Extracellular vesicles from microalgae, their biodistribution upon intranasal administration and uses thereof

Abstract

Compositions comprising microalgal extracellular vesicles (MEVs) formulated for intranasal delivery are provided, whereby upon intranasal administration, the MEVs are transported by a specific route, to specific regions in the brain via the olfactory nerve after intranasal administration, and to interconnected brain regions through the lateral olfactory tract (LOT). The MEVs are transported via neuronal axonal transport. The MEVs have the ability to cross synapses including: (i) synapses between olfactory sensory neurons (OSNs) and mitral / tufted neurons; (ii) synapses between mitral / tufted neurons and local neurons in multiple 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. The compositions contain extracellular vesicles (MEVs) from microalgae loaded with bioactive cargo for the treatment, detection, diagnosis, or monitoring of diseases, disorders, or conditions of or involving the brain, particularly neuronal delivery of the cargo. The compositions and methods have multiple applications as therapeutic and diagnostic agents for the treatment, diagnosis, and monitoring of diseases, disorders, or conditions of or involving the brain. The compositions can be used in methods and uses for the treatment of cancers involving the brain, and can be used, for example, to deliver therapeutic agents for psychiatric diseases, disorders, conditions, and to deliver therapeutic agents for neurodegenerative diseases, disorders, and conditions.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 517,083, filed August 01, 2023, entitled “Extracellular vesicles from microalgae, their biodistribution upon intranasal administration, and uses thereof,” inventors Lila Drittanti and Manuel Vega, applicant AGS Therapeutics SAS.

[0003] This application claims priority to U.S. Provisional Application Serial No. 63 / 480,264, filed January 17, 2023, entitled “Extracellular vesicles from microalgae, their biodistribution upon intranasal administration, and uses thereof,” inventors Lila Drittanti and Manuel Vega, applicant AGS Therapeutics SAS.

[0004] This application claims priority to U.S. Provisional Application Serial No. 63 / 418,959, filed October 24, 2022, entitled “Extracellular vesicles from microalgae, their biodistribution upon administration, and uses thereof,” inventors Lila Drittanti and Manuel Vega, applicant AGS Therapeutics SAS.

[0005] This application is related to PCT / EP2023 / 051650, filed January 24, 2023, published as International PCT Publication No. WO2023 / 144127 on August 03, 2023, entitled “Extracellular vesicles from microalgae, their biodistribution upon administration, and uses thereof,” inventors Lila Drittanti and Manuel Vega, applicant AGS Therapeutics SAS.

[0006] This application is related to PCT / EP2023 / 064751, filed June 01, 2023, entitled “Extracellular vesicles from genetically modified microalgae containing endogenously loaded cargo, their preparation and uses,” inventors Lila Drittanti and Manuel Vega, applicant AGS Therapeutics SAS.

[0007] This application is also related to International PCT Application No. PCT / EP2022 / 070371, filed July 20, 2022, published January 26, 2023, International PCT Publication No. WO 2023 / 001894, entitled “Extracellular vesicles from microalgae, their preparation and uses,” inventors Lila Drittanti, Juan Pablo Vega, Jeremy Pruvost, and Manuel Vega, applicant AGS Therapeutics SAS, 10 rue Greneta, 75003 Paris, France; AGS-M SAS, 41-43 Quai de Malakoff, 44000 Nantes, France; and Nantes Université, 1 Quai de Tourville, 44000 Nantes, France.

[0008] The subject matter of each of the applications referenced above is hereby incorporated by reference in its entirety to the extent permitted.

[0009] Incorporation by Reference of Sequence Listing Provided in Electronic Form

[0010] The electronic version of the sequence listing is submitted herewith and is hereby incorporated by reference in its entirety. The electronic file was created on October 11, 2023, is 589,338 bytes in size, and is named 5507SEQPC01.xml. TECHNICAL FIELD

[0011] Compositions comprising microalgal extracellular vesicles (MEVs) formulated for intranasal delivery are provided, whereby, upon intranasal administration, the MEVs are transported by a specific route after intranasal administration, via the olfactory nerve to specific regions in the brain, and through the lateral olfactory tract (LOT) to interconnected brain regions. The compositions can be administered in any form suitable for intranasal administration, whereby the MEVs are introduced into the olfactory nerve. The compositions contain extracellular vesicles (MEVs) from microalgae loaded with bioactive cargo for the treatment of diseases, disorders, or conditions of the brain or involving the brain. The compositions and methods have multiple applications as therapeutic and diagnostic agents for the treatment, diagnosis, and monitoring of diseases, disorders, or conditions of the brain or involving the brain. The compositions can be used in methods and uses for the treatment of cancers involving the brain, as well as therapeutic agents for psychiatric diseases, disorders, and conditions. BACKGROUND

[0012] Extracellular vesicles (EVs) are natural particles produced by most cells. EVs include exosomes (typically about 30-150 nm in size) that are released into the extracellular environment upon fusion of a multivesicular body with the plasma membrane, and microvesicles (about 50-1000 nm) that are produced by outward budding of membrane vesicles from the cell surface. Exosomes and microvesicles have similar properties and are often referred to as EVs. EVs facilitate intercellular communication through cell-cell transfer of proteins and nucleic acids, such as microRNAs (miRNAs), long non-coding RNAs (IncRNAs), and mRNAs. As such, EVs derived from mammals and plants have been used as carriers for short interfering RNA (siRNA) delivery, microRNAs (miRNAs), and small molecule drugs. There is a need for conveniently produced EVs that are easily delivered to cells and tissues. It is an object herein to provide such EVs. SUMMARY

[0013] Provided are cargo-loaded extracellular vesicles (EVs) for in vivo administration to a subject and in vitro administration to cells and cell lines. In particular, provided are EV-containing compositions formulated for intranasal delivery and for delivery of cargo to the brain. The EVs are loaded with cargo including biologically active molecules, including biomolecules, and small molecules, including diagnostic and / or therapeutic molecules. The EVs herein are derived from microalgae and are referred to as MEVs. Microalgae are single-celled green algae, including those belonging to the order Chlorellales, in particular the family Chlorellaceae, in particular those belonging to the genus Chlorella, such as Chlorella vulgaris. Microalgal extracellular vesicles (MEVs) can be manufactured at large scale.

[0014] By producing MEVs in genetically modified microalgae that encode or express proteins, polypeptides, small peptides, various RNA molecules, and / or other biomolecules, the MEVs can be endogenously loaded (internally loaded), which can be genetically programmed to express the biomolecules and thereby package in the MEVs.

[0015] MEVs can be exogenously loaded with biologically active molecule cargo after production. After isolation or partial purification / isolation of MEVs from microalgae, the MEVs can be exogenously loaded by contacting the MEVs with cargo to produce a composition in which substantially all of the MEVs (on average) have substantially the same exogenously loaded heterologous cargo. The biodistribution pattern is independent of the manner in which the MEVs are loaded (see, e.g., Example 14, in which exogenous and endogenous loading (as a control) deliver biologically active cargo). The MEVs provided herein have a unique biodistribution pattern that is a function of the route of administration. The biodistribution of the MEVs is different from that of mammalian EVs and other EVs and / or nanoparticles.

[0016] The MEVs herein are formulated for intranasal administration, typically as a liquid, such as a suspension or emulsion, or as a powder, or other formulation that can be administered intranasally. It is shown herein that, upon administration, the MEVs are distributed in the brain; they are transported to specific regions of the brain. By virtue of this mode of transport, they can deliver cargo to these regions of the brain for the treatment, detection, diagnosis, and / or treatment of diseases, disorders, and conditions involving these target regions.

[0017] As shown and described herein, upon intranasal (IN) administration, the MEVs take a unique route to the brain, thereby providing a unique route for the delivery of biologically active molecules. Upon IN administration, the MEVs are internalized by the olfactory sensory neurons (OSNs) from where they travel to the glomeruli. The MEVs that reach the glomeruli from the olfactory sensory neurons (OSNs) enter the mitral and tufted neurons and travel intracellularly in a clear route with clear kinetics throughout the lateral olfactory tract (LOT). The LOT is composed of long axons of the mitral and tufted neurons that travel from the olfactory bulb (OB) to various forebrain-hindbrain regions that are directly involved in the olfactory connectome network, including: the anterior olfactory nucleus, the olfactory tubercle, the lateral tenia tecta, the piriform cortex, the amygdala, and the entorhinal cortex. The lateral branches of the main long axons of the mitral and tufted neurons enter and colonize each of the brain regions, the anterior olfactory nucleus, the olfactory tubercle, the lateral tenia tecta, the piriform cortex, the amygdala, and the entorhinal cortex. Within these regions, the mitral / tufted axons connect (via synapses) with neurons from other regions with more secondary olfactory roles, including the frontal cortex, the hypothalamus, the thalamus, and the hippocampus.

[0018] The regions reached by the MEVs through IN administration include all and every brain region connected to the olfactory nerve and the lateral olfactory tract (LOT) in both hemispheres; ventral, lateral, and dorsal regions; external and internal regions; and along the anterior-posterior axis. These regions are: the anterior olfactory nucleus, the olfactory tubercle, the lateral tenia tecta, the piriform cortex, the amygdala, the entorhinal cortex, the primary motor cortex, the frontal cortex, the agranular insular cortex, the primary somatosensory cortex, the auditory cortex, the postpyriform cortex, the temporal association cortex, the basolateral amygdaloid nucleus, the hypothalamic arcuate nucleus, the corpus callosum, the internal capsule, the thalamus, and the hippocampus (the umbrella, the dentate gyrus). For example, the MEVs are delivered to or for delivery to the limbic system, such as the amygdala, the hippocampus, and the thalamus, or to the cortex, such as the frontal cortex or the parietal cortex.

[0019] MEVs are loaded with a variety of cargos (also referred to as “payloads” and described as biologically active molecules), including but not limited to RNA, such as inhibitory RNA and other RNA products, oligonucleotides, plasmids, peptides, proteins, small molecules.

[0020] As shown herein and elsewhere (see commonly owned International PCT Publication No. WO 2023 / 144127), MEVs can deliver cargos to organs, tissues, and cells, and can be targeted by route of delivery, where they can be delivered. It is shown herein that MEVs, including Chlorella MEVs, have a remarkable ability to cross strict natural barriers, such as the digestive tract and olfactory neurons, that are not shared by other extracellular vesicles (EVs) from other sources, including mammalian EVs.

[0021] As described herein, MEVs can be exogenously loaded (exo-loaded) with a variety of biologically active molecules, such as siRNA, mRNA, plasmids, ASOs, peptides, proteins, and / or small molecules, which allows for a variety of therapeutic, diagnostic, and other uses. MEVs can also be endogenously loaded by the microalgae that produce them (see U.S. Provisional Application Serial No. 63 / 349,006, filed June 03, 2022, and International PCT Application No. PCT / EP2023 / 064751). As shown herein, MEV biodistribution is determined by route of administration. Thus, MEVs can deliver their cargos to a variety of tissues and organs, including, for example, the lung, intestine, GALT, spleen, liver, and brain, depending on whether they are administered intratracheally, orally, intravenously, or intranasally.

[0022] As demonstrated herein, MEVs have a number of uses, including therapeutic uses, including delivery of therapeutic agents for the treatment and / or prevention (including reduction of risk or severity) of diseases, disorders, and conditions. These uses include therapeutic uses, including immunomodulation, immuno-oncology, treatment of genetic or metabolic disorders, neurological disorders, psychiatric disorders, respiratory disorders, and the like.

[0023] Cargos (also referred to as “payloads”) include but are not limited to RNA, such as inhibitory RNA and other RNA products, oligonucleotides, plasmids, peptides, proteins, and small molecules. Exogenously loaded MEVs can be loaded with virtually any molecule of interest; endogenously loaded MEVs, in which the microalgal cells are genetically modified to express or encode a product, produce MEVs containing cargos that are, for example, RNA, DNA, peptides, small peptides, polypeptides, and proteins produced by the microalgae and packaged in the EVs.

[0024] The present invention provides compositions containing MEVs, such as MEVs loaded with exogenous cargos, particularly MEVs produced by Chlorellales, particularly Chlorellaceae, particularly Chlorella, such as Chlorella vulgaris. The compositions include pharmaceutical compositions that can be formulated for particular routes of delivery.

[0025] Methods of loading MEVs are described and provided (see also International PCT Publication Nos. WO 2023 / 001894 and WO 2023 / 144127, which detail exogenous and endogenous loading of MEVs). The cargo is a biologically active molecule or combinations thereof, including biological molecules and small molecules. The cargo includes, for example, biological molecules, including biological polymers, such as DNA and RNA, proteins, protein complexes, protein-nucleic acid complexes, plasmids, and also or alternatively small molecules, such as small molecule drugs. The biologically active molecules include therapeutic agents, such as anti-cancer compounds and biological molecules, such as RNAi, oligonucleotides, and proteins, and complexes, as well as diagnostic molecules, such as detectable markers, cosmetic molecules, and molecules that act as anti-infective agents for humans, animals, and plants. Methods of treating diseases and conditions, including pathogen infections and cancer, as well as uses of MEVs for treating diseases and conditions, and diagnostic methods are provided.

[0026] Generally, cargo-loaded MEVs have applications in various fields, including diagnostics, prevention, treatment of human and other animal diseases, industrial uses, cosmetic uses, veterinary uses, and for the crop industry. MEVs with appropriate cargo for each application can be used as, for example, vaccines, gene therapy delivery vehicles, gene silencing, gene editing, transfection for industrial and research, analytical methods, cell-based assays, and other uses and applications. Cargo-loaded MEVs can be used to treat diseases, conditions, and disorders, as well as for industrial and cosmetic uses. Diseases, conditions, and disorders include, but are not limited to: genetic disorders; digestive tract disorders; respiratory tract disorders; central nervous system (CNS) disorders; skin disorders, including natural disorders and disorders induced by trauma; urogenital tract disorders; nose-oral cavity disorders; cardiovascular system disorders; immune and immunomodulatory disorders; cancer; eye disorders; liver disorders; systemic disorders; and diseases, conditions, and disorders caused by or involving a pathogen, such as a bacterium, virus, or parasite.

[0027] Target tissues for treatment and / or delivery include, for example, epithelial and mucosal cells (e.g., any kind of external or internal mucosa: oral, intestinal, uterine, tracheal, 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, immunomodulatory cells, effector cells), reproductive cells, secretory cells, glandular cells, muscle cells, stem cells (e.g., embryonic or tissue-specific stem cells), liver cells, infected cells (e.g., cells infected with a virus, bacteria, fungus, or other pathogen), native cells, and genetically engineered cells of the nervous system (NS). For purposes of intranasal administration of MEVs herein, the target tissue or organ is the brain. Of interest herein is delivery to the brain by intranasal administration.

[0028] Provided are compositions containing isolated microalgal extracellular vesicles (MEVs), wherein the microalga is a species of Chlorella; and the composition is formulated for administration to a subject. The Chlorella extracellular vesicles can contain a heterologous bioactive cargo molecule that has been introduced into the isolated extracellular vesicle, whereby the vesicles in the composition containing the heterologous bioactive molecule cargo contain the same bioactive molecule cargo, wherein: the cargo molecule is heterologous to the Chlorella; and the bioactive cargo is a biomolecule or a small molecule.

[0029] For all embodiments where the MEVs are from Chlorella, the Chlorella is any species of Chlorella, for example, but not limited to, Chlorella selected from the group consisting of Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. In specific embodiments, the Chlorella is Chlorella vulgaris.

[0030] Provided are compositions containing isolated microalgal extracellular vesicles (MEVs), wherein the microalga is a species of Chlorella; and the composition is formulated for administration to a subject. The Chlorella extracellular vesicles can contain a heterologous bioactive cargo molecule that has been introduced into the isolated extracellular vesicle, whereby the vesicles in the composition containing the heterologous bioactive molecule cargo contain the same bioactive molecule cargo, wherein: the cargo molecule is heterologous to the Chlorella; and the bioactive cargo is a biomolecule or a small molecule.

[0031] Cargo includes any molecule intended to be delivered to or on a plant or animal, and in particular herein to the brain. Due to the transport route following IN administration, the MEV and thus any cargo crosses the blood-brain barrier (BBB). Typically, the cargo is biologically active in that it can be used to treat or detect a disease, disorder, or condition. Biologically active cargo includes, for example, any molecule, such as biomolecules (including biopolymers) and small molecules, which can have an effect on a plant or animal when administered. Cargo includes, for example, proteins, peptides, and nucleic acids. Biologically active molecules can be synthetic, naturally occurring, and / or modified to alter properties or activities. Included are any molecules that have been used as pharmaceuticals or therapeutic agents or diagnostic agents or cosmetics or in industry. Cargo can be, but is not limited to, a therapeutic agent used to treat or prevent a disease or disorder or condition or to treat or prevent symptoms thereof. Cargo can be a nucleic acid molecule, a polypeptide, a protein, a plasmid, an aptamer, or an antisense oligonucleotide.

[0032] Cargo in the MEV in the composition can comprise a biopolymer. Biopolymers include naturally occurring biopolymers, or synthetic biopolymers, or modified biopolymers. Biopolymers can be include modified nucleic acids or proteins, where the modifications include insertions, deletions, substitutions, and translocations of nucleotide or amino acid residues, and / or in the case where the biopolymer is a protein, the modifications can also include post-translational modifications. Post-translational modifications include, but are not limited to, glycosylation, hyperglycosylation, pegylation, sialylation, albumination, other half-life extending moieties, and other modifications that improve or alter the pharmacological dynamics or kinetic properties of the protein.

[0033] Nucleic acids, such as DNA and RNA, are molecules that can be cargo. If the cargo is RNA or a protein, it can be provided as cargo, or it can be encoded by a nucleic acid that is then expressed in the organism to which it is administered. Examples of RNA are inhibitory RNA (RNAi) and mRNA, including modified mRNA. RNAi includes, for example, silencing RNA (siRNA) or short hairpin RNA (shRNA), microRNA (miRNA), small activating RNA (saRNA), and long non-coding RNA (lncRNA). RNA products also include double-stranded RNA and ribozymes. Cargo can also be an oligonucleotide, such as an antisense oligonucleotide or an allele-specific oligonucleotide. Cargo can include gene editing systems, such as CRISPR-Cas systems, as well as modified and improved gene editing systems, such as CRISPR- associated and CRISPR-like systems (see, e.g., published U.S. Patent Application Nos. 2020 / 0332273 and 2020 / 0332274 of Applicant Metagenomi).

[0034] The cargo includes therapeutic or diagnostic or therapeutic proteins or peptides, protein complexes, such complexes containing two or more proteins or a combination of proteins and nucleic acids, or proteins and aptamers, or proteins, nucleic acids, and other molecules. The cargo can be or can encode a protein that is an antibody or antigen-binding fragment thereof. The antibody can be in any form, including single chain forms, nanobodies, camelid antibodies, and other forms, such as scFv, bispecific antibodies, or antigen-binding fragments thereof. The antibody and antigen-binding fragments thereof include checkpoint inhibitor antibodies or antigen-binding fragments thereof, or tumor antigen-specific antibodies or antigen-binding fragments thereof, or anti-oncogene-specific antibodies or antigen-binding fragments thereof, or tumor-specific receptors, or signal 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, PD-1 / PDL1 pathway, PD-1 / PDL2 pathway, HER2, EGFR, TIM-3, LAG-3, BTLA-4, HHLA-2, CD28, and other checkpoints or immunosuppressive agents or tumor antigens.

[0035] The cargo in the MEV in the composition can include immunostimulatory products and antigens, and can be used as a vaccine to induce an immunoprotective response or an immune response upon administration. The cargo can be, but is not limited to, DNA, RNA, proteins, and viruses. The cargo can contain nucleic acids or proteins or nucleic acids encoding proteins that are therapeutic products for treating cancer, or infectious diseases, or neurodegenerative diseases or other CNS disorders, or aging, or aging-related diseases, or ophthalmic disorders, or immune disorders. The cargo can be a pharmaceutical cosmetic or a cosmetic or a cosmetically active product. The cargo can include small molecule biologically active molecules, such as small molecule biologically active molecules, such as small molecule drugs. Exemplary drugs include chemotherapeutic agents and prodrugs. The cargo in the MEV in the composition can be or comprise a diagnostic label or detectable product, such as, but not limited to, luciferase or nucleic acids encoding luciferase, fluorescent proteins or nucleic acids encoding fluorescent proteins, or luciferase operons. As described herein, the cargo includes any cargo that can be used to treat, detect, diagnose, and monitor any disease, disorder, or condition involving the brain.

[0036] The cargo can comprise DNA. The DNA can be a plasmid, such as a plasmid encoding a product for expression in the animal or plant to which it is administered. The plasmid can encode one or two or more cargo products. For expression of the cargo product, the encoding nucleic acid is operably linked to regulatory sequences recognized by eukaryotic cells. The cargo can comprise RNA, proteins, peptides, small molecules, and any other molecules that can be exogenously or by encoding them endogenously loaded into the MEV in the microalgae.

[0037] Exemplary products include, but are not limited to, therapeutic products and diagnostic products. These include protein and RNA products, including the RNA products listed above. Since MEVs are eukaryotes and are intended for administration to animals, e.g., humans, the plasmids typically encode the products under the control of eukaryotic regulatory signals and sequences, including eukaryotic promoters and translation sequences, e.g., RNA polymerase II and III promoters. Exemplary promoters include RNA polymerase II promoters, e.g., from animals, plants, and plant or animal viruses. Exemplary promoters include, but are not limited to, cytomegalovirus promoters, simian virus 40 promoters, herpes simplex virus promoters, Epstein Barr virus promoters, adenovirus promoters, synthetic promoters, actin promoters, and synthetic chimeric promoters. Other eukaryotic transcriptional sequences and eukaryotic translational sequences include, but are not limited to, one or more of enhancers, poly A sequences, and / or internal ribosome entry site (IRES) sequences.

[0038] Described herein are methods of making MEVs. The methods include introducing a cargo into an isolated MEV. The cargo includes any molecule that is desired to be delivered into or onto an animal or plant. Typically, the cargo is or contains or provides a biologically active molecule product, including small molecules and biopolymers. Biopolymers are naturally occurring, or synthetic, or modified, or combinations thereof. The cargo includes proteins, nucleic acids, or small molecules. The cargo can be loaded into the MEV by any method known to one of skill in the art; these methods include, e.g., one or more of electroporation, sonication, extrusion, and use of a surfactant. In some embodiments, the MEV is from a Chlorella, e.g., but not limited to, a Chlorella species selected from the group consisting of Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis. The MEVs produced by the methods or any of the MEVs provided herein, including compositions containing the MEVs, can be used as one or more of a diagnostic method, a vaccine, a therapeutic therapy, a disease diagnosis, a disease or disorder or condition treatment, a cosmetic, an industrial application, and / or any use known to one of skill in the art.

[0039] MEVs can be used in any such methods, including methods of treating a disease, disorder, or condition. Examples of diseases, disorders, and conditions are cancers, such as cancers comprising solid tumors or hematological malignancies or metastases thereof. Other diseases, disorders, and conditions include diseases, disorders, and conditions of the respiratory system or involving the respiratory system, the central nervous system or nervous system or involving the central nervous system or nervous system, the skin and exposed epithelium or mucosa or involving the skin and exposed epithelium or mucosa, the digestive tract or involving the digestive tract, and infectious agents or involving infectious agents. Infectious agents include bacteria, viruses, parasites, prions, oomycetes, and fungi.

[0040] Cargo can provide therapeutic molecules for treatment, or can induce an immune response for use as a vaccine. MEVs can contain cargo comprising or encoding an immunostimulatory protein or antigen, whereby the MEV is immunostimulatory and elicits an innate or adaptive immune response upon administration, or the MEV and / or cargo can elicit an immunoprotective response to prevent or treat a disease or disorder or condition.

[0041] Generally, MEVs can be used to treat diseases, disorders, or conditions arising from trauma. Trauma includes, but is not limited to, trauma from or involving wounds, burns, surgery, skin cuts, broken bones, hair loss, dermal exposure, mucosal exposure, fibrosis, lacerations, and ulcers. This includes brain or CNS trauma. MEVs can be used to elicit effects that treat disorders arising from natural aging or pathogenic or disease- or otherwise induced aging. For purposes herein, diseases, disorders, and conditions are those involving the brain or CNS that are treated or detected or monitored in the brain.

[0042] Generally, compositions containing MEVs can be formulated for administration by any route of administration. Routes include, but are not limited to, local, systemic, topical, parenteral, enteral, mucosal, inhaled lung or intranasal, vaginal, rectal, ear, oral, and other routes of administration. For purposes herein, MEVs are formulated for intranasal administration. They can be formulated in any form, including as tablets; as liquids, such as emulsions; as powders; or as aerosols; including for oral administration, for nebulization, or for inhalation, with respect to the form and formulation of the route of administration.

[0043] Compositions of MEVs can be used in any methods and processes described herein or known to one of skill in the art. Methods include, for example, any method described herein, including, for example, one or more of gene silencing, gene interference, gene therapy, gene / protein overexpression, gene editing, inhibition or stimulation of protein activity, and pathway signaling. Compositions and MEVs can be used for prevention and / or vaccination. They can be used for industrial purposes, such as for manufacturing, characterization, and calibration.

[0044] Methods for treating diseases, symptoms, and conditions are provided, wherein treatment can be achieved by delivering an active agent to the brain. A composition for intranasal delivery is provided. The composition contains microalgal extracellular vesicles containing the active agent. The microalgal extracellular vesicles can be loaded by any suitable method (see the methods and MEVs described in International Patent Publication No. PCT / EP2022 / 070371 and U.S. Provisional Application Serial No. 63 / 349,006), including exogenous loading after the production of said MEV and endogenous loading in vivo by microalgae that have been genetically modified to package nucleic acids or encoded products into the MEV.

[0045] EVs are derived from microalgae, which are single-celled green algae, and include those belonging to the order Chlorellales, particularly the family Chlorellaceae, and especially to the genus Chlorella, such as Chlorella vulgaris. MEVs are provided in compositions formulated for intranasal administration. MEVs can be exogenously loaded after isolation, or can be endogenously loaded via genetically modified microalgae that encode and package heterologous nucleic acids and / or proteins in vivo. The advantage of exogenously loading cargo (exogenous loading) into MEVs is that the cargo / MEV amount can be controlled, and the distribution of exogenous cargo in the MEV is predictable and substantially uniform, making it possible to know the average cargo molecules or amount per cargo / MEV. A variety of bioactive molecules, including biomolecules and small molecules such as pharmaceuticals and organic compounds, can be loaded into MEVs. MEVs can also be endogenously loaded via genetically modified microalgae to package heterologous nucleic acids and / or proteins.

[0046] The resulting MEVs, whether internally or externally loaded, are non-toxic; they can be administered into cells in vitro or in vivo and have a distribution pattern that depends on the route of administration.

[0047] MEV and delivery to the brain

[0048] For the purposes of this paper, MEVs are used for delivery to the brain via intranasal administration. This paper demonstrates that MEVs are transported to the brain via unique pathways and mechanisms following intranasal (IN) administration. These pathways and mechanisms are not shared by exogenous bodies or nanoparticles from other sources. This paper demonstrates that, following intranasal delivery, MEVs are transported via the olfactory nerve and across the lateral olfactory tract (LOT) to a large number of interconnected brain regions. MEVs are transported via neuronal axonal transport. MEVs have the ability to cross synapses at least (i) between olfactory sensory neurons (OSN) and mitral / clustered neurons, (ii) between mitral / clustered neurons and local neurons in various brain regions colonized by the lateral olfactory tract (LOT), and (iii) between neurons in brain regions colonized by the LOT and neurons from the frontal cortex, hippocampus, thalamus, and hypothalamus.

[0049] For all embodiments of the methods, uses, and compositions described and considered herein that relate to intranasal application and / or the brain or CNS, diseases, conditions, and illnesses include any diseases, conditions, and illnesses described herein and known to those skilled in the art that can be treated, detected, diagnosed, and / or monitored by delivering molecules to the brain.

[0050] The biodistribution of MEVs follows pathways and connections within the olfactory nerve and the neural networks of mitral / cluster neurons throughout the brain. Transport and pathways provide access to (biodistribution) brain regions (within 1 to 16 hours after IN administration), including: the preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, hypothalamic arcuate nucleus, corpus callosum, internal capsule, thalamus, and hippocampus (umbellate and dentate gyrus). Therefore, MEVs can deliver active agents, particularly bioactive payloads, to specific regions of the brain. The payload includes, but is not limited to, proteins, mRNA, DNA, small molecules, and any reagent that can be exogenously loaded (exogenously loaded) into a MEV or packaged in vivo by microalgae within the MEV, particularly genetically modified microalgae encoding or producing that reagent. Therefore, MEVs provide efficient 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 are used for therapeutic and diagnostic purposes, as well as for diagnostic and experimental applications. The examples illustrate delivery, demonstrating efficient delivery and expression in vitro and in vivo 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 / psilocine, harmine, temozolomide, rivastigmine, and rhodamine to neurons, astrocytes, glial cells, and / or neural stem cells.

[0051] Among many uses and methods, the MEV provides a unique vehicle for i) treating and / or preventing brain disorders, including but not limited to cognitive, emotional, behavioral, psychiatric, neurological, degenerative, and cancer disorders; (ii) studying brain disorders in vitro or in vivo; (iii) diagnosing brain disorders; and (iv) for recreational and therapeutic purposes.

[0052] This invention provides a method for delivering bioactive molecules to the brain via intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs), wherein the MEVs contain cargo containing bioactive molecules, thereby the MEVs travel via the olfactory nerve and throughout the lateral olfactory tract (LOT) to interconnected brain regions to deliver to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, mammillary body, hypothalamic arcuate nucleus, corpus callosum, internal capsule. The capsule, thalamus, and hippocampus, wherein: the bioactive molecule is any molecule that can affect the treatment of a disease, symptom, or condition, or can be used to detect a disease, symptom, or condition, or can be used to monitor the treatment of a disease, symptom, or condition; and the bioactive molecule is heterologous to the microalgae and / or MEV. Use of the MEV and composition formulated for intranasal administration for such delivery is also provided.

[0053] Delivery methods include treatment methods and uses for treating diseases, conditions, and illnesses involving the brain. This invention provides a method (and use of MEVs) for treating brain diseases, symptoms, or conditions, or involving brain diseases, symptoms, or conditions, by intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs), wherein the microalgal extracellular vesicles (MEVs) contain a cargo comprising bioactive molecules, thereby allowing the MEVs to travel via the olfactory nerve to the brain and throughout the lateral olfactory canal (LOT) to interconnected brain regions, to be delivered to the olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, mammillary body, hypothalamic arcuate nucleus, corpus callosum, and internal capsule. The bioactive molecule is any molecule that can achieve or involve the treatment of a disease, condition or illness of the brain; and the bioactive molecule is heterologous to microalgae and / or MEVs.

[0054] It also provides methods for detecting or treating diseases, symptoms, or conditions of the brain, or for monitoring or treating diseases, symptoms, or conditions of the brain (and the use of MEVs), which are performed by intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive molecules, whereby the MEVs travel via the olfactory nerve to the brain and through the lateral olfactory tract (LOT) to interconnected brain regions, to be delivered to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, mammillary body, hypothalamic arcuate nucleus, corpus callosum. The callosum, internal capsule, thalamus, and hippocampus (umbilicus, dentate gyrus), wherein the bioactive molecule comprises a reporter or detectable marker, wherein: the bioactive molecule is any molecule that can be used to detect or diagnose a disease, condition, or illness, or can be used to monitor a disease, condition, or illness for treatment, or can be used to detect or diagnose a disease, condition, or illness and treat the disease, condition, or illness; the disease, condition, or illness is a brain disease, condition, or illness involving the brain; and the bioactive molecule is heterologous to microalgae and / or MEVs.

[0055] A composition comprising microalgal extracellular vesicles (MEVs) is provided, the MEVs containing a cargo comprising bioactive molecules, wherein: the composition is formulated for delivery to the brain via the olfactory nerve intranasally and via the lateral olfactory tract (LOT) intranasally to interconnected brain regions, to deliver to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, mammillary body, hypothalamic arcuate nucleus, corpus callosum. The brain includes the callosum, internal capsule, thalamus, and hippocampus; the bioactive molecule is any molecule that can affect the treatment of a disease, symptom, or condition, or can be used to detect or diagnose a disease, symptom, or condition, or can be used to monitor the treatment of a disease, symptom, or condition, or to detect, diagnose, monitor, and / or treat a disease, symptom, or condition; the disease, symptom, or condition is a disease, symptom, or condition of the brain or involves the brain; and the bioactive molecule is heterologous to microalgae and / or MEVs. A composition is provided for treating, diagnosing, detecting, and / or monitoring diseases, symptoms, and conditions involving the brain and employing or targeting interconnected brain regions.

