be formulated as suspensions or emulsions, e.g., nanoemulsions or microemulsions. Those skilled in the art understand and are familiar with the properties of nanoemulsions and microemulsions and their formation. In the compositions, MEVs contain bioactive cargo. For example, MEVs can be prepared so that, on average, each MEV contains a predetermined amount of bioactive molecules, e.g., 1 to 100 bioactive molecules per MEV. The selection of the amount of cargo per MEV is within the skill of those in the art and will vary depending on factors known to those skilled in the art, such as the particular disease, disorder, or condition being treated or the use of the MEV, the subject, the particular cargo, and other such parameters and factors. Similarly, the concentration of the MEV will vary depending on the particular cargo and use. For example, the concentration of the MEV in the composition can be about 0.1 to 10 mg / mL or 0.1 to 10 mg / mL, as well as lower, higher, and intermediate concentrations. The compositions may be formulated for unit administration (direct administration without dilution) or for multiple dose administration in aliquots and / or for dilution to a desired concentration. 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 suitable volume. The compositions may be administered as a single dose or as a series of doses or other regimens. The compositions may be administered as part of a combination therapy protocol.

[0062] 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.

[0063] Compositions and methods include those in which MEVs are endogenously loaded with genetically modified microalgae encoding a bioactive molecule or a pathway for its production. MEVs also include those in which cargo is exogenously loaded into purified or partially purified MEVs. MEVs may also 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.

[0064] Microalgae used to generate MEVs for use in the present methods can be from a microalgae phylum selected from among Euglenophyta (euglenid algae), Chrysophyta (chrysophytes and diatoms), Pyrrophyta (fire algae), Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae), and Xanthophyta (yellow-green algae). For example, the microalgae is from the Chlorophyceae or Trebouxiophyceae or Chlorophyta phylum, e.g., a species of the genus Chlorella or Chlamydomonas.

[0065] Species of the genus Chlorella include, but are not limited to, Chlorella ellipsoidea, 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 species of the genus Chlorella, and the MEVs in the composition contain a heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEVs, such that the vesicles in the composition containing the heterologous bioactive molecular cargo contain, on average, the same heterologous cargo, where the cargo is heterologous to the genus Chlorella, and the cargo is a biomolecule or small molecule drug or any cargo for delivery to the brain as described herein and / or known to those of skill in the art. Also included are methods and compositions in which the MEVs are Chlorella extracellular vesicles, and the Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo that has been endogenously introduced into the extracellular vesicles by the microalgae, where the cargo molecule is heterologous to the genus Chlorella, and the bioactive cargo is a biomolecule for treating a disease, disorder, or condition of or involving the brain.

[0066] Methods and compositions are provided in which the MEVs are Chlorella extracellular vesicles, and the Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo introduced into the isolated extracellular vesicles, whereby vesicles in a 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, and the Chlorella extracellular vesicles contain a heterologous bioactive molecular cargo endogenously introduced into the extracellular vesicles by microalgae, whereby vesicles in a 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 MEV, such that, on average, the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same cargo, where the cargo is heterologous to the Chlorella genus and the cargo is a biomolecule or small molecule. In other embodiments, the cargo is endogenously introduced into the MEV by modifying the microalgae to express or produce the cargo, e.g., a nucleic acid or protein or the product of a biochemical pathway. In an exemplary embodiment, the Chlorella genus is Chlorella vulgaris.

[0067] Cargos include, but are not limited to, biomolecules, biopolymers, such as naturally occurring biopolymers, synthetic biopolymers, or modified biopolymers, such as nucleic acid molecules, polypeptides, proteins, plasmids, aptamers, or antisense oligonucleotides. Cargos include, but are not limited to, DNA or RNA, such as inhibitory RNA (RNAi), mRNA or modified mRNA, silencing RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), self-amplifying RNA, short activating RNA (saRNA), long non-coding RNA (lncRNA), ribozymes, or double-stranded RNA. Cargos include oligonucleotides, such as antisense oligonucleotides or allele-specific oligonucleotides or antisense oligonucleotides (ASOs), gene editing systems, such as CRISPR-CAS systems, CRISPR-related or CRISPR-like systems, etc. The cargo may comprise DNA, e.g., a plasmid, encoding a therapeutic and / or detectable or diagnostic product or an RNA product, e.g., RNAi and antisense oligonucleotides, or ribozymes, or forms of RNA such as those described above, including double-stranded RNA. The plasmid can 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, an actin promoter, or other promoters such as synthetic chimeric promoters. The plasmid can also contain other regulatory sequences for expression, e.g., other eukaryotic transcriptional and translational sequences. The MEV cargo can include a small molecule for effecting treatment, detection, diagnosis, or monitoring of a disease, disorder, or condition of or involving the brain.

[0068] Cargo includes any molecule of interest for delivery to the brain. This includes cargo that encodes or is an immunomodulator, such as an immunomodulator that increases or decreases production of one or more cytokines, upregulates or downregulates autoantigen presentation, masks MHC antigens, or promotes proliferation, differentiation, migration, or activation state of one or more types of immune cells. The cargo can comprise or encode a hormone, cytokine, or chemokine. The cargo can comprise 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 can comprise or encode an antibiotic, antiviral, antifungal, antiparasitic, or other anti-infective agent for treating infections of or involving the brain. The cargo can include a nucleic acid encoding a therapeutic nucleic acid or protein or a protein that is a therapeutic product for treating cancer or tumors in the brain, or infectious diseases in the brain, or neurodegenerative diseases or other central nervous system (CNS) disorders, or for treating 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, e.g., an scFv, a bispecific antibody or antigen-binding fragment thereof, etc. The cargo may comprise a nucleic acid for gene therapy.

[0069] The MEV may contain more than two cargo products. The cargo may include 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. Diagnostic agents may include 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. Bioactive molecule cargo may include any molecule that has an effect on a cell or organism to which it is delivered, or is detectable, or acts as a detectable marker or biomarker, thereby achieving treatment, detection, diagnosis, or monitoring of treatment of a disease, disorder, or condition of or involving the brain.

[0070] The cargo may include 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 the treatment of a disease, disorder, or condition of or involving the brain. The cargo may include, 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.

[0071] The disease, disorder, or condition may include a tumor in the brain. The cargo may include, for example, an oncolytic virus that infects a neural tumor, or may include a therapeutic agent for the treatment of a neural tumor. 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 tumor or other brain disorder) or a nervous system disorder (e.g., pain or seizures or 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, 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. The disease, disorder, or condition of or involving the brain may be cancer or a disease, disorder, or condition treated or prevented by a vaccine, and / or may be a disease, disorder, or condition caused by or involving an infectious pathogen, including one or more of bacteria, viruses, oomycetes, parasites, prions, and fungi.

[0072] For treatment, diagnosis, detection, or monitoring, MEVs, when administered intranasally, can deliver cargo to one or more of neurons, astrocytes, glial cells, and neural stem cells. 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. Compositions and methods are provided for the in vitro delivery of bioactive molecules into cells for cell therapy, diagnostics, and / or detection by introducing a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive cargo into cells, where the bioactive molecule is any molecule that can achieve treatment of a disease, disorder, or condition, or that can be used to detect a disease, disorder, or condition, or that can be used to monitor the treatment of a disease, disorder, or condition, and the bioactive molecule is heterologous to the microalgae and / or MEVs. The resulting cells containing the MEVs provided herein can be used for cell therapy to treat diseases, disorders, or conditions of the brain or involving the brain. Cells for cell therapy include, but are not limited to, stem cells, immune cells, or cell lines, provided that the stem cells are not one or more of embryonic, pluripotent, or totipotent stem cells in jurisdictions that exclude embryonic, pluripotent, or totipotent stem cells. The cells can be engineered cells, such as CAR-T cells, which are designed to target specific cells, tissues, and organs.

[0073] The diseases, disorders, and conditions include one or more of cognitive, emotional, 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 be caused by injury 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 cancer, or has anti-aging activity, or has brain regenerative activity.

[0074] The MEV can comprise a cargo for, for example, 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 brain and / or CNS cancers or tumors, genetic disorders, brain injury or trauma, and infectious diseases.

[0075] The cargo may be selected from antidepressants, antipsychotics, anxiolytics, analgesics, psychedelics, 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 can deliver drugs for the treatment of psychiatric disorders and / or psychoses.

[0076] MEVs can be used to deliver cargo, such as hydrophilic compounds that cannot reach the brain after hepatic first-pass metabolism or that have poor intestinal absorption when administered systemically or locally to a location other than the nose, and cargo 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.

[0077] 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 (OCD).

[0078] In some examples, the cargo comprises a bioactive molecule for the treatment of conditions such as:

[0079] JPEG2025505440000002.jpg25483 JPEG2025505440000003.jpg25083 JPEG2025505440000004.jpg25084 JPEG2025505440000005.jpg25082 JPEG2025505440000006.jpg8587

[0080] Diseases, disorders, and conditions involving or of the brain include, but are not limited to, genetic disorders, neurodegenerative diseases, and metabolic disorders, other brain-related conditions, and / or metabolic diseases that affect brain function. Exemplary 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, fear-related disorders, and generalized anxiety disorder; attention deficit hyperactivity disorder, such as inattentive type, hyperactive-impulsive type, and combined 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 disorder type I, bipolar disorder type II, bipolar with mixed features, bipolar with seasonal pattern major depression, cyclothymia, and rapid cycling bipolar; eating disorders, such as anorexia nervosa, bulimia nervosa, muscle dysmorphia, binge eating disorder, and other specific eating or feeding disorders. 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, and obsessive-compulsive disorder (OCD); post-traumatic stress disorder (PTSD), e.g., acute stress disorder, uncommon PTSD, complex PTSD, and comorbid PTSD; classic Rett syndrome, CDKL5-associated atypical Rett syndrome; schizophrenia disorders, e.g., catatonic schizophrenia, disorganized schizophrenia, paranoid schizophrenia, residual schizophrenia, and undifferentiated schizophrenia; and other such mind- 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 chorea, 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 drugs, 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, tomozolonide, rivastigmine, GABAB1A receptor, GABAB1A receptor siRNA, PTEN Neurotropic 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. acetam, licarbazepine, oxcarbazepine, valproic acid and derivatives, lamotrigine; antipsychotics such as Apriprazone, 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 modulating the cholinergic system such as biperiden, scopolamine; corticotropin-releasing factor (CRF) antagonists; drugs modulating the GABAergic system such as benzodiazepines, brexanolone, SAGE-217; glucocorticoid receptor agonists such as hydrocortisone;Drugs involved in glutamatergic modulation, e.g., AGN-241751, AV-101, AVP-786, AVP-923, AXS-05, D-cycloserine, dextromethorphan, rapastinel; glycine and glycine reuptake inhibitors, e.g., sarcosine; drugs modulating the hypothalamic-pituitary-adrenal (HPA) axis, e.g., fludrocortisone, metyrapone, mifepristone, and probiotics; drugs modulating the kynurenine pathway (KP); drugs modulating limbic and paralimbic brain regions. drugs that modulate the melatoninergic 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 (Malplan®), phenelzine (Nardil®), selegiline (Emsam®) and trametinib. Nircypromine (Parnate®); mood stabilizers, such as lithium salts, valproic acid, ebselen, and divalproex; multimodal antidepressants, such as vilazodone and vortioxetine; N-nitrosodimethylamine (NDMA)-receptor antagonists, such as amantadine, alketamine, ketamine, memantine, riluzole, esketamine; neurokinin-1 (NK1) receptor antagonists; neuropeptide Y (NPY) receptor agonists; drugs with neurotrophic effects, cilostazol, sildenafil norepinephrine-dopamine reuptake inhibitors (NDRIs), bupropion (Wellbutrin®, Zyban®, Aprendin®); 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;Psychedelic drugs, 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®, Paxeva®) and celvoxamine; traline (Zoloft®); selective norepinephrine transporter inhibitors, such as atomoxetine; serotonin-norepinephrine reuptake inhibitors (SNRIs), such as desvenlafaxine (Pristiq®), duloxetine (Cymbalta®), levomiraciran (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 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 one or more of a vasopressin 1B (V1B) receptor antagonist, such as nelivaptan (SSR149415). Exemplary cargoes for delivery to the brain may include, for example, catalase, GFP, luciferase, nerve growth factor (NGF), TrkA (tropomyosin kinase A), neurotrophic factors including, but not limited to, NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocybin / psilocin, harmine, tomozolonide, rivastigmine, and / or rhodamine. As described above, compositions containing MEV can be delivered to neurons, astrocytes, glial cells, and / or neural stem cells for gene therapy, via intranasal administration, or for administration to cells in vitro.

[0082] Delivery of Toll-like receptor (TLR) agonists and antagonists The drug delivery systems and compositions provided herein can be used to deliver agonists and / or antagonists of Toll-like receptors (TLRs). Drug delivery systems and compositions are provided that include microalgal extracellular vehicles (MEVs) containing cargos that include agonists or antagonists of Toll-like receptors (TLRs), internalized receptors, and / or intracellular (endosomal) receptors. For example, the cargo in the MEVs can include agonists or antagonists of Toll-like receptors (TLRs), internalized receptors, and / or intracellular (endosomal) receptors. The TLRs and their agonists can be one or more of the following:

[0083] JPEG2025505440000007.jpg100136

[0084] The TLR and antagonist may be one or more of the following:

[0085] JPEG2025505440000008.jpg84131

[0086] Thus, provided herein are MEVs containing cargo that can be used to deliver the cargo to organs, tissues, and / or cells involved in a particular disease, disorder, or condition. The cargo can be selected to treat, diagnose, and / or detect the disease, disorder, or condition, and / or to monitor treatment. Due to the unique transport of MEVs as described and demonstrated herein, MEVs provide a unique delivery vehicle. [Brief explanation of the drawings]

[0087] [Figure 1] FIG. 1 provides an exemplary profile of light intensity used in HECTOR PBR cultures. [Figure 2] Figure 2 shows an exemplary elution profile of a highly pure MEV preparation. MEVs, pre-concentrated by TFF, purified by ultracentrifugation, and formulated in PBS at concentrations of 10-10 per mL, were seeded onto a pre-packed column (qEV1) manufactured by IZON. MEVs were eluted using PBS solution. 0.5 mL elution fractions were collected. MEVs were recovered in the first fraction as indicated. The most concentrated fractions (4-5) were pooled and stored at 4 °C for subsequent use. [Figure 3] FIG. 3 provides an exemplary image of MEVs obtained using a transmission electron microscope (TEM). [Figure 4] FIG. 4 provides an electropherogram of a small RNA library. [Figure 5] FIG. 5 provides representative patterns of biodistribution by route of administration for intravenous (IV), intratracheal (IT), and oral (PO) routes. [Figure 6] FIG. 6 depicts in vivo whole-body imaging of a representative animal after intravenous administration as described in Example 5. [Figure 7] FIG. 7 depicts in vivo whole body imaging of a representative animal per os (oral) administration as described in Example 5. [Figure 8]FIG. 8 depicts in vivo whole-body imaging of a representative animal after intranasal administration as described in Example 5. [Figure 9] FIG. 9 depicts in vivo whole body imaging of a representative animal after intratracheal administration as described in Example 5. [Figure 10] FIG. 10 depicts the kinetics of accumulation in the liver, lungs, and spleen after intravenous administration as described in Example 5 (average of 6 animals). [Figure 11] FIG. 11 depicts the kinetics of accumulation in the lung, spleen, and intestine after oral administration as described in Example 5 (average of 6 animals). [Figure 12] FIG. 12 depicts the kinetics of accumulation in the lungs and kidneys (average of 4 animals) after intranasal administration as described in Example 5. [Figure 13] FIG. 13 depicts the kinetics of accumulation in the lung, spleen, and intestine (average of 3 animals) after intratracheal administration as described in Example 5. [Figure 14] Figure 14A-D represent ex vivo fluorescence analysis (total radiant efficiency) in isolated organs [A) liver; B) spleen; C) lung; and D) brain] 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration. [Figure 15] 15A and 15B show A) hematoxylin and eosin staining of the intestine (G=GALT tissue) and B) DAPI (nuclear) staining and MEV-PKH26 fluorescence. [Figure 16] FIG. 16 shows splenic pulp stained with DAPI (for nuclei) and MEV-PKH26 (red fluorescence), shown as white spots. [Figure 17] FIG. 17 shows a diagram illustrating the migration of MEVs from the GALT to the spleen. [Figure 18]Figures 18A-I show the results of evaluation of the toxicity of MEV in a mouse model after oral (PO) or intratracheal (IT) administration at various doses in four groups of mice for each parameter. MEV toxicity was assessed by 1) chemistry parameters: ALAT, ASAT, urea, and creatine (Figures 18A-D, respectively), and 2) hematology parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (Figures 18E-I, respectively). Groups were as follows: Group 1 mice received 100 μl of PBS via PO delivery (white bars); Group 2 mice received 100 μl of 4*10 MEV / mouse via PO delivery (white bars with black dots); Group 3 mice received 100 μl of 4*10 MEV / mouse via PO delivery (white bars with vertical lines); Group 4 mice received 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), and Figure 181 shows eosinophils. PO indicates per os (oral delivery), and IT indicates intratracheal administration. [Figure 19] FIG. 19 depicts in vivo delivery and expression of mRNA following local instillation of MEV into rabbit eyes. [Figure 20] Figures 20A and 20B depict the in vitro delivery of GFP protein to human monocytes. [Figure 21] 21A and 21B depict the in vitro delivery of GFP protein to human keratinocytes. [Figure 22]Figure 22 shows confocal microscopy observation of Hep-G2 cells containing GFP protein expression in Hep-G2 cells after 24 hours of incubation with GFP-protein loaded MEV (MEV-GFP) or mRNA-eGFP loaded MEV (MEV-mRNA). [Figure 23] FIG. 23 shows confocal microscopy of Huh7 cells containing GFP protein expression in Hep-G2 cells. [Figure 24] FIG. 24 shows the in vitro delivery of MEVs loaded with GFP-mRNA and mRNA encoding GFP into human fibroblasts. [Figure 25] Figures 25A-D show the results of flow cytometry analysis of MEV penetration and delivery studies using human fibroblasts. [Figure 26] FIG. 26 shows the antibacterial activity of Chlorella MEV exogenously loaded with siRNA directed against Pto DC3000 cfa6 and hrpL genes. [Figure 27] FIG. 27 shows delivery of exogenously loaded bioactive flg22 peptide in Chlorella MEVs. [Figure 28] Figure 28 is a schematic diagram showing the pathway through the olfactory epithelium. [Figure 29] Figure 29 shows positive control Dir-MEV on DAPI-stained brain section: a drop of MEV suspension placed on top of a brain tissue section. The dots are Dir-labeled MEVs. [Figure 30] Figure 30 is a schematic diagram of the insula and its connections (reproduced from Gogolla (2017) "The insular cortex," Current Biology:27(12): R580-R586). [Figure 31] FIG. 31 is a schematic diagram of the brain neuronal pathway from olfactory stimulating neurons (OSNs) through the olfactory bulb (OB) to the mitral and tufted neurons to the olfactory tract (OT). [Figure 32]Figure 32 is a schematic diagram showing the pathways and approximate average distances from the 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] Figure 33 is a 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] Figure 34 shows the transport of MEVs through the olfactory pathway. After IN administration, MEVs are taken up by the olfactory epithelium and transported by axonal transport by olfactory sensory neurons to the olfactory bulb, and then 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]Figure 35 depicts 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" (what-when-how.com)]. [Figure 36] Figures 36(a)-(g) show: (a) an overview of the experimental design for the brain biodistribution study; (b) the locations of the five brain sections studied; (c) the regions analyzed to determine the PK and biodistribution of MEV in each of the five brain sections studied; (d)-(g) represent and identify brain regions with reference to the diagram below showing MEV in the brain after IN administration. [Figure 37] Figure 37(a)-(d) show the pharmacokinetics (PK) and biodistribution of MEV in different regions of section 1 from Figure 36. Images of DiR-labeled MEV are black dots. [Figure 38] Figure 38 shows the PK and biodistribution of MEVs in different regions of section 1 and provides a graph of the total number of labeled MEVs with DiR spots per surface of the region of section 1 normalized by the total area analyzed. [Figure 39] Figure 39(a)-(d) shows the PK and biodistribution of MEV in different regions of section 2 (images of DiR-labeled MEV are shown). [Figure 40] Figure 40(a)-(d) shows the PK and biodistribution of MEVs in different regions of section 2 as a graph of the total number of labeled MEVs with DiR spots per surface of the region of section 2 normalized by the total area analyzed. [Figure 41] Figure 41(a)-(d) shows the PK and biodistribution of MEV in different regions of section 3; images of DiR-labeled MEV. [Figure 42]Figure 42(a)-(f) shows the PK and biodistribution of MEVs in different regions of section 3 in a graph of the total number of labeled MEVs with DiR spots per surface of the region of section 3 normalized by the total area analyzed. [Figure 43] Figure 43(a)-(d) shows the PK and biodistribution of MEV in different regions of section 4 as images of MEV labeled with DiR. [Figure 44] Figure 44(a)-(d) shows the PK and biodistribution of MEVs in different regions of section 4 and provides graphs of the total number of labeled MEVs with DiR spots per surface of the region of section 4 normalized by the total area analyzed. [Figure 45] Figure 45(a)-(d) shows the PK and biodistribution of MEV in different regions of section 5; images of DiR-labeled MEV. [Figure 46] Figures 46(a) and (b) show the kinetics of brain penetration by MEVs from the rostral to the distal part of the brain. [Figure 47] Figure 47(a) and (b) represent the blood-brain barrier [reproduced from Cecchelli et al. (2007) Nat Rev Drug Discov. 6(8):650-661]. [Figure 48] FIG. 48 shows microscopic images of mouse intestinal epithelium 8 hours after administration of PKH26-labeled MEV. [Figure 49] FIG. 49 shows whole-body bioluminescence images of a representative animal treated with MEVs loaded with luciferase mRNA. [Figure 50] FIG. 50 shows whole-body bioluminescence images of a representative animal treated with MEVs loaded with luciferase enzyme. [Figure 51] FIG. 51 depicts a timeline of image analysis using the Incucyte® Live Cell Analyzer. DETAILED DESCRIPTION OF THE INVENTION