[0056] Compositions comprising microalgal extracellular vesicles (MEVs) are also provided for the delivery of goods containing bioactive molecules to the brain for the treatment of or involving diseases, conditions, or illnesses of the brain, or for the diagnosis, detection, or monitoring of diseases, conditions, or illnesses of the brain, or for the treatment, diagnosis, detection, and / or monitoring of diseases, conditions, or illnesses of the brain, or for the treatment, diagnosis, detection, and / or monitoring of diseases, conditions, or illnesses of the brain, or for the treatment, diagnosis, detection, and / or monitoring of diseases, conditions, or illnesses of the brain, wherein: the MEVs contain bioactive molecules for delivery to the brain; the compositions are formulated for intranasal delivery to the brain via the olfactory nerve and for delivery via the lateral olfactory tract (LOT) to interconnected brain regions, to deliver to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala. The brain regions include the microalgae, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus; the bioactive molecules are any molecules that can affect the treatment of a disease, symptom, or condition, or that can be used to detect a disease, symptom, or condition, or that can be used to monitor the treatment of a disease, symptom, or condition; and the bioactive molecules are heterologous to microalgae and / or MEVs. Delivery and transport are achieved through pathways and interconnected brain regions.

[0057] According to these methods and compositions, the MEV travels in the brain via intraneural axonal transport and transsynaptic transport between neurons. For example, the MEV travels in the brain by means of neuronal axonal transport, and the MEV is delivered to the fimbriae or dentate gyrus of the hippocampus. The MEV follows pathways and connections in a neural network that includes olfactory nerves and mitral / clustered neurons throughout the brain. When administered intranasally, the MEV crosses one or more of the following: (i) synapses between olfactory sensory neurons (OSNs) and mitral / clustered neurons; (ii) synapses between mitral / clustered neurons and local neurons in brain regions colonized by LOTs; and (iii) synapses between neurons in brain regions colonized by LOTs and neurons originating from or reaching the frontal cortex, hippocampus, thalamus, and hypothalamus. For example, the MEV crosses (i), (ii), and (iii) or (i) and (ii), for example... Figure 35 The pathway through which the MEV passes when administered intranasally is described.

[0058] The methods and compositions described herein, as well as their uses, are for intranasal administration. Following intranasal administration, the MEV is transported and / or delivered, for example, to or intended for delivery to one or more of the corpus callosum, dorsal fornix, lateral hippocampal commissure, and fimbriae of the hippocampus.

[0059] The compositions used in the compositions and methods provided herein can be formulated as suspensions or emulsions, such as nanoemulsions or microemulsions. They can be formulated in any form, such as powders and liquids for intranasal administration via mucosal ingestion.

[0060] The composition can be formulated as, for example, a liquid, powder, tablet, granule, liquid, oil, suspension or emulsion, suitable for intranasal application or processing, for example, by dilution or dissolution for intranasal application.

[0061] Those skilled in the art understand and are familiar with the properties and formation of nanoemulsions and microemulsions. In the composition, the MEV contains bioactive cargo. For example, MEVs can be prepared such that each MEV contains, on average, a predetermined amount of bioactive molecules, such as 1 to 100, for example, but not limited to at least 1-10, 1-20, 1-30, 1-50, 10-20, and other amounts of bioactive molecules / MEV suitable for the indication and use. The selection of the amount of cargo per MEV is within the scope of those skilled in the art and depends on factors known to them, such as the specific disease, condition, or illness being treated, the use of the MEV, the subject, the specific cargo, and other such parameters and factors. Similarly, the concentration of the MEV depends on the specific cargo and the intended use. For example, the concentration of MEV in the composition can be, for example, from about 0.1 to 10 mg / mL, and lower or higher, as well as intermediate concentrations. The composition can be formulated for single-dose administration (direct administration without dilution) or multiple-dose administration by aliquoting and / or dilution to the desired concentration. Exemplary amounts of the composition for application are 0.1 to 100 mL, such as 1 to 10 mL, 1 to 5 mL, 0.1 to 1 mL, and any suitable amount for intranasal application. The composition may be administered as a single dose or as part of a series of doses or other regimens. The composition may be administered as part of a combination therapy regimen.

[0062] The compositions and methods include those in which the MEV is endogenously loaded by microalgae with genetic modifications encoding a bioactive molecule or a pathway for its production. MEVs also include those in which the cargo is exogenously loaded in purified or partially purified MEVs. MEEVs may contain a variety of different heterologous cargoes. For the purposes of this document, the cargo includes therapeutic agents for the treatment or prevention of diseases or conditions of the brain, or diseases or conditions involving the brain, or for the treatment or prevention of their symptoms.

[0063] The microalgae used to produce the MEV used in the method described above can be selected from the following microalgae phyla: Euglenophyta, Chrysophyta (golden brown algae and diatoms), Pyrrophyta (fire algae), Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae), or Xanthophyta (yellow-green algae). For example, the microalgae can be derived from species in the classes Chlorophyceae, Trebouxiophyceae, or Chlorophyta, such as species of the genera Chlorella or Chlamydomonas.

[0064] Species of the genus *Chlorella* include, but are not limited to: *Chlorella ellipsoidea*, *Chlorella pyrenoidosa*, *Chlorella sorokinina*, *Chlorella vulgaris*, and *Chlorella variantbilis*, such as *Chlorella vulgaris* and *Chlorella variantbilis*. In a specific embodiment, the *Chlorella* is *Chlorella vulgaris*. For example, methods and compositions include those in which the microalgae are *Chlorella* species; the MEVs in the composition contain heterologous bioactive molecular cargoes that have been exogenously introduced into isolated MEVs, such that, on average, the vesicles in compositions containing heterologous bioactive molecular cargoes contain the same heterologous cargoes, wherein: the cargoes are heterologous to the *Chlorella*; and the cargoes are biomolecules or small molecule drugs or any cargoes as described herein and / or known to those skilled in the art for delivery to the brain. It also includes methods and compositions, wherein the MEV is an extracellular vesicle of Chlorella; the extracellular vesicle of Chlorella contains a heterologous bioactive molecule cargo introduced into the extracellular vesicle by endogenous introduction of the microalgae, wherein the cargo molecule is heterologous to Chlorella; and the bioactive cargo is a biomolecule for treating or involving a disease, condition or symptom of the brain.

[0065] Methods and compositions are provided, wherein: the MEV is an extracellular vesicle of Chlorella; the Chlorella extracellular vesicle contains a heterologous bioactive molecular cargo introduced into the isolated extracellular vesicle, thereby the vesicles in the composition containing the heterologous bioactive molecular cargo contain, on average, the same bioactive molecular cargo, wherein: the cargo molecule is heterologous to Chlorella; and the bioactive cargo is a therapeutic or detectable molecule for treating, monitoring, and / or diagnosing brain diseases, symptoms, or conditions, or involving brain diseases, symptoms, or conditions. In other embodiments, the MEV is an extracellular vesicle of Chlorella; the Chlorella extracellular vesicle contains a heterologous bioactive molecular cargo introduced endogenously into the extracellular vesicle from the microalgae, thereby the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same bioactive molecular cargo, wherein: the cargo molecule is heterologous to Chlorella; and the bioactive cargo is a biomolecule or a small molecule. In other embodiments, the MEV in the composition contains heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEV, thereby, on average, the vesicles containing heterologous bioactive molecular cargo in the composition contain the same cargo, wherein: the cargo is heterologous to Chlorella; and the cargo is a biomolecule or a small molecule. In other embodiments, the cargo is endogenously introduced into the MEV by modifying the microalgae to express or produce the cargo, such as nucleic acids or proteins or biochemical pathway products. In an exemplary embodiment, the Chlorella is common Chlorella.

[0066] The goods include, but are not limited to, biomolecules and biopolymers, such as naturally occurring biopolymers, synthetic biopolymers, or modified biopolymers, such as nucleic acid molecules, peptides, proteins, plasmids, aptamers, or antisense oligonucleotides. The goods include, but are not limited to, DNA or RNA, such as repressive RNA (RNAi), mRNA or modified mRNA, silent RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), self-amplifying RNA, small activating RNA (saRNA), long non-coding RNA (lncRNA), ribozymes, or double-stranded RNA. The goods include oligonucleotides, such as antisense oligonucleotides or allele-specific oligonucleotides or antisense oligonucleotides (ASO), and gene editing systems, such as CRISPR-CAS systems, CRISPR-related systems, or CRISPR-like systems. The goods may contain DNA, such as plasmids encoding therapeutic and / or detectable or diagnostic products, or RNA products, such as RNAi and the aforementioned RNA forms, including antisense oligonucleotides or ribozymes or double-stranded RNA. Plasmids may encode cargo products controlled by eukaryotic promoters, such as RNA polymerase II or III promoters, eukaryotic viral promoters (e.g., cytomegalovirus promoters, simian virus 40 promoters, herpes simplex virus promoters, Epstein-Barr virus promoters, adenovirus promoters, synthetic promoters, other promoters (e.g., actin promoters), or synthetic chimeric promoters). Plasmids may also contain other regulatory sequences for expression, such as other eukaryotic transcriptional sequences and eukaryotic translational sequences. MEV cargoes may include small molecules for the treatment, detection, diagnosis, or monitoring of diseases, symptoms, or conditions of the brain, or diseases, symptoms, or conditions involving the brain.

[0067] The cargo includes any molecules of interest intended for delivery to the brain. This includes, for example, cargo encoding or encoding immunomodulators, such as those that increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells. The cargo may contain or encode hormones, cytokines, or chemokines. The cargo may contain a prodrug or a carrier encoding an enzyme that converts the prodrug into a drug for treating or involving a disease, condition, or illness of the brain. The cargo may contain or encode antibiotics, antiviral agents, antifungal agents, antiparasitic agents, or other anti-infective agents for treating or involving an infection of the brain. The cargo may contain therapeutic nucleic acids or proteins or nucleic acids encoding proteins that are therapeutic products for treating cancer or tumors in the brain, or infectious diseases of the brain, or neurodegenerative diseases or other central nervous system (CNS) conditions, or for treating dementia. The goods may contain chemotherapeutic agents for treating or involving brain diseases, conditions, or illnesses, and / or encoding or containing antibodies or antigen-binding fragments thereof, such as scFv, bispecific antibodies, or antigen-binding fragments thereof. The goods may contain nucleic acids for gene therapy.

[0068] MEVs may include two other distinct types of goods. Goods may include therapeutic products, or diagnostic products, or detectable products, or combinations thereof, for the detection, diagnosis, and / or monitoring of diseases, conditions, or illnesses of the brain, or diseases, conditions, or illnesses involving the brain, or combinations thereof. Diagnostics may contain luciferase or nucleic acids encoding luciferase, fluorescent proteins or nucleic acids encoding fluorescent proteins, or luciferase operons, or combinations thereof. Bioactive molecular goods may include any molecule that has an effect on the cells or organism to which it is delivered, or any molecule that is detectable or used as a detectable marker or biomarker, thereby enabling the treatment, detection, diagnosis, or monitoring or treatment of diseases, conditions, or illnesses of the brain or involving the brain.

[0069] The goods may contain one or more of the following: bioactive small molecules, peptides (polypeptides, proteins), RNA (mRNA, siRNA, miRNA, lncRNA), DNA (antisense oligonucleotides (ASO), plasmids, DNA fragments), and gene editing complexes. Bioactive molecules may be diagnostic or therapeutic agents or therapeutic diagnostic agents for the treatment, diagnosis, detection, and / or monitoring of diseases, conditions, or illnesses of the brain or involving the brain. The goods may include, for example, one or more of the following: hormones, growth factors, enzymes, immunomodulatory compounds, receptors, receptor agonists, or receptor antagonists, for the treatment of diseases, conditions, or illnesses of the brain or involving the brain.

[0070] Diseases, symptoms, or conditions may include tumors in the brain. Goods may include, for example, oncolytic viruses that infect gliomas, or may include therapeutic agents for treating gliomas. Diseases, symptoms, or conditions may be neurodegenerative diseases (such as Parkinson's disease, or Alzheimer's disease, or Huntington's disease, or Creutzfeldt-Jakob disease, or other neurodegenerative diseases), or cognitive impairments (such as dementia, or amnesia, or delirium, or other cognitive impairments), or brain disorders (such as encephalitis, or seizures, or tumors, or other brain disorders), or nervous system disorders (such as pain, or seizures, or infections, or other nervous system disorders), or genetic diseases (such as cystic fibrosis, thalassemia, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, Ty Sachs disease, or other genetic diseases), or brain tumors, or Niemann-Pick disease, or prions, or Parkinson's disease, or multiple sclerosis, or amyotrophic lateral sclerosis (ALS), or muscular dystrophy, or other diseases of the brain or involving the brain. Diseases, conditions, or illnesses of the brain or involving the brain can be cancers or diseases, conditions, or illnesses that are treated or prevented by vaccines, and / or can be diseases, conditions, or illnesses caused by or involving infectious agents. Infectious agents include one or more of, for example, bacteria, viruses, oomycetes, parasites, prions, and fungi.

[0071] For therapeutic, diagnostic, detection, or monitoring purposes, when administered intranasally, MEVs 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 described below, MEVs can also be used to deliver cargo to cells in vitro for cell therapy. The resulting cells can be administered.

[0072] Diseases, symptoms, and conditions include one or more of cognitive, emotional, behavioral, mental, neurological, degenerative, genetic, malignant (cancer), and / or traumatic brain diseases, symptoms, or conditions. Diseases, symptoms, or conditions involving the brain can be caused by damage to the brain or central nervous system (CNS). MEVs may contain therapeutic goods that are psychoactive or treat mental disorders, or are immunomodulatory products, or are detectable products, or treat brain injury or trauma, or treat cancer, or treat neurocranial disorders, or treat CNS disorders, or treat genetic brain disorders, or treat brain cancer, or have anti-aging activity, or have brain regeneration activity.

[0073] MEVs may include, for example, goods intended for one or more of the following: (i) treatment or prevention or risk reduction of brain diseases, conditions, and illnesses; (ii) in vitro and / or in vivo studies of brain diseases, conditions, and illnesses; (iii) diagnosis of brain diseases, conditions, and illnesses; and (iv) recreational use. Diseases, conditions, and illnesses include, but are not limited to, cognitive, emotional, behavioral, mental, neurological, and / or neurodegenerative diseases, conditions, and illnesses, or diseases, conditions, or illnesses caused by damage to the brain or central nervous system (CNS). Diseases, conditions, and illnesses are selected from brain and / or CNS cancers or tumors, genetic disorders, brain injuries or trauma, and infections.

[0074] 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 its direct route to brain regions, intranasally administered MEVs provide a carrier for delivering psychoactive agents. MEVs can deliver medications used to treat mental illnesses and / or disorders.

[0075] MEVs can be used to deliver cargo, such as hydrophilic compounds that, when administered systemically or locally to sites other than the nose, cannot reach the brain after first-pass metabolism in the liver, or have poor intestinal absorption, and cargoes that cannot cross the blood-brain barrier. Intranasal administration in MEVs provides delivery of such compounds, which are typically not otherwise administered to allow them to reach the brain.

[0076] Diseases, conditions, and illnesses treated with intranasally administered MEVs include, but are not limited to, boundary personality disorder, eating disorders, schizophrenia, attention deficit / hyperactivity disorder (ADHD), autism, bipolar disorder, anxiety, depression, obsessive-compulsive disorder (OCD), and post-traumatic stress disorder (PTSD).

[0077] In some examples, the goods include bioactive molecules used to treat conditions, as follows:

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Diseases, conditions, and illnesses involving the brain, including but not limited to genetic disorders, neurodegenerative diseases, metabolic disorders affecting brain function, and other brain-related conditions. Examples of diseases, conditions, and illnesses include, but are not limited to, human mental disorders; non-human animal brain disorders and central nervous system disorders; anxiety disorders such as panic disorder, social anxiety disorder, phobia-related disorders, and generalized anxiety disorder; attention deficit hyperactivity disorder such as inattentive, hyper-impulsive, and mixed types; autism spectrum disorders such as Asperger's syndrome, childhood disintegrative disorder (CDD), Kanner syndrome, and pervasive developmental disorder (PDD-NOS); bipolar disorders such as type I bipolar disorder, type II bipolar disorder, bipolar disorder with mixed features, bipolar disorder with seasonal pattern of major depressive disorder, cyclothymic disorder, and rapid-cycle bipolar disorder; eating disorders such as anorexia nervosa, bulimia nervosa, muscular dystrophy, binge eating disorder, and other specific eating disorders. Or eating disorders (OSFED), compulsive overeating, Prad-Willi syndrome, diabetic bulimia, neurotic health food obsession, selective eating, alcoholism, early eating obsession; personality disorders, including but not limited to antisocial personality disorder, borderline personality disorder, histrionic personality disorder, narcissistic personality disorder, avoidant personality disorder, dependent personality disorder, obsessive-compulsive disorder (OCD); post-traumatic stress disorder (PTSD), such as acute stress disorder, simple PTSD, complex PTSD, comorbid PTSD; classic Rett syndrome, CDKL5-related atypical Rett syndrome; schizophrenia, such as catatonic schizophrenia, disorganized schizophrenia, paranoid schizophrenia, residual schizophrenia, and undifferentiated schizophrenia; and other such mental illnesses and brain-related disorders. Other brain diseases, conditions, and illnesses include: Alzheimer's disease, prions such as Creutzfeldt-Jakob disease, Niemann-Pick disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, Ty Sachs disease, Wilson's disease, leukodystrophy, epilepsy, multiple sclerosis, encephalitis, and migraine.

[0085] MEV cargo intended for delivery to the brain includes, for example, cargo containing one or more psychoactive agents, enzymes, growth factors, and detectable products for the treatment, detection, or monitoring of diseases, conditions, or illnesses of the brain or involving the brain. Such goods include, for example, one or more of the following: TrkA (tropomyosin kinase A); neurotrophic factors selected from: NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocin and / or dephosphorylated psilocin, halamine, temozolomide, levamisole, GABAB1A receptor, GABAB1A receptor siRNA, PTEN siRNA (SEQ ID NO: 136-138); miR-17 (miRNA; SEQ ID NO: 139-141), MALAT1 (SEQ ID NO: 142); 5-HT1A and 5-HT3 receptor agonists, such as azaspirone, methylphenidate, dextromethorphan, ondansetron (e.g., as trademarked). Products sold); acetylcholinesterase inhibitors, such as donepezil, galantamine, and rivastigmine; alpha-1 receptor antagonists, such as prazosin; anticonvulsants, such as gabapentin, pregabalin, and topiramate (e.g., under trademarks). Products sold), carbamazepine, escricazepine, levetiracetam, licorice, oxcarbazepine, valproic acid and its derivatives, lamotrigine; antipsychotics, such as aripiprazole, asenapine, cariprazine, chlorpromazine, clozapine, haloperidol, luminidepiperone tosylate (e.g., as... Products sold include: olanzapine, paliperidone, quetiapine, risperidone, ziprasidone; beta-blockers, such as azaspiron and propranolol; drugs that regulate the cholinergic system, such as biperidone and scopolamine; corticotropin-releasing factor (CRF) antagonists; and drugs that regulate the GABAergic system, such as benzodiazepines. Brinolon, Sage-217; glucocorticoid receptor agonists, such as hydrocortisone; drugs involved in glutamatergic regulation, such as AGN-241751, AV-101, AVP-786, AVP-923, AXS-05, D-cycloserine, dextromethorphan, rapastin; glycine and glycine reuptake inhibitors, such as sarcosine; drugs regulating the hypothalamus-pituitary-adrenal (HPA) axis, such as fludrocortisone, metoprolol, mifepristone, and probiotics; drugs regulating the kynurenine pathway (KP); drugs regulating limbic and paralimbic brain regions, such as cannabidiol (CBD); drugs regulating the melatonin system, such as agomelatine; fatty acids, peptides, nucleic acids, and other precursor molecules, such as α-ω fatty acids, coenzyme Q10, inositol, methylfolate, S-adenosylmethionine, cysteine, and oxytocin; monoamine oxidase inhibitors (MAOIs), such as isocarboxazid. Phenylacetylhydrazine Sleigh Hills and anti-phenylcyclopropane Mood stabilizers, such as lithium salts, valproic acid, ebuselenium, and divalproic acid; multimodal antidepressants, such as vilazorone and vortioxetine; N-nitrosodimethylamine (NDMA) receptor antagonists, such as amantadine, alketamine, ketamine, memantine, riluzole, and esketamine; neurokinin-1 (NK1) receptor antagonists; neuropeptide Y (NPY) receptor agonists; drugs with neurotrophic effects, such as cilostazol, sildenafil, and vildagliptin; norepinephrine-dopamine reuptake inhibitors (NDRIs), such as bupropion. Drugs acting on the opioid system, such as ALKS-5461, AZD2327, BTRX-246040 (LY2940094), buprenorphine, JNJ-67953964, nalmefene, and naltrexone; protein kinase C inhibitors or anti-estrogenic drugs, such as indoxifene, tamoxifen, and verapamil; psychedelic drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), ayahuasca, lysergic acid diacetamide (LSD), and psilocybin; selective serotonin reuptake inhibitors (SSRIs), such as citalopram. Escitalopram Fluvoxamine, Paroxetine Heshequlin Selective norepinephrine transporter inhibitors, such as atoxetine; serotonin-norepinephrine reuptake inhibitors (SNRIs), such as desvenlafaxine. Duloxetine Zominaprun And venlafaxine; stimulants, including adenosine receptor antagonists and α-2-adrenergic receptor agonists, such as caffeine, clonidine, guanfaxine, extended-release amphetamine XR-OS, dextroamphetamine sulfate, lidextroamphetamine, methamphetamine, mixed amphetamine salts, racemic amphetamine sulfate, and triazine mixed amphetamine salts; substance P antagonists, such as aprepitant (MK0869) and fosaprepitant (MK-0517); tricyclic serotonin-norepinephrine reuptake inhibitors, such as amitriptyline. Amoxapine, Buspirone TM ), clomipramine, desipramine Doxepin, Imipramine Pamela tilapia, nortriptyline TM Examples of MEV-containing compositions include, for instance, protriptyline and trimipramine; and vasopressin 1B (V1B) receptor antagonists, such as neliptan (SSR149415). Exemplary goods 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), psilocin / dephosphorylated psilocin, halamine, temozolomide, rivastigmine, and / or rhodamine. As described above, compositions containing MEVs can be delivered to neurons, astrocytes, glial cells, and / or neural stem cells for intranasal or in vitro administration to cells for gene therapy.

[0086] The methods, uses, and compositions described herein may be used to treat one or more diseases, conditions, and illnesses involving the brain, such as, but not limited to, Alzheimer's disease, prions, Niemann-Pick disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, Ty-Sachs disease, Wilson's disease, leukodystrophy, epilepsy, multiple sclerosis, encephalitis, and migraine. For example, the disease, condition, or illness may be a neurodegenerative disease; the product is an ApoE4 inhibitor or an inhibitor of its expression in neurons, or an activator of ApoE2 and / or ApoE3 or its expression in neurons; or a gene editor cassette or system for modifying one or more ApoE2, ApoE3, or ApoE4 encoding genes in neurons; and the composition is formulated and administered via intranasal administration. In particular, the disease, condition, or illness to be treated is Alzheimer's disease or a condition or pathology associated with the risk of developing Alzheimer's disease. These can be treated by administering medications for risk-related conditions or Alzheimer's disease. For example, ApoE gene expression or expression levels or allele distribution can be modulated, such as by intranasal administration of cargo-loaded MEVs to alter Apo levels or expression, resulting in:

[0087] a) Physiological levels of ApoE lipidation modified with MEVs loaded with the following:

[0088] (i) peptides or small molecules known to increase the lipid-binding capacity of ApoE, or (ii) sequences of miRNA (miRNA-33) or miRNA-33 mimicking siRNA or ASO to increase ABCA1 levels or decrease Aβ levels, thereby enhancing the lipidation capacity of ApoE; and / or

[0089] b) Reduce the amount of ApoE4 in the brain by using MEVs loaded with a sequence of miRNA (miRNA146) or miRNA-146-mimicking siRNA or ASO, or loaded with other RNAi (such as siRNA or shRNA) that inhibit ApoE4 expression, thereby suppressing the immune response in the brain and / or reducing ApoE4 in the brain; and / or

[0090] c) Using MEVs loaded with (a) the ApoE2 protein, or (b) mRNA encoding the ApoE2 protein, or (c) a plasmid encoding the ApoE2 sequence to increase the expression of the ApoE2 isotype in the brain, thereby enhancing the protective effect of ApoE2 and compensating for the toxic effects of ApoE4; and / or

[0091] d) Editing the ApoE4 allele to produce ApoE3 and / or ApoE2 by using a MEV loaded with a gene-editing complex for genome editing.

[0092] Therefore, this document provides a MEV containing cargo and capable of delivering the cargo to organs, tissues, and / or cells involved in a specific disease, symptom, or condition. This document is of interest for delivery to the brain via intranasal administration. The cargo can be selected for the treatment, diagnosis, and / or detection of diseases, symptoms, or conditions, and / or for monitoring treatment. Due to the unique transport of the MEV described and demonstrated herein, the MEV provides a unique delivery vehicle. Brief description of the attached diagram

[0094] Figure 1 An exemplary curve of light intensity used in HECTOR PBR cultures is provided.

[0095] Figure 2 Exemplary elution profiles were plotted for a high-purity MEV formulation, wherein the previously concentrated TFF was purified by ultracentrifugation and then eluted at 10... 11 Up to 10 13 MEV prepared in PBS at a concentration of / mL was inoculated into a pre-packed column qEV1 from IZON. MEV was eluted with PBS solution. 0.5 mL of the elution fraction was collected. MEV was recovered from the first fraction as shown in the figure. The most concentrated fractions (4-5) were combined and stored at 4°C before use.

[0096] Figure 3 Exemplary images and approximate dimensions of MEVs obtained using transmission electron microscopy (TEM) are provided.

[0097] Figure 4 Electrophoresis images of the small RNA library are provided.

[0098] Figure 5 Representative patterns of biodistribution based on the route of administration are provided for intravenous (IV), intratracheal (IT), and oral (PO) routes.

[0099] Figure 6 Whole-body imaging of representative animals following intravenous administration as described in Example 5 was depicted.

[0100] Figure 7 In vivo whole-body imaging of representative animals after oral administration (oral administration) as described in Example 5 was depicted.

[0101] Figure 8 Whole-body imaging of representative animals after intranasal administration as described in Example 5 was depicted.

[0102] Figure 9 Whole-body imaging of representative animals after intratracheal administration as described in Example 5 was depicted.

[0103] Figure 10The accumulation kinetics in the liver, lungs, and spleen (average 6 animals) after intravenous administration were depicted as described in Example 5.

[0104] Figure 11 The accumulation kinetics in the lungs, spleen, and intestines (average 6 animals) after oral administration as described in Example 5 were depicted.

[0105] Figure 12 The dynamics of lung and kidney accumulation after intranasal administration were depicted (average 4 animals), as described in Example 5.

[0106] Figure 13 Accumulation kinetics in the lungs, spleen, and intestines (average 3 animals) after intratracheal administration as described in Example 5 were depicted.

[0107] Figure 14A -D depicts the ex vivo fluorescence analysis (total radiative efficiency) of organs [A) liver; B) spleen; C) lung; and D) brain isolated 3 days after intravenous (IV), intranasal (IN), oral (PO), and intratracheal (IT) administration.

[0108] Figure 15A and 15B The images depict A) hematoxylin and eosin staining of the intestine (G=GALT tissue) and B) DAPI (nuclear) staining and MEV-PKH26 fluorescence.

[0109] Figure 16 The spleen pulp stained with DAPI (for the nucleus) and MEV-PKH26 (red fluorescence) is depicted as white spots.

[0110] Figure 17 The graph shows the migration of MEV from GALT to the spleen.

[0111] Figure 18A -I shows the assessment results of MEV toxicity in mouse models after oral (PO) or intratracheal (IT) administration at different doses in four groups of mice for each parameter. This was achieved through chemical parameters: ALAT, ASAT, urea, and creatine (respectively...). Figure 18A -D); and 2) through hematological parameters: red blood cells, hemoglobin, hematocrit, MCV, and eosinophils (respectively). Figure 18E -I) Evaluation of MEV toxicity. Groups: Group 1 - Mice were administered 100 μl of PBS (white strip) via PO delivery; Group 2 - Mice were administered 100 μl of 4*10 PBS via PO delivery. 11 MEV / mice (white stripes with black dots); Group 3 mice were administered 100 μl of 4*10 via PO delivery. 12 MEV / mice (white bars with vertical lines); Group 4 mice were administered 100 μl of 4*10 via IT delivery.11 MEV / mouse (square bars). Data for each parameter were measured in 6 mice per group. Figure 18A This shows ALAT: alanine aminotransferase; Figure 18B ASAT: Aspartate aminotransferase is shown; Figure 18C Urea was shown; Figure 18D Creatine was shown; Figure 18E Red blood cells were shown; Figure 18F Hemoglobin was shown; Figure 18G Hematocrit was shown; Figure 18H The MCV (mean corpuscular volume) is shown; and Figure 18I Eosinophils are shown. PO indicates oral administration (oral delivery), and IT indicates intratracheal administration.

[0112] Figure 19 The in vivo delivery and expression of mRNA after local instillation of MEV into rabbit eyes are described.

[0113] Figure 20A and 20B The in vitro delivery of GFP protein into human monocytes is described.

[0114] Figure 21A and 21B The in vitro delivery of GFP protein into human keratinocytes was described.

[0115] Figure 22 Confocal microscopy of Hep-G2 cells was shown, including GFP protein expression in Hep-G2 cells after incubation with MEVs loaded with GFP-protein (MEV-GFP) or MEVs loaded with mRNA-eGFP (MEV-mRNA) for 24 h.

[0116] Figure 23 This image shows GFP protein expression in Huh7 cells, including Hep-G2 cells, under confocal microscopy.

[0117] Figure 24 The in vitro delivery of GFP mRNA and MEV loaded with GFP-encoding mRNA to human fibroblasts was demonstrated.

[0118] Figure 25A -D shows the results of flow cytometry analysis using human fibroblasts in MEV permeation and delivery studies.

[0119] Figure 26 The exogenous loading of siRNA from Chlorella MEV showed antibacterial activity against the Pto DC3000 cfa6 and hrpL genes.

[0120] Figure 27The delivery of the bioactive flg22 peptide loaded exogenously in Chlorella MEV was demonstrated.

[0121] Figure 28 It is a schematic diagram depicting the path through the olfactory epithelium.

[0122] Figure 29 The image shows a positive control, DiR-MEV, on a DAPI-stained brain slice: a drop of MEV suspension deposited on top of a brain tissue slide. The spot is DiR-labeled MEV.

[0123] Figure 30 This is a schematic diagram of the insula and its connections (copied from Gogolla (2017) "The insular cortex," Current Biology: 2712: R580-R586).

[0124] Figure 31 This is a schematic diagram of the neuronal pathway from the olfactory sensory neuron (OSN) through the olfactory bulb (OB) to the mitral neuron and cluster neuron, and finally to the olfactory tract (OT).

[0125] Figure 32 This is a schematic diagram showing the pathway and approximate average distance from the olfactory and respiratory epithelium to CNS targets (from...

[0126] 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 (2019 copy).

[0127] Figure 33 This is a schematic diagram of cortical projections of mitral cells and cluster cells, showing a ventrolateral view of the brain (copied from Imai (2014) "Construction of functional neuronal circuitry in the olfactorybulb," Seminars in Cell and Developmental Biology 35, DOI:10.1016 / j.semcdb.2014.07.012).

[0128] Figure 34This study demonstrates the transport of MEVs via the olfactory pathway. Following IN administration, MEVs are taken up by the olfactory epithelium via axonal transport, transported to the olfactory bulb via olfactory neurons, and then transported to the primary olfactory regions processing olfactory signals via mitral neurons and cluster neurons (from Selvaraj et al. (2018) Artificial Cells, Nanomedicine, and Biotechnology An International Journal 46:2088-2095).

[0129] (Copy from doi.org / 10.1080 / 21691401.2017.1420073).

[0130] Figure 35 The olfactory pathway used by the MEV after IN administration is depicted (a schematic diagram of the general pathway copied from "What-when-how in-Depthtutorials and information, Olfaction and Taste, Sensory system, part 1" (URL:what-when-how.com)).

[0131] Figure 36A-36G : Figure 36A This provides a general overview of experimental designs for studies on brain biodistribution. Figure 36B The locations of the five brain regions studied are shown; Figure 36C The regions analyzed to determine the PK and biodistribution of MEVs in each of the five brain regions studied were depicted. Figure 36D -G depicted and identified brain regions for reference to the following figure, which shows the MEV in the brain after IN administration.

[0132] Figure 37A-37D The pharmacokinetic (PK) and biodistribution of MEVs from different regions in the first part of Figure 36 are shown. Images of -MEVs labeled DiR are black dots.

[0133] Figure 38 The PK and biodistribution of MEVs in different regions of the first part are shown, and a graphical representation of the total number of MEV spots with DiR labels on each surface of the region of the first part is provided by total analysis area normalization.

[0134] Figure 39A-39D The second part shows the PK and biodistribution of MEVs in different regions (images of MEVs labeled with DiR are shown).

[0135] Figure 40A-40DThe second part shows the PK and biodistribution of MEVs in different regions, as derived from Figure 39A A graphical representation of the total number of MEV spots marked with DiR on each surface of the second part of the region of -D, normalized by total analysis area.

[0136] Figure 41A-41D The PK and biodistribution of MEVs in different regions of Part III are shown; images of MEVs labeled with DiR are also shown.

[0137] Figure 42A-42F From Figure 41A The graphical representation of the total number of MEV spots marked with DiR on each surface of region 3 in part 3 (normalized by total analytical area) shows the PK and biodistribution of MEVs in different regions of part 3.

[0138] Figure 43A-43D The PK and biodistribution of MEVs in different regions of Part IV are shown as images of MEVs labeled with DiR.

[0139] Figure 44A-44D Showing from Figure 43A The PK and biodistribution of MEVs in different regions of Part 4 of the -D dataset provides a graphical representation of the total number of MEV spots labeled with DiR on each surface of the region of Part 4, normalized by total analysis area.

[0140] Figure 45A-45D The pharmacokinetic and biodistribution of MEVs in different regions of Part 5 are shown; images of DiR-labeled MEVs are also shown.

[0141] Figure 46A and 46B The dynamics of MEV brain penetration from the cerebellar to the distal brain are shown.

[0142] Figure 47 The blood-brain barrier was described (copied from Cecchelli et al. (2007) Nat Rev Drug Discov. 6(8): 650-661).

[0143] Figure 48 Microscopic images of mouse intestinal epithelium 8 hours after administration of PKH26-labeled MEV are shown.

[0144] Figure 49 Whole-body bioluminescence imaging of a representative animal treated with MEV loaded with luciferase mRNA is shown.

[0145] Figure 50 Whole-body bioluminescence imaging of representative animals treated with MEV loaded with luciferase was depicted.

[0146] Figure 51 Depicting the use Timeline of image analysis performed by the live cell analyzer.

[0147] Figure 52 The structure of human ApoE3 is shown. The polypeptide chain of residue 299 is shown in a linear form, and the cysteine-arginine sites are interchanged at positions 112 and 158, which distinguish ApoE2 and ApoE4 from ApoE3. The protein folds into two separate domains, where the N-terminal residues 1 to 191 contain an antiparallel quadrature helical bundle, and the C-terminal residues 192 to 299 form a separate folded domain that interacts with the helical bundle. The segment spanning residues 135 to 150 (horizontal red arrow) in the N-terminal domain contains a cluster of basic amino acids that form the LDLR binding site. The C-terminal segment spanning residues 260 to 299 (horizontal red arrow) contains an amphiphilic α-helix that initiates protein binding to lipid surfaces. (Phillips MC. (2014) IUBMBLife 66: 616-623)). Detailed Implementation

[0148] Outline

[0149] A. Definition

[0150] B. Microalgae and Overview

[0151] C. Extracellular vesicles

[0152] 1. Types of extracellular vesicles (EVs)

[0153] a.Exosomes

[0154] b. Microvesicles

[0155] c. Apoptotic bodies

[0156] 2. EV intake

[0157] 3. General methods for separating EVs

[0158] a. Ultracentrifugation

[0159] b. Size-based technology

[0160] c. Immunoaffinity capture-based technologies

[0161] d. Exosome precipitation

[0162] e. Microfluidic-based separation technology

[0163] 4. Microalgae and microalgae-derived extracellular vesicles (MEVs)

[0164] 5. Green algae - Chlorella species

[0165] a. Lifecycle

[0166] b. Genome analysis of Chlorella species

[0167] c. Commercial and biotechnological uses of Chlorella

[0168] d. Chlorella MEV

[0169] D. Exogenously loaded microalgal extracellular vesicles (MEVs), cargo, and targets

[0170] 1. MEV separation

[0171] 2. MEV loading and cargo

[0172] 3. Generation of MEVs carrying payloads

[0173] a. Electroporation

[0174] b. Ultrasonic treatment

[0175] c. Extrusion

[0176] d. Surfactants

[0177] e. Other methods

[0178] 4. Exemplary cargo and exemplary uses of externally loaded MEVs

[0179] a. Goods

[0180] 1) RNA cargo

[0181] 2) Antibody cargo

[0182] b. Diseases and treatments

[0183] c. Agricultural and veterinary applications

[0184] d. Cosmetic and dermatological applications

[0185] E. Pharmaceutical compositions, formulations, reagent kits, articles, and combinations

[0186] 1. Pharmaceutical compositions and formulations

[0187] 2. Products / Reagent Kits and Combinations

[0188] 3. Application of exogenously loaded MEVs and application routes

[0189] 4. Combination therapy

[0190] F. Biodistribution of MEVs after administration via different routes

[0191] 1. Biodistribution of mammalian EVs

[0192] 2. Biodistribution of microalgae EVs

[0193] Oral administration

[0194] a) Components of the lymphatic system

[0195] b) Targeting GALT

[0196] 3. Diseases and symptoms treated with MEV

[0197] G. MEVs are biodistributed and delivered to the brain via intranasal (IN) administration for the treatment of diseases, symptoms, and conditions of the brain and central nervous system.

[0198] 1. Brain structure

[0199] brain structure

[0200] a. Proolal nucleus

[0201] b. Outer longitudinal stripes (Tenia Tecta)

[0202] c. Olfactory nodules

[0203] d. Piriform cortex

[0204] e. amygdala

[0205] f. Entorhinal cortex

[0206] g. Frontal cortex

[0207] h. Striatum: caudate nucleus and lenticular capsid

[0208] i. Nucleus accumbens

[0209] j. Thalamus

[0210] k. Hypothalamus

[0211] l. Substantia nigra compacta

[0212] m. seahorse

[0213] n. thalamus

[0214] o. Pontine raphe nuclei

[0215] 2. Blood-brain barrier

[0216] 3. Brain and target cells

[0217] 4. Differences in biodistribution between MEVs and other delivery vectors

[0218] 5. Intranasal (IN) administration

[0219] 6. After intranasal administration of MEV and delivery to the brain

[0220] 7. Transport and biodistribution of MEV after intranasal (IN) administration

[0221] 8. Primary and secondary circuits of the olfactory system and regions reached by the MEV during IN administration.

[0222] 9. Delivery of MEVs to the brain via IN administration – Exemplary bioactive cargoes and their uses

[0223] H. Preparations for administration and the diseases and conditions treated therefrom.

[0224] I.MEV-mediated intracellular signal transduction

[0225] J. Example

[0226] A. Definition

[0227] 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 pertains. Unless otherwise stated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other publicly available material referenced throughout this disclosure are incorporated herein by reference in their entirety. Where multiple definitions of terms exist herein, those in this section shall prevail. In the case of references to URLs or other such identifiers or addresses, it should be understood that such identifiers can change and specific information on the Internet can come and go, but equivalent information can be found by searching the Internet. References to such URLs demonstrate the availability and public dissemination of this information.

[0228] As used herein, cargo refers to exogenous molecules, such as bioactive molecules, including biomolecules and small molecules, which are loaded into microalgal extracellular vesicles (MEVs) after being isolated from the microalgal extracellular vesicles (MEVs) provided herein. This includes cargo that is heterologous to the MEV.

[0229] As used herein, heterologous cargo in a MEV generally refers to cargo that is not naturally present in the MEV but is exogenously loaded, as described above. It also refers to cargo in a MEV that is endogenously loaded into the MEV through genetically modified microalgae. A MEV with heterologous cargo contains cargo that is not naturally present in the MEV. Cargo heterologous to microalgae and / or MEV is cargo that is not naturally present in microalgae such that it is packaged in a MEV and / or is cargo that is not present in microalgae in an unmodified MEV or is exogenously loaded into an isolated MEV.

[0230] As used herein, a bioactive molecule or bioactive agent means any molecule or reagent that may have biological activity (such as therapeutic activity) or serve as a detectable marker or act on a subject in vivo. Bioactive agents and molecules include biomolecules such as DNA, RNA, proteins, other biopolymers, and small molecules such as small molecule drugs and pharmaceuticals, immunogens, and any molecule that will be delivered to a subject (e.g., human or other animal or plant or microorganism (bacteria or others)) in connection with a therapeutic, diagnostic application, or other such use (e.g., cosmetic). Bioactive agents or molecules may be used or have activity, such as therapeutic agents, immunogens, diagnostic agents, detectable markers, or cosmetics. The bioactive molecule used herein is any bioactive molecule that can be loaded into microalgal extracellular vesicles (MEVs).

[0231] As used herein, a biomolecule means any biologically active biopolymer or molecule that is present or may be present in a living organism or virus, or a modified form of such a biopolymer or molecule. Therefore, a biomolecule includes modified naturally occurring biomolecules, such as proteins comprising modified primary sequences, for example by altering the primary sequence through deletions, insertions, and / or substitutions of amino acids, and / or by modifications, such as post-translational modifications of proteins.

[0232] As used herein, when it is stated that MEVs have the same or substantially the same amount of cargo or their quantity, it should be understood that this refers to the average value of the group of MEVs in the composition. It should be understood that when MEVs are externally loaded, the cargo / MEV ratio can be selected such that each MEV has a predetermined amount of cargo on average. As a simple example, to load an average cargo / MEV of one molecule, a person skilled in the art can calculate the amount of cargo loaded into the composition of the MEVs and understand that in the composition of the MEVs, some will have more than one molecule of cargo / MEV, while others will not. On average, an MEV will have one molecule of cargo / MEV. Those skilled in the art will understand that, typically, the amount of cargo / MEV will be more than one molecule / MEV, and the amount of cargo depends on various parameters, including the cargo, the target tissue and / or cells, the disease, condition, or illness being treated, and the subject being treated. Typically, each MEV loads an average of more than one cargo molecule, for example, at least 10 or about 10 molecules / MEV. It is also possible to load substantially more cargo, 100, 500, 1000, 10 4 Individual molecules / MEVs, etc. The loading amount depends on the target, disease, symptom or condition, subject, cargo, and MEV capacity. The selected amount is within the scope of the art.

[0233] As used herein, subjects are any living organism, typically an animal or plant, on which or in which a composition containing MEV is introduced. Subjects include, but are not limited to, humans, plants, particularly crop plants, and animals, including farm animals and pets such as dogs and cats, as well as zoo animals.

[0234] As used herein, disease or symptom or condition means a pathological or undesirable condition in an organism caused by a cause or condition, including but not limited to infection, acquired and genetic conditions, and those characterized by identifiable symptoms.

[0235] As used in this article, treating a subject with a disease, symptom or condition means that the subject's symptoms or manifestations of the disease or condition are partially or completely relieved, or remain static after treatment.

[0236] As used herein, treatment means any effect that improves the symptoms of a disease or condition. Treatment includes prevention, treatment, and / or cure. Treatment also includes any pharmaceutical use of any MEV or composition provided herein.

[0237] As used herein, prevention refers to the prevention of potential disease and / or the prevention of the worsening of symptoms or progression of disease. Prevention or prophylaxis, and their grammatically equivalent forms, refer to methods that reduce or eliminate the risk or probability of the occurrence of a disease or condition, as well as products that reduce or eliminate the risk or probability of the occurrence of a disease or condition.

[0238] As used herein, a disease, symptom, or condition of the brain, or a disease, symptom, or condition involving the brain, is a disease, symptom, or condition in which the cause of the disease, symptom, or condition involves the brain, such that delivery of a therapeutic agent to the brain can achieve treatment, including improving or alleviating symptoms, and / or treating the cause or manifestation of the disease, symptom, or condition, or delivery of diagnostic molecules, such as marker molecules, reporter molecules, or enzymes, that can be used to aid or achieve diagnosis or to monitor the progress or effectiveness of treatment. Such diseases, symptoms, and conditions may affect or involve organs or tissues other than the brain, but treatment of the brain can at least improve symptoms, and the administration of detectable molecules or reporter molecules can be used to detect or monitor the disease, symptom, or condition or aspects thereof.

[0239] As used herein, modifications to the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule refer to and include the deletion, insertion, and substitution of amino acids or nucleotides. These include modifications to the primary sequence of the polypeptide or protein. Methods for modifying polypeptides and nucleic acid molecules are conventional to those skilled in the art, such as through the use of recombinant DNA methods. When referring to a polypeptide or protein but not its sequence, modification refers to post-translational or post-purification changes, such as the conjugation or linkage of portions that alter the properties of the polypeptide or protein, such as portions that extend half-life, glycosylation, purification tags, detectable reporters, and other such portions.

[0240] As used herein, modifications to the genome, plasmid, or gene include the deletion, substitution, insertion, and translocation of nucleic acids. These include any alteration to the natural or naturally occurring nucleic acid sequence.

[0241] As used in this article, RNA interference (RNAi) is a biological process in which RNA molecules suppress gene expression or translation by neutralizing target mRNA molecules to inhibit translation and thereby suppress the expression of the target gene.

[0242] As used in this article, RNA molecules that function via RNAi are referred to as repressive because they silence the expression of their target genes. Silencing expression means that the expression of the target gene is reduced, suppressed, or repressed.

[0243] As used in this article, gene silencing via RNAi is said to suppress, repress, disrupt, or silence the expression of a target gene. The target gene contains a nucleotide sequence that corresponds to a sequence in the repressive RNA, thereby silencing the expression of the mRNA. Small interfering RNA (siRNA) is a small piece of double-stranded (ds) RNA, typically about 21 nucleotides long, with a 3' overhang (2 nucleotides) at each end, which can be used to interfere with protein translation by binding to and promoting the degradation of messenger RNA (mRNA) at a specific sequence. 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 tight hairpin bend that can be used to silence the expression of a target gene via RNA interference (RNAi). shRNA expression in cells is typically achieved through delivery of plasmids or via viral or bacterial vectors.

[0244] As used herein, non-coding RNA is RNA that does not encode proteins. Categories of non-coding RNA include, but are not limited to, small interfering RNA (siRNA) and microRNA (miRNA). As used herein, inhibiting, repressing, disrupting, or silencing a target gene refers to altering the expression (such as translation) of a target gene, thereby reducing the activity or expression of the product encoded by the target gene. Reduction includes complete or partial knockout, thereby achieving therapeutic effects by referring to the MEVs and administration described herein.

[0245] As used in this article, the tumor microenvironment (TME) is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). Presence of these conditions includes, but is not limited to, increased angiogenesis, hypoxia, low pH, increased lactate concentration, increased pyruvate concentration, increased interstitial fluid pressure, and altered metabolites or metabolism, such as higher levels of adenosine, which indicates a tumor.

[0246] As used herein, the description of a target gene's nucleic acid or encoding RNA refers to its ability to inhibit, repress, or silence gene expression through any mechanism. Typically, such nucleic acid includes at least a portion complementary to the target gene, wherein that portion is sufficient to form a hybrid with the complementary portion.

[0247] As used herein, when referring to a nucleic acid or polypeptide sequence, a deletion refers to the absence of one or more nucleotides or amino acids compared to the sequence (e.g., target polynucleotide or polypeptide or natural or wild-type sequence).

[0248] As used herein, when referring to nucleic acid or amino acid sequences, an insertion describes the inclusion of one or more additional nucleotides or amino acids within the target, native, wild-type, or other relevant sequence. Therefore, a nucleic acid molecule containing one or more insertions contains one or more additional nucleotides within the linear length of the sequence compared to a wild-type sequence.

[0249] As used in this article, the addition to nucleic acid and amino acid sequences describes the addition of a nucleotide or amino acid at either end compared to another sequence.

[0250] 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 altering the length of the molecule (as described in terms of the number of residues). Therefore, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides in the molecule. Amino acid substitutions compared to a specific polypeptide can be expressed based on the number of amino acid residues along the length of the polypeptide sequence.

[0251] As used herein, the statement that a nucleotide or amino acid position corresponds to a nucleotide or amino acid position in a disclosed sequence (as described in the sequence listing) refers to the nucleotide or amino acid position identified when aligned with the disclosed sequence using a standard alignment algorithm (such as the GAP algorithm) to maximize identity. By aligning the sequences, those skilled in the art can identify the corresponding residues, for example, using conserved and identical amino acid residues as guidance. Typically, to identify the corresponding position, amino acid sequences are aligned to obtain the highest-order match (see, for example, 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).

[0252] As used herein, sequence alignment refers to the comparison of two or more nucleotide or amino acid sequences using homology. Typically, alignment is performed by comparing two or more sequences with 50% or higher identity correlation. A sequence alignment set refers to two or more sequences aligned at corresponding positions and may include RNA-derived sequences (e.g., ESTs and other cDNAs) aligned to genomic DNA sequences. Related or variant polypeptide or nucleic acid molecules can be aligned using any method known to those skilled in the art. Such methods typically maximize matching and include methods such as manual alignment and the use of many available alignment procedures (e.g., BLASTP) and other methods known to those skilled in the art. By aligning polypeptide or nucleic acid sequences, those skilled in the art can use conserved and identical amino acid residues as guidance to identify similar portions or positions. Furthermore, those skilled in the art can also use conserved amino acid or nucleotide residues as guidance to find corresponding amino acid or nucleotide residues between human and non-human sequences. Corresponding positions can also be based on structural alignment, for example, by using computer simulations of protein structures. In other cases, corresponding regions can be identified. Those skilled in the art can also use conserved amino acid residues as guidance to find corresponding amino acid residues between human and non-human sequences.

[0253] As used herein, the properties of a polypeptide, such as antibodies, refer to any properties exhibited by the polypeptide, including but not limited to binding specificity, structural configuration or conformation, protein stability, resistance to proteolysis, conformational stability, heat resistance, and tolerance to pH conditions. Changes in properties can alter the activity of a polypeptide. For example, changes in the binding specificity of an antibody polypeptide can alter its ability to bind to antigens, and / or various binding activities of the polypeptide, such as affinity or affinity, or in vivo activity.

[0254] As used herein, the activity or functional activity of a peptide, such as an antibody, refers to any activity exhibited by the peptide. Such activity can be determined empirically. Exemplary activities include, but are not limited to, the ability to interact with biomolecules, such as through antigen binding, DNA binding, ligand binding, or dimerization, or enzyme activity, such as kinase activity or proteolytic activity. For antibodies (including antibody fragments), activity includes, but is not limited to, the ability to specifically bind to a particular antigen, the affinity of antigen binding (e.g., high affinity or low affinity), the binding rate (on-rate), the dissociation rate (off-rate), effector function (e.g., the ability to promote antigen neutralization or clearance), viral neutralization, and in vivo activity (e.g., the ability to prevent pathogen infection or invasion or to promote clearance or penetration of a specific tissue or fluid or cell 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 to measure binding or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry, and binding assays (e.g., panning assays).

[0255] As used herein, bind, bound, and their grammatical variations refer to any interaction between a molecule and another molecule, or any interaction between molecules, resulting in a stable association where the molecules are brought close together. Binding includes, but is not limited to, non-covalent bonds, covalent bonds (such as reversible and irreversible covalent bonds), and includes interactions between molecules (such as, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and small molecules, such as compounds including drugs).

[0256] As used herein, an antibody refers to an immunoglobulin and immunoglobulin fragment, whether natural or partially or wholly synthetic, such as recombinant, including any fragment containing at least a portion of the variable heavy chain and light chain regions of an immunoglobulin molecule, sufficient to form an antigen-binding site and, upon assembly, specifically binding to an antigen. Therefore, an antibody includes any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). For example, an antibody is defined as an antibody containing two heavy chains (which may be denoted as H and H') and two light chains (which may be denoted as L and L'), wherein each heavy chain may be a full-length immunoglobulin heavy chain or a portion sufficient to form an antigen-binding site (e.g., heavy chains include, but are not limited to, the VH chain, the VH-CH1 chain, and the VH-CH1-CH2-CH3 chain), and each light chain may be a full-length light chain or a portion sufficient to form an antigen-binding site (e.g., light chains include, but are not limited to, the VL chain and the VL-CL chain). Each heavy chain (H and H') pairs with one light chain (L and L', respectively). Typically, antibodies include at least all or a portion of the variable heavy (VH) chain and / or variable light (VL) chain. Antibodies may also include all or a portion of the constant region.

[0257] For the purposes of this document, the term antibody includes full-length antibodies and portions thereof, including antibody fragments such as antitumor antibodies or antipathogen or gene-silencing fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. Antibodies also include synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and in vivo antibodies. The antibodies described 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 subclass (e.g., IgG2a and IgG2b).

[0258] As used herein, nucleic acid refers to at least two linked nucleotides or nucleotide derivatives typically linked together by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleic acid also includes nucleic acid analogs, such as peptide nucleic acids (PNA), phosphate-thioester 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 bonds, aminophosphate bonds, phosphate-thioester bonds, thioester bonds, or peptide bonds (peptide nucleic acids). The term also includes equivalents, derivatives, variants, and analogs of RNA or DNA made from nucleotide analogs, single (sense or antisense), and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For RNA, the uracil base is uridine.

[0259] As used herein, isolated nucleic acid molecules are nucleic acid molecules separated from other nucleic acid molecules present in natural sources of nucleic acid molecules. When produced by recombinant technology, isolated nucleic acid molecules (e.g., cDNA molecules) may be substantially free of other cellular material or culture medium, or when chemically synthesized, substantially free of chemical precursors or other chemicals. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding RNAi or therapeutic proteins.

[0260] As used herein, operatively linked nucleic acid sequences, regions, elements, or domains refer to nucleic acid regions that are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operatively linked to a nucleic acid encoding a polypeptide, thereby allowing the nucleic acid to be transcribed and translated to express a functional fusion protein, where the leader peptide influences the secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operatively linked to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed into 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 located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that when a partial amber-repressive cell is introduced, the resulting single mRNA transcript can be translated to produce a fusion protein containing both the first and second polypeptides, or can be translated to produce only the first polypeptide. In another instance, a promoter can be operatively linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of the nucleic acid.

[0261] As used herein, the synthesis of, for example, synthetic nucleic acid molecules, synthetic genes, or synthetic peptides refers to nucleic acid molecules or polypeptide molecules produced by recombination methods and / or by chemical synthesis methods.

[0262] As used in this article, naturally occurring α-amino acid residues are 20 α-amino acid residues found in nature that are incorporated into proteins by specifically recognizing charged tRNA molecules and their homologous mRNA codons in humans.

[0263] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms polypeptide and protein are used interchangeably in this document.

[0264] As used in this article, a peptide refers to a polypeptide with a length of 2 to approximately 40 amino acids.

[0265] As used herein, unless otherwise stated, references to proteins include all forms of peptides, polypeptides, small peptides, and proteins.

[0266] As used herein, an amino acid is an organic compound containing an amino and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes of this document, the amino acids contained in the antibodies provided include 20 naturally occurring amino acids (see the table below), non-natural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, amino acids present in the various amino acid sequences of the polypeptides appearing herein are identified according to their known three-letter or single-letter abbreviations (see the table below). Nucleotides present in various nucleic acid molecules and fragments are named using standard single-letter nomenclature conventionally used in the art.

[0267] As used herein, an amino acid residue refers to an amino acid formed during the chemical digestion (hydrolysis) of a polypeptide at its peptide bond. The amino acid residues described herein are typically in their L-isomer form. D-isomer residues may substitute for any L-amino acid residue, provided the polypeptide retains the desired functional properties. NH2 refers to the free amino group present at the amino terminus of the polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of the polypeptide. In accordance with the standard polypeptide nomenclature described in J. Biol. Chem., 243:3557-59 (1968) and adopted in 37 C. FR § § 1.821-1.822, the abbreviations for amino acid residues are shown in the table below:

[0268] Correspondence table

[0269]

[0270]

[0271] All amino acid residue sequences represented by the formula in this article have a left-to-right orientation in the conventional direction from the amino terminus to the carboxyl terminus. A phrase amino acid residue is defined as including the amino acids listed in the corresponding table above, as well as modified, non-natural, and unusual amino acids. A dash at the beginning or end of an amino acid residue sequence indicates a peptide bond with another sequence of one or more amino acid residues, or with an amino-terminal group such as NH2, or a carboxyl-terminal group such as COOH.

[0272] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0273] Such substitutions can be made according to the exemplary substitutions listed in the table below:

[0274] Exemplary Conserved Amino Acid Substitution

[0275]

[0276]

[0277] Other substitutions are also permitted and can be determined based on experience or based on other known conservative or non-conservative substitutions.

[0278] As used in this article, naturally occurring amino acids refer to the 20 L-amino acids present in a polypeptide.

[0279] As used herein, the term non-natural amino acid refers to an organic compound having a structure similar to that of a natural amino acid but having been structurally modified to mimic the structure and reactivity of a natural amino acid. Therefore, non-natural amino acids include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids, and include, but are not limited to, D-stereoisomers of amino acids. Exemplary non-natural amino acids are known to those skilled in the art and include, but are not limited to, 2-aminohexanoic acid (Aad), 3-aminohexanoic acid (bAad), β-alanine / β-aminopropionic acid (Bala), 2-aminobutyric acid (Abu), 4-aminobutyric acid / piperidinic acid (4Abu), 6-aminohexanoic 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), and 2,2'-diaminopimelic acid (Dpm). ), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allohydroxylysine (Ahyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesin (Ide), allo-isoleucine (Aile), N-methylglycine, sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), novovaline (Nva), novoleucine (Nle), and ornithine (Orn).

[0280] As used herein, a DNA construct is a single-stranded or double-stranded, linear or circular DNA molecule containing DNA fragments combined and juxtaposed in ways not found in nature. DNA constructs exist as the result of human manipulation and include clones and other copies of the manipulated molecule.

[0281] As used herein, a DNA fragment is a portion of a larger DNA molecule that has specific properties. For example, a DNA fragment encoding a particular polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5' to 3' direction, encodes the amino acid sequence of the particular polypeptide.

[0282] As used herein, the term polynucleotide refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' ends. 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 the form of nucleotides (abbreviated as nt) or base pairs (abbreviated as bp). Where the context permits, the term nucleotide is used for both single-stranded and double-stranded molecules. When the term is applied to a double-stranded molecule, it is used to indicate the total length and will be understood to be equivalent to the term base pair. Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide can be of slightly different lengths and their ends can be staggered; therefore, not all nucleotides within a double-stranded polynucleotide molecule will pair. Such unpaired ends are typically no more than 20 nucleotides in length.

[0283] As used in this article, "generating by recombination" refers to expressing proteins encoded by cloned DNA using well-known methods of molecular biology.

[0284] As used herein, heterologous nucleic acids are nucleic acids that encode products (i.e., RNA and / or proteins) not normally produced by the cells in which they are expressed in vivo, or nucleic acids located at loci in which they are not normally present, or nucleic acids that mediate or encode mediators that alter the expression of endogenous nucleic acids (such as DNA) by influencing transcription, translation, or other regulated biochemical processes. Heterologous nucleic acids, such as DNA, are also called foreign nucleic acids. Any nucleic acid, such as DNA, that a person skilled in the art will recognize or consider to be expressed by a heterologous or exogenous cell is covered herein as heterologous nucleic acids; heterologous nucleic acids include exogenously added nucleic acids that are also expressed endogenously. Heterologous nucleic acids are generally not endogenous to the cell in which they are introduced, but are obtained from another cell or synthesized or introduced into a genomic locus in which they are not naturally present, or whose expression is controlled by a regulatory sequence different from the natural regulatory sequence.

[0285] Examples of heterologous nucleic acids in this article include, but are not limited to, DNA molecules, RNA molecules, plasmids, and antisense oligonucleotides. In MEVs, heterologous nucleic acids may be encoded on plasmids. Heterologous nucleic acids, such as DNA, include nucleic acids that can mediate the expression of DNA encoding a therapeutic product in some way, or that can encode a product, such as a peptide or RNA, that mediates the expression of a therapeutic product directly or indirectly in some way.

[0286] As used herein, cell therapy involves delivering MEVs to a subject to treat a disease or condition. MEVs are exogenously loaded with cargo, such that they deliver or express the product upon introduction into the subject. MEVs may also be endogenously loaded with cargo (see, for example, co-pending U.S. Provisional Application Serial No. 63 / 349,006, filed June 3, 2022, which details the preparation of endogenously loaded MEVs and their production cell lines) and used as described herein. The transport of MEVs is generally independent of the manner in 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.

[0287] As used herein, genetic therapy involves transferring a heterologous nucleic acid (such as DNA) into certain cells (such as target cells) of a mammal (particularly humans) that has a disease or condition for which such therapy is sought. The nucleic acid (e.g., DNA) is introduced into the selected target cells in a manner that causes the heterologous nucleic acid (e.g., DNA) to be expressed and to produce a therapeutic product encoded therefrom. Genetic 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 cells to which they are introduced. The introduced nucleic acid may encode therapeutic compounds, such as growth factors or inhibitors thereof, or tumor necrosis factor or inhibitors thereof, such as their receptors, which are typically not produced in mammalian hosts or not produced at therapeutically effective amounts or at the time when therapy is useful. The heterologous nucleic acid (e.g., DNA) encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host to enhance or otherwise alter its product or expression. Genetic therapy may also involve delivering inhibitors or repressors or other regulators of gene expression.

[0288] As used herein, expression refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0289] As used herein, a host cell is a cell used to receive, maintain, replicate, and / or amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When a host cell divides, the nucleic acids contained in the vector are replicated, thereby amplifying the nucleic acids.

[0290] As used herein, a vector is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. References to vectors include those in which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically through restriction digestion and ligation. References to vectors also include those containing nucleic acids encoding polypeptides or RNA. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the amplification of nucleic acids or for the expression / display of polypeptides encoded by nucleic acids. Vectors are typically still free but can be engineered to achieve the integration of genes or portions thereof into chromosomes of the genome. Vectors for artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also considered. Such selection and use of vectors are well known to those skilled in the art. Vectors also include viral vectors or viral vectors. A viral vector is an engineered virus operatively linked to a foreign gene to transfer the foreign gene (as a vector or shuttle) into cells.

[0291] As used herein, expression vectors include vectors capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, capable of enabling the expression of such DNA fragments. Such additional regions may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, upon introduction into a suitable host cell, results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and / or prokaryotic cells, those that remain free, or those that are integrated into the host cell genome.

[0292] As used in this article, primary sequence refers to the amino acid residue sequence in a polypeptide or the nucleotide sequence in a nucleic acid molecule.

[0293] As used herein, sequence identity refers to the number of identical or similar amino acid 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 the purposes of this document, sequence identity is typically determined by alignment to identify identical residues. Alignment can be local or global. Matches, mismatches, and vacancies can be identified between compared sequences. A vacancy is an empty amino acid or nucleotide inserted between residues in the aligned sequences to align identical or similar characters. Typically, internal vacancies and terminal vacancies can be present. Sequence identity can be determined when a vacancy penalty is used, without penalty for terminal vacancies (e.g., no penalty for terminal vacancies). Alternatively, sequence identity can be determined without considering vacancies as the number of identical positions / total aligned sequence length x 100.