[0088] overview A.Definition B. Microalgae and Overview C. extracellular vesicles 1. Types of Extracellular Vesicles (EVs) a exosome b. Microvesicles C apoptotic bodies 2. EV uptake 3. General Methods for Isolating EVs a. 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. Fabrication of Payload-loaded MEVs a. Electroporation b. Sonication c. Extrusion d. surfactants e. Other methods 4. Exemplary Cargos 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 Administration of Exogenously Loaded MEVs 4. Combination therapy F. Biodistribution of MEV after administration by various routes 1. Mammalian EV Biodistribution 2. Microalgae EV distribution in the body Oral administration 1) Components of the lymphatic system 2) Targeting GALT 3. Diseases and Conditions Treated by MEV G. Formulations, Routes of Administration, and Diseases and Disorders H. 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. Lid string 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. Distance between biodistribution of MEVs and other delivery vehicles 5. Intranasal administration 6. Brain delivery after MEV and intranasal administration 7. Transport and Biodistribution of MEVs After Intranasal (IN) Administration 8. Primary and Secondary Circuitry of the Olfactory System and Areas Reached by MEV upon IN Administration 9. Delivery of MEVs to the Brain via IN Administration - Exemplary Bioactive Cargos and Uses Thereof I. MEV-mediated intracellular signaling J. Working Example

[0089] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, GenBank® sequences, databases, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. In the event that there are multiple definitions for terms herein, those in this section prevail. When a URL or other such identifier or address is referenced, it is understood that such identifiers may change, and information, particularly on the Internet, may fluctuate, but that equivalent information may be found by searching the Internet. Reference thereto demonstrates that such information is available and publicly distributed.

[0090] As used herein, cargo refers to exogenous molecules, e.g., bioactive molecules, including biomolecules and small molecules, that are loaded into the microalgal extracellular vesicles (MEVs) provided herein after the MEVs are isolated. This includes cargo that is heterologous to the MEVs.

[0091] As used herein, heterologous cargo generally refers to cargo that does not naturally occur in MEVs but is exogenously loaded in MEVs as discussed above. It also refers to cargo that is endogenously loaded in MEVs by genetically modified microalgae. MEVs with heterologous cargo contain cargo that does not naturally occur in MEVs.

[0092] As used herein, bioactive molecules or bioactive agents refer to any molecule or agent that can have biological activity, for example, therapeutic activity, or 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 molecules that can be delivered to a subject, for example, a human or other animal, or a plant, or a microorganism (bacteria or other), for therapy, diagnostic applications, or other such uses, for example, cosmetics. Bioactive agents or molecules can function or have activity as, for example, therapeutic agents, immunogens, diagnostic agents, detectable markers, or cosmetics. Bioactive molecules for use herein are any that can be loaded into microalgal extracellular vesicles (MEVs) from microalgae.

[0093] As used herein, a biomolecule refers to any biologically active biopolymer or molecule that occurs or may occur in a living organism or virus, or a biopolymer or molecule that is a modified form of such a biopolymer or molecule. Thus, a biomolecule includes modified naturally occurring biomolecules, such as proteins that contain a primary sequence that has been modified by deletion, insertion, and / or replacement of amino acids to alter the primary sequence, and / or by modifications such as post-translational modifications of the protein.

[0094] As used herein, when MEVs are described as having the same or substantially the same loaded cargo or amount, it is understood that this refers to the average value 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, generally, the amount of cargo / MEV will be more than one molecule / MEV, and that the amount of cargo will vary depending on various parameters, including the cargo, the target tissue and / or cells, the disease, disorder, or condition being treated, and the subject being treated. Generally, more than one molecule of cargo is loaded per MEV, for example, on average, at least 10 or about 10 molecules / MEV. Substantially more cargo, e.g., 100, 500, 1000, 10 4 It is possible to load 100 molecules / MEV or more. The amount loaded will vary depending on the target, disease, disorder or condition, subject and cargo and MEV capacity. Selecting the amount is within the skill of the art.

[0095] As used herein, a subject is any living organism, generally an animal or plant, into which or on which a composition containing MEV is introduced. Subjects include, but are not limited to, humans, plants, particularly crop plants, and animals, including livestock and pets, such as dogs and cats and zoo animals.

[0096] As used herein, a drug delivery system refers to a composition containing a MEV provided herein that contains a cargo for delivery to a tissue. As shown herein, the formulation and route of administration of a composition containing a MEV can select the transport route and / or final destination of the MEV upon administration. For example, as demonstrated herein, intranasal administration of a MEV results in transport of the MEV to the brain. Orally administered MEVs can target the gut-associated lymphoid tissue (GALT). Thus, the GALT is a target (effective compartment) and / or route by which MEVs and their therapeutic cargo can be used to deliver cargo. A delivery system refers to the combination of a formulation for a specific route of administration that targets specific tissues for the treatment of diseases, disorders, and conditions of these tissues or involving these tissues.

[0097] As used herein, a disease or disorder or condition refers to a pathological or undesirable or unwanted state in an organism resulting from a cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions and those characterized by identifiable symptoms or characteristics.

[0098] As used herein, treating a subject having a disease, disorder, or condition means that the symptoms or signs of the subject's disease or condition are partially or completely alleviated, or remain unchanged following treatment.

[0099] As used herein, treatment refers to any effect that ameliorates the symptoms of a disease or disorder. Treatment encompasses prophylaxis, therapy, and / or cure. Treatment also encompasses any medicinal use of any MEV or composition provided herein. Treatment refers to any effect that ameliorates, prevents, reduces, or eliminates any symptoms or signs of a disease or disorder. Treatment also encompasses any medicinal use of any MEV or composition provided herein.

[0100] As used herein, prevention refers to preventing potential disease and / or preventing worsening of symptoms or progression of disease. Prevention or prophylaxis and their grammatical equivalents refer to methods and products that reduce or eliminate the risk or probability of developing a disease or condition.

[0101] As used herein, modification refers to the deletion, insertion, and replacement of amino acids or nucleotides in the amino acid sequence of a polypeptide or in a nucleic acid molecule. These include modifications of the primary sequence of a polypeptide or protein. For example, methods for modifying polypeptides and nucleic acid molecules by using recombinant DNA methodology are routine for those skilled in the art. When referring to a polypeptide or protein rather than a sequence, modification refers to post-translational or post-purification changes, such as the conjugation or linkage of a moiety that changes the properties of the polypeptide or protein, for example, half-life extension moieties, glycosylation, purification tags, detectable reporters, and other such moieties.

[0102] As used herein, genome or plasmid or gene modifications include nucleic acid deletions, replacements, insertions and translocations, including any changes to a native or naturally occurring nucleic acid sequence.

[0103] As used herein, RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, neutralizing targeted mRNA molecules and inhibiting the translation and thereby expression of the targeted gene.

[0104] As used herein, the RNA molecule that acts by RNAi is called inhibitory due to its silencing of the expression of targeted gene.Silencing expression means that the expression of targeted gene is reduced or suppressed or inhibited.

[0105] As used herein, gene silencing by RNAi refers to inhibiting, suppressing, disrupting, or silencing the expression of a targeted gene. The targeted gene contains a sequence of nucleotides corresponding to a sequence in the inhibitory RNA, thereby silencing the expression of the mRNA. Small interfering RNA (siRNA) is a small piece of double-stranded (ds) RNA, usually about 21 nucleotides long, with a 3' overhang (2 nucleotides) at each end, which can be used to bind to messenger RNA (mRNA) at a specific sequence and promote its degradation, thereby interfering with protein translation. In doing so, siRNA prevents the production of a specific protein based on the nucleotide sequence of its corresponding mRNA. This process is called RNA interference (RNAi), also known as siRNA silencing or siRNA knockdown. Short-hairpin RNA or small-hairpin RNA (shRNA) is an artificial RNA molecule with a narrow hairpin turn that can be used to silence target gene expression by RNA interference (RNAi). Expression of shRNA in cells is usually achieved by delivery of plasmids or via viral or bacterial vectors.

[0106] As used herein, non-coding RNA is RNA that does not code for protein.Classes of non-coding RNA include but are not limited to small interfering RNA (siRNA) and microRNA (miRNA).As used herein, inhibiting, suppressing, destroying or silencing targeted gene refers to the process of changing the expression (for example, translation) of targeted gene, thereby reducing the activity or expression of the product coded by targeted gene.Reduction includes complete knockout or partial knockout, thereby achieving treatment with MEV provided herein and administration herein.

[0107] As used herein, tumor microenvironment (TME) refers to the cellular environment in which tumors exist, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and extracellular matrix (ECM).Existing conditions 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 high levels of adenosine, which indicate tumors.

[0108] As used herein, the statement that a nucleic acid or the encoded RNA targets a gene means that it inhibits, suppresses or silences the expression of the gene by any mechanism.Generally, such nucleic acid comprises at least a portion that is complementary to the targeted gene, and this portion is sufficient to form a hybrid with the complementary portion.

[0109] 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, e.g., a target polynucleotide or polypeptide or a native or wild-type sequence.

[0110] As used herein, insertion, when referring to nucleic acid or amino acid sequence, describes the inclusion of one or more additional nucleotides or amino acids in target, natural, wild-type or other related sequences.Therefore, the nucleic acid molecule that contains one or more insertions compared to wild-type sequence contains one or more additional nucleotides in the linear length of the sequence.

[0111] As used herein, additions to nucleic acid and amino acid sequences describe the addition of nucleotides or amino acids to either end relative to another sequence.

[0112] As used herein, substitution or replacement refers to replacing one or more nucleotides or amino acids in a natural, target, wild-type, or other nucleic acid or polypeptide sequence with alternative nucleotides or amino acids without changing the length of the molecule (as described by the number of residues).Thus, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides of the molecule.Amino acid replacements compared to a specific polypeptide can be expressed in terms of the number of amino acid residues along the length of the polypeptide sequence.

[0113] As used herein, a statement that a nucleotide or amino acid position corresponds to a nucleotide or amino acid position in a disclosed sequence, such as that shown in the sequence listing, or that a nucleotide or amino acid position corresponds to a nucleotide or amino acid position in a disclosed sequence, such as that shown 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, for example, 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 (see, 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).

[0114] As used herein, sequence alignment refers to the use of homology to align two or more nucleotide or amino acid sequences. 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 can be aligned by any method known to those skilled 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 skilled in the art. By aligning polypeptide or nucleic acid sequences, one skilled in the art can identify similar portions or positions using conserved and identical amino acid residues as a guide. Furthermore, one skilled in the art can also use conserved amino acid or nucleotide residues as a guide to find corresponding amino acid or nucleotide residues between and within human and non-human sequences. Corresponding positions may also be based on structural alignment, for example, by using computer-simulated alignments of protein structures. In other cases, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as a guide to find corresponding amino acid residues between and within human and non-human sequences.

[0115] As used herein, the term "properties" refers to any characteristic exhibited by a polypeptide, such as an antibody, including, but not limited to, binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, thermotolerance, and resistance to pH conditions. A change in the properties may alter the activity of the polypeptide. For example, a change in the binding specificity of an antibody polypeptide may alter the polypeptide's ability to bind to an antigen and / or various binding activities, such as affinity or avidity, or in vivo activity.

[0116] As used herein, the activity or functional activity of a polypeptide, e.g., 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, by antigen-binding, DNA-binding, ligand-binding, or dimerization, or enzymatic activity, such as kinase activity or proteolytic activity. For antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind to a specific antigen, antigen-binding affinity (e.g., high or low affinity), antigen-binding avidity (e.g., high or low avidity), on-rate, off-rate, effector function, e.g., antigen neutralization or clearance, the ability to promote virus neutralization, and in vivo activity, e.g., the ability to prevent infection or pathogen invasion or promote clearance, or the ability to penetrate specific tissues, fluids, or cells in the body. Activity can be assessed in vitro or in vivo using recognized assays, such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays for measuring on-rate or off-rate, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry and binding assays (e.g., panning assays).

[0117] As used herein, the terms "binding," "bound," and their grammatical differences refer to the involvement of a molecule in any interaction with or between other molecules, resulting in a stable association in which the molecules are in close proximity to each other. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (e.g., reversible and irreversible covalent bonds), and includes interactions between molecules, such as, but not limited to, compounds, including proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as drugs.

[0118] As used herein, antibody refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or partially or wholly synthetically produced, e.g., recombinantly produced, and includes any fragment containing at least a portion of the heavy chain variable and light chain regions of an immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, specifically bind to an antigen. Thus, an antibody includes any protein having a binding domain (antibody binding site) that is homologous or substantially homologous to an immunoglobulin antigen-binding domain. For example, an antibody refers to an antibody containing two heavy chains (which can be designated H and H') and two light chains (which can 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 contains, at a minimum, all or at least a portion of a variable heavy (VH) chain and / or a variable light (VL) chain. An antibody may also contain all or a portion of a constant region.

[0119] For purposes herein, the term "antibody" includes full-length antibodies and portions thereof, including antibody fragments, such as anti-tumor antibodies, anti-pathogens, 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 (dsFvs), Fd fragments, Fd' fragments, single-chain Fvs (scFvs), single-chain Fabs (scFabs), diabodies, anti-idiotypic (anti-Id) antibodies, or any of the above antigen-binding fragments. Antibodies also include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. 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).

[0120] As used herein, nucleic acid refers to at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically connected 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, as 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.

[0121] As used herein, isolated nucleic acid molecule is separated from other nucleic acid molecules that exist in the natural source of nucleic acid molecule.Isolated nucleic acid molecule, for example, cDNA molecule, can be substantially free from other cellular material or culture medium when produced by recombinant technology, or can be substantially free from chemical precursors or other chemicals when chemically synthesized.Exemplary isolated nucleic acid molecule provided herein includes the isolated nucleic acid molecule that encodes RNAi or therapeutic protein.

[0122] 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, thereby transcribing and translating the nucleic acid to express a functional fusion protein, where the leader peptide effects secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acids can be transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in the expression of one of the two polypeptides. For example, an amber stop codon can be positioned 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 containing 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 regulates or mediates transcription of the nucleic acid.

[0123] As used herein, synthetic, for example with respect to a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0124] As used herein, naturally occurring α-amino acid residues are residues of the 20 α-amino acids found in nature and which, in humans, are incorporated into proteins by specific recognition of a charged tRNA molecule using their cognate mRNA codon.

[0125] As used herein, a polypeptide refers to two or more amino acids joined by a covalent bond. The terms polypeptide and protein are used interchangeably herein.

[0126] As used herein, peptide refers to a polypeptide that is from 2 to about 40 or 40 amino acids in length.

[0127] As used herein, reference to protein includes peptides, polypeptides, small peptides and all forms of proteins, unless otherwise specified.

[0128] 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 herein, 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 occurring 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 various nucleic acid molecules and fragments are designated by standard one-letter names routinely used in the art.

[0129] 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 carboxy group present at the carboxyl terminus of a polypeptide. In accordance with standard polypeptide nomenclature as described in J. Biol. Chem., 243:3557-59 (1968) and adopted at 37 CFR §§ 1.821-1.822, abbreviations for amino acid residues are set forth in the following table:

[0130] JPEG2025505440000009.jpg151138

[0131] 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 corresponding table above, modified, non-natural, 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 the amino-terminal group, e.g., NH2, or to the carboxyl-terminal group, e.g., COOH.

[0132] In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled 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).

[0133] Such substitutions can be made in accordance with the exemplary substitutions shown in the table below:

[0134] JPEG2025505440000010.jpg99129

[0135] Other substitutions are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.

[0136] As used herein, naturally occurring amino acids refer to the 20 L-amino acids that occur in polypeptides.

[0137] As used herein, the term "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid, but is structurally modified to mimic the structure and reactivity of the natural amino acid. Thus, non-naturally occurring amino acids include, for example, amino acids or amino acid analogs 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, but are not limited to, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), β-alanine / β-amino-propionic 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-diamino These include propionic 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 ornithine (Orn).

[0138] 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 way not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of engineered molecules.

[0139] As used herein, a DNA segment is a portion of a larger DNA molecule having a specified characteristic. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read in the 5' to 3' direction, encodes the sequence of amino acids of the specified polypeptide.

[0140] 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 units 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 permits. When applied to double-stranded molecules, the term is used to denote the total length and is understood to be equivalent to the term base pair. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may be slightly different in length and their ends may be staggered, thus preventing all nucleotides in a double-stranded polynucleotide molecule from being paired. Such unpaired ends generally do not exceed 20 nucleotides in length.

[0141] As used herein, recombinant production refers to and means the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

[0142] 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 that is located in a locus in which it does not normally occur, or that mediates or encodes a mediator that alters the expression of an endogenous nucleic acid, e.g., DNA, by affecting transcription, translation, or other regulatable biochemical processes. Heterologous nucleic acids, e.g., DNA, are also referred to as foreign nucleic acids. Any nucleic acid, e.g., DNA, that a person skilled in the art would recognize or would consider 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 have been obtained from another cell or prepared synthetically, or have been 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 sequence(s).

[0143] Examples of heterologous nucleic acid herein include, but are not limited to, DNA molecules, RNA molecules, plasmids and antisense oligonucleotides.In MEV, heterologous nucleic acid can be encoded by plasmid.Heterologous nucleic acid, for example, DNA, includes the nucleic acid that can mediate the expression of the DNA that encodes therapeutic product in some way, or can encode the product, for example, peptide or RNA, that directly or indirectly mediates the expression of therapeutic product in some way.

[0144] 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, for example, 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 somatic cell lines) and used as described herein. Transport of MEVs is generally independent of the method by which the cargo is loaded. 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.

[0145] As used herein, gene therapy involves the transfer of heterologous nucleic acid, e.g., DNA, into certain cells, e.g., target cells, of a mammal, particularly a human, with a disorder or condition for which such therapy is sought. The nucleic acid, e.g., DNA, is introduced into the selected target cells in such a way that the heterologous nucleic acid, e.g., DNA, is expressed and the encoded therapeutic product 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, e.g., a growth factor or its inhibitor, or a 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. The heterologous nucleic acid, e.g., DNA, encoding a therapeutic product can be modified to enhance or otherwise alter the product or its expression before being introduced into the cells of the afflicted host. Gene therapy can also include the delivery of inhibitors or repressors or other modulators of gene expression.

[0146] As used herein, expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide.The expression level of a polypeptide can be evaluated by any method known in the art, including, for example, the method for determining the amount of polypeptide produced from host cells.Such methods can include, but are not limited to, ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay and Bradford protein assay to quantify polypeptide in cell lysate.

[0147] As used herein, a host cell is a cell used to receive, maintain, replicate, and / or amplify a vector. A host cell 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.

[0148] As used herein, a vector is a replicable nucleic acid that can express 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, usually 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 nucleic acid amplification or for expression / display of the polypeptide encoded by the nucleic acid. Vectors usually remain episomal, but may also be designed to achieve integration of a gene or a portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art. Vectors also include virus vectors or viral vectors. Viral vectors are engineered viruses that are operably linked (as vehicles or shuttles) to exogenous genes to transfer the exogenous genes into cells.

[0149] As used herein, an expression vector includes a vector capable of expressing DNA operably linked with a regulatory sequence, such as a promoter region, capable of achieving expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally originate from plasmid or viral DNA, or may contain both elements. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that results in expression of cloned DNA when introduced into an appropriate host cell. Suitable expression vectors are well known to those skilled in the art and include those that can replicate in eukaryotic and / or prokaryotic cells, as well as those that remain episomal or integrate into the host cell genome.

[0150] 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.

[0151] As used herein, sequence identity refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test 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 herein, 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 characters are aligned. Generally, internal and terminal gaps are possible. 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 without considering gaps as the number of identical positions / total length of aligned sequences x 100.

[0152] 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. The alignment is generated 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 identical in the entire alignment of two compared sequences, each 100 nucleotides in length. It is understood that global alignment can also be used in determining sequence identity even when the lengths of the aligned sequences are not identical. Differences at the ends of the sequences are taken into account in 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. Exemplary algorithms for performing global alignment include 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.

[0153] As used herein, local alignment refers to aligning two sequences, but only aligning the portions of the sequences that share similarity or identity. Thus, local alignment determines whether a subsegment of one sequence exists 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 local alignment means that in the full 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 identical in the region of similarity or identity.

[0154] For the purposes herein, sequence identity can be determined by using standard alignment algorithm programs with the default gap penalties established by each supplier.The default parameters of the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described by 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 that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, 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 and publicly available alignment databases and programs).Generally, for the purposes herein, sequence identity is determined by computer algorithms based on global alignment, such as the Needleman-Wunsch global sequence alignment tool available at NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign [William Pearson implementing the Huang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)]; and the program by Xiaoqui Huang available at deepc2.psi.iastate.edu / aat / align / align.html.Usually, the full-length sequences of each of the polypeptides or nucleotides being compared are aligned over the entire length of each sequence in global alignment.Local alignment can also be used when the sequences being compared are substantially the same length.

[0155] Thus, as used herein, the term identity refers to a comparison or alignment between a test 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. A level of identity of 90% or greater indicates that 10% or less (i.e., 10 out of 100) of the amino acids or nucleotides in the test polypeptide or polynucleotide differ from those in the reference polypeptide, assuming, for illustrative purposes, that test and reference polypeptides or polynucleotides are 100 amino acids or nucleotides in length. Similar comparisons can be made between test and reference polynucleotides. Such differences may be expressed as point mutations randomly distributed throughout the entire length of the amino acid sequence, or they may be clustered at one or more positions of varying length, up to a maximum allowable value, e.g., 10 / 100 amino acid difference (approximately 90% identity). Differences may also be due to deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. At levels of homology or identity above about 85-90%, depending on the length of the sequences being compared, results may be independent of the program and gap parameters set, and such levels of identity can often be easily assessed without reliance on software.

[0156] As used herein, a pharmaceutically active agent includes any therapeutic or bioactive agent, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, and conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0157] As used herein, a therapeutic effect means an effect resulting from treatment of a subject that alters, typically improves or reverses the symptoms of, or cures, a disease or condition.

[0158] As used herein, a therapeutically effective amount or therapeutically effective dose refers to at least the amount of an agent, compound, material, or composition containing a compound that is sufficient to produce a therapeutic effect after administration to a subject, and thus is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or disorder.

[0159] 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.

[0160] As used herein, prophylactically effective amount or prophylactically effective dose refers to the amount of drug, compound, material or composition containing compound that will have the intended preventive effect when administered to a subject, for example, prevent or delay the onset or recurrence of disease or symptoms, reduce the probability of the onset or recurrence of disease or symptoms, or reduce the incidence of viral infection.Complete preventive effect is not necessarily achieved by administering one dose, and may only be achieved after administering a series of doses.Therefore, prophylactically effective amount can be administered in one or multiple doses.

[0161] As used herein, ameliorating the symptoms of a particular disease or disorder by treatment, e.g., by administration of a pharmaceutical composition or other therapeutic agent, refers to any alleviation, whether permanent or temporary, persistent or transient, of symptoms that can result from or be associated with the administration of the composition or therapeutic agent.

[0162] As used herein, anticancer drug refers to any drug that is destructive or toxic to malignant cells and tissues.For example, anticancer drug includes the drug that kills cancer cells, or otherwise inhibits or impairs the growth of tumor or cancer cells.Exemplary anticancer drug is chemotherapeutic drug.

[0163] As used herein, therapeutic activity refers to the in vivo activity of a therapeutic polypeptide. Generally, a therapeutic activity is an activity associated with the treatment of a disease or condition.

[0164] As used herein, the term subject refers to animals, including mammals, for example, humans.

[0165] As used herein, a patient refers to a human subject.

[0166] As used herein, animals include any animals, such as, but not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; poultry, such as chickens; ruminants, such as goats, cows, deer, and sheep; and pigs and other animals. Non-human animals do not include humans as intended animals.

[0167] 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.

[0168] As used herein, combination refers to any association between or among two or more items.The combination can be two or more separate items, such as two compositions or two collections, or 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 related or correlated.

[0169] As used herein, combination therapy refers to the administration of two or more different therapeutic agents, which may be provided or administered separately, sequentially, intermittently, or may be provided in a single composition.

[0170] As used herein, a kit is a packaged combination that may include other elements, e.g., 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.

[0171] As used herein, unit dosage form refers to physically discrete units packaged individually suitable for human and animal subjects as is known in the art.

[0172] As used herein, a single unit dosage formulation refers to a formulation for direct administration.

[0173] 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 the course of minutes, hours, weeks, days, or months. Multi-dose formulations can allow for dose adjustment, dose storage, and / or dose splitting. Because multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.

[0174] As used herein, an article of manufacture is a product that is made and sold. As used throughout this application, this term is intended to encompass any of the compositions provided herein contained in a packaging article.

[0175] As used herein, fluid refers to any composition that can flow. Thus, fluids encompass compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0176] 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. Preparations can be determined to be substantially free if they are free of readily detectable impurities as determined by standard analytical methods used by those skilled in the art to assess such purity, such as thin-layer chromatography (TLC), gel electrophoresis, and high-performance liquid chromatography (HPLC), or appear sufficiently pure that further purification does not detectably alter the physical and chemical properties of the material, such as enzymatic and biological activity. Methods for purifying compounds to yield substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may enhance the specific activity of the compound.

[0177] As used herein, a cellular extract or lysate refers to a preparation or fraction made from lysed or disrupted cells.

[0178] 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, in the case of a plasma sample, may be obtained from a normal volunteer who is not afflicted with the condition of interest. A control may also be an internal control.

[0179] As used herein, psilocin is the active form of psilocybin. It is produced in the liver by oxidation of psilocybin. For purposes herein, in the context of MEV-mediated delivery, psilocybin and psilocin shall have the same meaning, and reference to "psilocybin" shall mean "psilocin," and vice versa.

[0180] As used herein, bregma is a unit of measurement of the distance between a location in the brain and the junction between the coronal and sagittal sutures of the skull.

[0181] As used herein, the tropism of a MEV refers to the cells, tissues and / or organs in which the MEV accumulates upon administration.

[0182] As used herein, native tropism in reference to the MEVs provided herein refers to the meaning that the MEVs have not been modified to provide a particular tropism or targeting property.

[0183] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a polypeptide comprising an immunoglobulin domain includes polypeptides having one or more immunoglobulin domains.

[0184] As used herein, the term "or" is used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive.

[0185] As used herein, ranges and amounts can be expressed as about a particular value or range. "About" also includes the exact amount. Thus, about 5 amino acids means about 5 amino acids and also 5 amino acids.

[0186] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, an optionally mutated moiety means that the moiety is mutated or non-mutated.

[0187] As used herein, the abbreviations for any protecting groups, amino acids and other compounds follow their common usage, recognized abbreviations or the IUPAC-IUB Biochemical Nomenclature Commission [see Biochem. (1972) 11(9):1726-1732] unless otherwise indicated.

[0188] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0189] B. Microalgae and Overview Algae are a complex, polyphyletic collection of primarily photosynthetic organisms. These organisms include microscopic and macroscopic forms. Macroalgae (seaweeds) are multicellular, large-sized algae visible to the naked eye. Microalgae are microscopic, unicellular organisms, including prokaryotes (e.g., cyanobacteria) and eukaryotes, such as green algae.

[0190] Compared to photosynthetic crops, microalgae have higher growth rates and can be cultivated in non-arable fields and in bioreactors. Many species of microalgae can be grown year-round in industrial-scale photobioreactors under controlled cultivation conditions [Adamo et al. (2021) Journal of Extracellular Vesicles 10:e12081].

[0191] Algae are generally classified into 11 major phyla: Cyanophyta, Chlorophyta, Rhodophyta, Glaucophyta, Euglenophyta, Chlorarachniophyta, Charophyta, Cryptophyta, Haptophyta, Heterokontophyta, and Dinophyta [Barkia et al. (2019) Mar. Drugs 17(5):304]. Different pigments occur in each algal group. Cyanobacteria (or Cyanobacteria) contain phycobiliproteins (proteins that capture light energy), phycocyanin, allophycocyanin, and phycoerythrin, as well as chlorophyll-a, -d, and -f. Glaucophytes contain chlorophyll-a and harvest light through phycobiliproteins. Chlorophytes contain chlorophyll-a and -b, as well as carotenoids including β-carotene and various xanthophylls (e.g., astaxanthin, canthaxanthin, lutein, and zeaxanthin). The primary pigments of the rhodophyta (red algae) 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].

[0192] Provided herein are extracellular vesicles produced by algae, particularly unicellular green algae such as Chlorella species, for use in delivering exogenously loaded cargo to animals and plants. Algae are unicellular eukaryotic organisms that are usually monoploid but may have a diploid life cycle. The algae can be cultured in a bioreactor and extracellular vesicles isolated therefrom. The resulting extracellular vesicles can be loaded with cargo, typically a heterologous bioactive molecule, by methods such as electroporation to produce compositions containing the extracellular vesicles for administration to animals and / or 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, intravaginal, 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. 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 purposes. 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 dermatological, pulmonary, and gastric diseases and conditions. Extracellular vesicles can be targeted to specific organs or tissues or administered locally.

[0193] Similar to extracellular vesicles (EVs) from other sources, such as mammalian EVs, microalgal EVs (MEVs) have evolved to efficiently transfer genetic material and other types of molecules from cell to cell. They orchestrate intracellular and intercellular communication across boundaries through the exchange of biologically active molecules. MEVs are natural nanoparticles. Because they are derived from cells, they are free of synthetic cargo and genetic modifications, lack synthetic components, and are therefore safe, eliminating the risk of endogenous viruses that are potentially dangerous to humans.

[0194] The MEVs provided herein include Chlorella MEVs, particularly Chlorella vulgaris, a freshwater microalga. Chlorella is a single-celled, monoploid alga that is a natural and efficient producer of extracellular vesicles. Chlorella vulgaris has been consumed worldwide as a dietary supplement for decades and is non-toxic, non-immunogenic, and can be cultivated on a large industrial scale at low cost. The MEVs provided herein can be used directly to protect, transport, and deliver a wide range of innovative therapeutic molecules into target cells associated with specific diseases.

[0195] 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 have the potential to have a longer clearance rate and, as a result, persist in targeted organs, tissues, and cells longer than those reported for other delivery systems, including mammalian EVs.

[0196] As shown herein, MEVs overcome natural body barriers (e.g., oral delivery or specific lymphatic tissue delivery or nose-to-brain delivery), which was not achieved with liquid nanoparticles and EVs of mammalian origin.

[0197] The MEVs provided herein address unmet needs, including the ability to carry and reliably deliver therapeutic molecules specifically to the site of treatment while avoiding premature degradation or inactivation of the therapeutic by the immune system or by enzymes for the treatment of diseases for which a therapeutic agent already exists but cannot be adequately delivered.

[0198] As demonstrated herein, purified or partially purified MEVs can be loaded by physical methods (exogenous loading; exoloading). Exoloading is scalable and industrially feasible. MEVs can be exoloaded with a variety of molecules, varying in size, hydrophobicity, and properties, including siRNA, mRNA, peptides, proteins, plasmids, oligonucleotides, and small molecules. The biological activity of exoloaded cargoes is preserved while simultaneously protecting them from degradation by enzymes and other substances present in vivo. MEVs can deliver their cargoes to recipient cells of numerous origins, including microalgae, bacteria, higher plants, mammals, and humans. MEVs can also deliver cargoes to the appropriate cellular compartments to ensure the proper expression and biological activity of cargo molecules, including those with complex biological pathways, such as siRNA, mRNA, and receptor-binding peptides, among others.

[0199] C. extracellular vesicles Extracellular vesicles (EVs) are biomolecular structures released from plant and animal cells that 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 cell (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 cells [Kuruvinashetti et al. (2020) 20 th International 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].

[0200] EVs contain endogenous lipids, nucleic acids, and proteins. Although results vary due to variations in isolation techniques and data analysis methods, EVs generally contain proteins associated with the cell membrane, cytosol, and those involved in lipid metabolism [see, e.g., Doyle and Wang (2019) Cells 8(7):727]. Proteins involved in EV biogenesis (e.g., ESCRT components), EV formation and release (e.g., RAB27A, RAB11B, and ARF6), signal transduction, and antigen presentation, as well as tetraspinins, 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-stranded non-coding RNAs, tRNA fragments, piwi-binding RNA, vault RNPs, and Y RNAs in EVs. Most naturally occurring RNAs in EVs are approximately 200 nucleotides in length (with small segments up to 4 kb) and are therefore fragmented in EVs. However, circular RNAs have 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 from EV research on the protein, nucleic acid, and lipid content of EVs (generally EVs of mammalian origin, e.g., human origin) as well as the isolation and purification procedures used [Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312].

[0201] EVs are used by cells to mediate several physiological processes, affect various pathologies associated with immune response activation or the spread of diseases or infections, and are involved in interspecies communication. They exist in all kingdoms of life. Sources of EVs include mammalian cells, bacteria, 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, which may be a physical barrier to EV release [Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a].

[0202] 1. Types of Extracellular Vesicles (EVs) a exosome There are three main 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 may transform into late endosomes, which accumulate ILVs by inward budding of the endosomal membrane. Late endosomes containing several small vesicles are called MVBs. MVBs either fuse with lysosomes and are degraded, or fuse with the plasma membrane to release ILVs into the extracellular space as exosomes. The endosomal sorting complex required for transport (ESCRT) pathway, which regulates MVB trafficking 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 proteins in the tetraspanin family, 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].

[0203] 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].

[0204] Exosomes are candidate drug delivery systems: they have a long circulating half-life; they are tolerated by the human body, can penetrate cell membranes, and can target specific cell types; and they can be loaded with genetic material, proteins, or small molecules [Doyle and Wang (2019) Cells 8(7):727].

[0205] 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 donor cells influence the number of MVs produced, while the physiological state and microenvironment of recipient cells influence the number of MVs consumed. MVs also possess several marker proteins, including cytoplasmic 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 (e.g., proteins, nucleic acids, and lipids) for delivery to another cell, thereby altering the function of the recipient cell (Doyle and Wang (2019) Cells 8(7):727).

[0206] C apoptotic bodies Apoptotic bodies are released into the extracellular space by dying cells and have diameters ranging from 50 nm to 5000 nm. They form when the cell membrane 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].