[0294] For the purposes of this article, sequence identity is determined by comparing a test peptide or nucleic acid molecule with a reference molecule and counting the number of differences (including vacancies and insertions). The number of differences is divided by the length of the reference molecule (typically the molecule for which the sequence is claimed to be protected) and a percentage is determined. For example, if the claimed peptide is 100 amino acids long and the variants include those with 90% sequence identity, then the variants could have 10 amino acid differences, including vacancies and insertions.

[0295] As used herein, global alignment is an alignment of two sequences from start to finish, comparing each letter in each sequence only once. Alignment is generated regardless of whether there is similarity or identity between the sequences. For example, 50% sequence identity based on global alignment means that in the alignment of the complete sequences of the two compared sequences, 50% of the residues are identical for every 100 nucleotides. It should be understood that global alignment can be used to determine sequence identity even if the aligned sequences are of different lengths. Differences at the ends of sequences are considered when determining sequence identity unless a no-terminal-vacancy penalty is chosen. Typically, 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 procedures for performing global alignments are publicly available, including a global sequence alignment tool available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov / ) and a procedure available at deepc2.psi.iastate.edu / aat / align / align.html.

[0296] As used herein, local alignment compares two sequences, but only those portions that share similarity or identity. Therefore, local alignment determines whether a sub-fragment of one sequence exists in another. If no similarity exists, no alignment is returned. Local alignment algorithms include BLAST or the Smith-Watterman algorithm (Adv. Appl. Math. 2:482 (1981)). For example, 50% sequence identity based on local alignment means that in the alignment of the complete sequences of two compared sequences of any length, a 100-nucleotide similarity or identity region has 50% of the same residues in that similarity or identity region.

[0297] For the purposes of this paper, sequence identity can be determined by a standard alignment algorithm procedure used with a default gap penalty established for each vendor. The default parameters of the GAP procedure may include: (1) a unary comparison matrix (1 for identical inclusion values ​​and 0 for non-identical inclusion values) and a weighted comparison matrix as described in Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for terminal gaps. Whether any two nucleic acid molecules have nucleotide sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or any two polypeptides have amino acid sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or other similar variants listing percentage identity, can be determined using known computer algorithms based on local or global alignment (see, for example, wikipedia.org / wiki / Sequence_alignment_software, which provides links to many known and publicly available alignment databases and programs). For the purposes of this paper, global alignment-based computer algorithms are typically used to determine sequence identity, such as the Needleman-Wunsch global sequence alignment tool available from NCBI / BLAST (blast.ncbi.nlm.nih.gov / Blast.cgi?CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson's implementation of the Huang and Miller algorithm (Adv. Appl. Math. (1991) 12:337-357)); and Xiaoqui Huang's program available from deepc2.psi.iastate.edu / aat / align / align.html. Generally, the full-length sequence of each compared polypeptide or nucleotide is aligned across the entire sequence in a global alignment. Local alignment can also be used when the lengths of the compared sequences are substantially the same.

[0298] Therefore, as used herein, the term identity refers to a comparison or alignment between a test peptide or polynucleotide and a reference peptide or polynucleotide. In a non-limiting example, at least 90% identity refers to a percentage of identity from 90% to 100% relative to the reference peptide or polynucleotide. A level of identity of 90% or higher indicates that, for illustrative purposes, assuming a test peptide or polynucleotide of 100 amino acids or nucleotides in length is compared to a reference peptide or polynucleotide, no more than 10% (i.e., 10 out of 100) of the amino acids or nucleotides in the test peptide or polynucleotide differ from the reference peptide. Similar comparisons can be made between the test polynucleotide and the reference polynucleotide. Such differences can be represented as point mutations randomly distributed across the entire length of the amino acid sequence, or they can cluster at one or more sites of different lengths up to a maximum permissible difference, such as 10 / 100 amino acid differences (approximately 90% identity). Differences can also be attributed to the deletion or truncation of amino acid residues. Differences are defined as nucleic acid or amino acid substitutions, insertions, or deletions. Depending on the length of the comparison sequences, results can be independent of the procedure and the set of empty parameters at homology or identity levels above approximately 85-90%; such high levels of identity can be readily assessed, typically without software dependence.

[0299] As used herein, pharmaceutically effective agents include any therapeutic or bioactive agents, including but not limited to, anesthetics, vasoconstrictors, dispersants, and conventional therapeutic agents, including small molecule drugs and therapeutic proteins.

[0300] As used in this article, therapeutic effect refers to the effect produced by the treated subject, which alters, usually improves or alleviates the symptoms of a disease or condition or cures the disease or condition.

[0301] As used herein, a therapeutically effective amount or therapeutically effective dose refers to an amount of agent, compound, material, or composition containing a compound that, when administered to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, stop, or partially stop the symptoms of a disease or condition.

[0302] 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 whom the drug, compound, material, or composition containing a compound has been administered.

[0303] As used herein, a preventive effective amount or preventive dose refers to the amount of a drug, compound, material, or composition containing a compound that, when administered to a subject, will have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom, or reducing the incidence of viral infection. A complete preventive effect does not necessarily occur with the administration of a single dose and may occur only after a series of doses. Therefore, a preventive effective amount can be administered in one or more administrations.

[0304] As used herein, improvement of symptoms of a particular disease or condition by treatment (such as by application of a pharmaceutical composition or other therapeutic agent) means any reduction of symptoms attributable to or related to the application of the composition or therapeutic agent, whether permanent or temporary, lasting or transient.

[0305] As used herein, an anticancer agent is any agent that is destructive or toxic to malignant cells and tissues. For example, anticancer agents include agents that kill cancer cells or otherwise inhibit or damage the growth of tumors or cancer cells. An exemplary anticancer agent is a chemotherapeutic agent.

[0306] As used in this article, therapeutic activity refers to the in vivo activity of a therapeutic peptide. Generally, therapeutic activity is activity associated with the treatment of a disease or condition.

[0307] As used in this article, the term "subject" refers to an animal, including mammals such as humans.

[0308] As used in this article, "patient" refers to a human subject.

[0309] As used herein, "animal" includes any animal, 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, cattle, deer, and sheep; and pigs and other animals. "Non-human animals" excludes humans as the intended animal.

[0310] As used herein, a composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0311] As used herein, a combination refers to any association between or among two or more items. A combination can be two or more individual items, such as two compositions or two collections, mixtures thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are typically functionally related or interconnected.

[0312] As used herein, combination therapy refers to the administration of two or more different therapeutic agents. The different therapeutic agents may be provided and administered individually, sequentially, intermittently, or in a single combination.

[0313] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof, for purposes including but not limited to activation, administration, diagnosis and evaluation of biological activity or properties.

[0314] As used herein, unit dosage form refers to a physically discrete unit that is suitable for human and animal subjects and is individually packaged as is known in the art.

[0315] As used in this article, a single-dose formulation refers to a formulation intended for direct administration.

[0316] As used herein, a multi-dose formulation is a formulation containing multiple doses of therapeutic agent and capable of being directly applied to provide several single doses of therapeutic agent. Dosage can be administered over minutes, hours, weeks, days, or months. Multi-dose formulations allow for dose adjustment, dose pooling, and / or dose splitting. Because multi-dose formulations are used over time, they typically contain one or more preservatives to prevent microbial growth.

[0317] As used herein, an article of manufacture is a product manufactured and sold. As used throughout this application, the term is intended to cover any composition provided herein contained in packaged articles of manufacture.

[0318] As used herein, fluid refers to any composition that is flowable. Therefore, fluids include compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0319] As used herein, isolated or purified polypeptides or proteins (e.g., isolated antibodies or their antigen-binding fragments) or their biologically active portions (e.g., isolated antigen-binding fragments) are substantially free of cellular material or other contaminating proteins from the cells or tissues from which the protein originates, or are substantially free of chemical precursors or other chemicals when chemically synthesized. If a formulation appears to be free of readily detectable impurities 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), it can be determined that the formulation is substantially free of, or sufficiently pure, impurities such that further purification will not detectably alter the physical and chemical properties of the substance, such as enzymatic and biological activities. Methods for purifying compounds to produce substantially chemically pure compounds are known to those skilled in the art. However, substantially chemically pure compounds can be mixtures of stereoisomers. In such cases, further purification may increase the specific activity of the compound.

[0320] As used herein, cell extracts or lysates refer to preparations or fractions made from lysed or destroyed cells.

[0321] As used in this article, a control is a sample that is substantially the same as the test sample, except that it has not been treated with the test parameters, or, if it is a plasma sample, it can be derived from healthy volunteers unaffected by the disease of interest. A control can also be an internal control.

[0322] As used herein, dephosphorylated psilocybin is the active form of psilocybin. Dephosphorylated psilocybin is produced by the oxidation of psilocybin in the liver. For the purposes of this article, in the context of MEV-mediated delivery, psilocybin and dephosphorylated psilocybin should have the same meaning: reference to “psilocybin” should mean “dephosphorylated psilocybin”, and vice versa.

[0323] As used in this article, the anterior fontanelle is a unit of measurement for the distance between a location in the brain and the junction between the coronal and sagittal sutures of the skull.

[0324] As used in this article, the tropism of MEVs refers to the accumulation of MEVs in cells, tissues, and / or organs upon administration.

[0325] As used in this article, the natural tendency of MEVS provided in this article refers to the following: not modifying the MEV to provide a specific tendency or targeting property.

[0326] As used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, references to polypeptides containing immunoglobulin domains include polypeptides having one or more immunoglobulin domains.

[0327] As used herein, the term “or” is used to mean “and / or” unless explicitly indicated to refer only to alternatives or that the alternatives are mutually exclusive.

[0328] As used herein, ranges and quantities can be expressed as approximately a specific value or range. “Approximately” also includes the exact quantity. Therefore, approximately 5 amino acids means approximately 5 amino acids as well as 5 amino acids.

[0329] As used herein, “optional” or “optionally” means that the event or situation subsequently described occurs or does not occur, and that the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur. For example, an “optionally variant” section means that the section is either a variant or a non-variant.

[0330] As used herein, unless otherwise stated, the abbreviations for any protecting groups, amino acids and other compounds are consistent with their common usage, recognized abbreviations or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. (1972) 1109): 1726-1732).

[0331] For the sake of clarity rather than limitation, the detailed description is divided into the following subsections.

[0332] B. Microalgae and Overview

[0333] Algae are complex, multi-sourced collections of primarily photosynthetic organisms. These organisms include both microscopic and macroscopic forms. Macroalgae (seaweed) are large, multicellular algae visible to the naked eye. Microalgae are microscopic, single-celled organisms, including prokaryotes (such as cyanobacteria) and eukaryotes, such as green algae.

[0334] Compared to photosynthetic organisms, microalgae exhibit higher growth rates and can be cultured in uncultivable land and in bioreactors. Many species of microalgae can grow year-round in industrial-scale photobioreactors under controlled culture conditions (Adamo et al. (2021) Journal of Extracellular Vesicle 10:e12081). Algae are generally classified into 11 major phyla: Cyanophyta, Chlorophyta, Rhodophyta, Glaucophyta, Euglenophyta, Chlorachniophyta, Charophyta, Cryptophyta, Haptophyta, Heterokontophyta, and Dinophyta (Barkia et al. (2019) Mar. Drugs 17(5):304). Different pigments exist within each algal group. In addition to phycobiliproteins (proteins that capture light energy), phycocyanin, allophycocyanin, and phycoerythrin, cyanobacteria (or cyanobacteria) also contain chlorophyll-a, -d, and -f. Glaucophanes contain chlorophyll-a and harvest light through phycobiliproteins. Chlorophyll includes chlorophyll-a and -b, as well as carotenoids, including β-carotene and various xanthophylls (e.g., astaxanthin, canthaxanthin, lutein, and zeaxanthin). The main pigments of Rhodophyta (red algae) are phycoerythrin and phycocyanin, which can mask chlorophyll-a; red algae also produce a broad spectrum of carotenoids and xanthophyll light-harvesting pigments (Barkia et al. (2019) Mar. Drugs 17(5):304).

[0335] This article describes extracellular vesicles produced by microalgae, particularly single-celled green algae such as species of the genus *Chlorella*, for delivering exogenously loaded cargo to animals and plants. Species of *Chlorella* include, for example, *Chlorella ellipsoidea*, *Chlorella pyrenoidosa*, *Chlorella sorokinina*, *Chlorella vulgaris*, *Chlorella variantbilis*, *Parachlorella kessleri*, *Parachlorella beijerinckii*, and *Parachlorella hussii*.

[0336] Algae are single-celled eukaryotic organisms, typically haploid, but can have a diploid stage in their life cycle. Algae can be cultured in bioreactors from which extracellular vesicles can be isolated. The resulting extracellular vesicles can be loaded with cargo, typically heterologous bioactive molecules, using methods such as electroporation, to produce compositions containing extracellular vesicles for application to animals and plants. The compositions can be formulated for any desired route of administration, including topical, systemic, parenteral, and oral. These routes include oral, intravenous, subcutaneous, inhalation, mucosal, rectal, vaginal, and other suitable routes. Cargo includes biomolecules such as DNA, RNA, proteins, protein complexes, protein-nucleic acid complexes, plasmids, and also includes small molecules, such as small molecule drugs. Extracellular vesicles can be formulated as liquids, powders, including lyophilized powders, tablets, capsules, emulsions, granules, sprays, gels, ointments, creams, and other formulations. They can be used for therapeutic, diagnostic, therapeutic diagnostic, cosmetic, and other purposes. Extracellular vesicles can be used to treat diseases and conditions, including cancer, inflammatory diseases and conditions where the immune system plays a role in the cause or symptoms, neurological disorders, and pathogen infections, including viruses, bacteria, and other pathogens. They can be used to treat dermatological diseases and conditions, lung diseases and conditions, and gastric diseases and conditions. Extracellular vesicles can be targeted to specific organs or tissues or can be applied topically.

[0337] Like extracellular vesicles (EVs) from other sources, such as mammalian EVs, microalgal extracellular vesicles (MEVs) have evolved to efficiently transfer genetic material and other types of molecules from cell to cell. They coordinate intercellular and cross-boundary communication by exchanging bioactive molecules. MEVs are natural nanoparticles. They are cell-derived, therefore, without synthetic cargo and genetic modifications, and without synthetic components; they are safe, for example, without the risk of endogenous viruses that are potentially dangerous to humans.

[0338] The MEVs provided in this article include MEVs of all Chlorella families, particularly *Chlorella vulgaris*, a freshwater microalga; *Chlorella pseudo-Chlorella* is also included within the Chlorellaceae family. Other members from the Chlorellaceae family, such as *Parachlorella pseudo-Chlorella*, include MEVs of *Parachlorella kessleri*, *Parachlorella beijerinckii*, and *Parachlorella hussii*.

[0339] Chlorella is a single-celled haploid algae that is a natural and efficient producer of extracellular vesicles. Common chlorella has been consumed worldwide as a food supplement for decades; it is non-toxic and non-immunogenic, and can be cultured on a large scale at low cost. The MEVs presented in this article can be used directly to protect, deliver, and transmit a broad spectrum of innovative therapeutic molecules to target cells associated with specific diseases.

[0340] As shown and described herein, MEVs possess numerous advantageous characteristics, including, for example, biodistribution patterns via administration routes, low toxicity, and favorable pharmacokinetic properties in vivo. They can be administered via a variety of routes, including oral, respiratory, intranasal, intravenous, and other routes. Depending on the route of administration, they reach specific organs such as the intestine, GALT, spleen, lung, liver, and brain. Based on the data presented herein and compared with data from other EVs and drug delivery systems, MEVs can exhibit longer clearance rates, resulting in longer durations of activity in target organs, tissues, and cells compared to other delivery systems, including mammalian EVs.

[0341] As demonstrated in this paper, MEVs overcome natural bodily barriers that have not yet been achieved with liquid nanoparticles and mammal-derived EVs (e.g., oral delivery, or delivery to specific lymphatic tissues, or nasal-to-brain delivery).

[0342] The MEVs described in this article address unmet needs. These include the ability to specifically deliver and reliably deliver therapeutic molecules to the treatment site while avoiding premature degradation or inactivation of the therapeutic agent by the immune system or enzymes; and for treating pre-existing diseases where therapeutic agents cannot be delivered correctly.

[0343] As illustrated in this article, purified or partially purified MEVs can be loaded using physical methods (exogenous loading; external loading). External loading is scalable and industrializable. MEVs can be externally loaded with a wide variety of molecules of different sizes, hydrophobicities, and properties, such as siRNA, mRNA, peptides, proteins, plasmids, oligonucleotides, and small molecules. The biological activity of the externally loaded cargo is preserved, while it is protected from degradation by enzymes and other agents present in the body. MEVs can deliver their cargo to recipient cells from a wide range of sources, such as microalgae, bacteria, higher plants, mammals, and humans. MEVs can also deliver cargo to appropriate cellular compartments, ensuring proper expression and biological activity of the cargo molecules, including those with complex biological pathways, such as siRNA, mRNA, receptor-binding peptides, etc.

[0344] C. Extracellular vesicles

[0345] Extracellular vesicles (EVs) are biomolecular structures released from plant and animal cells that play a role in cell-to-cell communication. Structurally, EVs are negatively charged lipid bilayer vesicles with a density of 1.13 to 1.19 g / mL. EVs are capable of crossing barriers such as the plasma (or cytoplasmic) membrane and the blood / brain barrier, and are able to transfer their functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor cells to recipient cells (Kuruvinashetti et al. (2020) 20). th International Conference on Nanotechnology 354-357). EVs are also naturally stable and immune-inert in various biofluids and can exhibit organ-specific targeting capabilities (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0346] EVs contain endogenous lipids, nucleic acids, and proteins. Although results vary due to variations in isolation techniques and methods of data analysis, EVs typically contain proteins associated with the plasma membrane, cytosol, and lipid metabolism (see, for example, Doyle and Wang (2019) Cells 8(7):727). Proteins involved in EV biogenesis (e.g., components of ESCRT), EV formation and release (e.g., RAB27A, RAB11B, and ARF6), signal transduction and antigen presentation, and tetraspan membrane proteins typically 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). th (International Conference on Nanotechnology 354-357). Although a few studies have identified genomic and mitochondrial DNA in EVs, EVs are primarily rich in endogenous small RNAs. Studies have identified mRNA, miRNA, rRNA, long and short noncoding RNAs, tRNA fragments, piwi-interacting RNAs, dome RNAs, and Y RNAs in EVs. Most RNAs naturally present in EVs are about 200 nucleotides long (with small segments up to 4 kb), and are therefore fragmented, although circular RNAs have also been shown to be enriched and stable in EVs. RNAs in EVs are protected from RNase digestion by RNases in the extracellular environment by a lipid bilayer (Abels and Breakefield (2016) Mol. Neurobiol. 36(3):301-312). The Exocarta, Vesiclepedia, and EVpedia databases are publicly available and provide data on the protein, nucleic acid, and lipid content of EVs (typically from mammalian sources, such as human EVs), as well as the isolation and purification procedures used in EV studies (Abels and Breakefield (2016) Mol. Neurobiol. 36(3): 301-312).

[0347] Extracellular vesicles are used by cells to mediate several physiological processes or influence various pathological conditions associated with the activation of immune responses or the spread of diseases or infections, and also constitute interspecies communication and are present in all living organisms. Sources of extracellular vesicles include mammalian cells, bacteria, milk, and plants (Adamo et al. (2021) J. Extracell. Vesicles 10:e12081). Although plants and algae possess a cell wall outside their plasma membrane (which may be a physical barrier to EV release), plants and algae release EVs (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0348] 1. Types of extracellular vesicles (EVs)

[0349] a.Exosomes

[0350] There are three main subtypes of EVs; they are classified based on their biogenesis, release pattern, size, abundance, and function: microvesicles (MVs), exosomes, and apoptotic bodies (Doyle and Wang (2019) Cells 8(7):727). Exosomes, or intraluminal vesicles (ILVs), are typically 30–150 nm in diameter and are released via multivesicles (MVBs) in the endosome pathway. In the endosome pathway, early endosomes are formed by inward budding of the plasma membrane and can transform into late endosomes, which accumulate ILVs through inward budding of the endosome membrane. Late endosomes containing numerous small vesicles are called MVBs. MVBs either fuse with lysosomes and are degraded, or fuse with the plasma membrane, releasing ILVs as exosomes into the extracellular space. The endosomal sorting complex (ESCRT) required for transport pathways regulates MVB transport and exosome formation and has been reported as a major driver of exosome biogenesis, although other mechanisms of exosome biogenesis exist, including those mediated by sphingolipid ceramides, which can promote invagination or proteins in the four-transmembrane protein family. ESCRT accessory proteins Alix, TSG101, HSC70, and HSP90β are often referred to as exosome marker proteins (Doyle and Wang (2019) Cells 8(7):727).

[0351] Exosomes are released into the extracellular space via the fusion of the MVB-restricted membrane with the plasma membrane. Many proteins are involved in the release of exosomes, including Rab GTPase, diacylglycerol kinase α, and SNARE protein (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312).

[0352] Exosomes are already candidates for drug delivery systems: they have long circulating half-lives; exosomes are tolerated by humans and can penetrate cell membranes and target specific cell types; and they can be loaded with genetic material, proteins or small molecules (Doyle and Wang (2019) Cells 8(7):727).

[0353] b. Microvesicles

[0354] Microvesicles (MVs or exosomes) form by budding or contraction of the cell's plasma membrane and have a diameter 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 mechanisms such as SNARE and binding factors. The physiological state and microenvironment of the donor cell influence the number of MVs produced, and the physiological state and microenvironment of the recipient cell influence the number of MVs consumed. MVs also possess a number of marker proteins, including cytoplasmic and plasma membrane-associated proteins, as well as cytoskeletal proteins, heat shock proteins, integrins, and proteins containing post-translational modifications, although no specific markers are known to distinguish MVs from exosomes. Like exosomes, MVs can load 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).

[0355] c. Apoptotic bodies

[0356] Apoptotic bodies are released into the extracellular space through stained cells and have a diameter of 50 nm to 5000 nm. Apoptotic bodies are formed when the cell membrane separates from the cytoskeleton due to the increased hydrostatic pressure after cell contraction. Unlike exosomes and MVs, apoptotic bodies contain intact organelles, chromatin, and a small amount of glycosylated proteins (Doyle and Wang (2019) Cells 8(7):727).

[0357] 2. EV intake

[0358] Cells internalize EVs through fusion with the plasma membrane, or more commonly through endocytosis (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312). Uptake via endocytosis can occur through several types of endocytosis processes, and different processes have been described in different cell types: clathrin-dependent endocytosis and phagocytosis have been described in neurons; macropinocytosis in microglia; phagocytosis and receptor-mediated endocytosis in dendritic cells; caveolin-mediated endocytosis in epithelial cells; and cholesterol and lipid raft-dependent endocytosis in tumor cells. Blocking heparan sulfate proteoglycans (HSPGs) on the plasma membrane with heparin reduced EV uptake in cell cultures, and blocking the B-1 scavenger receptor (SR-B1) with synthetic nanoparticle mimics of HDL also reduced EV uptake in cell cultures, suggesting the roles of HSPGs and SR-B1 in EV uptake (Abels and Breakefield (2016) Cell Mol. Neurobiol. 36(3):301-312). EV fusion with the plasma membrane is also an uptake method and requires low pH conditions; treatment of EVs with a combination of pH-sensitive fusion peptides and cationic lipids resulted in increased cellular uptake of exosomes and increased cytoplasmic release of cargo from exosomes (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), allowing EVs for delivering therapeutic payloads to tumor cells to enter cells via plasma membrane fusion.

[0359] Like cells, EVs have extracellular receptors and ligands on the outside and cytoplasmic proteins and nucleic acids on the inside, thus communicating with cells in different ways. EVs bind to the cell surface, undergo endocytosis, and / or fuse with the plasma membrane, releasing their cargo into the extracellular space. If they enter via endocytosis, the EV cargo must escape degradation pathways; late endosomes can fuse with lysosomes or the plasma membrane, so the cargo must leave before being degraded in lysosomes or re-released via fusion of MVBs 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).

[0360] 3. General methods for separating EVs

[0361] a. Ultracentrifugation

[0362] Ultracentrifugation is used to separate exosomes; alternative methods have also been developed. Separation methods can result in complex mixtures of EVs with other components of the extracellular space due to the complex nature of the biofluid from which exosomes are separated, the overlap of physicochemical and biochemical properties between exosomes and other types of EVs, and the heterogeneity among exosomes. Differential ultracentrifugation relies on the initial sedimentation of larger, denser particles from the extracellular matrix, leading to the enrichment of exosomes, but not complete separation of exosomes from other components of the extracellular space. Density gradient centrifugation is another ultracentrifugation method and is based on separation by size and density in the presence of a density gradient (usually made of sucrose or iodixanol) in the centrifuge tube. Density gradient centrifugation effectively separates EVs from protein aggregates and non-membrane particles, but has low exosome recovery rates, although purity can be improved by coupling differential ultracentrifugation with a different type of density gradient centrifugation (e.g., rate zoning or isodense centrifugation) (Doyle and Wang (2019) Cells 8(7):727).

[0363] b. Size-based technology

[0364] Numerous size-based techniques have been used for the separation of exosomes (Doyle and Wang (2019) Cells 8(7):727). Ultrafiltration separates particles based on size and the membrane's molecular weight cutoff, thereby retaining particles larger than the membrane's molecular weight cutoff and allowing particles smaller than the membrane's molecular weight cutoff to pass into the filtrate; however, low separation efficiency may occur if the filter becomes clogged and vesicles are trapped. ExoMir TM The kit (Bioo Scientific; Austin, TX) is a commercially available kit in which two membranes (200 nm and 20 nm) are placed in a syringe and the sample (usually pretreated with centrifugation and proteinase K) is passed through the syringe; larger vesicles are retained above the first 200 nm filter, the smallest vesicles pass through the syringe and are discarded, and vesicles between 20 and 200 nm are retained between the two filters in the syringe. Sequential filtration also relies on a series of filtration steps to separate exosomes (Doyle and Wang (2019) Cells 8(7):727).

[0365] Size exclusion chromatography (SEC) of exosomes is typically used in parallel with ultracentrifugation (in which the exosome precipitate obtained from ultracentrifugation is resuspended and further purified using SEC), similar to the method used for protein separation with SEC. In SEC, the column is packed with a porous stationary phase in which small particles can permeate and thus elute after larger particles. Typically, the SEC method requires several hours of run time; however, the qEV exosome isolation kit (iZON Science, New Zealand) allows for rapid and accurate exosome separation via SEC in as little as 15 minutes (Doyle and Wang (2019) Cells 8(7):727).

[0366] In flow field-flow separation (FFFF), in addition to the flow perpendicular to the parabolic flow (lateral flow), the sample injected into the chamber is also subjected to parabolic flow as it is pushed downwards into the chamber to separate particles from the sample. Larger particles are more affected by the lateral flow and are pushed toward the chamber wall, which has a slower parabolic flow, while smaller particles are retained in the center. In FFFF, smaller particles are eluted earlier and larger particles are eluted later (Doyle and Wang (2019) Cells8(7):727).

[0367] In hydrostatic filtration dialysis (HFD), hydrostatic pressure forces the sample through a dialysis tube with a membrane that has a molecular weight cutoff of 1000 kDa. As a result, small solutes can pass through the tube, but larger particles (including exosomes and EVs) are retained in the tube and can then be further separated using, for example, ultracentrifugation (Doyle and Wang (2019) Cells 8(7):727).

[0368] c. Immunoaffinity capture-based technologies

[0369] Immunoaffinity-based capture techniques can isolate exosomes based on the expression of antigens on the exosome surface and allow for the isolation of exosomes derived from specific sources. In these methods, antibodies specific to the target antigen can be attached to plates (e.g., in enzyme-linked immunosorbent assays, ELISA), 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 the protein / antigen used for exosome isolation to be expressed on the exosome surface, and its specificity is limited by the specificity of the antibody used, generally resulting in low yields but high purity of isolated exosomes. These methods can also be used to isolate exosomes within mixed EV populations. Immunoaffinity-based capture techniques are typically used after ultracentrifugation or ultrafiltration (Doyle and Wang (2019) Cells 8(7):727).

[0370] d. Exosome precipitation

[0371] Methods for precipitating exosomes include precipitation with polyethylene glycol (PEG) and lectins. In PEG precipitation, the PEG polymer binds water molecules, causing other particles, including exosomes, to precipitate from the solution. PEG precipitation is rapid and not limited by the initial volume of solution, but it lacks selectivity because other EVs, extracellular proteins, and protein aggregates precipitate along with the EVs. Sample pretreatment using filtration and / or ultracentrifugation can improve exosome yield. Commercially available kits for separating exosomes using precipitation include, for example, (System Biosciences, Palo Alto, CA) and Invitrogen TM Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA). Alternatively, lectin precipitation can usually be used after ultracentrifugation, whereby the lectin binds to carbohydrates on the surface of the exosome, altering their solubility and causing them to precipitate from the solution (Doyle and Wang (2019) Cells 8(7):727).

[0372] e. Microfluidic-based separation technology

[0373] Microfluidic-based techniques separate exosomes based on both their physical and biochemical properties, offering rapid, efficient separation with small initial volumes. In acoustic nanofilters, a matrix containing EVs and other cellular components is injected into a chamber and exposed to ultrasound. Depending on particle size and density, the response of particles to the radiative forces exerted by the waves varies; larger particles experience stronger forces and migrate towards pressure nodes more quickly. Immunoassay-based microfluidic separation techniques are similar to ELISA, although unlike ELISA, they do not require prior ultrafiltration or ultracentrifugation of exosomes (Doyle and Wang (2019) Cells 8(7):727). ExoChip (Kanwar et al. (2014) Lab Chip. 1411): 1891-1900) and ExoSearch Chip (Zhao et al. (2016) Lab Chip. 1653): 489-496) have been developed to separate exosomes using microfluidic techniques.

[0374] 4. Microalgae and microalgae-derived extracellular vesicles (MEVs)

[0375] The taxonomy and classification of microalgae can vary. According to some schemes, there are seven (7) divisions of microalgae: Euglenophyta (golden algae), Chrysophyta (golden brown algae and diatoms), Dinophyta (fire algae), Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae), and Xanthophyta (yellow-green algae). This paper is of interest to photosynthetic microalgae, such as species of the genera *Chlorella* and *Chlamydomonas*. The methods and uses described herein utilize MEVs commonly derived from green algae. Examples of such algae are the genera *Chlamydomonas* and *Chlorella*, which belong to the classes Chlorophyta and Coccococci, respectively.

[0376] Microalgae are a biological resource for producing EVs for nanomedicine and other fields. The mechanisms by which microalgae secrete EVs are known and involve primary and motile cilia / flagellates (Picciotto et al. (2021) Biomate. Sci.).

[0377] doi:10.1039 / d0bm01696a). Chlamydomonas flagella lack MVBs; therefore, ciliated EVs detached from Chlamydomonas are classified as exosomes. Studies have shown that exosomes detach from the flagella and ciliary tips of the green plant *Chlamydomonas reinhardtii*. EVs along the ciliary length have also been observed in the *Chlamydomonas* genus. Membrane budding and ciliated EV formation are mediated by components of the transport-dependent endosome sorting complex (ESCRT), which are present in the isolated ciliary transition zone, ciliary membrane, and ciliated EVs in *Chlamydomonas*, and can act as sensors of membrane curvature. Ciliated EV formation may also occur when ciliary membrane transport is disrupted or during ciliary reabsorption (Wang and Barr (2018) Essays Biochem. 62(2):205-213). Ciliated exosomes from *Chlamydomonas* contain lysins that digest the parent cell wall and are required for the release of daughter cells. The flagellated Ift88-nucleate mutant could not be released from the parent cell, and the addition of ciliated extranuclear bodies from wild-type cells rescued the phenotype, suggesting the role of flagella and intraflagellate transport (IFT) mechanisms in EV production (Wang and Barr (2016) Cell Mol. Neurobiol. 36(3):449-457).