[0207] 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 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 cell membrane with heparin reduced EV uptake in cell culture, as did blocking scavenger receptor type B1 (SR-B1) with synthetic nanoparticle mimics 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 a method of uptake, requiring low pH conditions; treatment of EVs with a pH-sensitive membrane-fusogenic peptide in combination with a cationic lipid 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 can enter the cells via fusion with the cell membrane to deliver therapeutic payloads to tumor cells.

[0208] Like cells, EVs possess extracellular receptors and ligands on the outside and cytoplasmic proteins and nucleic acids on the inside, thus communicating with cells in various 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 escape the degradative pathway; since late endosomes can fuse with lysosomes or the plasma membrane, the cargo must exit before being degraded in lysosomes or re-released via MVB fusion with the plasma membrane. EVs containing cargo, including mRNA and non-coding RNA, can be transferred to 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].

[0209] 3. General Methods for Isolating EVs a. Ultracentrifugation Ultracentrifugation methods are 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 and other components of the extracellular space. Differential ultracentrifugation relies on the initial sedimentation of large, dense particles of 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 (usually made with sucrose or iodixanol) in a centrifuge tube. Density gradient centrifugation effectively separates EVs from protein aggregates and non-membranous particles, but exosome recovery 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].

[0210] b. Size-based technology There are several size-based techniques for isolating exosomes [Doyle and Wang (2019) Cells 8(7):727]. Ultrafiltration separates particles based on size and the molecular weight cutoff of the membrane, whereby particles larger than the membrane's molecular weight cutoff are retained and particles smaller than the membrane's molecular weight cutoff pass through into the filtrate; however, low isolation efficiency can occur if the filter becomes clogged and vesicles become trapped. The ExoMir™ kit (Bioo Scientific; Austin, Texas) is a commercially available kit in which two membranes (200 nm and 20 nm) are placed in a syringe, and a sample (usually pretreated with centrifugation and proteinase K) is passed through the syringe; large vesicles remain on the first 200 nm filter, the smallest vesicles are passed through the syringe and discarded, and vesicles between 20 and 200 nm 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].

[0211] Size-exclusion chromatography (SEC), a commonly used technique for separating proteins, is similar to the use of SEC for ultracentrifugation (where the exosome pellet obtained from ultracentrifugation is resuspended and further purified using SEC). In SEC, a column is packed with a porous stationary phase that allows small particles to permeate and therefore elute after larger particles. While SEC methods typically require several hours of runtime, 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].

[0212] In flow field-flow fractionation (FFFF), a sample injected into a chamber is subjected to a parabolic flow as it is pushed down the chamber, in addition to a crossflow, which is a flow perpendicular to the parabolic flow, to separate particles in the sample. Larger particles are more affected by the crossflow and are pushed toward the walls of the chamber with the 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].

[0213] In hydrostatic filtration dialysis (HFD), hydrostatic pressure forces a sample through dialysis tubing equipped with a membrane with a molecular weight cutoff of 1000 kDa. The result is that small solutes can pass through the tubing, while larger particles, including exosomes and EVs, remain in the tubing and can be further separated, for example, using ultracentrifugation [Doyle and Wang (2019) Cells 8(7):727].

[0214] 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 derived 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. This assay requires that the protein / antigen for exosome isolation be expressed on the surface of the exosome, and its specificity is limited by the specificity of the antibody used. As a result, the yield of isolated exosomes is often low, but the purity is high. 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].

[0215] d. Exosome precipitation Methods for precipitating exosomes include polyethylene glycol (PEG) and lectin precipitation. In PEG precipitation, PEG polymers bind water molecules, allowing other particles, including exosomes, to precipitate from solution. PEG precipitation is rapid and not limited by the starting volume of the solution, but lacks selectivity, as other EVs, extracellular proteins, and protein aggregates are precipitated along with EVs. Sample pretreatment using filtration and / or ultracentrifugation can improve exosome yield. Commercially available kits for isolating exosomes using precipitation include ExoQuick® (System Biosciences, Palo Alto, CA) and Invitrogen® Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA). Alternatively, lectin precipitation can be used, usually after ultracentrifugation, whereby lectins bind to carbohydrates on the surface of exosomes, altering their solubility and leading to their precipitation from solution [Doyle and Wang (2019) Cells 8(7):727].

[0216] e. Microfluidic-based isolation techniques Microfluidic-based techniques isolate exosomes simultaneously based on their physical and biochemical properties, 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 according to their size and density, with larger particles experiencing stronger forces and moving more quickly 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]. To isolate exosomes using microfluidic technology, 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.

[0217] 4. Microalgae and Microalgae-Derived Extracellular Vesicles (MEVs) The taxonomy and classification of microalgae can vary. According to some schemes, there are seven (7) phyla of microalgae: Euglenophyta (Euglenophyta), Chrysophyta (Chrysophyta and Bacillariophyta), Pyrrophyta (Pyrrophyta), Chlorophyta (Green Algae), Rhodophyta (Red Algae), Phaeophyta (Brown Algae), and Xanthophyta (Yellow-Green Algae). Of interest herein are photosynthetic microalgae, such as species of the genera Chlorella and Chlamydomonas. The methods and uses described herein generally use MEVs derived from green algae. Exemplary of such algae are the genera Chlamydomonas and Chlorella, which belong to the classes Chlorophyceae and Trebouxiophyceae, respectively.

[0218] 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 / flagellum [Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a]. The flagella of Chlamydomonas lack MVBs, and therefore, the ciliary-like EVs excreted from Chlamydomonas are classified as ectosomes. Studies have demonstrated the excretion of ectosomes from the flagella and ciliary tips of the green alga Chlamydomonas reinhardtii. EVs have also been observed along the length of cilia in Chlamydomonas. Membrane budding and ciliary-like 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-like EVs in Chlamydomonas and may act as sensors of membrane bending. Ciliary-like 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]. Ciliary-like ectosomes from Chlamydomonas contain lytic enzymes that digest the mother cell wall and are necessary for the release of daughter cells. Ift88 null mutants lacking flagella were unable to egress from mother cells, and the phenotype was rescued by the addition of ciliated ectosomes from wild-type cells, suggesting a role for flagella and the intraflagellar transport (IFT) machinery in EV generation [Wang and Barr (2016) Cell Mol. Neurobiol. 36(3):449-457].

[0219] EVs were extracted from algal cells using ultracentrifugation [Kuruvinashetti et al. (2020) 20 thInternational 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 sink to the bottom of the ultracentrifuge tube and can be collected using a pipette. Extracted algal EVs can be characterized by size and concentration using nanoparticle tracking analysis (NTA). Studies using this method have isolated green algal EVs ranging in size from 25 to 200 nm and at concentrations of 0.89E8 to 0.94E8 particles / mL [Kuruvinashetti et al. (2020) 20 th International Conference on Nanotechnology 354-357].

[0220] 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 between 100 and 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 chuii, termed nanoalgosomes. Similar results were obtained using sEVs isolated from batch cultures of two other microalgae species, the chlorophyte Dunaliella tertiolecta and the dinoflagellate Amphidinium species, suggesting that nanoalgosome production is an evolutionarily conserved trait within microalgae strains. Nanoargosomes were isolated using differential centrifugation (dUC) and tangential flow filtration (TFF), and gradient ultracentrifugation was used to further purify samples enriched for small EVs by TFF or dUC. The isolated nanoargosomes 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 significantly higher than the reported number of isolated EVs, approximately 10%. 9The EV particle density was consistent with that of EV proteins 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 exosomes derived from mammalian cells, which have a density of 1.15–1.19 g / mol, nanoargosomes had a slightly lower density of 1.13 g / mol. Electron microscopy demonstrated that nanoargosomes 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 nanoargosomes 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 hepatocellular carcinoma Hep G2 cell line showed no cytotoxic or genotoxic effects after nanoargosome treatment. Furthermore, nanoargosomes were internalized by the MDA-MB-231 and 1-7 HB2 cell lines [Adamo et al. (2021) J. Extracell. Vesicles 10:e12081].

[0221] EVs were collected from at least 18 microalgae strains from the main microalgae lineages studied, including strains with various characteristics, e.g., saltwater and freshwater inhabitants, small and large cell sizes, colonies and unicellular strains, and strains with sequenced genomes [Ankistrodesmus spp., Brachiomonas spp., Chlamydomonas reinhardtii, Dunaliella tertiolecta, Tetraselmis cheui, Chloromonas spp., Rhodella violacea, Kirchneriella spp., Pediastrum spp., Nannochloropsis spp., Cyanophora paradoxa, Cryptomonas pyrenoidifera, and others]. pyrenoidifera, Phaeodactylum tricornutum, Phaeothamnion species, Diacronema species, Isochrysis galbana, Stauroneis species and Amphidinium species.

[0222] MEVs can be isolated using a differential ultracentrifugation protocol and characterized according to the International Society for Extracellular Vesicles (ISEV) guidelines. All strains tested demonstrated 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 paradoxa, Tetraselmis chuui, Amphidinium species, Rodella violacea, Diacronema species, Dunaliella tertiary orecta, Phaeodactylum tricornutum, Asclepias species, and Phaeothamnion species [Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a]. Data for Cyanophora paradoxa show that approximately 2 × 10 9 The sEV particles exhibited the strongest positive signal for EV markers and had a size distribution consistent with plant-derived vesicles, 130 ± 5 nm. Cytotoxicity and genotoxicity studies showed that sEVs isolated from the freshwater gray alga Cyanophora paradoxa were not toxic to tumorigenic MDA-MB-231 breast cancer or C2C12 myoblast cell lines over time or at various concentrations, and sEV-treated MDA-MB-231 cells also did not exhibit morphological nuclear changes associated with apoptotic events [Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a].

[0223] EVs have also been isolated from Synechocystis species PCC6803 (cyanobacteria), Chlamydomonas reinhardtii (green microalgae), Euglena gracilis (euglenophyte), and Haematococcus pluvialis (chlorophyte) in a study conducted by Zhao et al., who also performed rnomic and proteomic analyses on EVs isolated from C. 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 resuspension showed them to contain membranous structures with small aggregates of particles ranging in diameter from 110 to 120 nm, consistent with the reported diameters of exosomes and small MVs, although there were differences in diameter between microalgae species. Specifically, EVs from Chlamydomonas reinhardtii had diameters ranging from 37 to 710 nm, with an average particle size of 120.1 nm. EVs from Synechocystis had diameters ranging from 24 to 450 nm, with an average particle size 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 a fluorescent lipophilic dye inside the microalgae cells. Thus, microalgae 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 numerous flagella-associated membrane proteins in microalgal EVs [Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / v1].

[0224] These studies demonstrate that microalgae produce EVs that can be isolated using traditional or standard methods, that microalgae-derived EVs are similar in size and concentration and display similar markers compared to EVs isolated from other species, that EVs isolated from microalgae do not display cytotoxic or genotoxic effects in vitro, and that microalgae-derived EVs can be taken up by cells.

[0225] EVs of mammalian origin have been shown to be capable of delivering cargo to target cells and thus have therapeutic potential for the delivery of various cargoes for use in the treatment of several diseases or conditions, a potential lack of therapeutic potential for MEVs. However, mammalian EVs, with the exception of bovine milk EVs, cannot be administered orally because they do not survive the harsh environment 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 potentially be attached to the exosome surface and tracked using NTA, fluorescence, or bioluminescence.

[0226] References in publications that microalgae EVs can be used to deliver drugs of interest to targeted cells, tissues, or organs [Kuruvinashetti et al. (2020) 20 thOther than the International Conference on Nanotechnology 354-357, there is no published evidence or technical description of the use of MEVs for delivery to treat mammalian diseases, disorders, or conditions. There are no publications or technical descriptions describing knowledge, whether it is possible, or how to apply EV technology to microalgae-derived extracellular vesicles. Previous studies have not considered Chlorella species, nor have previous studies generally evaluated the biodistribution and related properties of MEVs. Therefore, methods such as the oral delivery method exemplified herein using Chlorella can use MEVs derived from other microalgae.

[0227] However, as described and demonstrated herein, microalgae EVs offer several advantages over existing drug delivery systems, such as the use of 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 are used in drug delivery, such as anti-cancer vaccines, because they have enhanced passive targeting (a method of preparing a drug carrier system so that it remains circulating in the bloodstream). Mesenchymal stem cell-derived EVs have the ability to actively target due to their small size, unique properties, and ability to cross biological barriers. However, mesenchymal stem cells have limited exosome secretion, and scaling up exosome production is challenging due to the need to optimize purification, increase exosome homogeneity, and establish efficient transfection strategies. Nanoparticles can lead to toxicity, and current technologies for synthesizing nanoparticles are limited in their ability to scale for manufacturing purposes. Nanoparticle- and liposome-based drug delivery methods can also lead to 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 derived from curcumin, ginger, grapefruit, and lemon, have been used for drug delivery, but their extraction process and use in treatments have yet to be optimized. The generation of EVs from agricultural products, such as fruit and milk, is economically impractical and requires 3–4 months to grow, compared to algal EVs, which can grow 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, high-quality, well-characterized EVs can be obtained [Kuruvinashetti et al. (2020) 20 thInternational Conference on Nanotechnology 354-357]. Kuruvinashetti et al. did not describe the use of Chlorella species as a source of EVs or their advantages as a source. The prior art does not describe the biodistribution of MEVs per se, nor does it relate the administration of MEVs to drugs targeted to specific organs, tissues, or systems.

[0228] 5. Green Algae – Chlorella species Previous research and discussion of EVs has not focused on or evaluated species in the genus Chlorella 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, nonmotile, contain a single chloroplast, and some have rigid cell walls [Blanc et al. (2010) Plant Cell 22(9):2943–2955].

[0229] Molecular analysis has separated Chlorella species into two classes of green algae: the Trebouxiophyceae, which contains the true Chlorella genus, and the Chlorophyceae. As used herein, Chlorella species includes any that can be or be used as a food complement, or that can be consumed by humans or other animals, e.g., livestock. Exemplary species include, but are not limited to, the species: Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis.

[0230] True Chlorella species are characterized by glucosamine as a major component of their robust cell walls. While most Chlorella species are free-living in nature, the Trebouxiophyceae include 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 photobiont) of the unicellular protozoan Paramecium 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].

[0231] a. Life cycle In unicellular organisms, such as microalgae, the life cycle is identical to the cell cycle. The genus Chlorella is a monoploid organism that reproduces asexually by autosporulation. The cell cycle and growth of Chlorella vulgaris have been investigated by Rioboo et al. using flow cytometry analysis of algal cells stained with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Their results indicate that, as commonly described for microalgae, C. vulgaris mother cell growth occurs during the light period, while cytokinesis and release of daughter cells occur during the dark period. C. vulgaris also exhibits a distinct light / dark cycle, indicated by increases in cell size, cell complexity, and autofluorescence during the light period, measured over a 96-hour period. Monoparametric histograms of CFSE-stained C. vulgaris cells, showing only one peak of daughter cells, indicate that each mother cell undergoes only one division cycle over the 96-hour period, further indicating that cytokinesis occurs during the dark period. Thus, the C. vulgaris strain used exhibits three life cycle phases: 1) mother cell growth, 2) cell division, and 3) daughter cell release. C. vulgaris cells grow during two light periods and begin to divide during the following dark period; cell division occurs when the mother cell doubles the size of the daughter cell. Furthermore, C. vulgaris cells exposed to the herbicide terbutryn require a longer growth period to reach a cell size large enough to divide. This suggests that there is a critical threshold size required for C. vulgaris to complete the growth phase and initiate the division phase, and that this critical threshold may control progression through the G1 phase of the C. vulgaris cell cycle. Finally, this study demonstrated 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, C. vulgaris cells undergo a first mitosis, followed by cytokinesis and then two other simultaneous mitoses, which result in the release of four daughter cells [Rioboo et al. (2009) doi:10.1016 / j.aquatox.2009.07.009].

[0232] b. Genome analysis of Chlorella species Although Chlorella species have been reported to be nonmotile and lack a reproductive 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 and consists of 12 chromosomes. NC64A encodes the meiosis-specific protein dosage suppressor MCk1 DMC1, the homologous pairing proteins HOP1 and HOP2, the meiotic recombination protein MER3, the meiotic nuclear division protein MND1, and the mutS homolog MSH4, genes that are also present in most other sequenced chlorophyte algae. Nineteen homologs of the Chlamydomonas gametolysin protein, which promotes gamete cell wall degradation and enables gamete fusion, were also identified in NC64A. Furthermore, an orthologue 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]. In addition to a gene encoding gametolysin (g3347) and a gene encoding a protein containing a domain with putative GCS1 / HAP2 function, key genes involved in meiosis are also present in the 40 Mb genome of Chlorella vulgaris 211 / 11P (GenBank® accession number SIDB00000000) [Cecchin et al. (2019) Plant J. 100(6):1289-1305]. Thus, although Chlorella species have only been observed in the monoploid phase, the presence of meiotic genes indicates that the Chlorella life cycle may include a diploid phase.

[0233] Similarly, although flagella have not been observed in NC64A, orthologs of Chlamydomonas flagellar proteins have been identified in the NC64A genome, including orthologs of 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 outer arm of axonemal dynein [Blanc et al. (2010) Plant Cell 22(9):2943-2955].

[0234] Sequencing of three Chlorella sorokiniana strains, strains 1228, UTEX 1230, and DOE1412, reveals the presence of sex- and flagellum-related genes [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 conductrix; GenBank® accession number LHPF00000000) [Arriola et al. (2018) Plant J. 93(3):566-586]; and Chlorella vulgaris strain UTEX395 [Guarnieri et 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].

[0235] 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 possess antioxidant, antibacterial, and antitumor activity. High-yield production of Chlorella is routine, and MEV can be isolated from cell culture media, as detailed herein. For its use as a pharmaceutical, oral ingestion of Chlorella is known to be nontoxic and nonimmunogenic in humans.

[0236] Chlorella species have been used in various biotechnological applications, including biofuels, CO2 capture, production of high-economic-value molecules, 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 disturbances and can use nutrients, such as organic carbon and minerals, directly from wastewater for growth. Among microalgae, Chlorella species have a high photosynthetic efficiency that exceeds that of 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].

[0237] Chlorella species can also be genetically modified by Agrobacterium-mediated transformation. A study by Cha et al. developed a method for genetically transforming Chlorella vulgaris using Agrobacterium tumefaciens strain LBA4404, and compared with wild-type uninfected Chlorella, the presence of a gene fragment in 30% of the transgenic strains indicated integration of the T-DNA into the Chlorella genome [Cha et al. (2012) World J. Microbiol. Biotechnol. 28:1771-1779].

[0238] d. Chlorella MEV As described herein, Chlorella species, such as C. vulgaris, are advantageous species for the production of EVs, referred to herein as MEVs, for use in the delivery of biomolecules and small molecules for numerous applications, including therapeutic, diagnostic, and cosmetic uses. Of particular interest herein are MEVs produced by Chlorella species. Chlorella EVs have not previously 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 monoploid organism, meaning that specific, targeted mutants can be easily genetically modified or loaded to produce or contain biologically active molecules and small molecules, which can be generated by genetic engineering. Stable cell lines containing stable producers of the encoded products can be generated. These are defined products, and when exogenously loaded, the resulting compositions contain EVs containing the same cargo.

[0239] Detailed genetic maps have been obtained, allowing correlations between genotype and phenotype to be established. The Chlorella genome has been fully sequenced, allowing the structure and function of various genes to be determined. Phylogenetically, Chlorella lies at the crossroads between higher plants and microalgae. As such, Chlorella shares a significant (and useful) number of molecular biological and metabolic features with higher plants, yet remains a unicellular, monoploid microalga. Exemplary molecular biological features shared with eukaryotes include the intracellular machinery, including the Dicer enzyme system, which processes exogenous RNA into siRNA. Unlike mammalian and other animal cells, Chlorella is autotrophic and therefore can be cultivated and replicated without the need for nutrients or factors of animal origin.

[0240] Regarding the use of its EVs as therapeutic agents, Chlorella species are not toxic. For example, tablets (i.e., compressed whole Chlorella cells) made from Chlorella vulgaris biomass have been regularly consumed by people around the world as a dietary supplement for many years without any limitations associated with toxicity or immunogenicity. Japan is a world leader in the consumption of Chlorella biomass. For example, in Japan, Chlorella biomass has been used for medical treatment, as it has been shown to have immunomodulatory properties and purported anti-cancer activity, as well as for anti-aging applications, such as for cardiovascular disease, hypertension, and cataracts, reduce the risk of atherosclerosis, and stimulate collagen synthesis in the skin.

[0241] Chlorella cells naturally produce extracellular vesicles (EVs) that meet the "standard specifications" of more well-known EVs (e.g., mammalian EVs). Plant-derived EVs have several characteristics that make them more promising / favorable 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 closely related to plants, it provides a source of EVs with characteristics similar to those of plant EVs. At the same time, large-scale mass production of Chlorella is easier and less expensive than that of higher plants. The glycosylation pattern of membrane proteins in Chlorella is similar / identical to that present in higher plants.

[0242] The size of Chlorella MEVs ranges from about 50 nm to 200 nm, with an average size of about 130 nm. Their morphology resembles plant and mammalian exosomes. For administration, MEVs can be separated by size and selected to achieve a more uniform size distribution, which may vary depending on the intended use and route of administration.

[0243] 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 and the Examples below describe the biodistribution of MEVs after administration by various routes, as well as related uses and methods for targeting or treating specific diseases, disorders, and conditions, and for formulating and administering MEVs.

[0244] 1. MEV Isolation Methods of isolation are discussed in the sections above and detailed in the Examples.

[0245] 2. MEV Load and Cargo MEVs can be loaded with any desired cargo (also called 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, act as vaccines, and be used in human and other animal health, agricultural applications, gene therapy applications, including delivery genes, gene modification using gene editing systems, and gene silencing nucleic acids, cosmetic applications, dermatology applications, diagnostic applications, industrial uses, and others. MEVs can deliver regulators of gene pathways to produce nutrients or beneficial products, and can be used to achieve gene editing, such as delivering gene editing systems, e.g., CRISPR / Cas. MEVs can be used to deliver gene therapy vectors, including, but not limited to, adeno-associated (AAV) viral vectors, adenovirus vectors, vaccinia virus-derived vectors, and other substances and products.

[0246] Treatable diseases and conditions include any known to those skilled in the art, including, but not limited to, cardiovascular disease, metabolic disease, respiratory infections, bladder infections and other urinary tract infections, infectious diseases including viral diseases such as hepatitis, HIV, and coronaviruses including SARS-Cov-2, CNS diseases, ocular diseases, and liver diseases. As discussed, delivered cargoes include protein products such as antibodies and their antigen-binding forms, 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 the products, such as plasmids, nucleic acid products such as DNA encoding antisense oligonucleotides, and also antisense oligonucleotides and small molecule drugs.

[0247] MEVs can carry cargo including reporter genes and proteins and 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).

[0248] Other cargoes can target genes or products involved in disease, such as, 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).Disease-related genes, such as oncogenes and checkpoints, can be modulated by cargoes that encode products that inhibit or agonize the expression of genes or that inhibit or agonize gene products.Exemplary of such modulators include RNAi-type modulators, 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.

[0249] 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, immunomodulator 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, for example, WO2009 / 082606, JP2014-240428A, WO2011 / 072292A2, WO2010 / 141724, and WO2020 / 097540). These types of products can be delivered into or encoded in the MEV to activate genes or pathways or provide a therapeutic effect. Certain cytokines can be used to treat diseases / disorders in which immunosuppression plays a role, such as certain cancers.

[0250] Extracellular vesicles and exosomes can also be used to transport therapeutic agents, such as nucleic acids, e.g., microRNA, mRNA, tRNA, rRNA, siRNA, regulatory RNA, non-coding and coding RNA, DNA fragments and DNA plasmids (see, e.g., CN105821081A and CN110699382A), nucleotides or amino acids that contain detectable moieties or toxins or that disrupt 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). For nucleic acid cargo, non-limiting examples of proteins that may be encoded by the nucleic acid cargo molecule 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).

[0251] For example, a cocktail of three siRNA oligonucleotides targeting human MYCN with two thymidine residues (dTdT) at the 3' end of the sequence (purchased from B-Bridge International Inc., Sunnyvale, California) 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 sequences of the siRNAs (see also SEQ ID NOS: 13-35) are provided in the table below.

[0252] JPEG2025505440000011.jpg1641521 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 lung cancer 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

[0253] The cargo bioactive molecule can target central nervous system diseases, such as neurodegenerative diseases, such as Alzheimer's disease. Exemplary of such is FKBP52 and tetratricopeptide derivatives thereof. The full sequence of human peptidyl-prolyl cis-trans isomerase FKBP4 is (SEQ ID NO: 1): MTAEEMKATESGAQSAPLPMEGVDISPKQDEGVLKVIKREGTGTEMPMIGDRVFVHYTGW LLDGTKFDSSLDRKDKFSFDLGKGEVIKAWDIAIATMKVGEVCHITCKPEYAYGSAGSPP KIPPNATLVFEVELFEFKGEDLTEEEDGGIIRRIQTRGEGYAKPNEGAIVEVALEGYYKD KLFDQRELRFEIGEGENLDLPYGLERAIQRMEKGEHSIVYLKPSYAFGSVGKEKFQIPPN AELKYELHLKSFEKAKESWEMNSEEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWL EYESSFSNEEAQKAQALRLASHLNLAMCHLKLQAFSAAIESCNKALELDSNNEKGLFRRG EAHLAVNDFELARADFQKVLQLYPNNKAAKTQLAVCQQRIRRQLAREKKLYANMFERLAE EENKAKAEASSGDHPTDTEMKEEQKSNTAGSQSQVETEA

[0254] Tetratricopeptide repeat (TPR) domains 260-400 are (SEQ ID NO: 2): MNSEEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWLEYESSFSNEEAQKAQALRLA SHLNLAMCHLKLQAFSAAIESCNKALELDSNNEKGLFRRGEAHLAVNDFELARADFQKVL QLYPNNKAAKTQLAVCQQRI

[0255] A relatively simple in vitro model was developed and described by a group from the National Institute of Health and Medical Sciences at Paris XI University (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 examined by introducing two different small interfering RNA (siRNA) duplexes specific for rat FKBP52, designated RNAi 1 and RNAi 2. Oligonucleotide duplexes with the sense sequence of the siRNA and a scrambled sequence corresponding to RNAi 1 were used as negative controls. In these experiments, FKBP52 levels analyzed by Western blot were substantially reduced after 48 hours 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 resulted in the formation of elongations. Thus, these cells acquired a differentiated phenotype comparable to that of NGF-treated PC12 cells. No significant changes were observed in control-transfected cells. In another study, Chambraud et al. (Proc. Natl. Acad. Sci. USA, cited above) reported that FKBP52 prevented 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 positively tested clones, one clone, designated H7C2, was selected and used to further investigate the relationship between FKBP52 and tau by studying the effects of FKBP52 overexpression on PC12 cells. 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. The effect of FKBP52 on tau accumulation was then examined. Tau protein levels were determined by Western blotting of extracts from cultures of either PC12 or H7C2 cells treated or untreated with nerve growth factor (NGF) (50 nM) with or without Dox for 5 days. In PC12 cells, FKBP52 expression was unchanged after treatment with NGF. As expected, an increase in tau was observed after NGF treatment in both PC12 and H7C2 cells. When H7C2 cells were exposed to Dox in addition to NGF so that they overexpressed FKBP52, no additional accumulation of tau protein occurred. In PC12 cells treated with NGF and Dox, an increase in tau protein was still observed, 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.

[0256] Because one role of tau is to stimulate neurite outgrowth, the effects of FKBP52 overexpression on neurite length in PC12 and H7C2 cells were also investigated. In H7C2 cells, no neurite outgrowth was observed in the absence of NGF, regardless of whether the cells 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 controls (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 outgrowth process. The inhibition of neurite outgrowth due to FKBP52 overexpression is consistent with a previous report from Chambraud showing that loss of FKBP52 in PC12 cells results in the formation of neurite outgrowth. The FKBP52 effect on neurite length may be explained by tau binding to FKBP52 and removing tau from microtubules. Prevention of tau accumulation by overexpression of FKBP52 coincides with a decrease 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 for the treatment of Alzheimer's disease by preventing tau accumulation.

[0257] The reporter gene, reporter protein and / or its modulator can be delivered in the MEV.

[0258] Reporter proteins Target sequences in the form of siRNA, miRNA, antisense oligonucleotides (ASO), peptides and / or tetratricopeptides for modulating (inhibiting or stimulating) each marker gene, e.g., GFP protein, eukaryotic luciferase or prokaryotic luciferase, e.g., Lux operon (luxCDABE) and lux operon (luxABCDE), can be used, e.g., for diagnostics and gene expression assessment (SEQ ID NOs: 5-6, 7 and 62-65, respectively):

[0259] JPEG2025505440000012.jpg49157

[0260] Other exemplary cargoes include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (available under the trademark Cytoxan®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzino filin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; aromatase inhibiting agents such as tamoxifen, raloxifene, 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, antiestrogens, including onapristone and toremifene (sold under the trademark Fairston®); antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pterouterin, and trimetrexate; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; folic acid supplements, such as folinic acid;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, 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 and 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 Oncology, Princeton, NJ] and docetaxel [Taxotere®, Rhone-Poulenc Rorer, Antony, France]; topoisomerase inhibitors RFS 2000; thymidylate synthase inhibitors (e.g., Tomudex); additional chemotherapy drugs including aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; Bestravcil; bisantrexate; edatrexate; defosfamide; demecolcine; diaziquone; difluoromethylornithine (DFMO); eflornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lenalidomide Chinane; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol;Chemotherapeutic agents may include 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 under the trademark Xeloda®); ibandronate; CPT-11; retinoic acid; esperamicin; capecitabine; and topoisomerase inhibitors, such as irinotecan. Pharmaceutically acceptable salts, acids, or derivatives of any of the above may also be used.

[0261] Chemotherapeutic agents include, but are not limited to, prodrugs that can be converted to more active cytotoxic free drugs, 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.

[0262] Other cargoes include, for example, anti-angiogenic agents, which 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, but are not limited to, antibodies that bind to vascular endothelial growth factor (VEGF) or that bind to 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, solimastat, 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.

[0263] Other cargoes include, but are not limited to, tyrosine kinase inhibitors, such as quinazolines, e.g., PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, e.g., 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, e.g., CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); imatinib mesylate (STI571, Gleevec®; Novartis); PKI 166 (Novartis); GW2016 (Glaxo) SmithKline; CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Sugen); ZD6474 (AstraZeneca); IMC-1C11 (ImClone); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; PCT WO99 / 09016 (American Cyanamid); PCT WO98 / 43960 (American Cyanamid); PCT WO97 / 38983 (Warner-Lambert); PCT WO99 / 06378 (Warner-Lambert); PCT WO99 / 06396 (Warner-Lambert); PCT WO96 / 30347 (Pfizer, Inc.); PCT WO96 / 33978 (AstraZeneca); PCT WO96 / 33979 (AstraZeneca); PCT WO96 / 33980 (AstraZeneca), gefitinib (Iressa®, ZD1839, AstraZeneca), and OSI-774 (Tarceva®, OSI Pharmaceuticals / Genentech).

[0264] Other cargoes include immunomodulatory agents that increase or decrease the production of one or more cytokines, upregulate or downregulate autoantigen 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, prednisone, prednisolone, triamcinolone, azulfidine eicosanoids, such as prostaglandins, thromboxanes, and leukotrienes; and topical steroids, such as anthralin, calcipotriene, clobetasol, and tazarotene); cytokines, such as , TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-10; antibodies, soluble receptors and receptor-Fc fusions, including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab), B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15 Cytokines, chemokines, or receptor antagonists, including CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, 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, and T-cell receptors; heterologous antilymphocyte globulin;Other immunomodulatory molecules include, for example, 2-amino-6-aryl-5-substituted pyrimidines, anti-idiotypic antibodies of MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide, cyclosporin A, D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitrilamide (e.g., leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasalazine;

[0265] Other cargoes include cytokines, including, but not limited to, lymphokines, monokines, and traditional 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), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; 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 factors, such as NGF-beta; platelet growth factors; transforming growth factors (TGFs), such as 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).

[0266] 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.

[0267] The cargo includes antibiotics for the treatment of infectious diseases, 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 that can be treated by topical administration, e.g., by inhalation of an aerosol containing the MEV. Antibiotic treatment for lung infections in subjects with cystic fibrosis can be combined with gene therapy using the same or different MEVs, comprising nucleic acids, DNA or RNA, that encode the cystic fibrosis transmembrane conductance regulator (CFTR) protein or that provide a gene editing system to correct a defect in the CFTR protein.

[0268] Antibiotics that can be loaded as cargo into MEVs include, but are not limited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycin, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), aminocyclitols (e.g., spectinomycin), amphenicol antibiotics (e.g., azidamphenicol, chloramphenicol, ansamycin antibiotics (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, and tobramycin), monobactams (e.g., aztreonam, carumonam, and tigemonam); mupirocin; oxacephems (e.g., flomoxef, latamoxef, and moxalactam); penicillins [e.g., amdinocillin, amdinocillin pivoxil, cil, amoxicillin, bacampicillin, benzylpenicillinate, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamecillin, penethamate hydroiodide, 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 (such as 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); streptogramins (e.g., quinupristin and dalfopristin); sulfonamides (sulfanilamide and sulfamethoxazole); and tetracyclines (chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin®, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin).

[0269] 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, MEVs loaded with the cargos described herein are administered with one or more antiviral agents, including, but not limited to, 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, protease inhibitors, reverse transcriptase inhibitors, and others.

[0270] In all cases, the cargo form includes a protein, a nucleic acid encoding the protein, e.g., a plasmid, or an mRNA. The nucleic acid may be operably linked to regulatory elements recognized in the particular subject, e.g., mammal, to be delivered.

[0271] JPEG2025505440000013.jpg74149

[0272] 3. Preparation of Payload-loaded MEV As demonstrated herein, isolated Chlorella can be loaded with cargo for delivery to humans via 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, but not limited to, lipid nanoparticles, vectors, therapeutic bacteria, and therapeutic viruses. Upon administration, the MEV is 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 targeted cells, such as when the cargo is a plasmid encoding a therapeutic product. Transcriptional regulatory signals can be selected so that the encoded product is expressed in targeted 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 targeted cells, for example, in 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.

[0273] 4. Exemplary Cargos and Exemplary Uses of Exogenously Loaded MEVs a. Cargo As described above, MEVs are loaded with cargo that can be used for any purpose of interest, including those for which other delivery vehicles are used. These uses include the delivery of mRNAs, such as mRNAs encoding coronavirus spike proteins and modified spike proteins to improve immune responses to viruses, genes, such as bacterial and viral pathogen virulence genes, antibiotic resistance genes, antimicrobial resistance genes, genes that suppress the immune system, oncogenes, and RNAi, such as siRNA and antisense RNA or antisense DNA (ASO), to silence host factors for viral infection, such as targeting angiotensin-converting enzyme-2 (ACE2), transmembrane protein serine 2 (TMPRSS2), and other such genes. The cargo may 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.

[0274] 1) RNA cargo The mechanism of RNA interference, or RNAi, was originally 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, e.g., Dicer and ARGONAUTE).

[0275] In the plant kingdom, RNAi is involved in antiviral defense mechanisms and defense mechanisms against plant pathogenic fungi and oomycetes.Small regulatory RNA can be active in silencing genes in bacterial cells that lack the RNAi mechanism.The silencing activity of siRNA has been demonstrated to be inter-kingdom (see, for example, PCT / EP2019 / 072169, published as International PCT Publication No. WO2020 / 035619, PCT / EP2019 / 072170, published as WO2020 / 035620; Singla et al. (2019c) bioRxiv, doi: doi.org / 10.1101 / 863902).

[0276] RNAi-mediated modulation of gene expression has been used for several years in biotechnology 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 / parasites and to induce protection from these organisms.

[0277] 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 the virus. Recent studies have shown that plant EVs (loaded by EV-producing plant cells) naturally loaded with small RNAs from human food plants 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].

[0278] 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 and are both 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 are distinct. The major difference between siRNA and miRNA is that the former is highly specific and has only one mRNA target, while the latter has multiple targets. Both siRNA and miRNA play roles in gene regulation and serve as targets for drug discovery and development. Compared to traditional small therapeutic molecules, siRNA and miRNA offer the potential to be highly potent, can act against "undruggable" targets (e.g., proteins lacking enzymatic function), and furthermore, RNAi can be designed to target and / or affect the expression of any gene of interest.