[0378] EVs have been extracted from algal cells using ultracentrifugation (Kuruvinashetti et al. (2020) 20). thInternational Conference on Nanotechnology 354-357). According to this method, algal cells were cultured; the cultured algal cells were collected and centrifuged; the supernatant was collected (and further centrifuged); a sucrose solution was added to the supernatant; and the algal supernatant with the sucrose solution was ultracentrifuged; due to the sucrose solution, high-density EVs settled at the bottom of the ultracentrifuge tube and could be collected using a pipette. The size and concentration of the extracted algal EVs could be characterized using nanoparticle tracking analysis (NTA). Studies using this method isolated green algal EVs with sizes ranging from 25 to 200 nm and concentrations of 0.89E8 to 0.94E8 particles / mL (Kuruvinashetti et al. (2020) 20). th International Conference on Nanotechnology354-357).

[0379] Ultracentrifugation protocols have also been used to isolate extracellular vesicles (EVs) from marine microalgae grown under various conditions; NTA revealed that the nanoparticles were distributed between 100 and 200 nm in size, and Western blot analysis of proteins confirmed the presence of EV markers (VES4US, extracellular vesicles of a natural origin from specially formulated nanomaterials, 2020). Subsequent studies have identified small extracellular EVs (sEVs) isolated from the marine photosynthetic microalga *Tetraselmis chuii*, termed nanoalgosomes. The generation of nanoalgosomes is an evolutionarily conserved trait within microalgal strains, as similar results were obtained using sEVs isolated from batch cultures of two other microalgal species: *Dunaliella tertiolecta* and *Amphidinium sp.*. Nanoalgal-derived exovesicles were isolated using differential ultracentrifugation (dUC), tangential flow filtration (TFF), and gradient ultracentrifugation, which were then used to further purify and enrich samples of small EVs via TFF or dUC. The isolated nanoalgal-derived exovesicles exhibited characteristics of EVs from other sources. EV yields from dUC and TFF (measured by sEV protein content and sEV number) were consistent with reported isolated EV numbers, approximately 10. 9EV particles / μg EV protein. Biophysical analysis of particle size using multi-angle dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), fluorescence nanoparticle tracking analysis (F-NTA), and fluorescence correlation spectroscopy (FCS) yielded a consistent size distribution, with the most frequently occurring size from DLS (DLS mode) being approximately 70 nm. The density of nanoalgal exovesicles was slightly lower at 1.13 g / mol compared to exosomes derived from mammalian cells, which have a density of 1.15–1.19 g / mol. Electron microscopy revealed that the nanoalgal exovesicles were spherical, non-uniform in size and shape, and possessed a lipid bilayer structure. Compared to microvesicles (or large EVs, lEVs) and lysates, sEVs were enriched in three of the four target protein biomarkers (Alix, enolase, HSP70, and β-actin). DLS measurements showed that the nanoalgal exovesicles were resistant to pH changes and stable in human plasma. No cytotoxic or genotoxic effects were observed in the tumorigenic MDA-MB-231 breast cancer cell line, the non-tumorigenic 1-7HB2 cell line, and the human hepatocellular carcinoma Hep G2 cell line after treatment with nano-algal-derived exovesicles. Furthermore, the nano-algal-derived exovesicles were absorbed by the MDA-MB-231 and 1-7HB2 cell lines (Adamo et al. (2021) J. Extracell. Vesicles 10:e12081).

[0380] EVs have been derived from at least eighteen microalgal strains from major microalgal lineages (Sp. *Ankistrodesmus*, Sp. *Brachiomonas*, Sp. *Chlamydomonas reinhardtii*, Sp. *Dunaliella tertiolecta*, Sp. *Tetraselmis chuii*, Sp. *Chloromonas*, Sp. *Rhodella violacea*, Sp. *Kirchneriella*, Sp. *Pediastrum*, Sp. *Nannochloropsis*, Sp. *Cyanophora paradoxa*, Sp. *Cryptomonas pyrenoidifera*, Sp. *Phaeodactylum tricornutum*, Sp. *Phaeothamnions*, Sp. *Diacronema*, Sp. *Isochrysis galbana*, and Sp. *Stauroneis*). The strains studied were isolated from and investigated in the genera *Amphidinium* and *Amphidinium* sp. . The strains studied included those with a variety of characteristics, such as saltwater and freshwater inhabitants, small and large cells, colonies and single cells, and species with sequenced genomes.

[0381] MEVs were isolated using differential ultracentrifugation and characterized following the International Society for Extracellular Vesicles (ISEV) guidelines. All tested strains showed the presence of MEVs in the culture medium. EV-producing microalgae strains were established based on EV protein content, expression of EV protein markers (e.g., Alix, Hsp70, enolase, and β-actin), total scattering signal (measured by dynamic light scattering, DLS) or total particle number (measured by NTA), and mean and size range of sEVs. These EV-producing strains included *Cyclocarya scabra*, *Tetracarya zhui*, *Amphidinium* sp., *Rhodella violacea*, *D. tegmentosum*, *D. triquetrum*, *Phaeothamnion* sp. (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a). Data from *Cyanobacterium moniliforme* showed approximately 2 × 10⁻⁶ mmol / L of microalgae conditioned medium. 9The sEV particles exhibited strong positive signals for EV markers and a size distribution pattern of 130±5 nm, consistent with data from plant-derived vesicles. Cytotoxicity and genotoxicity studies showed that sEVs isolated from *Glaucophyte* (a freshwater cyanobacterium) did not exhibit toxicity over time or at different concentrations to the tumorigenic MDA-MB-231 breast cancer or C2C12 myoblast cell lines. Furthermore, MDA-MB-231 cells treated with sEVs did not show any morphological or nuclear changes associated with apoptotic events (Picciotto et al. (2021) Biomater. Sci. doi:10.1039 / d0bm01696a).

[0382] EVs have also been isolated from *Syntrophus cytozoa* PCC6803 (cyanobacteria), *Chlamydomonas reinhardtii* (green microalgae), *Euglena gracilis* (euglena), and *Haematococcus pluvialis* (green plants) in the work of Zhao et al. Zhao et al. also performed RNA and proteomic analyses on EVs isolated from *Chlamydomonas reinhardtii* at different stages of cell growth and under different types of abiotic stress (Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / v1). EVs were separated using differential ultracentrifugation and filtration, and the resuspension showed a membrane structure with small particle clusters of 110–120 nm in diameter, consistent with reported exosome diameters and small MVs, although diameters varied among microalgal species. Specifically, the diameters of *Chlamydomonas reinhardtii* EVs ranged from 37–710 nm, with an average particle size of 120.1 nm. EVs from the genus *Synostemma* ranged in diameter 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 take up EVs, as evidenced by the presence of EVs labeled with fluorescent lipophilic dyes within microalgal cells. Therefore, microalgal EVs can be absorbed by recipient cells. Non-coding RNAs were detected in microalgal EVs at different growth stages and under various treatments (biological stress, nitrogen consumption, and nitrogen recovery), and proteomic analysis identified numerous flagella-associated membrane proteins in microalgal EVs (Zhao et al. (2020) doi:10.21203 / rs.3.rs-38027 / v1).

[0383] These studies demonstrate that microalgae produce EVs that can be isolated using conventional or standard methods; microalgae-derived EVs are similar in size and concentration and exhibit similar biomarkers compared to EVs isolated from other species; EVs isolated from microalgae do not show cytotoxic or genotoxic effects in vitro; and microalgae-derived EVs can be absorbed by cells.

[0384] It has been shown that mammalian EVs can deliver cargo to target cells and therefore have therapeutic uses for delivering a variety of cargoes to treat a variety of diseases or conditions; this has not typically been shown for MEVs. However, mammalian EVs, except for bovine milk EVs, cannot be administered orally because they cannot survive the harsh conditions of the stomach. Small molecules, such as hydrophobic and hydrophilic drugs, can be injected into exosomes, or large proteins and nucleic acids can be embedded within them. Nucleic acids may include those encoding target genes. Specific targeting ligands, imaging probes, and covalent bonds can be attached to the exosome surface and tracked using NTA, fluorescence, or bioluminescence.

[0385] In addition to mentioning in publications that microalgae EVs may be used to deliver targeted drugs to target cells, tissues, or organs (Kuruvinashetti et al. (2020) 20) th Aside from the International Conference on Nanotechnology (354-357), there is no publicly available evidence or technical description of using MEVs for delivery to treat mammalian diseases, conditions, or illnesses. No publications or technical specifications describe how to understand the application of EV technology to extracellular vesicles derived from microalgae, nor do they describe whether or how this can be done. Previous studies have not considered Chlorella species, nor have previous studies generally assessed the biodistribution and associated properties of MEVs. Therefore, this paper uses Chlorella as an example, suggesting that oral delivery methods could utilize MEVs from other microalgae.

[0386] However, as described and shown in this paper, microalgal EVs offer numerous advantages over existing drug delivery systems such as exosomes derived from mesenchymal stem cells (MSCs), gold nanoparticles, liposomes, and other plant and animal-derived EVs. MSCs are a common source of exosomes, and MSC-derived exosomes are used for drug delivery, such as anticancer vaccines, due to their enhanced passive targeting (methods for preparing drug carrier systems to maintain circulation in the bloodstream). MSC-derived EVs possess passive targeting capabilities due to their small size, inherent properties, and ability to cross biological barriers. However, MSCs have limited exosome secretion, and scaling up exosome production is challenging due to the need for optimized purification, increased exosome homogeneity, and the development of efficient transfection strategies. Nanoparticles can cause toxicity, and current technologies used to synthesize nanoparticles limit their scale-up capabilities for manufacturing purposes. Nanoparticles and liposome-based drug delivery methods can also lead to teratoma formation (tumors composed of several different tissue types). Liposome-based drug delivery methods have further demonstrated lower efficiency for internalization into specific cells, tissues, or organs compared to exosomes. Plant-derived EVs, such as those from curcumin, ginger, grapefruit, and lemon, have been used for drug delivery, but their extraction processes and therapeutic applications have not yet been optimized. Producing EVs from agricultural products (such as fruits and milk) is economically impractical compared to algal EVs, which can grow anywhere and within days, and require 3–4 months of growth. Algal EVs avoid phagocytosis or degradation by macrophages, have a prolonged circulation time in vivo, and exhibit low immunogenicity. Algal EVs also have a lower risk of teratoma formation. Therefore, algae provide a source from which high-quality, pure, and well-characterized EVs can be obtained (Kuruvinashetti et al. (2020) 20). th (International Conference on Nanotechnology 354-357). Kuruvinashetti et al. did not describe the use of Chlorella species as a source of EVs, nor did they describe the advantages of using them as a source. The prior art does not describe the biodistribution of MEVs themselves, nor does it describe the implications of administering MEVs in conjunction with drugs targeting specific organs, tissues, or systems.

[0387] 5. Green algae - Chlorella species

[0388] Previous research and consideration of EVs have not focused on, nor evaluated, Chlorella species as sources of EVs. Chlorella and the resulting EVs offer advantages for growth, manipulation, and drug application that other species and EVs do not provide. Green algae belong to the Chlorophyta phylum and include different groups of photosynthetic eukaryotes. Green algae include both unicellular and multicellular organisms. The algae originally included in the Chlorella genus are among the most widespread and frequently encountered algae in freshwater. These algae exist in aquatic environments and on land. They are typically small (approximately 2 to 10 μm in diameter), unicellular, spherical, immobile, and contain a single chloroplast, and some have rigid cell walls (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0389] Molecular analysis has classified Chlorella species into two classes of green plants: the class Chlorococci, which includes true Chlorella; and the class Chlorophyta. For the purposes of this document, Chlorella species include any species that can be or be used as a food supplement or that can be consumed by humans or other animals (e.g., livestock). Exemplary species include, but are not limited to, the following: Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokinina, Chlorella vulgaris, and Chlorella variantabilis.

[0390] True Chlorella species are characterized by glucosamine as a major component of their rigid cell walls. Although most Chlorella species are naturally free-living, the class Chlorella includes most known endosymbiotic green algae living in lichens, single-celled eukaryotes, plants, and animals (e.g., mussels and water snakes). For example, Chlorella variant NC64A is a genetically photosynthetic endosymbiotic (or symbiotic photosynthetic) of Paramecium bursaria (a single-celled protozoan), and NC64A is also a host of a family of large double-stranded DNA viruses that occur in freshwater (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0391] a. Lifecycle

[0392] In unicellular organisms such as microalgae, the life cycle is identical to the cell cycle. Chlorella is a haploid organism that reproduces asexually through autospore formation. Rioboo et al. investigated the cell cycle and proliferation of Chlorella vulgaris using flow cytometry analysis of algal cells stained with 5(6)-carboxyfluorescein diacetate N-succinimide ester (CFSE). Their results showed that, as generally described in microalgae, the growth of Chlorella vulgaris mother cells occurs during the light period, while cytoplasmic division and the release of daughter cells occur during the dark period. Chlorella vulgaris also exhibited a distinct light / dark cycle, which was marked by an increase in cell size, cell complexity, and autofluorescence during the photoperiod measured over 96 hours. A single-parameter histogram of CFSE-stained Chlorella vulgaris cells showing only a peak in daughter cells indicated that each mother cell underwent only one division cycle over 96 hours; further evidence showed that cytoplasmic division occurred during the dark period. Thus, the Chlorella vulgaris strains used exhibited three life cycle phases: 1) mother cell growth, 2) cell division, and 3) daughter cell release. Chlorella cells grow during two bright cycles and begin dividing during the subsequent dark cycle; cell division occurs once the mother cell is twice the size of the daughter cells. Furthermore, Chlorella cells exposed to the herbicide terbufos require a longer growth period to reach a sufficiently large cell size for division. This suggests that Chlorella requires a critical threshold size to complete its growth phase and begin division, and that this critical threshold controls the progression of the G1 phase of the Chlorella cell cycle. Finally, this study shows that the intensity of the CFSE-fluorescence peak in the mother cell is four times that of the daughter cell, indicating that each mother cell produces four daughter cells. Therefore, Chlorella cells undergo a first mitosis, followed by cytoplasmic division, and then two more simultaneous mitosis, resulting in the release of four daughter cells (see, for example, Rioboo et al. (2009) doi:10.1016 / j.aquatox.2009.07.009).

[0393] b. Genome analysis of Chlorella species

[0394] Although Chlorella species have been reported to be nonmotile and lack sexual cycles, variant Chlorella

[0395] Genome analysis of NC64A (NC64A) and Chlorella vulgaris 211 / 11P (211 / 11P) revealed the presence of genes involved in 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. Meiosis-specific protein dose inhibitors of MCk1DMC1, homologous pairing proteins HOP1 and HOP2, meiotic recombinant protein MER3, meiotic nucleomitotic protein MND1, and mutS homolog MSH4 are encoded in NC64A; these genes are also present in most other sequenced chlorophyll-bearing algae species. Nineteen homologs of the Chlamydomonas gametolysin protein, which promotes the breakdown of gamete cell walls and allows gamete fusion, were also identified in NC64A. Additionally, an ortholog of the Chlamydomonas GCS1 protein, essential for cell fusion, was found in NC64A (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Besides the gene encoding gametolysin (g3347) and the gene encoding a protein containing a domain with the putative GCS1 / HAP2 function, major genes involved in meiosis were also found in the Chlorella 211 / 11P 40Mb genome (GenBank accession number SIDB00000000) (Cecchin et al. (2019) Plant J.100(6):1289-1305). Therefore, although Chlorella species were observed only in the haploid stage, the presence of meiotic genes suggests that the life cycle of Chlorella may include a diploid stage.

[0396] Similarly, although no flagella were observed in NC64A, orthologs of Chlamydomonas flagellar proteins were identified in the NC64A genome, including orthologs of intraflagellate transport (IFT) proteins IFT52, IFT57 and IFT88, kinin-2 motor protein FLA8, kinin-associated protein KAP, and proteins involved in the dynein arm of the axial filament (Blanc et al. (2010) Plant Cell 22(9):2943-2955).

[0397] Sequencing of three Chlorella sorokiniana strains (strain 1228, UTEX1230 and DOE1412) revealed the presence of sex and flagella-related genes (Hovde et al. (2018) Algal Research 35:449-461). The genomes of several other Chlorella species have been sequenced: Chlorella protothecoides sp. 0710 (Gao et al. (2014) BMC Genomics 15(1):582; GenBank accession number APJO00000000); Chlorella sorokin UTEX1602 (GenBank accession number LHPG00000000) and Chlorella sp. strain SAG241.80 (Micractinium conductrix; GenBank accession number LHPF00000000) (Arriola et al. (2018) Plant J.93(3):566-586); and Chlorella guarnieri UTEX395 (Guarnieri et al. (2018) Front. Bioeng. Biotechnol. 6:37; GenBank login number LDKB00000000), UMT-M1 (Tehe et al. (2019) Data Brief 27:104680; GenBank login number VJNP00000000), UTEX 259 (GenBank login number VATW00000000) and NJ-7 (Wang et al. (2020) Mol. Biol. Evol. 37(3):849-863; GenBank login number VATV00000000).

[0398] c. Commercial and biotechnological uses of Chlorella

[0399] Commercial cultivation of microalgae for food purposes began in the 1960s with the production of common Chlorella in Japan and Taiwan, China. Due to reports of high protein content, nutritional value, and health benefits, dried biomass products from the genera *Arthrospira* and *Chlorella* are included in dietary supplements. For example, Chlorella extract has been shown to lower cholesterol and possess antioxidant, antibacterial, and antitumor activities. High-yield production of Chlorella is routine, and MEVs can be isolated from cell culture media, as detailed herein. For its medicinal use, ingestion of Chlorella is known to be non-toxic and non-immunogenic in humans.

[0400] Chlorella has been used in a variety of biotechnological applications, including biofuels, CO2 chelation, the production of high-value molecules, and the removal of heavy metals from wastewater (Blanc et al. (2010) Plant Cell 22(9):2943-2955). Chlorella species exhibit metabolic flexibility in response to environmental disturbances and are able to utilize nutrients directly from wastewater, such as organic carbon and minerals, for growth. Among microalgae, Chlorella species have higher photosynthetic efficiency than other photosynthetic organisms. Furthermore, common Chlorella can grow under autotrophic, heterotrophic, or fascicic conditions (…). et al. (2016) Plant Physiol. 172(1):589-602).

[0401] Chlorella species can also be genetically modified through Agrobacterium-mediated transformation. A study by Cha et al. developed a method for genetically transforming common Chlorella using Agrobacterium tumefaciens strain LBA4404, and the presence of gene fragments in 30% of the transgenic lines compared to wild-type uninfected Chlorella indicated that T-DNA was integrated into the Chlorella genome (Cha et al. (2012) World J. Microbiol. Biotechnol. 28: 1771-1779).

[0402] d. Chlorella MEV

[0403] As described herein, Chlorella species (e.g., common Chlorella) are advantageous species for producing EVs (referred to herein as MEVs) for delivering biomolecules and small molecules for numerous applications, including therapeutic, diagnostic, and cosmetic uses. This paper is of particular interest to MEVs produced by Chlorella species. Chlorella EVs have not yet been used as a source of MEVs for the exogenous loading of biomolecule products or small molecule drugs or diagnostic agents. Chlorella offers several advantages as a source of EVs for such applications. Chlorella is a haploid organism, meaning that specific and targeted variants can be produced through genetic engineering; it can be readily genetically modified or loaded to produce or contain bioactive molecules and small molecules. Stable cell lines can be produced, including stable producers encoding products. These are defined products, and when exogenously loaded, the resulting compositions contain EVs containing the same cargo.

[0404] Detailed genetic maps are available, and correlations between genotypes and phenotypes can be established. The Chlorella genome has been fully sequenced, so the structure and function of various genes are known. In plant genetics, Chlorella occupies a highly intersecting position between higher plants and microalgae. Therefore, Chlorella shares a large number (and useful) molecular biological and metabolic features with higher plants, but remains a single-celled haploid microalga. An example of molecular biological features shared with eukaryotes is the intracellular machinery involved in the cleatase system for processing exogenous RNA into siRNA. Chlorella is autotrophic; unlike mammalian and other animal cells, it can therefore be cultured and reproduced without the need for animal-derived nutrients or factors.

[0405] Regarding the use of its EVs as a therapeutic agent, Chlorella species are non-toxic. For example, tablets made from common Chlorella biomass (i.e., compressed whole Chlorella cells) have been regularly consumed by the global public as a dietary supplement for many years without restrictions related to toxicity or immunogenicity. Japan is a world leader in the consumption of Chlorella biomass. It is also used in medical treatments, such as in Japan, because it has shown immunomodulatory properties and claimed anticancer activity; in anti-aging applications, such as for cardiovascular disease, hypertension, and cataracts; it reduces the risk of atherosclerosis and stimulates collagen synthesis in the skin.

[0406] Chlorella cells naturally produce extracellular vesicles (EVs) that respond to the “standard specifications” of better-known EVs (e.g., mammalian EVs). Plant-derived EVs possess several characteristics that make them more promising / convenient than synthetic nanoparticles or semi-synthetic EVs for use as drug delivery systems in humans. These include, for example, higher stability, lower toxicity, and lower immunogenicity. As closely related to plants, Chlorella provides a source of EVs with similar characteristics to plant EVs. Furthermore, large-scale production of Chlorella is easier and cheaper than that of higher plants. The glycosylation patterns of membrane proteins in Chlorella are similar to / identical to those present in higher plants.

[0407] Chlorella microcystis MEVs range in size from approximately 50 nm to 200 nm, with an average size of approximately 130 nm. Their morphology resembles that of plant and mammalian exosomes. For application purposes, a more uniform size distribution can be achieved by isolating MEVs by size and selecting those of the size of interest, which can vary depending on the intended use and route of application.

[0408] D. Exogenously loaded microalgal extracellular vesicles (MEVs), cargo, and targets

[0409] Targets and cargoes (see discussion below) include anything known to those skilled in the art. Sections F and G, as well as the examples below, describe the biodistribution of MEVs after administration via various routes, and the meaning, uses, and methods for targeting or treating specific diseases, symptoms, and conditions, as well as for the formulation and administration of MEVs.

[0410] 1. MEV separation

[0411] The methods used for separation are discussed in the above sections and detailed in the embodiments.

[0412] 2. MEV loading and cargo

[0413] MEVs can carry any desired cargo (also known as payloads), including but not limited to nucleic acid molecules, such as 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 combinations thereof. MEVs can deliver therapeutic molecules, can be used as vaccines, and can be used for human and other animal health, agricultural applications, gene therapy applications (including gene delivery, gene modification with gene editing systems, and gene silencing nucleic acids), cosmetic applications, dermatological applications, diagnostic applications, and industrial uses. MEVs can deliver nutrients or regulators of gene pathways to produce beneficial products and can be used to deliver gene editing systems, such as CRISPR / Cas, and to achieve gene editing. MEVs can be used to deliver gene therapy vectors, such as, but not limited to, adeno-associated virus (AAV) vectors, adenovirus vectors, vaccinia virus-derived vectors, and products.

[0414] Treatable diseases and conditions include any diseases known to those skilled in the art, including but not limited to cardiovascular diseases, metabolic diseases, infections (including respiratory infections, bladder infections, and other urinary tract infections), infectious diseases (including viral diseases such as hepatitis and HIV), coronaviruses (including SARS-CoV-2), CNS diseases, eye diseases, and liver diseases. As discussed, the delivered goods 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 antisense oligonucleotides; and small molecule drugs.

[0415] MEVs can carry cargo including reporter genes and proteins as well as other detectable products, such as fluorescent proteins, including but not limited to enhanced green fluorescent protein (EGFP; SEQ ID NO:10), luciferase gene (SEQ ID NO:11), luxA (SEQ ID NO:8), luxB (SEQ ID NO:9), and Lux ​​operons (luxCDABE and luxABCDE; SEQ ID NO:12).

[0416] Other goods may target genes or products involved in diseases, such as, but not limited to, peptidyl-prolyl cis-trans isomerases.

[0417] FKBP4 or FKBP52 (SEQ ID NO:1); γ-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 NO: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 regulated by cargoes encoding products that repress or activate gene expression or products that repress or activate gene expression. Examples of such regulators are RNAi-type regulators, such as siRNA, miRNA, shRNA, antisense oligonucleotides (ASO), peptides, and / or tetrapeptides. For example, this article illustrates siRNA and ASO targeting EGFP (SEQ ID NO:5 and 6), firefly luciferase (SEQ ID NO:7), MYCN (SEQ ID NO:13-19), RAS (SEQ ID NO:20-27), BCL2 (SEQ ID NO:29-31), and PLK1 (SEQ ID NO:32-35), as well as microRNA-34A targeting MYC and BCL2 (SEQ ID NO:28).

[0418] 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 spiral family members, Hox family members, cytokines / lymphokines and their receptors, growth / differentiation factors and their receptors, and neurotransmitters and their receptors; oncogenes; tumor suppressor genes; enzymes; genes associated with pathological conditions; genes associated with autoimmune diseases; anti-angiogenic genes; angiogenic genes; immunomodulatory genes; genes associated with ethanol metabolism and liver function; genes associated with neurological diseases; genes associated with tumorigenesis or cell transformation; and genes associated with metabolic diseases and conditions (see, for example, WO2009 / 082606, JP2014-240428A, WO2011 / 072292A2, WO2010 / 141724, and WO2020 / 097540). These types of products can be delivered or encoded in MEVs to activate genes or pathways or provide therapeutic effects. Some cytokines can be used to treat diseases / conditions that involve immunosuppression, such as certain cancers.

[0419] Extracellular vesicles and exosomes can also be used to transfer therapeutic agents, such as nucleic acids, including microRNA, mRNA, tRNA, rRNA, siRNA, regulatory RNA, non-coding and coding RNA, DNA fragments, and DNA plasmids (see example).

[0420] CN105821081A and CN110699382A); respectively containing nucleotides or amino acids that can detect partial or toxin- or disrupt transcription or translation; polypeptides (e.g., enzymes); lipids; carbohydrates; and small molecules (e.g., small molecule drugs and toxins) (see US Patent No. 10,195,290). Non-limiting examples of proteins that can be encoded by nucleic acid cargo molecules include, but are not limited to: antibodies, intracellular antibodies, single-stranded variable fragments, affibodies, enzymes, transport proteins, tumor inhibitors, viral or bacterial inhibitors, cellular component proteins, DNA and / or RNA-binding proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transport proteins, nucleotide-binding proteins, heat shock proteins, CRISPR-related proteins, cytokines, cytokine receptors, caspases, and any combination and / or derivatives thereof (see, for example, AU2018365299).

[0421] For example, as summarized in the table below, a mixture of three siRNA oligonucleotides targeting human MYCN with two thymidine residues (dTdT) at the 3' end of the sequence can be used (purchased from B-Bridge International Inc. (Sunnyvale, CA)). Anti-MYCN siRNA (si MYCN) and negative control siRNA (non-target control library) (siNeg) (ON-TARGET plus siRNA, Dharmacon, Cambridge, UK) are used (see Reference 1). Exemplary target oncogenes and siRNA sequences are provided in the table below (also see SEQ ID NO: 13-35).

[0422]

[0423]

[0424]

[0425] 1 Nara et al. (2007) Int.J.Oncol.30(5):1189-1196; Silencing of MYCN by RNA interference induces growth inhibition, poptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification

[0426] 2 Maeshima et al. (2020) Nucleic Acid Ther.30(4):237-248; MYCN Silencingby RNAi Induces Neurogenesis and Suppresses Proliferation in Models ofNeuroblastoma with Resistance to Retinoic Acid

[0427] 3 Veas-Perez de Tudela et al.(2010)J.Neurochem.113(4):819-825;Humanneuroblastoma cells with MYCN amplification are selectively resistant tooxidative stress by transcriptionally up-regulating glutamate cysteine ligase

[0428] 4 Watson et al.(1991)Cancer Res.51(15):3996-4000;Inhibition of c-mycexpression by phosphorothioate antisense oligonucleotide identifies acritical role for c-myc in the growth of human breast cancer

[0429] 5 Yoshikawa et al.(2019)Mol.Ther.Methods Clin.Dev.13:290-302;Anti-cancer Effects of a Chemically Modified miR-143on Bladder Cancer by EitherSystemic or Intravesical Treatment

[0430] 6 Tsujino et al.(2019)Cancer Sci.110(7):2189-2199;MicroRNA-143 / Musashi-2 / KRAS cascade contributes positively to carcinogenesis in humanbladder cancer

[0431] 7 Tirella et al.(2019)Int.J.Pharm.561:114-123;CD44 targeted deliveryof siRNA by using HA-decorated nanotechnologies for KRAS silencing in cancertreatment

[0432] 8 Nakada et al.(2001)Pancreatology 1(4):314-319;Antisenseoligonucleotides specific to mutated K-ras genes inhibit invasiveness ofhuman pancreatic cancer cell lines

[0433] 9 Adams et al.(2015)Expert Opin.Ther.Targets 20(6):737-753;The Tumor-Suppressive and Potential Therapeutic Functions of miR-34a in EpithelialCarcinomas

[0434] 10 Poeck et al.(2008)Nat.Med.14(11):1256-1263;5'-Triphosphate-siRNA:turning gene silencing and Rig-I activation against melanoma

[0435] 11 Szegedi et al.(2008)Pathol.Oncol.Res.14(3):275-279;Bcl-2 AntisenseOligonucleotide Inhibits the Proliferation of Childhood Leukemia / lymphomaCells of the B-cell Lineage

[0436] 12 Ripoll et al.(2018)RSC Adv.8:20758-20763;Co-delivery of anti-PLK-1siRNA and camptothecin by nanometric polydiacetylenic micelles results in asynergistic cell killing

[0437] 13 Liu et al. (2012) BMC Cancer 12(1):519-529; MicroRNA-100 is apotential molecular marker of non-small cell lung cancer and functions as tumor suppressor by targeting polo-like kinase 1

[0438] 14 et al. (2008) Neoplasia 10(3):223-234; Downregulation of Plk1expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles.

[0439] The bioactive molecules in the cargo can target central nervous system diseases, such as neurodegenerative diseases like Alzheimer's disease. An example of this is FKBP52 and its tetratricopeptide derivatives. The complete sequence of the human peptidyl-prolyl cis isomerase FKBP4 is (SEQ ID NO:1):

[0440] MTAEEMKATESGAQSAPLPMEGVDISPKQDEGVLKVIKREGTGTEMPMIGDRVFVHYTGWLLDGTKFDSSLDRKDKFSFDLGKGEVIKAWDIAIATMKVGEVCHITCKPEYAYG SAGSPPKIPPNATLVFEVELFEFKGEDLTEEEDGGIIRRIQTRGEGYAKPNEGAIVEVALEGYYKDKLFDQRELRFEIGEGENLDLPYGLERAIQRMEKGEHSIVYLKPSYAFGS VGKEKFQIPPNAELKYELHLKSFEKAKESWEMNSEEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWLEYESSFSNEEAQKAQALRLASHLNLAMCHLKLQAFSAAIESCNK ALELDSNNEKGLFRRGEAHLAVNDFELARADFQKVLQLYPNNKAAKTQLAVCQQRIRRQLAREKKLYANMFERLAEEENKAKAEASSGDHPTDTEMKEEQKSNTAGSQSQVETEA

[0441] The 34-peptide repeat (TPR) domain 260-400 is (SEQ ID NO:2):

[0442] MNSEEKLEQSTIVKERGTVYFKEGKYKQALLQYKKIVSWLEYESSFSNEEAQKAQALRLASHLNLAMCHLKLQAFSAAIESCNKALELDSNNEKGLFRRGEAHLAVNDFELARADFQKVLQLYPNNKAAKTQLAVCQQRI

[0443] The in vitro model was developed and described by a group from the National Institute of Health and Medical Research, Paris XI University (see Chambraud et al. (2007) FASEB J. 21(11):2787-97; and Chambraud et al. (2010) Proc. Natl. Acad. Sci. USA 107(6):2658-63). The role of FKBP52 depletion in PC12-cultured cells was examined by introducing two distinct small interfering RNA (siRNA) duplexes (named RNAi1 and RNAi2) specific to rat FKBP52. The sense sequence of the siRNA and an oligonucleotide duplex with a scrambled sequence corresponding to RNAi1 were used as negative controls. In these experiments, FKBP52 levels, as analyzed by Western blot, decreased significantly after 48 hours and remained at low levels for 72 hours post-transfection. Tubulin and FKBP52 staining were performed 72 hours post-transfection. FKBP52 staining was significantly lower in cells transfected with RNAi 1 or 2 than observed in control cells, and tubulin staining revealed changes in the PC12 cell phenotype, particularly the loss of FKBP52 in PC12 cells leading to cell elongation. Therefore, these cells acquired a differentiation phenotype comparable to NGF-treated PC12 cells. No significant modifications were observed in cells transfected with controls. In another study, Chambraud et al. (Proc. Natl. Acad. Sci. USA, cited above) reported that FKBP52 prevents Tau accumulation and neurite growth in PC12 cells. They used a tetracycline-responsive element-based FKBP52-inducible expression system. This system allowed for the generation of stably transformed PC12 cell lines to determine the cellular role of FKBP52. From the positive test clones, one clone (so-called H7C2) was selected to investigate the effect of FKBP52 overexpression on PC12 cells and to further investigate the relationship between FKBP52 and Tau. Under basal conditions, H7C2 cells expressed endogenous FKBP52, and treatment with doxycycline (Dox) resulted in a significant increase in recombinant FKBP52 protein expression. After 5 days of Dox treatment, FKBP52 induction in H7C2 cells was approximately four-fold. Next, the effect of FKBP52 on Tau accumulation was examined. The amount of Tau protein was determined by Western blotting of extracts from cultures of PC12 cells or H7C2 cells treated with or without nerve growth factor (NGF) (50 nM) for 5 days, with or without Dox.In PC12 cells, FKBP52 expression remained unchanged after NGF treatment. As expected, an increase in Tau was observed in both PC12 and H7C2 cells after NGF treatment. No additional accumulation of Tau protein occurred when H7C2 cells were exposed to Dox (in addition to NGF) to overexpress FKBP52. The increase in Tau protein was still observed in PC12 cells treated with both NGF and Dox, ruling out the possibility of Dox causing a reduction in Tau deficiency. This report concludes that FKBP52 prevents NGF-induced Tau accumulation in PC12 cells.