[0279] 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 those 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, and SJ-749; anti-αVβ3 integrin antibodies, such as abciximab, CNTO-95, Mab-17E6, and Vitaxin®; anti-complement factor 5 (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 trademark Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies, e.g., IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies, e.g., oncolysin B and oncolysin CD6; anti-CD7 antibodies, e.g., HB2; anti-CD19 antibodies, e.g., B43, MT-103 and oncolysin B; anti-CD20 antibodies, e.g., 2H7, 2H7.v16, 2H7.v114, 2H7.v115, products sold or offered under the trademark Bexar® (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, e.g., SGN-40 and toralizumab; anti-CD40L antibodies, e.g., 5c8, Antova®, and IDEC-131; anti-CD44 antibodies, e.g., bivatuzumab; anti-CD46 antibodies; anti-CD52 antibodies, e.g., alemtuzumab (sold under the trademark Campus®); anti-CD55 antibodies, e.g., SC-1; anti-CD56 antibodies, e.g., h uN901-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, e.g., Crucell's anti-EpCAM, ING-1, and KS-IL-2; anti-ephrin B2 / EphB4 antibodies; anti-Her2 antibodies, e.g., trastuzumab (trademark Herceptin®), MDX-210; anti-FAP (fibroblast activation protein) antibodies, e.g., sibrotuzumab; anti-ferritin antibodies, e.g., NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250 antibodies, e.g., WX-G250 and girentuximab b) [sold under the trademark Rencarex®]; anti-GD2 ganglioside antibodies, such as EMD-273063 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 and pertuzumab; anti-HLA antibodies [for example, products sold under the trademark Oncolym®], Smart™ 1D10; anti-HM1.24 antibodies; anti-ICAM antibodies, e.g., ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies, e.g., CP-751871 and EM-164; anti-IGF-1R antibodies, e.g., IMC-A12; anti-IL-6 antibodies, e.g., CNTO-328 and elsilimomab; 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, e.g., Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1 antibodies, e.g., BravaRex and TriAb®; anti-NCAM antibodies, e.g., ERIC-1 and ICRT; anti-PEM antigen antibodies, e.g., Theragyn® and Therex®; anti-PSA antibodies; anti-PSCA antibodies, e.g., IG8; anti-Ptk antibodies; anti-PTN antibodies; anti-RANKL antibodies, e.g., AMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies, e.g., 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, can also be used. Bispecific antibodies, including but not limited to the anti-CD3 / CD20 antibody Bi20, can also be used.

[0280] Further exemplary cargoes, uses and treatments that can be achieved with cargo-loaded MEVs are described, by way of example, below.

[0281] 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 others, and combinations thereof, for delivering therapeutic molecules, acting as vaccines, and for use in human and other animal health, agricultural, cosmetic, dermatological, and diagnostic applications, industrial, and other uses. MEVs can deliver regulators of gene pathways to produce nutrients or beneficial products, gene editing systems, such as CRISPR / cas to achieve gene editing, and gene therapy vectors and products.

[0282] MEVs can carry cargos, 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 using, for example, siRNA or microRNA (see, for example, International Publication No. WO 2013 / 048734) include cancer (e.g., lung cancer, leukemia and lymphoma, pancreatic cancer, colon cancer, prostate cancer, glioblastoma, ovarian cancer, breast cancer, head and neck cancer, liver cancer, skin cancer, and uterine cancer), cardiovascular disease, ocular diseases (e.g., age-related macular degeneration, herpes stromal keratitis, glaucoma, dry eye syndrome, diabetic retinal disease, and the like). and conditions associated with ocular neovascularization and ocular hypertension), 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 (MS), diabetes, Examples include type II diabetes and diabetic conditions, arthritis (e.g., rheumatoid or psoriatic), 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., International Publication No. WO 2006 / 029161, W (See WO2007 / 022470, WO2007 / 130604, WO2008 / 021157, WO2009 / 104051, WO2009 / 142822, WO2019 / 217459, WO2020 / 123083; European Publication No. EP2504435 and U.S. Patent Publication Nos. US2011 / 0223665, US2012 / 0116360, US2012 / 0071540, US2016 / 0257956, US2015 / 0196648 and US2017 / 0304459).RNAi molecules can target genes encoding, for example, oncogenes, transcription factors, receptors, enzymes, structural proteins, cytokines, cytokine receptors, lectins, selectins, immunoglobulins, kinases, and phosphatases.

[0283] Other cargoes and uses are contemplated. For example, the MEV can carry a cargo to treat conditions resulting from trauma, such as wounds, burns, open skin lesions, fractures, hair loss, exposed dermis, exposed mucous membranes, fibrosis, lacerations, and ulcers. The MEV can carry a cargo to treat conditions resulting from natural or induced aging, particularly of the skin or of the eye.

[0284] MEV can be used to deliver cargo to treat infectious diseases, for example, by gene silencing, or can be prevented by vaccination.For example, MEV derived from antigen-pulsed macrophages or dendritic cells has been shown to induce 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-related proteins, such as viral proteins involved in host immunosuppression, pathogen replication, pathogen spread or infection maintenance; or host proteins that promote pathogen invasion into the host, pathogen or host drug metabolism, pathogen genome replication or integration, the establishment or spread of infection in the host, or the assembly of the next generation of pathogen.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; bacteria; fungi; helminths; schistosomes; trypanosomes; malaria parasites (e.g., Plasmodium vivax) malariae) and others]; and mammalian transposable elements (e.g., International Publication Nos. WO2010 / 141724, WO2011 / 071860, WO2011 / 072292, WO2013 / 126803, WO2020 / 035620 and WO2020 / 097540; Australian Publication Nos. AU2004257373 A1, AU2013203219 B2 and AU2016225873 A1; European Publication Nos. EP2395012 and EP2888240; U.S. Patent Publication Nos. US2011 / 0223665, US2014 / 0256785 and US2019 / 0032051; Japanese Publication No. JP 2018-197239A; and Taiwan Publication No. TW (See 201204351A).

[0285] MEV can also be used to deliver DNA or mRNA sequences that encode therapeutically useful polypeptides.For example, if a subject lacks a specific gene product, the gene can be encoded in a nucleic acid molecule, for example, a DNA or RNA molecule.The nucleic acid molecule that encodes the gene product can be loaded into MEV and delivered to the subject that lacks the gene product.For example, diseases resulting from the absence or deficiency of gene products include, but are not limited to, lysosomal storage disorders; metabolic disorders of the urea cycle; SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders, including cystinuria; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbach disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS) IIIB); CTNS-associated cystinosis; FMR1-related disorders including 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; neuronal ceroid lipofuscinosis-related disorders including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Hartia-Santa Vulgaris disease, Jansky-Bielschowski disease, and 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 idiopathic ataxia type 2; MECP2-associated disorders including classic Rett syndrome, ME These include CP2-related severe neonatal encephalopathy and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A- and SCN1B-related seizure disorders; Alpers-Hutten-Locker syndrome, POLG-related sensory ataxic neuropathy, including dysarthria and ophthalmoplegia, polymerase G-related disorders and 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., International Publication Nos. WO2011 / 068810, WO2019 / 243574, WO2019 / 092287, and WO2020 / 099682).

[0286] MEVs can be loaded with CRISPR / Cas systems to achieve 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 integrate sequences from invading foreign nucleic acids, such as viral or plasmid DNA, between CRISPR repeat sequences encoded within the host genome. Transcripts from the CRISPR repeat sequences are processed into CRISPR RNAs (crRNAs). Each crRNA contains a variable sequence transcribed from the foreign DNA and a portion of the CRISPR repeat. Each crRNA hybridizes with a second trans-activating CRISPR RNA (tracrRNA), and these two RNAs complex with Cas9 nuclease, directing it to cleave the target DNA sequence. Cas nucleases complexed with synthetic guide RNAs (gRNAs) consisting of a fusion of crRNA and tracrRNA can be delivered into cells to cleave the cell's genome at desired locations in vivo, allowing existing genes to be removed and / or new ones to be added [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 achieve genome modification and can be delivered in lipid nanoparticles, EVs, and other vesicles (see, e.g., International Publication Nos. WO2017 / 161010, WO2019 / 238626, and WO2020 / 097540).

[0287] MEVs can also be used to treat diseases, including but not limited to those listed above, by the introduction of 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 tumor therapeutics (e.g., chemotherapeutic, hormonal, immunotherapeutic, and radiotherapeutic agents), lipid-lowering agents for treating lipid disorders, antivirals, antifungals, anticholinergics, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, antiangiogenic agents, cytovascular agents, and the like. agents), antifibrotics, antihypertensives, aromatase or esterase inhibitors, signal transduction inhibitors, synthase inhibitors, cardiovascular agents such as antiarrhythmic agents, hormones or hormone antagonists, ion channel modifiers, antineoplastic agents, neuroactive agents, vasoconstrictors, cytotoxic agents, nucleolytic compounds, radioisotopes, prodrug-activating enzymes, and steroids (see, e.g., International Patent Publication Nos. WO2015110957A2 and WO2019018349A1; and U.S. Patent Publication Nos. US2019 / 0032051, US2012 / 0315324, US2019 / 0388347, and US2019 / 0175506). The therapeutic agent can also be a biological therapeutic selected from an allergen, adjuvant, antigen or immunogen, antibody (e.g., whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and 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), cytokine, hormone, factor, cofactor, subcellular protein, metabolic enzyme, immunomodulatory enzyme, interferon, interleukin, gastrointestinal enzyme, enzyme or factor involved in hemostasis, growth regulatory enzyme, vaccine, antithrombolytic agent, toxin, antitoxin, or diagnostic or imaging biological agent (see, e.g., International Publication No. WO 2017 / 203260,See WO2018 / 102397, WO2019 / 081474, WO2019 / 155060, WO2020 / 041720; Australian Publication No. AU2018365299A1; Singapore Publication No. SG11201811149TA; and U.S. Patent Publication No. US2019 / 0202892). For example, MEV therapy has been used to treat Crohn's disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, acute respiratory distress syndrome (ARDS), and rheumatoid arthritis. blood clots, meningitis, encephalitis, liver failure, non-alcoholic steatohepatitis (NASH), 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, type 2 diabetes and cancers [e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related Cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, cerebellar or cerebral basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain stem glioma, brain cancer, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor (pediatric, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma, cerebellar astrocytoma / malignant glioma , cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic 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, heart cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kidney cancer (renal cell carcinoma), pharyngeal cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell leukemia), lip and oral cavity cancer, cavity cancer, liposarcoma, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma, AIDS-related lymphoma, Burkitt lymphoma, skin T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's, medulloblastoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic / myeloproliferative disorders, myeloid leukemia, chronic myelocytic leukemia, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumor), ovarian germ cell tumor , ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell carcinoma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer (non-melanoma, melanoma), small intestine cancer, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's hypergammaglobulinemia and / or Wilms' tumor] can be treated (see, e.g., International Publication Nos. WO2017 / 203260 and WO2019 / 155060A1; and U.S. Patent Publication No. US2019 / 0388347).

[0288] c. Agricultural-veterinary applications MEV carrying biomolecule cargo can be used for agricultural-veterinary applications.For example, immune ribonucleic acid can be used to treat and prevent poultry diseases, and selective modulation of miRNA pathway can enhance resistance to pathogenic pathogens in plants and animals (see International Publication No. WO2008 / 087562).Therefore, cargo-loaded MEV can be used to treat diseases in animals, including livestock.

[0289] Cargo-loaded MEVs can be used to treat plant diseases. Illustratively, MEVs can be loaded with therapeutic molecules, such as siRNAs that target pathogenic genes in plant pathogens, including bacteria and viruses, and delivered to plants, such as by painting or spraying onto leaves and / or other surfaces, to target the gene and eliminate or control the pathogen.

[0290] d. Cosmetic and dermatological applications MEVs carrying pharmacological drug payloads 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, containing stem cell-derived EVs can be used to improve and / or reduce symptoms and problems such as dry skin, elasticity, wrinkles, folds, bumps, and / or vertical skin grooves (see, for example, Singapore Publication No. SG11201811149TA). Stem cell EVs, which inherently carry cytokines, growth, and transcription factors among their cargo, have also been shown to control inflammation, promote skin cell migration and proliferation, control wound scarring, improve angiogenesis, and reverse 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, playing a role in skin cell rejuvenation [da Fonseca Ferreira, A. and Gomes, D. (2019) Bioengineering (Basel) 6(1):4]. Therefore, MEVs loaded with desired cargo can be used for cosmetic and dermatological applications.

[0291] E. Pharmaceutical Compositions, Formulations, Kits, Articles of Manufacture and Combinations 1. Pharmaceutical Compositions and Formulations The MEVs and compositions containing the loaded MEVs provided herein can be formulated as pharmaceutical compositions provided for administration by a desired route, e.g., oral, mucosal, intravenous, and others. Pharmaceutically acceptable compositions are prepared for use in animals and humans, and for agricultural applications, taking into consideration regulatory or other authority approvals provided for in accordance with generally accepted pharmacopoeias for 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).

[0292] Pharmaceutical compositions can be used for therapeutic, prophylactic, cosmetic, and / or diagnostic applications. The MEVs and cargo-loaded MEVs provided herein can be formulated using 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, compatible with the other components, and not toxic to the subject to which it is administered. Formulations can be provided in unit dosage forms, including, but not limited to, tablets, pills, powders, liquid solutions or suspensions (e.g., injectable, ingestible, topical formulations, including eye drops, gels, pastes, creams, or ointments), aerosols (e.g., nasal sprays and inhalants), liposomes, suppositories, pessaries, injectable and insoluble solutions, and sustained-release forms, and can be prepared by methods well known in the art of pharmacy. For example, Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; See 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, therapeutic compositions are sterile, pyrogen-free, and generally free of particulate matter, and are in a parenterally acceptable solution having due regard for pH, isotonicity, and stability, conditions which are known to those skilled in the art.Methods for preparing parenterally administrable compositions are well known, or will be apparent to those skilled in the art, and are described in more detail, for example, in "Remington: The Science and Practice of Pharmacy (Formerly Remington's Pharmaceutical Sciences)", 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0293] The pharmaceutical compositions provided herein can be in various forms, for example, solid, semi-solid, liquid, powder, aqueous and lyophilized form.The example of suitable pharmaceutical carrier is known in the art, and includes but is not limited to, among others, water, buffer solution, physiological saline solution, phosphate buffered physiological saline solution, various types of wetting agent, 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, perfume oil, fatty acid monoglyceride and diglyceride, pentaerythritol fatty acid ester, hydroxymethylcellulose and powder. The pharmaceutical compositions provided herein can contain, for example, antioxidants, preservatives, antimicrobial 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, emulsifying and suspending agents, e.g., gum arabic, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl ... Other additives may be included, including propylmethylcellulose, methylcellulose, octoxynol-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum and its derivatives, solubilizers, and a variety of ingredients, such as crystalline cellulose, 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, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and / or additives can be formulated by conventional methods and administered to subjects at appropriate doses.Stabilizing agents, such as lipids, nuclease inhibitors, polymers, and chelating agents, can preserve the composition from degradation within the body.

[0294] The route of administration is consistent with known methods, such as injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation, or intralesional routes, topical, rectal, mucosal, and sustained-release systems. MEVs or cargo-loaded MEVs can be administered continuously by infusion or by bolus injection. MEVs or cargo-loaded MEVs can be administered by local or systemic methods.

[0295] 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, such as a liquid or powder aerosol (lyophilized), can be administered intravenously or through the nose or lungs. The composition can also be administered parenterally or subcutaneously, as desired. When administered systemically, the therapeutic composition must be sterile, pyrogen-free, and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those of skill in the art.

[0296] Pharmaceutical compositions suitable for use include compositions containing an amount of MEV or cargo-loaded MEV effective to achieve its intended purpose. Determining a therapeutically effective amount is well within the capabilities of one skilled 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.

[0297] Therapeutic formulations can be administered in a number of conventional dosage forms. The dosage forms of the MEVs and cargo-loaded MEVs provided herein are prepared for storage or administration by mixing the compound having the desired degree of purity with a physiologically acceptable carrier, excipient, or stabilizer. Such materials are non-toxic to recipients at the dosages and concentrations employed and may include buffers such as Tris HCl, phosphates, citrates, acetates 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 or 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 or sorbitol; counterions such as sodium and / or non-ionic surfactants such as polysorbate (TWEEN), Pluronic, polyethylene glycol, etc.

[0298] In certain examples, pharmaceutical compositions containing a stabilizer are provided herein.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 a 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 may be 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, inclusive. to 500 mM or about 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.

[0299] 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 the 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.

[0300] When used for in vivo administration, the formulation must be sterile and can be formulated according to conventional pharmaceutical practice. This is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and reconstitution. MEVs or cargo-loaded MEVs can be stored in lyophilized form or in solution; they can be frozen or refrigerated. Other vehicles, such as naturally occurring vegetable oils like sesame, peanut, or cottonseed oil, or synthetic fatty vehicles like ethyl oleate, can be included. Buffers, preservatives, and antioxidants may also be incorporated according to accepted pharmaceutical practice.

[0301] The MEVs or cargo-loaded MEVs provided herein may have a concentration of at or about 0.1-10 mg / mL or, depending on the application and subject, at least 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 more or a concentration that is 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 more. The volume of the solution can be at or about 1 to 100 mL, such as at or about at least 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.

[0302] 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 (see, 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; 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, and 5,128,326; and PCT 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.

[0303] In some examples, the pharmaceutical composition contains a MEV or cargo-loaded MEV provided herein and one or more additional agents for combination therapy, e.g., an antibody or other therapeutic agent.

[0304] 2. Manufactured Products / Kits and Combinations Pharmaceutical compositions of MEVs or cargo-loaded MEVs can be packaged as articles of manufacture containing packaging materials, a pharmaceutical composition that is effective to treat a disease or condition treatable 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, e.g., 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 a metered 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.

[0305] The product provided herein contains packaging material.Packaging material for use in packaging pharmaceutical products is well known to those skilled in the art (see, for example, U.S. Patent No. 5,323,907, U.S. Patent No. 5,052,558 and U.S. Patent No. 5,033,252).Examples of pharmaceutical packaging material include but are not limited to blister pack, bottle, tube, inhaler (for example, pressurized metered dose inhaler (MDI), dry powder inhaler (DPI), nebulizer (for example, jet or ultrasonic nebulizer) and other single-breath liquid system), pump, bag, vial, container, syringe, bottle and any packaging material suitable for selected formulation and intended administration mode and treatment.

[0306] MEVs or cargo-loaded MEVs can be provided as combinations and as kits. Kits may include one or more components, such as instructions for use, devices and additional reagents (e.g., sterile water or saline solution for diluting compositions and / or reconstituting lyophilized proteins), as well as components for carrying out methods, such as tubes, containers, and syringes. Exemplary kits can include MEVs or cargo-loaded MEVs provided herein, and may also include instructions for use, a device for administering the MEVs or cargo-loaded MEVs to a subject, a device for detecting the MEVs or cargo-loaded MEVs in a sample obtained from the subject, and a device for administering additional therapeutic agents to the subject.

[0307] The kit may include instructions for use. The instructions typically include specific language describing the MEV or cargo-loaded MEV, optionally other components included in the kit, and methods of administration, including methods for determining the appropriate subject condition, appropriate dosage, administration schedule, and appropriate administration method for administering the MEV or cargo-loaded MEV. The instructions may also include guidance for monitoring the subject over the duration of treatment.

[0308] Kits can also include pharmaceutical compositions and diagnostic items 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.

[0309] 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).

[0310] The kit provided herein can also include a device for administering MEV to a subject.Any of the various devices known in the art for administering medicines to a subject can be included in the kit provided herein.Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, nebulizers, and inhalers.Usually, the device for administering the composition is compatible with the desired method of administering the composition.

[0311] 3. Administration and Route of Administration 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 parenterally (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intracavity routes), topically, epidurally, or mucosally (e.g., topically, intranasally, orally, intravaginally, vulvovaginally, esophageal, oroesophageal, bronchial, rectal, and pulmonary routes). The cargo-loaded MEVs can be administered externally to a subject at the site of disease for local or transdermal effect. Compositions containing cargo-loaded MEVs can be administered by any conventional route, for example, by injection, inhalation, bolus injection, or by absorption through epithelial or mucocutaneous layers (e.g., topically, orally, intravaginally, rectally, and intestinal mucosa). Compositions containing cargo-loaded MEVs can be administered in conjunction with or sequentially with other biologically active agents, for example, cargo-loaded MEVs can be administered by infusion delivery, such as by an infusion pump or syringe pump, and can be administered in combination with another therapeutic agent or as monotherapy.

[0312] The method and / or route of administration can be modified to reduce 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 can 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 can be temporarily or permanently discontinued.

[0313] In some cases, if a subject experiences adverse side effects, such as severe skin toxicity, for example, a severe acne-like rash, treatment adjustments can be made. For example, administration can be delayed for, for example, 1 to 2 weeks after the onset of adverse side effects, or until the adverse side effects have improved. In some cases, the dosage can be reduced after additional adverse side effects occur. Specific regimens and treatment protocols can be established by a physician or other practitioner skilled in the art.

[0314] An appropriate method for delivery can be selected by one of skill in the art based on the dosage characteristics of the cargo-loaded MEV or pharmaceutical composition containing the cargo-loaded MEV, including, but not limited to, solubility, hygroscopicity, crystallization characteristics, melting point, density, viscosity, flow, stability, and degradation profile.

[0315] 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 dose(s) and timing and method of administration of each therapeutic regimen or agent empirically or by considering the pharmacokinetics and mode of action of the agent. The additional therapeutic regimen or agent may improve the efficacy or safety or other properties of the cargo-loaded MEV. In some examples, the additional therapeutic regimen or agent can treat the same disease or coexisting disease. In some examples, the additional therapeutic regimen or agent can ameliorate, 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.

[0316] For example, 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, and cargo-loaded MEVs can be administered in conjunction with other anti-pathogen therapeutics and treatments. 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 inhibitory agents, antihormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotective agents, immunostimulants, 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.

[0317] The one or more additional agents can be administered simultaneously with the cargo-loaded MEV, sequentially, or intermittently. The agents can be co-administered, for example, as part of the same pharmaceutical composition or the same delivery method. In some examples, the agents can be co-administered simultaneously with the cargo-loaded MEV, but by a different delivery means. The agents can also be administered at different times 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 following or prior to 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.

[0318] F. Biodistribution of MEV after administration by various routes 1. Mammalian EV Biodistribution 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]. Treatments using mammalian cell-derived EVs are generally based on intravenous or intraperitoneal administration routes. The main target organs during systemic administration of mammalian EVs are the liver, spleen, and lungs. A comprehensive study of the tissue distribution of fluorescently labeled mammalian EVs derived from various cell sources [see Wiklander et al. (2015) J. Extracellular Vesicles 4:26316] demonstrated that 24 hours 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 due to saturation of the mononuclear phagocyte system (MPS). Comparison of intraperitoneal (i.p.), subcutaneous (sc), and i.v. administration showed that i.p. and sc doses resulted in reduced EV accumulation in the liver and spleen and enhanced accumulation in the pancreas and gastrointestinal tract compared with i.v. injection. Systemically administered EVs have been reported to be rapidly taken up by the mononuclear phagocyte system (MPS), particularly in the liver and spleen. The mechanism of clearance is similar to that described for synthetic nanoparticles, e.g., liposomes [Van der Meel et al. (2014) J. Control. Release 195:72-8].The majority of splenic accumulation is due to EV storage in the spleen rather than splenic uptake [Lai CP et al. (2014) ACS Nano 8:483-494]. The biodistribution of mammalian EVs after other administration routes has also been investigated. For central nervous system targeting, intranasal administration of curcumin-loaded mammalian EVs resulted in EV localization in the brain. Drug levels peaked 1 hour after administration, and significant amounts were detected 12 hours later, but no toxic effects were observed [Zhuang et al. (2011) Mol. Ther. 19:1769-1779].

[0319] Mammalian EVs are generally not utilized for oral delivery due to their low stability at various pH and temperatures, rapid degradation of biomolecules in the gastrointestinal tract, and limitations on industrial-scale production for oral administration [Cheng et al. (2019) Protein Cell 10:295-299]. The only exception to date is bovine milk-derived EVs, which, upon oral delivery to mice, showed a distribution pattern analyzed using a whole-body in vivo imaging system (IVIS), which included rapid accumulation in the intestine. EVs were detectable after 2 and 6 hours, followed by fluorescent signals in the liver, spleen, lungs, kidneys, heart, and gastrointestinal tract at 24 hours. After 48 hours, the fluorescent signal subsided within most of the organs, indicating clearance of the nanovesicles from the system [Samuel et al. (2021) Nat Commun 12:3950, doi.org / 10.1038 / s41467-021-24273-8]. Therefore, mammalian EVs (derived from sources other than milk) cannot be absorbed by and from the intestinal tract and become bioavailable in target organs [Zhong et al. (2021) Biomaterials. 277:121126. doi: 10.1016 / j.biomaterials.2021.121126].

[0320] Treatments using mammalian cell-derived EVs generally use intravenous or intraperitoneal routes for systemic administration, with the target organs being the liver, spleen, and lungs. As previously described, most mammalian EVs have not been used for oral delivery due to their low stability at various pH levels and temperatures, rapid degradation of biomolecules in the gastrointestinal tract, and limitations in industrial-scale production for oral administration [Cheng et al. (2019) Protein Cell 10(4):295-299]. The only exception is bovine milk-derived EVs, which, upon oral delivery to mice, showed a distribution pattern that included rapid accumulation in the intestine, as analyzed using a whole-body in vivo imaging system (IVIS). EVs were detectable after 2 and 6 hours, followed by fluorescent signals in the liver, spleen, lungs, kidneys, heart, and gastrointestinal tract at 24 hours. After 48 hours, the fluorescent signal subsided within most of the organs, indicating clearance of the nanovesicles from the system.

[0321] As shown in the Examples and discussed below, MEVs have properties that differ from those derived from mammalian EVs. For example, they are more stable in the harsh environment of the gastrointestinal tract than EVs derived from mammalian cells. Therefore, microalgal EVs as described herein are particularly suitable for oral administration and drug delivery, as well as other routes of delivery as described herein.

[0322] 2. Microalgae EV distribution in the body MEVs, including those provided herein from Chlorella, are shown herein to have properties that are distinct from mammalian EVs, including bovine milk EVs. For example, a notable difference, discussed below, is that MEVs can be administered orally and their primary target is the spleen, presumably the white pulp of the spleen (white spleen).

[0323] The MEVs provided herein can deliver a variety of bioactive molecules, such as RNAs (e.g., mRNA, siRNA, and miRNA), proteins, peptides, and small molecules, which can be loaded exogenously or endogenously. These include tissue-specific and / or disease-specific products. As discussed below, each route can be used to target specific organs and treat specific diseases. MEVs can be formulated for administration by each route. Thus, compositions containing MEVs for treating specific diseases and for specific administration routes are provided.

[0324] It has been shown herein that the route of administration determines the fate of MEVs, and that their ultimate location is a function of the route of administration. Targets and endpoints of MEVs include, but are not limited to, the liver, spleen, lungs, intestine, and brain. Routes of administration include, but are not limited to, respiratory (nose, lung), oral (digestive), intravenous, central nervous system (CNS), and topical. The choice of route depends on the ultimate target and payload. It has been shown herein that intranasal administration leads to the lungs, intratracheal administration via spray leads to the lungs, intravenous administration leads to accumulation in the spleen and liver, and oral administration leads to the digestive tract and spleen. In contrast, mammalian EVs cannot be taken orally.

[0325] MEVs are readily internalized by human cells. For example, when administered to cultured cells, such as A549 cells, at a ratio of 1000 / 1 MEVs / cell in vitro, 93% of the cells internalized the MEVs within 24–48 hours of contacting the cells with the MEVs.

[0326] DIR-labeled MEVs were administered to mice via four routes: intranasal (IN), intratracheal (IT), intravenous (IV), and oral. The fate of the MEVs was visualized by whole-body imaging over time for 3 days. Subsequently, the mice were sacrificed and organs were harvested for study. As shown in the Examples, intravenous administration targets the liver approximately 4-12 hours after administration and the spleen 10-30 hours later, where it appears to be located in the red pulp of the spleen. Oral administration targets the intestine and spleen. Herein, it has been shown that MEVs are orally available, survive passage through the stomach, reach the intestine 0.5-4 hours later, and then reach the spleen 0.5-10 hours later. Of interest is the route to the spleen, which involves targeting and delivering cargo to the immune system, where it accumulates for 4 to 28 hours: via the blood (to the red spleen) and via lymphocytes (to the white spleen). This can be achieved by internalization by activated lymphocytes that reach the spleen and expand there, and / or by non-activated lymphocytes that phagocytose the MEVs, which progress to the white pulp of the spleen (white spleen) and are disseminated from there by the immune system.

[0327] Oral administration Thus, orally ingested MEVs travel to the intestine and then, as shown, end up in the spleen, presumably the white pulp. The spleen is involved in initiating immune responses to blood-borne antigens and filtering foreign material and old or damaged red blood cells from the blood. These functions are carried out by two distinct compartments in the spleen: the white pulp and the red pulp. The two compartments differ significantly in structure, vascular organization, and cellular composition [see, e.g., Cesta (2006) Toxicologic Pathology 34:455-465 for a review of the structure, function, and histology of the spleen].

[0328] White blood cells, abundant in the intestine, migrate to the white spleen. When ingested orally, MEVs can be internalized by intestinal cells and, as discussed below, by intestinal lymphocytes, leading to their transport to the spleen. This contrasts with mammalian vesicles, which cannot be administered orally. Thus, MEVs provide a delivery vehicle for immune system-targeted drugs, e.g., immunomodulatory cargo. As discussed above, the route to the white spleen can occur via, for example, activated lymphocytes and / or phagocytic lymphocytes. Lymphocytes can phagocytose MEVs and home them to the spleen. Unlike mammalian EVs, MEVs provide a method for orally delivering small molecule drugs and proteins and other therapeutics, e.g., nucleic acid therapeutics, that cannot be administered orally. In particular, orally administered MEVs provide a route for the treatment of diseases, e.g., cancer and inflammatory diseases, in which the immune system is involved or where treatment can be achieved by targeting the immune system. Such diseases include, but are not limited to, infectious diseases, autoimmune diseases, cancer, and prevention of organ transplant rejection, which are treated by suppressing or enhancing immune cell activity.

[0329] 1) Components of the lymphatic system The lymphatic system includes lymph, lymphatic vessels, and lymphatic organs [see discussion in Zgair et al., (2016) Targeting Immunomodulatory Agents to the Gut-Associated Lymphoid Tissue. In: Constantinescu C., Arsenescu R., Arsenescu V. (eds) Neuro-Immuno-Gastroenterology. Springer, Cham. (doi.org / 10.1007 / 978-3-319-28609-9_14) and summarized below].

[0330] lymph Lymph is generally a clear, colorless fluid that drains from the interstitium and contains collected fluid and plasma proteins, and may also contain lipids, immune cells, hormones, bacteria, viruses, cellular debris, and cancer cells.

[0331] Lymphatic vessels The lymphatic system is the body's second circulatory system. It is a unidirectional, blind-ended, thin-walled system of capillaries through which lymph is pumped. Lymphatic capillaries drain into afferent collecting ducts, which then pass through one or more collections of lymph nodes. Lymph fluid then passes through efferent collecting ducts, larger trunks, and then lymphatic vessels, which drain lymph into the systemic circulation. Primary lymphoid organs include the thymus and bone marrow, which produce mature lymphocytes that identify and respond to antigens, while secondary lymphoid organs include the lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT). Within secondary lymphoid organs, lymphocytes initiate immune responses. MALT is distributed throughout the mucosa and provides a defense mechanism against a wide variety of inhaled or ingested antigens. According to their anatomical location, MALT is categorized as bronchus-associated lymphoid tissue (BALT), nasal cavity-associated lymphoid tissue (NALT), salivary duct-associated lymphoid tissue (DALT), conjunctiva-associated lymphoid tissue (CALT), lacrimal duct-associated lymphoid tissue (LDALT), and gut-associated lymphoid tissue (GALT).

[0332] Gut-associated lymphoid tissue (GALT) The GALT consists of effector and inductive sites. Effector sites include lymphocytes distributed throughout the lamina propria (LP) and intestinal epithelium, while inductive sites include tissues such as mesenteric lymph nodes (MLNs), PPs, and smaller isolated lymphoid follicles (ILFs). The mesenteric lymph nodes (MLNs), located at the base of the mesentery, are the largest collection of lymph nodes in the body. MLNs are structurally divided into two regions: the medulla and the cortex. The cortex is primarily composed of T cell regions and B cell follicles. Within the T cell regions, circulating lymphocytes enter the lymph nodes, where dendritic cells (DCs) present antigens to T cells. Lymph (containing cells, antigens, and chylomicrons) is collected from the intestinal mucosa and reaches the MLNs via afferent lymphatic vessels. The lymph fluid then leaves the MLNs through efferent lymphatic vessels, reaches the thoracic duct, and flows into the bloodstream.

[0333] Peyer's patches (PPs) are collections of lymphoid nodules distributed throughout the intestinal mucosa and submucosa. They contain B cell follicles dispersed among subepithelial dome regions and T cell regions. A single layer of epithelial cells called follicle-associated epithelium (FAE) separates the lymphoid regions of the PP from the intestinal lumen. The FAE are permeated by specialized enterocytes called microfold (M) cells. These cells are the gate for transport of luminal antigens into the PP.

[0334] Isolated lymphoid follicles (ILFs) are a combination of lymphoid cells in the intestinal LP. ILFs consist of germinal centers surrounded by FAEs containing M cells. ILFs are a complementary system to PPs for the induction of intestinal immunity.

[0335] The GALT is the largest lymphoid organ in the human body and contains more than half of the body's lymphocytes. The GALT is exposed to more antigens than any other part of the body, in the form of commensal bacteria and dietary antigens, in addition to those derived from invasive pathogens. Intestinal lymphatic transport avoids first-pass metabolic loss in the liver by diverting lipophilic drug absorption toward intestinal lymphatics rather than the portal vein. The intestinal immune system must distinguish between antigens requiring a protective immune response and generate a state of immune hyporesponsiveness (oral tolerance) to harmless antigens. This is achieved by sampling of luminal antigens in the intestinal epithelium by DCs. Antigens can cross the epithelium via M cells, specialized epithelial cells in the follicle-associated epithelium of the GI tract. Antigens interact with DCs in the underlying subepithelial dome region. DCs then present antigens to local T cells in the PP.

[0336] DCs also migrate to the draining MLNs, where they present antigens to local lymphocytes. Alternative routes of antigen transport across intestinal epithelial cells include receptor-mediated transport and direct sampling from the lumen by DC processes. Antigen-loaded DCs then migrate via afferent lymphatics to the MLNs, where they present antigens to T cells. Differentiated lymphocytes then migrate from the MLNs via the thoracic duct and bloodstream, eventually accumulating in the mucosa for appropriate immune responses.

[0337] 2) Targeting GALT Orally administered MEVs can target the gut-associated lymphoid tissue (GALT). Thus, the GALT is a target (effective compartment) and / or pathway through which MEVs and their therapeutic cargoes can be used to deliver cargo to organs, tissues, and / or the systemic circulation. The GALT is a favorable target for various pharmacological agents, such as immunomodulators, chemotherapeutic agents, and anti-infectives. The lymphatic system is a major pathway for intestinal and other tumor metastasis, and therefore targeting cytotoxic agents to intestinal lymphatics can be used to treat tumor metastasis. The GALT is a delivery target for antiviral agents, as some viruses, such as human immunodeficiency virus (HIV), morbillivirus, canine distemper virus, or severe acute respiratory syndrome (SARS)-associated coronavirus, hepatitis B, and hepatitis C, spread and develop within the lymphatic system.

[0338] Thus, MEVs, including the exemplified Chlorella MEVs herein, can be used to target immune cells during oral delivery. As noted above, microalgal MEVs exhibit distinct patterns of biodistribution when administered orally, including initial intestinal accumulation followed by targeting to the spleen, where they are detectable for up to 24 hours (see, e.g., Figure 7).

[0339] Because microalgae MEVs are delivered to the spleen, the mechanism of delivery may be based on cells of the immune system. Immune cells are abundant in the intestinal epithelium of the gut-associated lymphoid tissue (GALT) and the underlying single-cell layer of the lamina propria. Immune cells include T cells, plasma cells, mast cells, dendritic cells, and macrophages [Luongo et al. (2009) Current perspectives. International Reviews of Immunology 28(6):446-464, doi.org / 10.3109 / 08830180903236486]. Macrophages, dendritic cells, neutrophils, and also B cells perform phagocytosis. Therefore, immune cells in the intestine may engulf MEVs and deliver them to the spleen. After phagocytosis, the fate of MEV cargo may vary depending on the type of cargo. For example, macrophages and dendritic cells are involved in antigen presentation, and proteins present in or products delivered in the MEV may be secreted, or products such as RNA may be translated.

[0340] Immune cells present in the intestinal epithelium and lamina propria of the intestine migrate to the spleen and home to the intestine. This homing to the spleen may be involved in MEV transport from the intestine to secondary lymphoid organs, particularly the spleen. T cells exhibit a specific lymphocyte recirculation pathway that may be part of MEV transport to the spleen upon oral delivery [Mackay et al. (1990) J Exp Med 171:801-17]. Thus, cells of the immune system are targeted by orally administered MEV, and this phenomenon contributes to MEV localization in the spleen within hours of administration.

[0341] As shown herein, upon oral administration, MEVs travel to the intestine and then migrate to the spleen. The route to the spleen could be via absorption into the blood and / or internalization by immune cells in the intestine. The blood route is an unlikely route because MEVs should then appear in the liver, as shown for intravenous administration. When MEVs are administered intravenously, they reach primarily the liver (in large quantities) and, to a much lesser extent, the spleen. It has been shown herein that clearance of MEVs from the spleen follows different kinetics depending on their origin (oral or IV). Thus, migration to the spleen after oral administration uses a different route than intravenously administered MEVs. When MEVs are administered by mouth, they reach the spleen after passing through the intestine. These results indicate that MEVs are localized to "different compartments" within the spleen depending on their route of arrival, either from the intestine or from the blood. As discussed, upon oral administration, a likely pathway is that MEVs in the intestine are internalized by lymphocytes present in the GALT, and subsequent movement of MEVs from the intestine / GALT to the spleen occurs because MEVs are transported by lymphocytes. From the intestine / GALT, MEVs end up in the white spleen compartment. Thus, MEVs offer a method of delivering cargo to different organs than mammalian EVs, which cannot be administered orally.