[0444] Because one of Tau's roles is to stimulate neurite growth, the effects of FKBP52 overexpression on neurite length in PC12 and H7C2 cells were also investigated. In the absence of NGF, no neurite growth was observed in H7C2 cells, regardless of whether they were treated with Dox for one week. In H7C2 cells treated with 50 nM NGF and Dox, a 40% (±7) reduction in neurite length was observed compared to the control (H7C2 cells not treated with Dox). The same effect of Dox on neurite length was observed in H7C2 cells treated with 10 or 20 nM NGF. Dox itself is not involved in the process of neurite growth, as no difference in neurite length was observed between Dox-treated and untreated PC12 cells. The inhibition of neurite growth induced by FKBP52 overexpression is consistent with previous reports from Chambraud et al., showing that loss of FKBP52 in PC12 cells leads to neurite elongation. The effect of FKBP52 on neurite length can be explained by the removal of Tau from microtubules through the binding of Tau to FKBP52. Overexpression of FKBP52 prevents tau accumulation, consistent with a reduction in neurite length, and suggests a potential role for this immunophile protein in tau function. Therefore, the aforementioned targets and sequences could be delivered or encoded in the MEV for the treatment of Alzheimer's disease by preventing tau accumulation.

[0445] Pathogens involved in AD pathology

[0446] Microbial, viral, and fungal infections have been reported to increase the risk of Alzheimer's disease (AD), trigger AD, or participate in the pathology of AD (Catumbela et al. (2023) Transl. Neurodegener. 37, doi:10.1186 / s40035-023-00369-7). For example, oral bacteria have been identified in the brains of AD patients. *Porphyromonas gingivalis*, an oral bacterium, has been identified in the brains of patients with Alzheimer's disease (AD). *Porphyromonas gingivalis* has been identified as a risk factor for AD, and its components (gingipains and lipopolysaccharide) have been shown to induce AD-like neurodegeneration in infected neurons derived from induced pluripotent stem cells in an in vitro culture system continuously expressing active gingipains. *Porphyromonas gingivalis* has been detected in the brain tissue of AD patients and associated with pathological changes. The MEV described in this article can be loaded with agents that inhibit *Porphyromonas gingivalis* and / or gingival proteases for the prevention or treatment of Alzheimer's disease (AD). The amyloid pathway has been proposed as an inflammatory response to infection and toxic products, including gingival proteases. The resulting amyloid plaques and the aberrant protein tau can be sources of neuroinflammation and neurodegeneration (see, for example, Seymour et al. (2022) J. Exploratory Res. in Pharmacology 7:45-53). The MEV provided in this article can be loaded with bacterial inhibitors and / or gingival proteases inhibitors, such as atuzaginstat, and delivered to the brain, including neurons, via intranasal administration. Treatment of infections with antiviral agents (e.g., acyclovir, famciclovir, ganciclovir, idoxuridine, penciclovir, troamantadine, valacyclovir, and valganciclovir), antimicrobial agents, and antifungal agents has been shown to reduce the risk of developing AD. Some antibiotic regimens have resulted in cognitive improvement. For example, a Helicobacter pylori eradication regimen using omeprazole (a proton pump inhibitor), clarithromycin (a macrolide antibiotic), and amoxicillin (a penicillin-type antibiotic) was used to treat Helicobacter pylori-positive AD patients. At the end of the study, AD patients successfully treated with the Helicobacter pylori eradication regimen showed significantly improved cognitive scores on the Cambridge Assessment of Cognition in Older Adults and the Functional Rating Scale for Dementia Symptoms, compared to patients who failed to show reduced Helicobacter pylori levels after similar treatment. Therefore, Helicobacter pylori infection contributes to AD pathology, and eradication of the pathogen can improve disease progression. Therefore, various therapeutic agents for AD-associated pathogens can be administered via intranasal MEV, where the cargo contains therapeutic agents such as antiviral agents or antibiotics, or other antimicrobial agents.

[0447] ApoE and Alzheimer's disease

[0448] As described in Example 33, the ApoE gene, particularly the ApoE4 allele, has been identified as a risk factor for late-onset Alzheimer's disease (AD). The ApoE gene and its alleles are therapeutic targets for AD treatment. As described herein, orally administered MEVs cross neurons in the brain; MEVs may be loaded with cargo to alter ApoE expression or levels in neurons to treat, prevent, or reduce the risk of AD. For example, as detailed in the examples, MEVs may be loaded with RNAi that inhibits ApoE4 expression for intranasal administration, thereby delivering the RNAi directly to neurons. Other treatment methods include, for example, using a gene-editing system within the MEV to convert the ApoE4 allele to ApoE3 or ApoE2, among others. Intranasally administered MEVs provide a direct pathway to neurons; other treatment regimens do not offer this pathway.

[0449] Reporter genes, reporter proteins, and / or their regulators can be delivered in MEVs.

[0450] Reporter protein

[0451] Each marker gene, such as GFP protein, eukaryotic luciferase, or prokaryotic luciferase, can be regulated (inhibited or stimulated) using target sequences in the form of siRNA, miRNA, antisense oligonucleotides (ASO), peptides, and / or 34 peptides, for example: the Lux operon (luxCDABE) and the lux operon (luxABCDE), for example, for diagnostic and gene expression assessment (SEQ ID NO: 5-6, 7, and 62-65, respectively).

[0452]

[0453] Other exemplary goods may include chemotherapy agents, including but not limited to alkylating agents such as thiotepa and cyclophosphamide (which may be trademarked). (obtained); alkyl sulfonates, such as busulfan, indomethacin, and piperosulfan; androgens, such as calcitestosterone, drotaloferrin propionate, epithioandrostanol, meandrolone, and testrolide; antiadrenergic drugs, such as aminoglutethimide, mitotane, and tramostan; antiandrogens, such as flutamide, niglutethimide, bicalutamide, leuprorelin, and goserelin; antibiotics, such as aclarubicin, actinomycin, amiodarone, diazoserine, bleomycin, actinomycin, chachomycin, carborubicin, erythromycin, carcinomamycin, chromomycin, daunorubicin, and daunorubicin. Toxoplasm, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esopixin, idarubicin, metharubicin, mitomycin, mycophenolic acid, nogamycin, olivomycin, pepromycin, pofibromycin, puromycin, triamcinolone acetonide, rodopilline, streptozotocin, streptozotocin, tuberculin, ubenimex, fenestrated statin, and zolrubicin; anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazole, 4-hydroxytamoxifen, trivoxifen, raloxifene, LY 117018, onasone, and toremifene (by trademark). (For sale); antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, methotrexate, pteroxate, and trimethyltroxa; aziridines, such as benzotipane, carboquinone, metopepiphenate, and uratepiphenate; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylene melamine, triethylene ethylphosphamide, triethylene thiophosphamide, and trimethylol melamine; folic acid supplements, such as folinic acid; nitrogen mustard, such as chlorambucil, naphthiamethoxam, chlorphosphamide, estradiol, ifosfamide, nitrogen mustard, nitrogen oxychloride, melphalan, neonitrogen mustard, benzyl mustard, and cholesterol. Alcohols, prednisolone, trazophos and uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, nimustine and ramustine; platinum analogs, such as cisplatin and carboplatin; vincristine; platinum; proteins, such as arginine deiminase and asparaginase; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazoline and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine, 5-FU; taxanes, such as paclitaxel (e.g., under trademarks) Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS2000; thymidine synthase inhibitors (e.g., Tomudex) TMOther chemotherapy agents include glucuronolactone; aldehyde phosphoramidone glycoside; aminolevulinic acid; acridine; amustine; bifenthrin; edaraxazole; desphosphonamide; colchicine; desaccharin; difluoromethylornithine (DFMO); efornithine; erythritol; itogluconate; gallium nitrate; hydroxyurea; lentinan; clonidine; mitoxantrone; mopiperol; nitropropamide; pentostatin; methamidophos; pirarubicin; podophyllotoxin; 2-ethylhydrazine; procarbazine; Propylene; zearalenone; germanium spiroamine; tinuzonic acid; triaminoquinone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromoeutherol; piperbbromide; gascitocin; cytarabine (Ara-C); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine (as trademarked) Novantrone (selling vinorelbine tartrate); TM Teniposide; Daunomycin; Aminopterin; Capecitabine (e.g., as a glycoside) (For sale); ibandronic acid; CPT-11; retinoic acid; esperamycin; capecitabine; and topoisomerase inhibitors such as irinotecan. Pharmaceutically acceptable salts, acids, or derivatives of any of the above substances may also be used.

[0454] Chemotherapy agents include prodrugs, including but not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted into more active, non-cytotoxic drugs.

[0455] Other goods include, for example, anti-angiogenic agents. Anti-angiogenic agents can be small molecules or proteins that bind to growth factors or growth factor receptors involved in promoting angiogenesis, such as antibodies, Fc fusions, and cytokines. Examples of anti-angiogenic agents include, but are not limited to, antibodies that bind to vascular endothelial growth factor (VEGF) or VEGF-R, RNA-based therapeutics that reduce VEGF or VEGF-R expression levels, VEGF-toxin fusions, Regeneron's VEGF-trap, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay12-9566, BeneFin, bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitors (CDI), CAI, CD59 complement fragment, CEP-7055, Col3, cobbitastatin A-4, and other similar products. Pistatin (collagen XVIII fragment), farnesyltransferase inhibitor, fibronectin fragment, GRO-β, halofopontone, heparinase, heparin hexose fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon α, interferon β, interferon γ, interferon inducible protein 10 (IP-10), interleukin-12, kringle5 (plasminogen fragment), marimasitol, metalloproteinase inhibitors (e.g., TIMP), 2-methoxyestradiol, MMI270 (CGS27023A), plasminogen activator inhibitor (PAI), platelet factor-4 (PF4), prolactin 16kDa fragment, proliferation protein-associated protein (PRP), PTK 787 / ZK 222594, retinoic acid, solistat, squalamine, SS3304, SU5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombin-sensitive protein-1 (TSP-1), TNP470, transforming growth factor β (TGF-β), vasculostatin, angiostatin (troponin fragment), ZS6126 and ZD6474.

[0456] Other goods include tyrosine kinase inhibitors, including but not limited to quinazolines such as PD153035, 4-(3-chloroaniline)quinazoline; pyridopyrimidine; pyrimidine, pyrrolopyrimidine, such as CGP59326, CGP60261 and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo(2,3-d)pyrimidine; curcumin (bisferoylmethane, 4,5-bis(4-fluoroaniline)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoded nucleic acids); quinazoline. Phosphorus (US Patent No. 5,804,396); tyrphostins (US Patent No. 5,804,396); PTK-787 (Novartis / Schering AG); pan-ErbB inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS3521; Isis / Lilly); imatinib mesylate (STI571, 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: US Patent No. 5,804,396; PCT WO 99 / 09016 (American Cyanamid); PCT WO 98 / 43960 (American Cyanamid); PCT WO 97 / 38983 (Warner-Lambert); PCT WO 99 / 06378 (Warner-Lambert); PCT WO 99 / 06396 (Warner-Lambert); PCT WO 96 / 30347 (Pfizer, Inc.); PCT WO 96 / 33978 (AstraZeneca); PCT WO 96 / 33979 (AstraZeneca); PCT WO 96 / 33980 (AstraZeneca), gefitinib ( ZD1839,AstraZeneca), and OSI-774( OSIPharmaceuticals / Genentech).

[0457] Other goods include immunomodulators that increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells. Examples of immunomodulators include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodoxacin, fenprofen, indomethacin, ketorolac, oxapazol, nabumetone, sulindac, tolmetine, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g., glucocorticoids), dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, triamcinolone, etc. Sulfasalazine; arachidic acids such as prostaglandins, thromboxanes, and leukotrienes; and topical steroids such as anthraquinone, calcipotriene, clobetasol, and tazarotene; cytokines such as TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, and IL-10; cytokines, chemokines, or receptor antagonists, including antibodies, soluble receptors and receptor-Fc fusion compounds, B7, CCR2, CCR5, CD2, and CD3. CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D), CTLA4, eosinophil activation chemokine, 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, integrin, LFA-1, LFA-3, MHC, selectin, TGFβ, TNFα, TNFβ, TNF-R1, T cell receptor, including (Inerasipu) (adalimumab) and (Infliximab); heterologous anti-lymphocyte globulin; other immunomodulatory molecules, such as 2-amino-6-aryl-5-substituted pyrimidines, anti-idiotypic antibodies for MHC-binding peptides and MHC fragments, azathioprine, buquina, bromocriptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyguanidine, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitrile amide (e.g., leflunomide), methotrexate, minocycline, imidazolidin, mycophenolate mofetil, rapamycin, and sulfasalazine.

[0458] Other goods include cytokines, including but not limited to lymphokines, monokines, and conventional polypeptide hormones. Among the cytokines are 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); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and-β; Müller duct inhibitors; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-β; platelet-derived growth factor; and transforming growth factor (TGF), such as TGF-β. -α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); bone-inducing factor; interferons, such as interferon-α, -β and -γ; 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-1α, 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 factor, such as TNF-α or TNF-β; and other peptide factors, including LIF and kit ligand (KL).

[0459] Other exemplary goods include cytokines and other agents that stimulate immune system cells and enhance desired effector functions. For example, agents that stimulate NK cells include IL-2; agents that stimulate macrophages include, but are not limited to, C5a and formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine. Goods include agents that stimulate neutrophils, such as G-CSF and GM-CSF. Additional agents include, but are not limited to, interferon-γ, IL-3, and IL-7.

[0460] The goods include antibiotics for treating infections, particularly difficult-to-treat bacterial infections, including urinary tract infections, respiratory infections, especially *Pseudomonas aeruginosa* or *Staphylococcus aureus* infections in subjects with cystic fibrosis, and sinus infections, which can be treated by topical application, such as by inhalation of an aerosol containing a MEV. Antibiotic treatment for lung infections in subjects with cystic fibrosis can be combined with gene therapy using the same or different MEVs, said MEVs containing nucleic acids, DNA, or RNA encoding the cystic fibrosis transmembrane conduction regulator (CFTR) protein, or providing a gene-editing system to correct defects in the CFTR protein.

[0461] Antibiotics that can be loaded as cargo in an MEV include, but are not limited to: aminoglycoside antibiotics (e.g., apramycin, abexacin, babemycin, butirocin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), aminocyclohexol (e.g., spectinomycin), amphenicol antibiotics (e.g., chloramphenicol, chloramphenicol, florfenicol, and thiamphenicol), anisoxam antibiotics (e.g., rifamycin and rifampin), carbapenems (e.g., imipenem, meropenem, and panipenem); cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, ceftrazine, cefoxitin, cefazolin, cefazolin, cefimidazole, cefpirome, cefpirome). Cephalosporins, cefuroxime, cefixime, cephalexin, and cefadroxil; cephalosporins (cefadroxil, cefoxitin, cefminox, cefmetazole, and cefotetan); lincosamides (e.g., clindamycin and lincomycin); macrolides (e.g., azithromycin, brefidobacterium A, clarithromycin, erythromycin, roxithromycin, and tobramycin); monocyclic β-lactams (e.g., aztreonam, carumonam, and tegafur); mupirocin; oxacephalosporins (e.g., fluoxetine, latamoxetine, and latamoxetine); penicillins (e.g., adenomidine, pimecrolimus, amoxicillin, bamectin, benzylpenicillin, penicillin sodium, ipilimil, fenbecilil, flucloxacillin, pentazocine, penicillin hydroiodide, and benzethonamic acid penicillin G). o-benethamine), penicillin O, penicillin V, penicillin V benzoate, penicillin V phenazine, penicillin-perazine, and fenestrone potassium; polypeptides (such as bacitracin, colistin, polymyxin B, teicoplanin, and vancomycin); quinolones (amloxacin, sinofloxacin, ciprofloxacin, enoxacin, enflufloxacin, fleroxacin, flumethylquine, gatifloxacin, gemimifloxacin, guaparfloxacin, loperamide, etc.). Dextromethorphan, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxoriprine, pefloxacin, pipemidic acid, rosofloxacin, rufloxacin, sparfloxacin, temafloxacin, tofloxacin, and trovafloxacin; rifampin; streptozotocins (such as quinupristin and dalfopristin); sulfonamides (sulfonamides and sulfamethoxazole); and tetracyclines (chlortetracycline, demethylchlortetracycline hydrochloride, demethylchlortetracycline, doxycycline, ... Minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, and vancomycin.

[0462] The cargo also includes antifungal agents, including but not limited to amphotericin B, ciclopirox ol, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, nystatin, terbinafine, teconazole, and tiaconazole. In some instances, the MEVs carrying the cargo described herein are administered in combination with one or more antiviral agents, including but not limited to protease inhibitors, reverse transcriptase inhibitors, and other antiviral agents, including type I interferon, viral fusion inhibitors, neuraminidase inhibitors, acyclovir, adefovir, amantadine, ampranasvir, clavidine, enfuviride, entecavir, foscarnet, ganciclovir, idoxuridine, indicavir, lopinavir, prazolam, ribavirin, amantadine, ritonavir, saquinavir, trifluralin, vidarabine, and zidovudine.

[0463] In all cases, the cargo takes the form of proteins, as well as nucleic acids encoding those proteins, such as plasmids, and mRNA. Nucleic acids can be operatively linked to regulatory elements recognized in a specific subject (e.g., a mammal) in which they will be delivered.

[0464]

[0465]

[0466] 3. Generation of MEVs carrying payloads

[0467] As illustrated 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 routes known to those skilled in the art for delivery of vectors (e.g., lipid nanoparticles, carriers, therapeutic bacteria, and therapeutic viruses). Upon administration, the MEV is absorbed by cells. Any cargo currently delivered in carriers, bacteria, exosomes, nanoparticles, and other such delivery vectors can be loaded into the MEV provided herein. The loaded cargo can be selected such that it is expressed or produced only in target cells, for example, in the case where the cargo is a plasmid encoding a therapeutic product. Transcriptional regulatory signals can be selected such that the encoded product is expressed in target cells. For example, for expression in the liver, the encoded product can be expressed under the control of a liver-specific promoter, or the product can target a receptor or target expressed in target cells, for example, in a tumor or in the tumor microenvironment. The loading methods in the above and following examples include, but are not limited to:

[0468] a. Electroporation

[0469] b. Ultrasonic treatment

[0470] c. Extrusion

[0471] d. Surfactants

[0472] e. Other methods known to those skilled in the art for introducing exosomes into cells.

[0473] 4. Exemplary cargo and exemplary uses of externally loaded MEVs

[0474] a. Goods

[0475] As described above, the MEV carries cargo that can be used for any purpose of interest, including any cargo delivered using other delivery vectors. These uses include delivering mRNA, such as mRNA encoding the coronavirus spike protein and modified spike proteins to improve the immune response to viruses; RNAi, such as siRNA; and antisense RNA, or antisense DNA (ASO); to silence genes, such as bacterial and viral pathogen virulence genes, antibiotic resistance genes, antimicrobial resistance genes, genes that suppress the immune system; oncology genes, such as oncogenes; and host factors of viral infection, such as those targeting angiotensin-converting enzyme-2 (ACE2), transmembrane protein serine 2 (TMPRSS2), and other such genes. The cargo may also include any therapeutic antibodies. Therapeutic antibodies include, but are not limited to, anticancer antibodies, antibodies for treating autoimmune or inflammatory diseases, antibodies for treating graft rejection, antibodies for treating graft-versus-host disease (GVHD), and antibodies for treating infectious diseases.

[0476] 1) RNA cargo

[0477] The mechanism of RNA interference, or RNAi, was initially described as a sequence-specific silencing process 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 by which small RNAs target (and silence) messenger RNAs involves specific RNAi mechanisms (including silencing factors such as DICER and ARGONAUTE).

[0478] In the plant kingdom, RNAi is involved in antiviral defense mechanisms, as well as defense mechanisms against plant pathogenic fungi and oomycetes. Small regulatory RNAs can be active in silencing genes within bacterial cells lacking the aforementioned RNAi mechanisms. The silencing activity of siRNAs has been demonstrated to be cross-species (see, e.g., Singla, Navarro., 2019a, PCT / EP2019 / 072169; Singla, Navarro., 2019b, PCT / EP2019 / 072170; Singla et al. (2019c) bioRxiv, doi:doi.org / 10.1101 / 863902).

[0479] RNAi-mediated gene expression regulation has been developed in the biotechnology field for many years to confer resistance to viruses (Baulcombe (2015) Current Opinion in Plant Biology 26:141-146). Cross-kingdom RNAi has been used to characterize the function of genes in eukaryotic pathogens and / or eukaryotic parasites and to induce protection against these organisms.

[0480] In Drosophila and Caenorhabditis elegans, RNAi plays a crucial role in antiviral defense by directly targeting viral RNA via small RNAs produced by the host in response to the virus. Recent work has shown that plant EVs (naturally loaded with small RNAs from edible 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).

[0481] Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are non-coding RNAs that play important roles in gene regulation. They have recently been investigated as novel therapeutic agents for a wide range of conditions, including cancer and infections. Clinical trials of siRNA and miRNA-based drugs have begun. While siRNAs and miRNAs share many similarities—both are short, double-stranded RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNAs (mRNAs)—their mechanisms of action and clinical applications differ. A key difference between siRNAs and miRNAs is that the former has only one highly specific mRNA target, while the latter has multiple targets. siRNAs and miRNAs function in gene regulation and serve as targets for drug discovery and development. Compared to conventional small therapeutic molecules, siRNAs and miRNAs offer highly potent potential and can act on “drug-insoluble” targets (e.g., proteins lacking enzymatic function); furthermore, RNAi can be engineered to target and / or affect the expression of any gene of interest.

[0482] 2) Antibody cargo

[0483] Examples of anticancer antibodies and other antibodies include, but are not limited to, antibodies against the 17-1A cell surface antigen, such as those marketed under trademarks. Antibodies sold or supplied (ejaculumab); anti-4-1BB antibody; anti-4Dc antibody; anti-A33 antibody, such as A33 and CDP-833; anti-α1 integrin antibody, such as natezumab; anti-α4β7 integrin antibody, such as LDP-02; anti-αVβ1 integrin antibody, such as F-200, M-200 and SJ-749; anti-αVβ3 integrin antibody, such as abciximab, CNTO-95, Mab-17E6 and... Anti-complement factor 5 (C5) antibodies, such as 5G1.1; anti-CA125 antibodies, such as those marketed under trademarks. Antibodies (Ogovacuumab) sold or supplied; anti-CD3 antibodies, such as those marketed under trademarks. (Vesizumab) and Rexomab TM Antibodies sold or supplied; anti-CD4 antibodies, such as IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies, such as Oncolysin B and Oncolysin CD6; anti-CD7 antibodies, such as HB2; anti-CD19 antibodies, such as B43, MT-103, and Oncolysin B; anti-CD20 antibodies, such as 2H7, 2H7.v16, 2H7.v114, 2H7.v115, and others under trademarks. (Tosimomab) Products sold or supplied under the trademark (rituximab) antibodies sold or supplied under trademarks (Tiemomumab) antibodies sold or supplied; anti-CD22 antibodies, such as those sold or supplied under the following generic names, trade names or trademarks: (Epazolizumab); anti-CD23 antibodies, such as IDEC-152; anti-CD25 antibodies, such as baliximab and (Dacroizumab); anti-CD30 antibodies, such as AC10, MDX-060, and SGN-30; anti-CD33 antibodies, such as gemtuzumab / ozomicin (trademarked). (sales), Oncolysin M and Smart M195; anti-CD38 antibodies; anti-CD40 antibodies, such as SGN-40 and toliszumab; anti-CD40L antibodies, such as 5c8, And IDEC-131; anti-CD44 antibodies, such as bivalizumab; anti-CD46 antibodies; anti-CD52 antibodies, such as alenzusumab (trademarked) (For Sale); anti-CD55 antibodies, such as SC-1; anti-CD56 antibodies, such as huN901-DM1; anti-CD64 antibodies, such as MDX-33; anti-CD66e antibodies, such as XR-303; anti-CD74 antibodies, such as IMMU-110; anti-CD80 antibodies, such as galiximab and IDEC-114; anti-CD89 antibodies, such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodies, such as B-B4-DM1; anti-CD146 antibodies, such as AA-98; anti-CD148 antibodies; anti-CEA antibodies, such as cT84.66, labezizumab, and Pentacea; anti-CTLA-4 antibodies, such as MDX-101; anti-CXCR4 antibodies; anti-EGFR antibodies, such as ABX-EGF, cetuximab (e.g., by trademark). Products sold), IMC-C225 and MerckMab425; anti-EpCAM antibodies, such as Crucell's anti-EpCAM, ING-1 and KS-IL-2; anti-EphrinB2 / EphB4 antibodies; anti-Her2 antibodies, such as trastuzumab (trademark). ), MDX-210; anti-FAP (fibroblast activation protein) antibodies, such as sirocillinumab; antiferritin antibodies, such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250 antibodies, such as WX-G250 and gereximab (trademarked) (For sale); Anti-GD2 ganglioside antibodies, such as EMD-273063 and Tri Anti-GD3 ganglioside antibodies, such as BEC2, KW-2871, and mitumomab; anti-gpIIb / IIIa antibodies, such as... Anti-heparinase antibodies; anti-Her2 / ErbB2 antibodies, such as trastuzumab, MDX-210, and pertuzumab; anti-HLA antibodies (e.g., marketed by trademark). Products for sale: Smart1D10; anti-HM1.24 antibody; anti-ICAM antibody, such as ICM3; anti-IgA receptor antibody; anti-IGF-1 antibody, such as CP-751871 and EM-164; anti-IGF-1R antibody, such as IMC-A12; anti-IL-6 antibody, such as CNTO-328 and elsilimomab; anti-IL-15 antibody (e.g., under trademarks). - IL15 products (products sold); anti-KDR antibodies; anti-lamin 5 antibodies; anti-Lewis Y antigen antibodies, such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1 antibodies, such as BravaRex and TriAb TM Anti-NCAM antibodies, such as ERIC-1 and ICRT; anti-PEM antigen antibodies, such as Theragyn and Therex; anti-PSA antibodies; anti-PSCA antibodies, such as IG8; anti-Ptk antibodies; anti-PTN antibodies; anti-RANKL antibodies, such as AMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies, such as MonopharmC; 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., by trademark). Products sold) and infliximab (e.g., under trademarks) Products sold); anti-TRAIL-R1 and anti-TRAIL-R2 antibodies; anti-VE-cadherin-2 antibody; and anti-VLA-4 antibody (e.g., under trademarks). (Products sold). In addition, anti-idiotype antibodies may be used, including but not limited to the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7. Furthermore, bispecific antibodies may be used, including but not limited to the anti-CD3 / CD20 antibody Bi20.

[0484] Additional exemplary cargo, uses, and handling that can be achieved using an MEV carrying cargo are described below by way of example.

[0485] b. Diseases and treatments

[0486] As described above, MEVs can carry any desired cargo, including but not limited to nucleic acid molecules, detectable marker proteins and tags, small molecule drugs, gene editing systems, and combinations thereof, for the delivery of therapeutic molecules, as vaccines, and for human and other animal health, agriculture, cosmetics, dermatology and diagnostic applications, industrial use, and other uses. MEVs can deliver nutrients or regulators of gene pathways to produce beneficial products, gene editing systems such as CRISPR / Cas to achieve gene editing, and gene therapy vectors and products.

[0487] MEVs can carry cargo, for example, for treating diseases characterized by genetic defects leading to the deficiency of functional proteins, or for treating diseases characterized by peptide overexpression. Non-limiting examples of diseases that can be treated by silencing target genes, such as using siRNA or microRNA (see, for example, International Publication No. WO2013 / 048734) include cancers (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 diseases, and eye diseases (e.g., age-related macular degeneration, herpetic stromal keratitis, glaucoma, dry eye syndrome, diabetic retinopathy, and diseases related to ocular angiogenesis and ocular hypertension). Related conditions), neurological diseases (e.g., amyotrophic lateral sclerosis, Alzheimer's disease, myasthenia gravis), Huntington's disease, spinocerebellar ataxia, frontotemporal dementia, Parkinson's disease, prions, and Lafora disease, as well as those caused by ischemic or hypoxic conditions), kidney diseases, inflammatory or autoimmune diseases (e.g., ischemic or reperfusion injury, restenosis, rheumatoid arthritis, inflammatory bowel diseases such as Crohn's disease or ulcerative colitis, lupus, multiple sclerosis, diabetes (e.g., type 2 diabetes) and diabetic conditions, arthritis (e.g. Rheumatoid arthritis or psoriasis), respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, acute respiratory distress syndrome (ARDS), emphysema, and acute lung injury), hearing impairment, epilepsy, spinal cord injury, stomatitis, male infertility, uterine conditions, endometrial conditions or disorders, and conditions related to metabolism (e.g., obesity), ischemia, stroke, alcohol metabolism, and liver function (see, for example, International Publication Nos. WO2006 / 029161, WO2007 / 022470, WO2007 / WO2008 / 021157, WO2009 / 104051, WO2009 / 142822, WO2019 / 217459, WO2020 / 123083; European Publication No. EP2504435; and US Patent Publications 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, select proteins, immunoglobulins, kinases, and phosphatases.

[0488] Other cargo and uses are envisioned. For example, the MEV could carry cargo for treating conditions caused by trauma, such as wounds, burns, skin cuts, broken bones, hair loss, exposed dermis, exposed mucous membranes, fibrosis, lacerations, and ulcers. The MEV could also carry cargo for treating conditions caused by natural or induced aging, particularly skin or visual conditions.

[0489] MEVs can be used to deliver goods for purposes such as treatment through gene silencing or prevention of infectious diseases through vaccination. For example, when introduced into juvenile animals, MEVs derived from antigen-pulsating macrophages or dendritic cells show signs of evoking an immune response. (et al. (2015) Annu. Rev. Pharmacol. Toxicol. 55: 439-464). Gene silencing can also be used to target pathogen-associated proteins, such as viral proteins involved in host immunosuppression, pathogen replication, pathogen transmission, or infection maintenance; or host proteins that promote pathogen entry into the host, pathogen or host drug metabolism, pathogen genome replication or integration, establishment or spread of infection in the host, or assembly of the next generation of pathogens. Pathogens can include, for example, RNA and DNA viruses such as arenaviruses, coronaviruses, influenza viruses, paramyxoviruses, flaviviruses (e.g., West Nile virus), small RNA viruses (e.g., Coxsackievirus, poliovirus, and rhinovirus), rod-shaped viruses, filoviruses, retroviruses (e.g., lentiviruses and Rous sarcoma virus), adenoviruses, poxviruses, herpesviruses, human papillomaviruses, cytomegaloviruses, hepatotropic DNA viruses (e.g., hepatitis B and hepatitis C viruses), rotaviruses, respiratory syncytial virus, polyomaviruses, etc.; bacteria; fungi; worms; schistosomiasis; trypanosomes; and parasites, including Plasmodium (e.g., Plasmodium malariae). (malariae, etc.); and mammalian repositionable elements (see, for example, International Publications WO2010 / 141724, WO2011 / 071860, WO2011 / 072292, WO2013 / 126803, WO2020 / 035620 and WO20097540; Australian Publications AU2004257373A1, AU2013203219B2 and AU201622587A1; European Publications EP2395012 and EP2888240; US Patent Publications US2011 / 0223665, US2014 / 0256785 and US2019 / 0032051; Japanese Publication JP202018TW; and Taiwan Publication TW 201204351A).

[0490] MEVs can also be used to deliver DNA or mRNA sequences encoding therapeutically useful peptides. For example, in cases where a subject lacks a specific gene product, the gene may be encoded in a nucleic acid molecule, such as DNA or RNA. The nucleic acid molecule encoding the gene product can be loaded into an MEV and delivered to the subject who lacks the gene product.For example, diseases resulting from the lack or defect 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 adrenospinal neuropathy; Friedreich ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS) IIIB); CTNS-related cystine storage disorders; FMR1-related disorders, including fragile X syndrome, fragile X-related tremor / ataxia syndrome, and fragile X-related premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia; Niemann-Pick disease type C1; neuronal ceroid lipofuscin deposition disorders, including juvenile neuronal ceroid lipofuscin deposition disease (JNCL), juvenile Batten disease, Haltia-Santavuori disease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies; childhood ataxia associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5, with decreased myelination / white matter loss in the central nervous system; type 2 paroxysmal ataxia associated with CACNA1A and CACNB4; MECP2-related disorders, including classic Rett syndrome. MECP2-related severe neonatal encephalopathy and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy's disease (SBMA); Notch-3-related autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related epilepsy; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxia neuropathy, dysarthria and oculomotor palsy, and autosomal dominant and recessive progressive oculomotor palsy with mitochondrial DNA deletion; X-linked adrenal dysplasia; X-linked agammaglobulinemia; and Wilson's disease (see, for example, International Publication Nos. WO 2011 / 068810, WO 2019 / 243574, WO 2019 / 092287 and WO 2020 / 099682).

[0491] MEVs can be loaded with a CRISPR / Cas system for gene editing. Clustered, regularly spaced short palindromic repeats (CRISPR) technology allows for genome modification in living organisms and is based on the bacterial CRISPR / Cas9 antiviral defense system. This system allows DNA cleavage at target sites. Type II CRISPR systems incorporate sequences from invading foreign nucleic acids, such as DNA from viruses or plasmids, between CRISPR repeat sequences encoded within the host genome. Transcripts from the CRISPR repeat sequences are processed into CRISPR RNA (crRNA). Each crRNA contains a variable sequence transcribed from foreign DNA and a portion of the CRISPR repeat sequence. Each crRNA hybridizes with a second trans-activating CRISPR RNA (tracrRNA), and both RNAs complex with the Cas9 nuclease, directing the Cas9 nuclease to cleave the target DNA sequence. By delivering Cas nucleases, which are complexed with synthetic guide RNA (gRNA) consisting of a fusion of crRNA and tracrRNA, into cells, the cellular genome can be cleaved at desired locations, allowing for the removal of existing genes and / or the addition of new genes in vivo (Sander and Joung (2014) Nat. Biotechnol. 32(4):347-355). CRISPR technology can be used in conjunction with Cas peptides or the single RNA-guided endonuclease Cpf1 for genome modification and can be delivered in lipid nanoparticles, EVS, and other vesicles (see, for example, International Patent Publications WO2017 / 161010, WO2019 / 238626, and WO2020 / 097540).

[0492] Examples of gene therapy methods include gene replacement, such as replacing the gene encoding a defective product. An example of a disease that can be treated with gene replacement is Rett syndrome. Rett syndrome symptoms are caused by a mutation in a single gene, MECP2, which in turn produces a mutated protein. As demonstrated in animal models, restoration of MECP2 protein levels reversed symptoms, and if delivered to the brain, it could influence treatment. Gene replacement adds a healthier MECP2 gene to the brain, resulting in more unmutated MECP2 protein. MECP2 is encoded on the X chromosome. The goal is to replace as many defective genes as possible in the brain. MEVs offer a method for achieving gene replacement in the brain. This can be achieved by delivering a gene-editing machine (such as a CRISPR-Cas system) into an MEV to perform gene editing in the brain. By editing existing genes to correct mutations, the regulatory mechanisms controlling expression remain unaffected.

[0493] RNA editing is an alternative to DNA editing. Because unused RNA molecules degrade rapidly, any errors introduced by the treatment are not permanent. RNA can be introduced via RNA transsplicing. RNA transsplicing is a technique that hijacks this naturally occurring phenomenon to remove the mutated portion of the MECP2 protein RNA and replace it with a healthy version. A single RNA transsplicing treatment can treat 97% of all Rett syndrome patients, and it avoids any possibility of producing excessive amounts of MECP2 protein.

[0494] Another alternative treatment that can be delivered via MEV is the administration of the MECP2 protein to compensate for the mutated protein. Protein replacement is a well-established modality for treating cancer, diabetes, autoimmune diseases, blood disorders, and many other conditions. Protein replacement in Rett syndrome allows for titration of the dosage administered to an individual, allowing only the correct amount to improve their symptoms.

[0495] These methods for treating the genetic conditions described for Rett syndrome can be applied to the brain or any disease, condition, or ailment involving a defective protein product. These include the diseases, conditions, and ailments listed above, and any such disease, condition, or ailment in which the substitution or editing of a defective gene or gene product can enable therapeutic delivery to the brain. Nucleic acids and / or proteins can be delivered via intranasal administration of a MEV carrying nucleic acids and / or proteins as cargo.

[0496] MEVs can also be used to treat diseases, conditions, and illnesses by delivering payloads in the form of therapeutic proteins, peptides, or small organic molecules or compounds to target cells, including but not limited to those listed above. Non-limiting examples of such therapeutically effective agents or drugs include oncology drugs (e.g., chemotherapy drugs, hormone therapy agents, immunotherapy agents, and radiotherapy agents), lipid-lowering agents for treating lipid disorders, antiviral drugs, antifungal agents, anticholinergics, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cellular angiogenesis agents, antifibrotic drugs, antihypertensive drugs, aromatase or esterase inhibitors, signal transduction inhibitors, synthase inhibitors, and cardiovascular drugs such as antiarrhythmics. Arrhythmic agents, hormones or hormone antagonists, ion channel modulators, antitumor agents, neuroactive agents, vasoconstrictors, cytotoxic agents, nucleolysing compounds, radioisotopes, prodrug activators, and steroids (see, for example, International Patent Publications WO2015110957A2 and WO2019018349A1; and US Patent Publications US2019 / 0032051, US2012 / 0315324, US2019 / 0388347 and US2019 / 0175506). Therapeutic agents can also be biological therapeutic agents selected from allergens, adjuvants, antigens, immunogens, antibodies (e.g., whole antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and also including single-chain antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab)2, Fv, dAb and Fd fragments, biantibodies and antibody-associated peptides) Cytokines, hormones, factors, cofactors, cellular component proteins, metabolic enzymes, immunomodulatory enzymes, interferons, interleukins, gastrointestinal enzymes, enzymes or factors involved in hemostasis, growth-regulating enzymes, vaccines, antithrombolytic agents, toxins, antitoxins, diagnostic or imaging biological agents (see, for example, International Patent Publications WO2017 / 203260, WO2018 / 102397, WO2019 / 081474, WO2019 / 155060, WO2020 / 041720; Australian Patent Publication AU2018365299 A1; Singapore Patent Publication SG11201811149TA; and US Patent Publication US2019 / 0202892). For example...MEV therapy can be used to treat Crohn's disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist (DIRA) deficiency, endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, meningitis, encephalitis, liver failure, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). Kidney failure, heart failure, or any acute or chronic organ failure and related underlying causes; graft-versus-host disease; Duchenne muscular dystrophy and other muscular dystrophy; lysosomal storage diseases; neurodegenerative diseases; cancer-induced cachexia; anorexia; type 2 diabetes and cancer (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma), anal cancer, appendiceal cancer, astrocytoma, cerebellum or brain, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, brainstem glioma, brain cancer, brain tumor (cerebellar astrocytoma), astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primary tumor. Neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenoma / carcinoid tumors, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), cancers of unknown primary central nervous system lymphoma, cerebellar astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumors, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumors, gonadal extragerminoma, extrahepatic bile duct cancer, eye cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors. (Extracranial, extragonadal, or ovarian), gestational trophoblastic tumors, gliomas (brainstem gliomas, astrocytomas, visual pathway and hypothalamic gliomas), gastric carcinoid tumors, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma (endocrine pancreas), renal cell carcinoma (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia), lip and oral cancer, cavity cancer, liposarcoma, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma.Hodgkin's lymphoma, non-Hodgkin's lymphoma, medulloblastoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occlusion primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic disorders, myeloid leukemia, chronic myeloid leukemia, myeloma, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian epithelial carcinoma with surface epithelial-stromal tumor, ovarian germ cell tumor, low-potency ovarian tumor, pancreatic cancer, islet cell carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineal blastoma and supratentorial primitive neuroectodermal tumor, pituitary Somatic adenoma, pleural pulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma (renal carcinoma), retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma (Ewing family of tumor sarcomas, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma), Cézari syndrome, skin cancer (non-melanoma, melanoma), small bowel cancer, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal 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, Waldenström macroglobulinemia, and / or Wilms' tumor (see, for example, International Patent Publications WO2017 / 203260 and WO2019 / 155060A1; and US Patent Publication US2019 / 0388347).

[0497] c. Agricultural and veterinary applications

[0498] MEVs carrying biomolecules can be used in agro-veterinary applications. For example, immune RNAs can be used to treat and prevent poultry diseases, and can enhance plant and animal resistance to toxic pathogens by selectively regulating miRNA pathways (see International Publication No. WO2008 / 087562). Therefore, cargo-loaded MEVs can be used to treat diseases in animals, including livestock.

[0499] MEVs carrying cargo can be used to treat plant diseases. As illustrated, MEVs can be loaded with therapeutic molecules, such as siRNAs that target virulence genes in plant pathogens (including bacteria and viruses), and delivered to the plant, such as by application or spraying onto leaves and / or other surfaces, to target genes to eliminate or control pathogens.

[0500] d. Cosmetic and dermatological applications

[0501] MEVs carrying pharmaceutical payloads can also be used in cosmetic and dermatological applications. For example, skincare products containing EVs, particularly those derived from stem cells, such as creams, lotions, gels, emulsions, ointments, pastes, powders, liniments, sunscreens, and shampoos, can be used to improve and / or alleviate symptoms and problems such as dry skin, elasticity, wrinkles, folds, ridges, and / or skin folds (see, for example, Singapore Patent Publication No. SG11201811149 TA). Stem cell EVs inherently carrying cytokines as well as growth and transcription factors have also been shown to control inflammation, accelerate skin cell migration and proliferation, control wound scarring, improve angiogenesis, and improve signs of skin aging. Although the exact mechanisms are being elucidated, the effects of stem cell EVs on wound healing may depend on the vertical transfer of microRNAs or proteins into skin cells. Angiogenesis, a component of wound healing, can be induced by stem cell EVs. Evs (e.g., stem cells) also have beneficial effects on cell matrix maintenance and collagen production, and have been shown to function in regenerating skin cells (da Fonseca Ferreira, A. and Gomes, D. (2019) Bioengineering (Basel) 6(1):4). Therefore, MEVs loaded with the desired cargo can be used for cosmetic and dermatological applications.

[0502] E. Pharmaceutical compositions, formulations, reagent kits, articles, and combinations

[0503] 1. Pharmaceutical compositions and formulations

[0504] The compositions containing MEVs and loaded MEVs provided herein can be formulated as pharmaceutical compositions for administration via desired routes, such as oral, mucosal, intravenous, etc. Pharmaceutically acceptable compositions are prepared according to regulatory or other approvals, prepared in accordance with generally accepted pharmacopoeias for use in animals and humans, and also for agricultural and botanical applications. Typically, compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).

[0505] Pharmaceutical compositions may be used for therapeutic, preventative, cosmetic, and / or diagnostic applications. The MEVs and cargo-carrying MEVs described herein may be formulated with pharmaceutically acceptable carriers or diluents. Typically, such pharmaceutical compositions comprise components that do not significantly impair the biological or other properties of the cargo. Each component is pharmaceutically and physiologically acceptable, making it compatible with other ingredients and harmless to the subject to whom it will be administered. Formulations may be provided in unit dosage forms and may be prepared by methods known in the pharmaceutical field, including but not limited to tablets, pills, powders, liquid solutions or suspensions (e.g., including injectable, ingestible, and topical formulations such as eye drops, gels, pastes, creams, or ointments), aerosols (e.g., nasal sprays and inhalers), liposomes, suppositories, pessaries, injectable and infusionable solutions, and sustained-release forms. See, for example, Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8 th Ed.,Pergamon Press; and Remington's Pharmaceutical Sciences,17 th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. When administered systemically, the therapeutic composition is sterile, pyrogen-free, generally free of particulate matter, and in a parenteral acceptable solution taking into account pH, isotonicity, and stability. These conditions are known to those skilled in the art. Methods for preparing parenterally applicable compositions are well known or obvious 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)," 19 th ed., Mack Publishing Company, Easton, Pa. (1995).

[0506] The pharmaceutical compositions described herein can be in various forms, such as solid, semi-solid, liquid, powder, aqueous, and lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art and include, but are not limited to, water, buffers, saline solutions, phosphate-buffered saline solutions, various types of wetting agents, sterile solutions, alcohols, gum arabic, vegetable oils, benzyl alcohol, gelatin, glycerol, carbohydrates such as lactose, sucrose, amylose or starch, magnesium stearate, talc, silica, viscous paraffin, fragrance oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethyl cellulose, and powders, etc. The pharmaceutical compositions described herein may contain other additives, including, for example, antioxidants, preservatives, antimicrobial agents, analgesics, binders, disintegrants, colorants, diluents, excipients, thickeners, flow aids, solubilizers, stabilizers, tensiating agents, solvents, viscosity modifiers, flavoring agents, emulsions (e.g., oil / water emulsions), emulsifiers and suspending agents (e.g., gum arabic, agar, alginate, sodium alginate, bentonite, carbomer, carrageenan, carboxymethyl cellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, octylbenzenesilol-9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitol ester, stearyl alcohol, tragacanth gum, xanthan gum and their derivatives), solvents and various ingredients such as crystalline cellulose, microcrystalline cellulose, citric acid, dextrin, dextran, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate and starch, etc. (see Alfonso for more details). R.Gennaro(2000)Remington:The Science andPractice of Pharmacy,20 th Edition. Baltimore, MD: Lippincott Williams & Wilkins). Such carriers and / or additives can be formulated using conventional methods and administered to subjects at appropriate doses. Stabilizers such as lipids, nuclease inhibitors, polymers, and chelating agents can protect the composition from degradation in vivo.

[0507] The route of administration is consistent with known methods, such as intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, local, rectal, mucosal, and via injection or infusion through sustained-release systems. MEVs or cargo-loaded MEVs can be administered continuously by infusion or bolus injection. MEVs or cargo-loaded MEVs can be administered locally or systemically.

[0508] MEVs, or cargo-carrying MEVs, can be prepared by mixing with pharmaceutically acceptable carriers. The formulation and administration techniques of the compounds are known to those skilled in the art (see, for example, "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.). The therapeutic composition can be administered intravenously or via the nose or lung, for example, as a liquid or powder aerosol (lyophilized). The composition can also be administered parenterally or subcutaneously as needed. When administered systemically, the therapeutic composition should be sterile, pyrogen-free, and in a parenterally acceptable solution that takes into account pH, isotonicity, and stability. These conditions are known to those skilled in the art.

[0509] Suitable pharmaceutical compositions include those contained in an amount appropriate to effectively achieve their intended purpose in a MEV or a cargo-loaded MEV. The determination of therapeutically effective amounts is entirely within the capabilities of those skilled in the art. Therapeuticly effective doses can be determined by using in vitro and in vivo methods and / or by a technician.

[0510] Therapeutic formulations can be administered in many conventional dosage forms. Dosage forms of MEVs and cargo-loaded MEVs described herein are prepared for storage or administration by mixing compounds of desired purity with physiologically acceptable carriers, excipients, or stabilizers. Such materials are non-toxic to receptors at the doses and concentrations used and may include buffers such as TrisHCl, 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 derivatives thereof, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbate (TWEEN), pronicadin, polyethylene glycol, etc.

[0511] In specific examples described herein, pharmaceutical compositions containing stabilizers are provided. Stabilizers can be amino acids, amino acid derivatives, amines, sugars, polyols, salts, or surfactants. In some examples, the stabilizing co-formulation contains a single stabilizer. In other examples, the stabilizing co-formulation contains two, three, four, five, or six different stabilizers. For example, stabilizers can be sugars or polyols such as glycerol, sorbitol, mannitol, inositol, sucrose, or trehalose. In a specific example, the stabilizer is sucrose. In other examples, the stabilizer is trehalose. The concentration of the sugar or polyol is approximately 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, each included.

[0512] In this example, the stabilizer can be a surfactant, such as polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer, or polysorbate. For instance, the surfactant can be polypropylene glycol, polyethylene glycol, glycerin, sorbitol, poloxamer, or polysorbate, such as poloxamer 188, polysorbate 20, and polysorbate 80. In this specific example, the stabilizer is polysorbate 80. As a percentage of the mass concentration (w / v) in the formulation, the concentration of the surfactant is between 0.005% and 1.0%, 0.01% and 0.5%, 0.01% and 0.1%, 0.01% and 0.05%, or 0.01% and 0.02%, or about 0.005% and 1.0%, 0.01% and 0.5%, 0.01% and 0.1%, 0.01% and 0.05%, or 0.01% and 0.02%, each of which is included.

[0513] When intended for in vivo administration, the formulation should be sterile and formulated according to standard pharmaceutical practices. This can be easily achieved by filtration through a sterile filter membrane before or after lyophilization and reconstitution. MEVs, or cargo-loaded MEVs, can be stored in lyophilized or solution form; they can be frozen or refrigerated. Other carriers may be included, such as naturally occurring vegetable oils like sesame oil, peanut oil, or cottonseed oil, or synthetic fatty acid carriers like ethyl oleate. Buffers, preservatives, and antioxidants may be incorporated according to accepted pharmaceutical practices.

[0514] Depending on the application and the subjects, the concentration of the MEV or cargo-carrying MEV provided herein in the composition may be from or about 0.1 to 10 mg / mL or higher or lower, for example, at least or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 mg / mL or higher. The volume of the solution can be 1 to 100 mL or about 1 to 100 mL, for example, at least or about 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 MEV or the MEV carrying the cargo is supplied in phosphate-buffered saline.

[0515] The MEVs or cargo-loaded MEVs provided herein can be provided as controlled-release or sustained-release compositions. Polymer 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, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer and Peppas (1983) J. Macromol. Sci. 23:61; also see Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al. al. (1989) J. Neurosurg. 71:105; US Pat. Nos. 5,679,377,5,916,597,5,912,015,5,989,463,5,128,326; and International Pat. 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), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolic acid) (PLGA), and polyorthoesters. Typically, polymers used in sustained-release formulations are inert, free of leaching impurities, stable during storage, sterile, and biodegradable. Any techniques known in the art for producing sustained-release formulations can be used to produce sustained-release formulations containing the MEVs provided herein or MEVs loaded onto cargo.

[0516] In some instances, the pharmaceutical composition contains the MEV provided herein or a cargo-loaded MEV and one or more additional pharmaceutical agents, such as antibodies or other therapeutic agents, for combination therapy.

[0517] 2. Products / Reagent Kits and Combinations

[0518] Pharmaceutical compositions of MEVs or MEVs loaded with cargo may be packaged as articles containing packaging materials, pharmaceutical compositions that are effective in treating diseases or conditions (e.g., those described herein or known in the art) by administration of a specific MEV or MEV loaded with cargo, and labels indicating that the cargo (e.g., antibodies or nucleic acid molecules) will be used to treat an infection, disease, or condition. The pharmaceutical compositions may be packaged in unit dosage forms containing an amount of the pharmaceutical composition for single or multiple doses. Packaged compositions may contain lyophilized powders of the pharmaceutical composition containing the MEV loaded with cargo, which may be reconstituted prior to administration (e.g., with water or saline).

[0519] The articles described herein include packaging materials. Packaging materials used for packaging pharmaceutical products are well known to those skilled in the art (see, for example, U.S. Patents 5,323,907, 5,052,558, and 5,033,252). Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubing, inhalers (e.g., pressurized metered-dose inhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other single-breathing liquid systems), pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and the intended administration and mode of treatment.

[0520] MEVs or cargo-loaded MEVs may be provided as combinations and kits. Kits may optionally include one or more components, such as instructions for use, devices, and additional reagents (e.g., sterile water or saline solutions for diluting the composition and / or reconstituted lyophilized proteins), and components for performing the method, such as tubes, containers, and syringes. Exemplary kits may include the MEVs or cargo-loaded MEVs provided herein and may optionally include instructions for use, devices for administering the MEVs or cargo-loaded MEVs to a subject, devices for detecting the MEVs or cargo-loaded MEVs in samples obtained from the subject, and devices for administering additional therapeutic agents to the subject.

[0521] The kit may optionally include instructions for use. Instructions for use typically include a tangible description of the MEV or the cargo-loaded MEV, and optionally, other components included in the kit, and methods of administration, including methods for determining the appropriate status of the subject, appropriate dosage, dosing regimen, and appropriate methods of administration for administering the MEV or the cargo-loaded MEV. Instructions may also include guidance for monitoring the subject throughout the duration of treatment.

[0522] The kit may also include the pharmaceutical compositions described herein and articles for diagnostic purposes. For example, such a kit may include articles for measuring the concentration, amount, or activity of MEVs in a subject and in cargo.

[0523] In some examples, the MEV or cargo-loaded MEV is provided in the diagnostic kit for detecting the MEV or cargo-loaded MEV or cargo in isolated biological samples (e.g., tumor cells, such as circulating tumor cells obtained from a subject or tumor cells removed from a subject).

[0524] The kits provided herein may also include a device for administering the MEV to a subject. Any of a variety of devices known in the art for administering drugs to a subject may be included in the kits provided herein. Exemplary devices include, but are not limited to, subcutaneous needles, intravenous needles, catheters, nebulizers, and inhalers. Generally, the device for administering the composition is compatible with the desired method of administration of the composition.

[0525] 3. Application of exogenously loaded MEVs and application routes

[0526] The cargo-loaded MEVs provided herein can be administered to subjects by any method known in the art for administering peptides, including, for example, systemic or local administration. The cargo-loaded MEVs can be administered via routes such as parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intracavitary routes), local, epidural, or mucosal (e.g., local, intranasal, oral, vaginal, vulvovaginal, esophageal, oroesophageal, bronchial, rectal, and pulmonary routes). The cargo-loaded MEVs can be administered externally to the subject, at the site of disease, to exert a local or transdermal effect. Compositions containing cargo-loaded MEVs can be administered via any convenient route, such as by infusion, inhalation, bolus injection, or absorption through the epithelial or mucosal lining of the skin (e.g., local, oral, vaginal, rectal, and intestinal mucosa). Compositions containing cargo-loaded MEVs can be administered together with or sequentially with other bioactive agents. For example, cargo-loaded MEVs can be administered via infusion delivery (e.g., via an infusion pump or syringe pump) and can be administered in combination with another therapeutic agent or as a monotherapy.

[0527] The method and / or route of administration may be modified to mitigate adverse side effects associated with the administration described herein. For example, if a patient experiences a mild or moderate (i.e., Grade 1 or 2) infusion reaction, the infusion rate may be reduced (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more). If a patient experiences a severe (i.e., Grade 3 or 4) infusion reaction, the infusion may be temporarily or permanently interrupted.

[0528] In some cases, treatment adjustments may be made if a subject experiences adverse side effects, such as severe skin toxicity, like a severe acne-like rash. For example, administration may be delayed, for example, for 1 to 2 weeks or until the adverse side effect improves, following the occurrence of an adverse side effect. In some instances, the dose may be reduced after another adverse side effect occurs. Specific protocols and treatment regimens may be established by a skilled physician or other practitioner.

[0529] Those skilled in the art can select an appropriate delivery method based on the properties of the MEV loaded with the cargo or the dosage of the pharmaceutical composition containing the MEV loaded with the cargo. These properties include, but are not limited to, solubility, hygroscopicity, crystallinity, melting point, density, viscosity, flowability, stability, and degradation profile.

[0530] 4. Combination therapy

[0531] The cargo-loaded MEV described herein may be administered before, after, or simultaneously with one or more other treatment regimens or agents. A skilled medical practitioner may determine this based on experience or by considering the pharmacokinetics and mode of action of the agent, the appropriate dosage of each treatment regimen or agent, and the appropriate timing and method of administration. Additional treatment regimens or agents may improve the efficacy, safety, or other properties of the cargo-loaded MEV. In some examples, additional treatment regimens or agents may treat the same disease or comorbidity. In some examples, additional treatment regimens or agents may improve, reduce, or eliminate one or more side effects known in the art or described herein associated with the administration of the cargo-loaded MEV or cargo.

[0532] For example, the cargo-loaded MEV described herein can be administered in combination with chemotherapy, radiotherapy, or both chemotherapy and radiotherapy, or for antiviral or antibacterial or other pathogen therapy. The cargo-loaded MEV can also be administered in combination with other antipathogenic therapies and treatments. The cargo-loaded MEV can be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitors, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotective agents, immunostimulants, immunosuppressants, agents that promote blood cell proliferation, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, FcγRIIb or other Fc receptor inhibitors, or other therapeutic agents.

[0533] One or more additional agents may be administered simultaneously, sequentially, or intermittently with the cargo-loaded MEV. The agents may be administered, for example, as part of the same pharmaceutical composition or the same delivery method. In some examples, the agents may be administered simultaneously with the cargo-loaded MEV, but via different delivery methods. The agents may also be administered at different times from the administration of the cargo-loaded MEV, but close enough in time to have a combined preventive or therapeutic effect. In some examples, one or more additional agents are administered at selected time intervals, after or before the administration of the cargo-loaded MEV. In some examples, the time intervals are 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, one or more additional agents are administered multiple times and / or the cargo-loaded MEV provided herein is administered multiple times.

[0534] F. Biodistribution of MEVs after administration via different routes

[0535] 1. Biodistribution of mammalian EVs

[0536] The pharmacokinetics and biodistribution of mammalian EVs in organs and tissues have been extensively studied (Vader et al. (2016) Advanced drug delivery reviews 106(Pt A):148–156, doi.org / 10.1016 / j.addr.2016.02.006; Morishita et al. (2017) Journal of pharmaceutical sciences 106(9):2265–2269, hdoi.org / 10.1016 / j.xphs.2017.02.030). Treatment with mammalian cell-derived EVs is typically based on intravenous or intraperitoneal administration. The main target organs after systemic administration of mammalian EVs are the liver, spleen, and lungs. A comprehensive study of the tissue distribution of fluorescently labeled mammalian EVs from multiple cellular sources (see Wiklander et al. (2015) J. Extracellular Vesicles 4:26316) showed that, 24 hours after intravenous (iv) injection in mice, the highest fluorescence signal was observed in the liver, followed by the spleen, gastrointestinal tract, and lungs. Furthermore, cell source, EV dose, and route of administration were shown to influence EV distribution; for example, higher EV doses resulted in relatively lower hepatic accumulation compared to lower doses, likely due to saturation of the mononuclear phagocyte system (MPS). Comparisons between intraperitoneal (ip), subcutaneous (sc), and intravenous administration showed that intraperitoneal and subcutaneous doses resulted in reduced EV accumulation in the liver and spleen, and enhanced accumulation in the pancreas and gastrointestinal tract compared to intravenous administration. Systemically administered EVs have been reported to be rapidly taken up by the mononuclear phagocyte system (MPS), particularly in the liver and spleen. The clearance mechanism is similar to that described for synthetic nanoparticles (e.g., liposomes) (Van der Meel et al. (2014) J. Control. Release 195:72–8). Most splenic accumulation is due to EV storage in the spleen rather than splenic uptake (Lai CP et al. (2014) ACS Nano 8:483–494). Biodistribution of mammalian EVs following other routes of administration was also investigated. For targeting the central nervous system, intranasal administration of curcumin-loaded mammalian EVs resulted in EV localization in the brain. Drug levels peaked 1 hour after administration and were detectable at a significant level after 12 hours, with no toxic effects observed (Zhuang et al. (2011) Mol. Ther. 19:1769–1779).

[0537] Typically, mammalian Evs are not used for oral delivery due to their low stability across a variety of pH and temperature ranges, rapid degradation of the biomolecules in the digestive tract, and limitations in industrial-scale production for oral administration (Cheng et al. (2019) Protein Cell 10:295-299). The only exception to date is milk-derived Evs, which has shown a distribution pattern upon oral delivery to mice, analyzed using a whole-body in vivo imaging system (IVIS), including rapid accumulation in the intestine, where Evs were detectable at 2 and 6 hours, followed by fluorescent signals observed in the liver, spleen, lung, kidney, heart, and gastrointestinal tract at 24 hours. After 48 hours, the fluorescent signals faded in most 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 the intestine and from the intestine, thus failing to become bioavailable in target organs (Zhong et al. (2021) Biomaterials. 277: 121126. Doi: 10.1016 / j.biomaterials. 2021. 121126).

[0538] Treatment with mammalian cell-derived Evs typically involves intravenous or intraperitoneal administration for systemic delivery, with target organs including the liver, spleen, and lungs. As noted, most mammalian Evs have not yet been used for oral delivery due to their low stability at various pH and temperature levels, rapid degradation of the biomolecules in the digestive tract, and limitations in industrial-scale production for oral administration (Cheng et al. (2019) Protein Cell 10(4):295-299). The sole exception is milk-derived Evs, which has shown a distribution pattern upon oral delivery to mice, analyzed using a whole-body in vivo imaging system (IVIS), including rapid accumulation in the intestine, where Evs were detectable at 2 and 6 hours, followed by fluorescent signals observed in the liver, spleen, lungs, kidneys, heart, and gastrointestinal tract at the 24-hour timepoint. After 48 hours, the fluorescent signals faded in most organs, indicating clearance of the nanovesicles from the system.

[0539] As illustrated in the embodiments herein and discussed below, MEVs possess properties distinct from mammalian Evs. For example, they are stable in the harsh environment of the gastrointestinal tract compared to mammalian cell-derived Evs. Therefore, the microalgal Evs described herein are particularly suitable for oral administration and drug delivery, as well as other delivery routes described herein.

[0540] 2. Biodistribution of microalgae EVs

[0541] This article shows that MEVs (including those derived from Chlorella provided in this article) have properties that differ from mammalian Evs (including bovine Evs). For example, a significant difference discussed below is that MEVs can be administered orally, with the primary target being the spleen, possibly the white pith of the spleen (white spleen).

[0542] The MEVs provided herein can deliver a variety of bioactive molecules, such as RNA (e.g., mRNA, siRNA, and miRNA); proteins; peptides; and small molecules, which can be loaded exogenously or endogenously. These include products such as tissue-specific products and / or disease-specific products. As described below, each route can be used to target a specific organ and treat a specific disease. MEVs can be formulated for administration via each route. Therefore, compositions containing MEVs for treating a specific disease and for a specific route of administration are provided.

[0543] This article shows that the route of administration determines the fate of the MEV, and the final location of the MEV is a function of the route of administration. The targets and endpoints of the MEV include, but are not limited to, the liver, spleen, lungs, intestines, and brain. Routes of administration include, but are not limited to, inhalation (nasal, lungs), oral administration (digestive), intravenous administration, central nervous system (CNS), and local administration. The choice of route depends on the final target and payload. This article shows that intranasal administration reaches the lungs, intratracheal administration via atomization reaches the lungs, intravenous administration accumulates in the spleen and liver, and oral administration (perOs) enters the digestive tract and spleen. Conversely, mammalian effervescents (EVs) cannot be administered orally.