[0342] 3. Diseases and Conditions Treated by MEV Depending on the organ being targeted, various diseases and disorders can be treated by MEV. MEV can be loaded and produced to contain therapeutic agents for treating these diseases and conditions. The appropriate administration route for the targeted organ and disease is selected. For example, to target the spleen and intestine, oral administration is selected, and to target the lung, inhalation or nasal administration is selected. Based on biodistribution and pharmacokinetic data, the following organs can be targeted to treat the following exemplary diseases:

[0343] Liver: cancer, cancer metastasis, metabolic syndrome, genetic disorders (gene therapy delivery), alpha-antitrypsin (AAT) deficiency and other inborn errors of metabolism, hemophilia, hypercholesterolemia, liver inflammation, steatohepatitis and other diseases and disorders that can be treated by delivery of therapeutic agents to the liver;

[0344] Spleen: Diseases treated by immune modulation, including cancer and immune cell disorders, and cancer and other diseases that may be treated by administration to the spleen, particularly by immune cells that originate in or traffic to the white spleen;

[0345] Intestinal: diseases and disorders treated or prevented by vaccines, intestinal infections, microbiota modulation, Crohn's disease, cancer, ulcers, diseases treated by orally administered drugs, e.g., small molecules and proteins, and other such diseases, disorders, and conditions; and

[0346] Pulmonary: infectious diseases, especially respiratory diseases, chronic obstructive pulmonary disease (COPD), pulmonary hypertension, asthma, other inflammatory lung diseases, cystic fibrosis, ATT deficiency, lung diseases, cancer, cancer metastasis and other such diseases and disorders.

[0347] G. Formulations, Routes of Administration, and Diseases and Disorders Compositions containing MEV in an amount suitable for achieving treatment of a particular disease or disorder are provided. The amount may vary depending on the therapeutic cargo, the disease or disorder, and the subject being treated. It is within the level of ordinary skill in the art to determine the individual dosage of MEV. Formulations include any known to those skilled in the art, for example: Injectable solution for intravenous administration to reach the liver and spleen, Oral, such as tablets, capsules, films and lozenges; Oral drops for administration, e.g. vaccines, which reach the intestines, the immune system (immune cells) and the spleen; Compositions for inhalation, such as for intratracheal and intrapulmonary administration to reach the lungs, e.g. emulsions (microemulsions and nanoemulsions); Drops for intranasal administration, and Preparations, such as creams, oils, gels, lotions, ointments for the skin and mucous membranes Examples include:

[0348] Pharmaceutical compositions containing microalgal extracellular vesicles (MEVs) in a pharmaceutically acceptable medium are provided. The MEVs may contain drugs, generally therapeutic or biologically active agents, such as nucleic acids, particularly RNA, proteins, small molecules, and other such drugs. The compositions contain MEVs in an amount that can be diluted to deliver a therapeutically effective amount of the drug, or are formulated for direct administration without dilution. The specific concentration of MEVs will vary depending on various parameters within the skill of those in the art, including, for example, the indication being treated; the active agent; the route of administration; the disease, disorder, or condition being treated; and the regimen. Routes of administration include systemic and topical routes, oral, rectal, intravenous, intramuscular, subcutaneous, mucosal, inhalation, nasal, ocular, peritoneal, intratracheal, intravitreal, vaginal, and any other suitable route known to those skilled in the art.

[0349] Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.

[0350] Exemplary Formulations Pharmaceutical compositions containing MEV can be formulated in any conventional manner by mixing a selected amount of active compound with one or more physiologically acceptable carriers or excipients. The choice of carrier or excipient is within the purview of the administering professional and can vary depending on several parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical, or any other mode) and the disorder being treated. The formulation can also be co-formulated with other active agents for combination therapy.

[0351] A selected amount of MEV is formulated in a vehicle suitable for administration by a selected route. Pharmaceutical compositions can be formulated in any conventional manner by mixing a selected amount of MEV with one or more physiologically acceptable carriers, excipients, or vehicles. The pharmaceutical compositions can be used for therapeutic, prophylactic, cosmetic, and / or diagnostic applications. The concentration of MEV in the composition will vary depending on various factors, including those described above and the absorption, inactivation, and excretion rates of the active drug cargo, release of the cargo, mechanism of release, dosage schedule and amount administered, age and size of the subject, and other factors related to the characteristics of the MEV known to those skilled in the art.

[0352] The pharmaceutical compositions provided herein may be in various forms, including, but not limited to, solid, semisolid, liquid, emulsion, powder, aqueous, and lyophilized forms. The pharmaceutical compositions provided herein may be formulated for single-dose (direct) administration or for dilution or other regimens. The concentration of the co...

Claims

1. 1. A composition comprising microalgal extracellular vesicles (MEVs) for use in treating or preventing a disease, disorder, or condition, comprising: formulated for inhalation, oral, intravenous, intramuscular, intranasal, intratracheal, or ophthalmic administration; the formulation is selected for a route of administration that delivers the MEV to a target tissue of or involved in the disease, disorder, or condition; the MEV comprises a heterologous bioactive cargo for the treatment of a disease, disorder, or condition of or involving a target tissue; The composition, wherein the microalgae is a species of the genus Chlorella.

2. 2. The composition of claim 1, wherein the target tissue is selected from among lung, liver, spleen, intestine, gut-associated lymphoid tissue (GALT), brain, nasal-buccal mucosa, heart, kidney, and retina and / or choroid tissue of the eye.

3. The Chlorella extracellular vesicles comprise a heterologous bioactive molecular cargo that has been exogenously introduced into the isolated extracellular vesicles; The composition of claim 1 , wherein the biologically active cargo is a biomolecule or a small molecule.

4. comprising microalgal extracellular vesicles (MEVs) in a pharmaceutically acceptable vehicle for oral administration; 2. The composition of claim 1, wherein the target tissue is gut-associated lymphoid tissue (GALT).

5. 5. The composition of claim 4, wherein the MEV comprises a heterologous bioactive cargo that is immunomodulatory.

6. the composition is formulated for oral administration and the disease, disorder, or condition involves the gastrointestinal tract or the immune system; The composition of claim 1 , wherein the heterologous cargo targets the gastrointestinal tract or the immune system or the white spleen for treatment.

7. The composition of claim 6, wherein the cargo is for the treatment of a disease, disorder or condition by immune modulation.

8. the composition is formulated for intranasal administration; the target tissue is the brain; 10. The composition of claim 1, wherein the cargo comprises a biologically active molecule for the treatment, detection or diagnosis of a disease, disorder or condition of or involving the brain, or for monitoring the treatment of a disease, disorder or condition of or involving the brain.

9. the composition is formulated for instillation into the eye as eye drops; The composition of claim 1 , wherein the target tissue is the choroid and / or retina.

10. 1. A composition that is a vaccine, comprising: the composition is formulated for intramuscular or oral administration; The composition of claim 1 , wherein the cargo comprises an antigen or an immunomodulatory protein or product.

11. 10. The composition of claim 1, wherein the composition is formulated for oral administration and the disease, disorder, or condition involves or is of an organ or tissue accessible via the bloodstream.

12. the composition is formulated for intratracheal administration or for inhalation or nebulization; the disease involves or is of the respiratory tract; 10. The composition of claim 1, wherein the cargo is a product for treating the airways and / or lungs or for treating a respiratory disease or a disease involving the airways.

13. The composition of claim 1 , wherein the cargo is a nucleic acid molecule, a polypeptide, a protein, a plasmid, an aptamer, an antisense oligonucleotide, or a small molecule drug.

14. The composition of claim 1 , wherein the cargo is a nucleic acid that is DNA or RNA.

15. The composition of claim 14 , wherein the cargo comprises a plasmid.

16. Cargo, Increase or decrease the production of one or more products; up- 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; 10. The composition of claim 1 comprising or encoding an immunomodulatory agent.

17. The composition of claim 1 , wherein the cargo comprises or encodes a hormone, cytokine, or chemokine.

18. The composition of claim 13 , wherein the cargo comprises a nucleic acid encoding a product for gene therapy.

19. 19. The composition of claim 18, wherein the gene therapy is for treating an inborn error of metabolism.

20. 2. The composition of claim 1, wherein the MEV cargo comprises or encodes a protein that is an antibody or an antigen-binding fragment thereof.

21. 21. The composition of claim 20, wherein the antibody is an scFv, a bispecific antibody, or an antigen-binding fragment thereof.

22. 1. A composition that is a vaccine, comprising:

2. The composition of claim 1, wherein the cargo comprises DNA or RNA encoding an antigen or an immunomodulatory protein, wherein the cargo is a protein that elicits an immune response or is an antigen.

23. 23. The composition of claim 22, wherein the composition is formulated for oral administration.

24. 2. The composition of claim 1, wherein the MEV cargo comprises a nucleic acid or protein or a nucleic acid encoding a protein that is a therapeutic product for treating cancer or an infectious disease or a neurodegenerative disease or other central nervous system (CNS) disorder, or for ameliorating the effects of aging or slowing or reversing aging, or for treating an age-related disease or an ocular disorder or an immunological disorder.

25. The composition of claim 1 , wherein the cargo comprises an agonist or antagonist of an intracellular endosomal receptor.

26. 26. The composition of claim 25, wherein the receptor is a Toll-like receptor (TLR) and the cargo comprises an agonist or antagonist of the TLR.

27. 27. The composition of claim 26, wherein the cargo comprises an agonist of a TLR, wherein the TLR and TLR agonist are selected from one or more of the following: TLR1, and an agonist that is the triacyl lipopeptide Pam3CSK4; TLR2 and an agonist that is zymosan, porin, modulin, lipoprotein, lipoteichoic acid, diacyl lipopeptide, atypical LPS, peptidoglycan, and / or triacyl lipopeptide; TLR3, and an agonist that is dsRNA; TLR4 and an agonist that is mannan, taxol, and / or lipopolysaccharide (LPS); TLR5, and an agonist that is bacterial flagellin, profilin, HMGB1, and / or the small molecule agonist CBLB502; TLR6 and an agonist that is zymosan, porin, modulin, lipoprotein, lipoteichoic acid, diacyl lipopeptide (Pam2CSK4), atypical LPS, and / or peptidoglycan; TLR7 and an agonist that is an imidazoquinoline, loxoribine, ssRNA, bropirimine, and / or resiquimod; agonists that are TLR8, ssRNA, and / or small molecule synthetic compounds; TLR9, and an agonist that is CpG DNA; TLR10, and an agonist that is a diacyl and / or triacyl lipopeptide; and TLR11, and agonists that are profilin-like proteins or non-pathogenic bacteria.

28. 27. The composition of claim 26, wherein the cargo comprises an antagonist of a TLR, the TLR and antagonist being selected from one or more of the following: TLR1, and an agonist that is CU-T12-9 and / or MMG-11; TLR2 and an agonist that is AT1-AT8, CU-CPT22, CU-T12-9, MMG-11, NPT1220-312, phloretin, and / or a sulfoglycolipid; TLR3, and agonists that are CU-CPT4a, monoclonal antibody CNTO4685, and / or monoclonal antibody CNTO5429; TLR4 and an agonist that is norbinaltorphimine, T4Ics, T5342126, and / or simvastatin; TLR5, and an agonist that is TH1020; TLR6, and an agonist that is simvastatin; TLR7 and an agonist that is chloroquine, hydroxychloroquine, and / or quinacrine; an agonist that is TLR8, and CU-CPT8m, and / or CU-CPT9a; and TLR9, and agonists that are NPT1220-312, chloroquine, hydroxychloroquine, quinacrine, and / or inhibitory or inhibitory oligonucleotides.

29. 27. The composition of claim 26, wherein the TLR is TLR9; and the cargo comprises an oligonucleotide or RNAi that modulates TLR9.

30. 27. The composition of claim 26, wherein the TLR is TLR5; the cargo is a TLR5 agonist; and the disease, disorder, or condition comprises a bacterial infection.

31. 31. The composition of claim 30, formulated for oral administration.

32. 10. The composition of claim 1, formulated for oral administration, for use in treating a disease, disorder or condition involving the immune system, or for preventing a condition involving the immune system.

33. 10. The composition of claim 1, formulated for inhalation, for use in treating a disease, disorder or condition of the respiratory system or a disease, disorder or condition involving the lungs and / or respiratory system.

34. 10. The composition of claim 1, which is formulated for administration to the eye to treat a disease, disorder, or condition involving or related to the choroid or retina of the eye.

35. 35. The composition of claim 34, formulated as eye drops or for injection.

36. 10. The composition of claim 1, wherein the disease is selected from among cancer, cancer metastasis, metabolic syndrome, genetic disorders, alpha-antitrypsin (AAT) deficiency, other inborn errors of metabolism, hemophilia, hypercholesterolemia, liver inflammation, steatohepatitis, and other diseases and disorders that can be treated by delivery of therapeutic agents to the liver.

37. 10. The composition of claim 1, wherein the MEV comprises or encodes a therapeutic agent for treating a disease, disorder, or condition involving the liver, and wherein the composition is formulated for intravenous administration, and wherein the disease involves the liver or the treatment targets the liver.

38. the composition is formulated for oral administration and the disease involves the intestine or spleen or the treatment targets delivery to the intestine or spleen; or the composition is formulated for oral administration and the disease involves or the treatment targets the gastrointestinal tract, immune system or white spleen; or the composition is formulated for intratracheal administration or inhalation and the disease involves a pulmonary or respiratory disease; or the composition is formulated for intranasal administration and the disease, disorder or condition is a disease of the brain or a disease involving the brain; or 10. The composition of claim 1, wherein the composition is formulated for administration to the eye and the disease, disorder, or condition is age-related macular degeneration, herpes stromal keratitis, glaucoma, dry eye syndrome, diabetic retinopathy, a condition associated with ocular neovascularization, or ocular hypertension.

39. 10. The composition of claim 1, wherein the disease or condition is a disease of or involving the brain, central nervous system or nervous system, and the composition is formulated for intranasal administration.

40. 10. The composition of claim 1, wherein the disease is treated by immune modulation and the MEV comprises a vaccine.

41. 2. The composition of claim 1, wherein the disease is cancer and / or immune cell disorder, or a disease treated or prevented by a vaccine.

42. 10. The composition of claim 1, wherein the composition is formulated for oral administration and the disease, disorder, or condition is an intestinal infection, Crohn's disease, or cancer.

43. 10. The composition of claim 1, wherein the composition is formulated for oral administration and the disease involves the gastrointestinal tract, immune system, or white spleen, or the treatment targets the gastrointestinal tract, immune system, or white spleen.

44. 10. The composition of claim 1, wherein the disease or condition is associated with an infectious pathogen that is a bacterium, a virus, an oomycete, or a fungus.

45. 2. The composition of claim 1, wherein the disease, disorder, or condition is selected from among chronic obstructive pulmonary disease (COPD), pulmonary hypertension, asthma, other inflammatory lung diseases, cystic fibrosis, alpha-antitrypsin (AAT) deficiency, inborn errors of metabolism, lung disease, cancer, and cancer metastasis involving the lungs and / or respiratory system.

46. 10. The composition of claim 1, wherein the disease, disorder, or condition is a disease, disorder, or condition of the brain or a disease, disorder, or condition involving the brain; and the composition is formulated for intranasal administration.

47. 10. The composition of claim 1, wherein the composition is formulated for oral administration; and the MEV comprises a biologically active molecule for use in modulating the immune system or treating or preventing a disease, disorder, or condition of the digestive system.

48. 10. The composition of claim 1, wherein the disease, disorder, or condition is selected from one or more of the following: Brain or central nervous system disorders; genetic disorders; multifactorial disease; cancer; hypertension; metabolic diseases; immune disorders; respiratory diseases; diseases of the digestive tract; Mucous membrane diseases; Infectious disease; Smell and taste disorders; allergy; Diseases of the muscle or neuromuscular tissue; bone diseases; Diseases of the endocrine system; Diseases of the hematopoietic system or lymphatic tissue; Bone marrow malignancies.

49. 49. The composition of claim 48, wherein the disease, disorder or condition is a neurodegenerative disease, a cognitive disorder, a brain disorder, a nervous system disorder, schizophrenia, or a bipolar disorder.

50. 49. The composition of claim 48, wherein the disease, disorder, or condition is selected from the following: a neurodegenerative disease selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, or Creutzfeldt-Jakob disease; cognitive or psychiatric disorder selected from dementia, amnesia, or delirium; a brain disorder selected from encephalitis, seizures, or tumors; a nervous system disorder selected from pain, seizures, or infection; a genetic disorder selected from cystic fibrosis, thalassemia, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, and Tay-Sachs disease; multifactorial diseases, such as diabetes or chronic obstructive pulmonary disease; breast, ovarian, bowel, prostate, or skin cancer or other cancers; immune disorders, such as arthritis or rheumatoid arthritis; a metabolic disorder that is high cholesterol, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, maple syrup urine disease, metachromatic leukodystrophy, MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), Niemann-Pick disease, phenylketonuria (PKU), porphyria, Tay-Sachs disease, or Wilson disease; a respiratory disease that is asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, lung cancer, cystic fibrosis, bronchiectasis, or pneumonia; A disease of the digestive tract that is irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), celiac disease, or diverticulitis; Mucous membrane diseases such as Behçet's disease, burning mouth syndrome, oral lichen planus, pemphigus and pemphigoid, recurrent aphthous stomatitis, or Sjögren's syndrome; infections that are urogenital or sinusitis; a disease of the muscle or neuromuscular tissue that is amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, muscular dystrophy, myasthenia gravis, muscle disease, myositis, or peripheral neuropathy; bone disease, which is cervical spondylosis, osteoporosis, metatarsalgia, polymyalgia rheumatica, or bone cancer; immune or autoimmune disorders, such as rheumatoid arthritis or osteoarthritis; Diseases of the endocrine system, such as acromegaly, adrenal insufficiency, Addison's disease, Cushing's syndrome, Graves' disease, Hashimoto's disease, Creutzfeldt-Jakob disease, hyperthyroidism, hypothyroidism, multiple endocrine neoplasia, polycystic ovary syndrome (PCOS), or primary hyperparathyroidism; A myeloid malignancy that is Fanconi anemia, thrombocytopenia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, chronic granulomatous disease, or Gaucher disease, myeloproliferative neoplasm, myelodysplastic disorder, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), a disease of the hematopoietic system or lymphoid tissue that is lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphadenitis, lymphangitis, lymphedema, or lymphocytosis.

51. 51. The composition of any one of claims 1 to 50, wherein the microalgae is a species of the genus Chlorella selected from Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis.

52. 52. The composition of claim 51, wherein the Chlorella species is Chlorella vulgaris.

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