[0544] MEVs are readily internalized by human cells. For example, in vitro, when MEVs are applied to cultured cells (e.g., A549 cells) at a ratio of 1000 / 1 MEV / cell, 93% of the cells internalize the MEVs, and this occurs within 24 to 48 hours after the cells come into contact with the MEVs.

[0545] Mice were administered DIR-labeled MEVs via four routes: intranasal (IN), intratracheal (IT), intravenous (IV), and oral, and the fate of the MEVs was visualized for 3 days using whole-body imaging as a function of time, after which mice were sacrificed to harvest organs for the study. As shown in the examples, intravenous administration targeted the liver approximately 4–12 hours after administration, and the spleen appeared to be in the red pulp (red spleen) at 10–30 hours. Oral administration targeted the intestine and spleen. This paper shows that MEVs are orally available; they are resistant and pass through the stomach, reaching the intestine in 0.5 to 4 hours, and then the spleen in 0.5 to 10 hours. The pathways to the spleen are of interest; two possible pathways to the spleen exist, via the blood (to the red spleen) and via lymphocytes (to the white spleen), which have an impact on targeting and delivery of cargo to the immune system, accumulating from 4 to 28 hours. This can be achieved by the internalization of activated lymphocytes and their eventual entry into the spleen (where they proliferate), and / or by the phagocytosis of lymphocytes of unactivated MEVs, which enter the white pulp of the spleen (white spleen) and are then spread through the immune system.

[0546] Oral administration

[0547] Therefore, orally ingested MEVs enter the intestine and then, as shown, eventually reach the spleen (likely the white spleen). The spleen is responsible for initiating an immune response to blood-derived antigens and for filtering foreign substances and old or damaged red blood cells from the blood. These functions are performed by two distinct compartments within the spleen: the white spleen and the red spleen. These two compartments differ significantly in structure, vascular tissue, and cellular composition (see, for a review of the structure, function, and histology of the spleen, e.g., Cesta (2006) Toxicologic Pathology 34:455-465).

[0548] In the intestine, abundant leukocytes migrate to the white spleen. When orally ingested, MEVs can be internalized by intestinal cells, including, as described below, by intestinal lymphocytes carrying the MEVs to the spleen. This is in contrast to mammalian vesicles, which cannot be administered orally. Thus, MEVs serve as agents for the immune system, acting as delivery carriers for immunomodulatory cargoes. As mentioned above, the pathway to the white spleen can occur, for example, through activated lymphocytes and / or phagocytic lymphocytes. Lymphocytes can engulf MEVs and home to the spleen. Unlike mammalian EVs, MEVs provide a means of oral delivery of small molecule drugs and proteins, as well as other therapeutic agents such as nucleic acid therapeutics, that cannot be administered orally. In particular, orally administered MEVs provide a pathway to treat diseases such as cancer and inflammatory diseases, which involve the immune system 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. These diseases are treated by inhibiting or enhancing the activity of immune cells.

[0549] a) Components of the lymphatic system

[0550] The lymphatic system includes lymph, lymphatic vessels, and lymphatic organs (see 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) for discussion and summary below).

[0551] lymph

[0552] Lymph is a normally clear and colorless fluid that drains from the interstitium and contains recycled fluid and plasma proteins, and may also contain lipids, immune cells, hormones, bacteria, viruses, cell debris and cancer cells.

[0553] Lymphatic vessels

[0554] The lymphatic system is the body's second circulatory system. It is a unidirectional, blind-ended, thin-walled capillary system that drives lymph. Lymphatic capillaries drain into afferent collecting ducts and then flow through one or more lymph nodes. The lymph then flows through efferent collecting ducts, larger trunks, and finally lymphatic vessels, which drain the lymph into the systemic circulation. Primary lymphatic organs include the thymus and bone marrow, which produce mature lymphocytes that recognize and respond to antigens; secondary lymphatic organs include lymph nodes, the spleen, and mucosa-associated lymphoid tissue (MALT). In secondary lymphatic organs, lymphocytes initiate an immune response. MALT is distributed throughout the mucous membranes, providing a defense mechanism against various inhaled or ingested antigens. Based on their anatomical location, MALT can be divided into: bronchus-associated lymphoid tissue (BALT), nasal-associated lymphoid tissue (NALT), salivary gland duct-associated lymphoid tissue (DALT), conjunctival-associated lymphoid tissue (CALT), lacrimal duct-associated lymphoid tissue (LDALT), and intestinal-associated lymphoid tissue (GALT).

[0555] Gut-associated lymphoid tissue (GALT)

[0556] GALT consists of effector sites and immune-inducing sites. Effector sites include lymphocytes distributed in the lamina propria (LP) and intestinal epithelium; inducing sites involve tissues such as mesenteric lymph nodes (MLN), prostate cells (PP), and smaller, isolated lymphoid follicles (ILF). The mesenteric lymph nodes (MLN), located at the base of the mesentery, are the largest lymph node aggregation sites in the body. The MLN is structurally divided into two regions: the medullary region and the cortical region. The cortical region is mainly composed of T-cell areas and B-cell follicles. In the T-cell area, circulating lymphocytes enter the lymph nodes, and dendritic cells (DCs) present antigens to T cells. Lymph (containing cells, antigens, and chylomicrons) is collected from the intestinal mucosa and reaches the MLN via afferent lymphatic vessels. The lymph then leaves the MLN via efferent lymphatic vessels, reaches the thoracic duct, and is drained into the bloodstream.

[0557] Pell's cluster lymph nodes (PPs) are small lymph nodes distributed in the intestinal mucosa and submucosa. They contain a lower epithelial fornix and B-cell follicles scattered throughout the T-cell area. A layer of epithelial cells called follicle-associated epithelium (FAE) separates the lymphatic area of ​​the Pell's cluster lymph nodes from the intestinal lumen. The FAE is filled with specialized intestinal cells called microfold (M) cells. These cells act as gateways for transporting intestinal antigens to the Pell's cluster lymph nodes.

[0558] Isolated lymphoid follicles (ILFs) are a collection of lymphoid cells in the intestinal lymphoid follicle (LP). ILFs consist of germinal centers covered by fecal follicles (FAEs) containing M cells. ILFs are a complementary system to progesterone (PP) and are used to induce intestinal immunity.

[0559] The gastrointestinal tract (GALT) is the largest lymphoid organ in the human body, containing more than half of the body's lymphocytes. Compared to any other part of the body, the GALT is exposed to more antigens in the form of symbiotic bacteria and gastrointestinal antigens, in addition to antigens from invasive pathogens. Intestinal lymphatic transport avoids first-pass metabolic loss from the liver by diverting the absorption of lipophilic drugs to the intestinal lymph nodes rather than the portal vein. The intestinal immune system must distinguish antigens that require a protective immune response and generate a state of low immune response to harmless antigens (oral tolerance). This is achieved by dendritic cells (DCs) sampling luminal antigens from the intestinal epithelium. Antigens can cross the epithelium via M cells, which are specialized epithelial cells of the follicle-associated epithelium of the gastrointestinal tract. Antigens interact with dendritic cells (DCs) in the fornix region of the underlying epithelium. Dendritic cells present the antigens to local T cells within the PP.

[0560] DCs also migrate to the draining MLN, where they present antigens to local lymphocytes. Alternative pathways of antigen transport across intestinal epithelial cells involve receptor-mediated transport, as well as direct sampling from the lumen via DC projection. Antigen-loaded DCs then migrate to the MLN via afferent lymphocytes, where they present antigens to T cells. Subsequently, differentiated lymphocytes migrate from the MLN through the thoracic duct and blood flow, eventually accumulating in the mucosa for appropriate immune responses.

[0561] b) Targeting GALT

[0562] Orally administered MEVs can target the gut-associated lymphoid tissue (GALT). Therefore, the GALT is a target (effective compartment) and / or route for delivering drugs to organs, tissues, and / or systemic circulation using MEVs and their therapeutic agents. The GALT is a favorable target for various pharmacological agents such as immunomodulators, chemotherapeutic agents, and anti-infective agents. The lymphatic system is a major route of metastasis for the intestine and other tumors; therefore, targeting the intestinal lymph nodes with cytotoxic drugs can be used to treat tumor metastases. The GALT is a delivery target for antiviral agents because some viruses spread and develop within the lymphatic system, such as human immunodeficiency virus (HIV), measles virus, canine distemper virus, severe acute respiratory syndrome (SARS)-associated coronavirus, hepatitis B, and hepatitis C.

[0563] Therefore, microalgal MEVs, including the Chlorella MEV exemplified herein, can be used to target immune cells upon oral delivery. As mentioned above, microalgal MEVs exhibit a unique biodistribution pattern upon...

Claims

1. A composition comprising microalgal extracellular vesicles (MEVs) for delivering bioactive molecules to the brain for the treatment, detection, or monitoring of diseases, conditions, or illnesses of the brain, wherein: The MEV contains the bioactive molecule, and the composition is formulated for intranasal administration. When administered intranasally, the MEV travels via the olfactory nerve to the brain and through the lateral olfactory tract (LOT) to interconnected brain regions, delivering to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral longitudinal striatum (teniatecta), piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal association cortex, basolateral amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus; The bioactive molecule is any molecule that can affect the treatment of a disease, condition, or disease of the brain, or can be used to detect a disease, condition, or disease of the brain, or to monitor the treatment of a disease, condition, or disease of the brain; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

2. A method of delivering a bioactive molecule to the brain, comprising intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs), said microalgal extracellular vesicles containing a cargo containing said bioactive molecule, wherein said MEVs travel via the olfactory nerve to the brain and through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral longitudinal striae, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal lobe 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 affect or treat a disease, symptom or condition, or that can be used to detect a disease, symptom or condition, or that can be used to monitor the treatment of a disease, symptom or condition. The disease, condition, or symptom may be one that can be treated, detected, or monitored by delivering the bioactive molecule to one or more of the interconnected regions; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

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

4. The composition or method of any one of claims 1-3, wherein the bioactive molecule achieves the treatment of neurodegenerative diseases, symptoms, or conditions.

5. The composition or method of any one of claims 1-4, wherein the disease, symptom or condition is Alzheimer's disease or a mental illness, symptom or condition.

6. The composition or method according to any one of claims 1-5, for treating diseases, symptoms or conditions of the brain or involving diseases, symptoms or conditions of the brain.

7. A method of treating a disease, condition, or symptom of the brain, the method comprising intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs), the microalgal extracellular vesicles containing a cargo comprising bioactive molecules, wherein the MEVs travel via the olfactory nerve to the brain and through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insula cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal lobe 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 capable of treating or involving diseases, symptoms, or conditions of the brain; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

8. A method for detecting or treating a disease, symptom, or condition of the brain, or involving a disease, symptom, or condition of the brain, the method comprising intranasal administration of a composition comprising microalgal extracellular vesicles (MEVs) containing bioactive molecules, wherein the MEVs travel via the olfactory nerve to the brain and through the lateral olfactory tract (LOT) to interconnected brain regions for delivery to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral striatum, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal lobe association cortex, basolateral amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus, wherein the bioactive molecules comprise reporter molecules or detectable markers, wherein: The bioactive molecule is any molecule that can be used to detect or diagnose a disease, condition or illness, or to monitor the treatment of a disease, condition or illness, or to detect or diagnose a disease, condition or illness and treat the disease, condition or illness. The disease, symptom, or condition is a disease, symptom, or condition of the brain or involving one or more interconnected brain regions; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

9. A composition comprising microalgal extracellular vesicles (MEVs) containing cargo, said cargo comprising bioactive molecules, wherein: The composition is formulated for delivery via the olfactory nerve into the brain and through the lateral olfactory tract (LOT) to interconnected brain regions, specifically to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral longitudinal striae, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal lobe association cortex, basolateral amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus; The bioactive molecule is any molecule that can achieve the treatment of a disease, symptom or condition, or can be used to detect or diagnose a disease, symptom or condition, or can be used to monitor the treatment of a disease, symptom or condition. The disease, symptom, or condition is a disease, symptom, or condition of the brain or involving one or more interconnected brain regions; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

10. The composition of claim 9, used for delivering a cargo containing a bioactive molecule to the brain to treat a disease, condition, or illness of the brain or involving the brain, or for diagnosing, detecting, or monitoring a disease, condition, or illness of the brain or involving the brain, or for treating, diagnosing, detecting, and / or monitoring a disease, condition, or illness of the brain or involving the brain, wherein: The MEV contains bioactive molecules for delivery to the brain; and The composition is formulated for delivery via the olfactory nerve into the brain and through the lateral olfactory tract (LOT) to interconnected brain regions, specifically to one or more of the following: olfactory bulb, preolfactory nucleus, olfactory tubercle, lateral longitudinal striae, piriform cortex, amygdala, entorhinal cortex, primary motor cortex, frontal cortex, agranular insular cortex, primary somatosensory cortex, auditory cortex, posterior granular cortex, temporal lobe association cortex, basolateral amygdala, mammillary bodies, arcuate nucleus of the hypothalamus, corpus callosum, internal capsule, thalamus, and hippocampus; The bioactive molecule is any molecule capable of treating a disease, symptom, or condition, or capable of detecting a disease, symptom, or condition, or capable of monitoring the treatment of a disease, symptom, or condition; and The bioactive molecules are heterologous to the microalgae and / or the MEV.

11. The method or composition of any one of claims 1-10, wherein the MEV travels in the brain via intraneural axonal transport.

12. The method or composition of claims 1-11, wherein the MEV is delivered to or used for delivery to the limbic system, such as the amygdala, hippocampus, and thalamus, or the cortex, such as the frontal cortex or parietal cortex.

13. The method or composition of any one of claims 1-12, wherein the MEV, after intranasal administration, follows the pathways and connections in a neural network, or is used to follow the pathways and connections in the neural network comprising olfactory nerves and mitral / cluster neurons throughout the brain.

14. The method or composition according to any one of claims 1-13, wherein, When administered intranasally, the MEV crosses one or more of the following: (i) synapses between olfactory sensory neurons (OSN) and mitral / cluster neurons; (ii) synapses between mitral / cluster neurons and local neurons in brain regions colonized by LOT; and (iii) synapses between neurons in brain regions colonized by LOT and neurons originating from or reaching the frontal cortex, hippocampus, thalamus, and hypothalamus.

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

16. The method or composition of any one of claims 1-15, wherein the pathway through which the MEV passes when administered intranasally is depicted in Figure 35.

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

18. The method or composition of any one of claims 1-17, wherein the disease, symptom, or condition relates to neurons.

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

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

21. The method or composition of any one of claims 1-20, wherein each MEV in the composition contains an average of 1 to 100 bioactive molecules.

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

23. The method or composition of any one of claims 1-22, wherein: The composition is formulated for single-dose or multiple-dose administration, and the amount of the composition is from 0.1 to 100 mL; and A single dose contains approximately or between 10e10 and 10e14 MEV particles.

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

25. The method or composition of any one of claims 1-24, wherein: The MEVs are extracellular vesicles derived from microalgae of the Chlorella family; The Chlorella family extracellular vesicles contain heterologous bioactive molecular cargoes introduced from isolated extracellular vesicles, thereby the vesicles containing the heterologous bioactive molecular cargoes in the composition contain, on average, the same bioactive molecular cargoes, wherein: The cargo molecules are heterologous to the Chlorella family; and The bioactive cargo is a therapeutic or detectable molecule used for the treatment, monitoring, and / or diagnosis of diseases, conditions, or illnesses involving the brain.

26. The method or composition of any one of claims 1-24, wherein: The MEV is an extracellular vesicle of Chlorella; The extracellular vesicles of *Chlorella* contain heterologous bioactive molecular cargo, which is introduced into the extracellular vesicles endogenously by the microalgae. Thus, the vesicles containing the heterologous bioactive molecular cargo in the composition contain the same bioactive molecular cargo, wherein: The cargo molecules are heterologous to Chlorella; and The bioactive cargo is a therapeutic or detectable molecule used for the treatment, monitoring, and / or diagnosis of diseases, conditions, or illnesses involving the brain.

27. The method or composition of any one of claims 1-24, wherein: The microalgae mentioned are species of microalgae belonging to the Chlorellaceae family; The MEV in the composition contains a heterologous bioactive molecular cargo, which has been exogenously introduced into the isolated MEV, thereby the vesicles in the composition containing the heterologous bioactive molecular cargo contain the same cargo, wherein: The goods described are heterologous to Chlorella; and The goods are biomolecules or small molecules.

28. The method or composition of any one of claims 1-24, wherein: The MEV is an extracellular vesicle of Chlorella; The extracellular vesicles of *Chlorella* contain heterologous bioactive molecular cargo, which is introduced into the extracellular vesicles endogenously by the microalgae. Thus, the vesicles containing heterologous bioactive molecular cargo in the composition contain the same bioactive molecular cargo, wherein: The cargo molecules are heterologous to Chlorella; and The bioactive cargo is a biomolecule.

29. The method or composition of any one of claims 1-24, wherein the MEV is derived from the microalgae selected from the following phyla: Euglenophyta, Chrysophyta (golden brown algae and diatoms), Pyrrophyta (fire algae), Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyta (brown algae), and Xanthophyta (yellow-green algae).

30. The method or composition of any one of claims 25-28, wherein the Chlorella is selected from species of the Chlorellaceae family: Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella variabilis, or from species of the Parachlorella genus: Parachlorella kessleri, Parachlorella beijerinckii, and Parachlorella hussii.

31. The method or composition of claim 30, wherein the Chlorella is Chlorella vulgaris or Chlorella kJ.

32. The method, composition, or drug delivery of any one of claims 1-24, wherein the MEV is derived from microalgae, said microalgae being species of the class Chlorophyceae or Trebouxiophyceae.

33. The method or composition of any one of claims 1-24, wherein the MEV is derived from the phylum Chlorophyta.

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

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

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

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

38. The method or composition of any one of claims 1-37, wherein each MEV containing cargo comprises a variety of different heterogeneous cargoes.

39. The method or composition of any one of claims 1-38, wherein the goods are therapeutic agents for treating or preventing diseases or conditions of the brain or involving the brain, or for treating or preventing their symptoms.

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

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

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

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

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

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

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

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

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

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

50. The method or composition of claim 48, wherein the gene editing system comprises a CRISPR-related or CRISPR-like system.

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

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

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

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

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

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

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

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

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

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

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

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

63. The method or composition of any one of claims 1-62, wherein the cargo code is an immunomodulator.

64. The method or composition of any one of claims 1-63, wherein the goods comprise or encode an immunomodulator to increase or decrease the production of one or more cytokines; upregulate or downregulate self-antigen presentation; mask MHC antigens; or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells.

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

66. The method or composition of any one of claims 1-65, wherein the cargo comprises a prodrug or a carrier encoding an enzyme that converts the prodrug into a medicament for treating diseases, symptoms, or conditions of the brain or involving the brain.

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

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

69. The method or composition of any one of claims 1-68, wherein the goods comprise a therapeutic nucleic acid or protein or nucleic acid encoding a protein, said therapeutic nucleic acid or protein or nucleic acid encoding a protein being a therapeutic product for treating cancer or tumors in the brain, or for treating infectious diseases in the brain, or for treating neurodegenerative diseases or other central nervous system (CNS) disorders, or for treating dementia.

70. The method or composition of any one of claims 1-69, wherein the goods comprise a chemotherapy drug for treating diseases, conditions or illnesses of the brain or involving the brain.

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

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

73. The method or composition of any one of claims 1-72, wherein the goods comprise nucleic acids for gene therapy.

74. The method or composition of any one of claims 1-73, wherein the disease, symptom or condition includes a tumor.

75. The method or composition of any one of claims 1-74, wherein the cargo comprises an oncolytic virus for infecting gliomas or comprises a therapeutic agent for treating gliomas.

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

77. The method or composition of any one of claims 1-76, wherein the goods comprise a diagnostic or detectable product for detecting, diagnosing, and / or monitoring diseases, conditions, or illnesses of the brain or involving the brain.

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

79. The method or composition of any one of claims 1-78, wherein the bioactive molecular cargo comprises any molecule that has an effect on the cell or organism to which it is delivered, or is detectable or can be used as a detectable marker or biomarker, thereby achieving treatment, detection, diagnosis or monitoring of diseases, symptoms or conditions of the brain or involving the brain.

80. The method or composition of claims 1-79, wherein the bioactive molecular cargo comprises one or more of bioactive small molecules, peptides (polypeptides, proteins), RNA (mRNA, siRNA, dsRNA, miRNA, lncRNA), DNA (antisense oligonucleotides (ASO), plasmids, DNA fragments), and gene editing complexes.

81. The method or composition of any one of claims 1-80, wherein the bioactive molecule is a diagnostic agent or therapeutic agent or therapeutic diagnostic agent for the treatment, diagnosis, detection and / or monitoring of diseases, conditions or illnesses of the brain or involving the brain.

82. The method or composition of any one of claims 1-81, wherein the disease, condition, or illness is a neurodegenerative disease (such as Parkinson's disease, or Alzheimer's disease, or Huntington's disease, or Creutzfeldt-Jakob disease, or other neurodegenerative diseases), or cognitive impairment (such as dementia, or amnesia, or delirium, or other cognitive impairment), or brain disorder (such as encephalitis, or seizures, or tumors, or other brain disorders), or nervous system disorder (such as pain, or seizures, or infections, or other nervous system disorders), or genetic disease (such as cystic fibrosis, thalassemia, sickle cell anemia, Huntington's disease, Duchenne muscular dystrophy, Ty Sachs disease, Rett syndrome, or other genetic diseases), or brain tumor, or Niemann-Pick disease, or prions, or Parkinson's disease, or multiple sclerosis, or amyotrophic lateral sclerosis (ALS), or muscular dystrophy, or other diseases of the brain or involving the brain.

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

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

85. The method or composition of claim 84, wherein the infectious agent is one or more of bacteria, viruses, oomycetes, and fungi.

86. The method or composition of any one of claims 1-76, wherein the MEV, after intranasal administration, delivers the cargo to one or more of neurons, astrocytes, oligodendrocytes, microglia, ependymal cells, and / or neural stem cells.

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

88. The method or composition of any one of claims 1-87, wherein the disease, symptom or condition is one or more of cognitive, emotional, behavioral, psychotic, neurological, degenerative, genetic, malignant (cancer) and / or traumatic brain diseases, symptoms or conditions.

89. The method or composition of claim 88, wherein the brain disease, symptom or condition is caused by damage to the brain or central nervous system (CNS).

90. The method or composition of any one of claims 1-89, wherein the MEV comprises a therapeutic product, said therapeutic product being psychoactive or treating mental disorders, or an immunomodulatory product, or a detectable product, or treating brain injury or trauma, or treating cancer, or treating neurocranial disorders, or treating CNS disorders, or treating genetic brain disorders, or treating brain cancer, or having anti-aging activity, or having brain regeneration activity.

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

92. The method or composition of any one of claims 1-91, wherein the MEV comprises goods for one or more of the following: (i) treating or preventing or mitigating the risk of brain diseases, symptoms and conditions; (ii) in vitro and / or in vivo studies of brain diseases, symptoms and conditions; (iii) diagnosis of brain diseases, symptoms and conditions; and (iv) recreational use.

93. The method or composition of claim 92, wherein the disease, symptom, and condition are selected from cognitive, emotional, behavioral, psychotic, neurological, and / or neurodegenerative diseases, symptoms, or conditions, or diseases, symptoms, or conditions caused by brain or CNS damage.

94. The method or composition of claim 92, wherein the disease, condition, and illness are selected from brain and / or CNS cancer or tumors, genetic disorders, brain injury or trauma, and infection.

95. The method or composition of any one of claims 1-94, wherein the MEV cargo comprises small molecules.

96. The method or composition of any one of claims 1-95, wherein the goods are selected from antidepressants, antipsychotics, anxiolytics, analgesics, hallucinogens, and memory enhancers.

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

98. The method or composition of any one of claims 1-97, wherein the cargo cannot cross the blood-brain barrier when not in the MEV (naked).

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

100. The method or composition of any one of claims 1-99, wherein: As a naked molecule or in a MEV, the cargo is one that, when administered systemically or locally via a route other than the nose, does not reach the brain after first-pass metabolism in the liver or has poor intestinal absorption; but When administered intranasally in the MEV, the cargo reaches the brain.

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

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

103. The method or composition of any one of claims 1-102, wherein the goods comprise a bioactive molecule for treating the following conditions:

104. The method or composition of any one of claims 1-103, wherein the brain-related diseases, conditions, and illnesses are selected from human mental disorders or neurological disorders; non-human animal brain disorders, central nervous system (CNS) disorders; anxiety disorders, such as panic disorder, social anxiety, phobia-related disorders, and generalized anxiety disorder; attention deficit hyperactivity disorder, such as inattentive, hyper-impulsive, and mixed types; autism spectrum disorders, such as Asperger's syndrome, childhood disintegrative disorder (CDD), Kanner syndrome, and pervasive developmental disorder (PDD-NOS); epilepsy, drug-resistant epilepsy; bipolar disorder, such as type I bipolar disorder, type II bipolar disorder, bipolar disorder with mixed features, bipolar disorder with seasonal pattern of major depressive disorder, cyclothymic disorder, and rapid-cycle bipolar disorder; and eating disorders, such as anorexia nervosa.

1. Bulimia nervosa, muscle dysmorphic disorder, binge eating disorder, other specific eating or eating disorders (OSFED), compulsive overeating, Prad-Willi syndrome, diabetic bulimia, neurotic health food obsession, selective eating, alcoholism, early eating obsession; 2. Personality disorders, including but not limited to antisocial personality disorder, borderline personality disorder, histrionic personality disorder, narcissistic personality disorder, avoidant personality disorder, dependent personality disorder, obsessive-compulsive disorder (OCD); 3. Post-traumatic stress disorder such as PTSD, acute stress disorder, simple PTSD, complex PTSD, comorbid PTSD; 4. Classic Rett syndrome, CDKL5-related atypical Rett syndrome; 5. Schizophrenia, catatonic schizophrenia, disorganized schizophrenia, paranoid schizophrenia, residual schizophrenia, and undifferentiated schizophrenia; and 6. Other such mental illnesses and brain-related disorders.

105. The method or composition of any one of claims 1-103, wherein the diseases, conditions and illnesses involving the brain are selected from genetic diseases, neurodegenerative diseases, neurological diseases and metabolic diseases affecting brain function, as well as other brain-related metabolic diseases, conditions or illnesses.

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

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

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

109. The method or composition of any one of claims 105-108, wherein modification of the Apo level or expression is achieved by intranasal administration of a cargo-loaded MEV, said MEV resulting in: a) Physiological levels of ApoE lipidation modified with MEVs loaded with: (i) peptides or small molecules known to increase the lipid-binding capacity of ApoE, or (ii) sequences of miRNA (miRNA-33) or miRNA-33 mimicking siRNA or ASO to increase ABCA1 levels or decrease Aβ levels, thereby enhancing the lipidation capacity of ApoE; and / or b) Reduce the amount of ApoE4 in the brain by using MEVs loaded with a sequence of miRNA (miRNA146) or miRNA-146-mimicking siRNA or ASO, or loaded with other RNAi (such as siRNA or shRNA) that inhibit ApoE4 expression, thereby suppressing the immune response in the brain and / or reducing ApoE4 in the brain; and / or c) Using MEVs loaded with (a) the ApoE2 protein, or (b) mRNA encoding the ApoE2 protein, or (c) a plasmid encoding the ApoE2 sequence to increase the expression of the ApoE2 isotype in the brain, thereby enhancing the protective effect of ApoE2 and compensating for the toxic effects of ApoE4; and / or d) Editing the ApoE4 allele to produce ApoE3 and / or ApoE2 by using a MEV loaded with a gene-editing complex for genome editing.

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

111. The method or composition of any one of claims 1-110, wherein the goods in the MEV comprise one or more of the following: TrkA (tropomyosin kinase A), neurotrophic factors selected from: NT-3, NT-4, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), psilocin and / or dephosphorylated psilocin, halamine, temozolomide, levamisole, GABAB1A receptor, GABAB1A receptor siRNA, PTEN siRNA (SEQ ID NO: 136-138); miR-17 (miRNA; SEQ ID NO: 139-141), MALAT1 (SEQ ID NO: 142); 5-HT1A (5-HT1A) and 5-HT3 (5-HT3) receptor agonists, such as azaspirone, methylphenidate, dextromethorphan, ondansetron (e.g., as marketed under trademarks). Products sold); acetylcholinesterase inhibitors, such as donepezil, galantamine, and rivastigmine; alpha-1 receptor antagonists, such as prazosin; anticonvulsants, such as gabapentin, pregabalin, and topiramate (e.g., under trademarks). Products sold), carbamazepine, escricazepine, levetiracetam, licorice, oxcarbazepine, valproic acid and its derivatives, lamotrigine; antipsychotics, such as aripiprazole, asenapine, cariprazine, chlorpromazine, clozapine, haloperidol, luminidepiperone tosylate (e.g., as... Products sold include olanzapine, paliperidone, quetiapine, risperidone, and ziprasidone; beta-blockers such as azaspiron and propranolol; and drugs that regulate the cholinergic system such as biperidone and scopolamine. Corticotropin-releasing factor (CRF) antagonists; drugs that regulate the GABAergic system, such as benzodiazepines. Brinolon, Sage-217; glucocorticoid receptor agonists, such as hydrocortisone; drugs involved in glutamatergic regulation, such as AGN-241751, AV-101, AVP-786, AVP-923, AXS-05, D-cycloserine, dextromethorphan, rapastin; glycine and glycine reuptake inhibitors, such as sarcosine; drugs that regulate the hypothalamus-pituitary-adrenal (HPA) axis, such as fludrocortisone, metoprolol, mifepristone, and probiotics; drugs that regulate the kynurenine pathway (KP); drugs that regulate limbic and paralimbic brain regions, such as cannabidiol (CBD); drugs that regulate the melatonin system, such as agomelatine. Fatty acids, peptides, nucleic acids, and other precursor molecules, such as α-ω fatty acids, coenzyme Q10, inositol, methylfolate, S-adenosylmethionine, cysteine, and oxytocin; monoamine oxidase inhibitors (MAOIs), such as isocarboxazid (Marplan), phenelzine (Nardil), selegiline (Emsam), and parnate; mood stabilizers, such as lithium salts, valproic acid, ebuselenium, and divalproic acid; multimodal antidepressants, such as vilazorone and vortioxetine; N-nitrosodimethylamine (NDMA) receptor antagonists, such as amantadine, alketamine, ketamine, memantine, riluzole, and esketamine; neurokinin-1 (NK1) receptor antagonists; neuropeptide Y (NPY) receptor agonists; drugs with neurotrophic effects, such as cilostazol, sildenafil, and vildagliptin; norepinephrine-dopamine reuptake inhibitors (NDRIs), such as bupropion. Drugs acting on the opioid system, such as ALKS-5461, AZD2327, BTRX-246040 (LY2940094), buprenorphine, JNJ-67953964, nalmefene, and naltrexone; protein kinase C inhibitors or anti-estrogenic drugs, such as indoxifene, tamoxifen, and verapamil; psychedelic drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), ayahuasca, lysergic acid diacetamide (LSD), and psilocybin; selective serotonin reuptake inhibitors (SSRIs), such as citalopram. Escitalopram Fluvoxamine, Paroxetine Heshequlin Selective norepinephrine transporter inhibitors, such as atoxetine; serotonin-norepinephrine reuptake inhibitors (SNRIs), such as desvenlafaxine. Duloxetine Zominaprun And venlafaxine; stimulants, including adenosine receptor antagonists and α-2-adrenergic receptor agonists, such as caffeine, clonidine, guanfaxine, extended-release amphetamine XR-OS, dextroamphetamine sulfate, lidextroamphetamine, methamphetamine, mixed amphetamine salts, racemic amphetamine sulfate, and triazine mixed amphetamine salts; substance P antagonists, such as aprepitant (MK0869) and fosaprepitant (MK-0517); tricyclic serotonin-norepinephrine reuptake inhibitors, such as amitriptyline. Amoxapine, Buspirone TM ), clomipramine, desipramine Doxepin, Imipramine Pamela tilapia, nortriptyline TM Protriptyline and trimethoprim; and vasopressin 1B (V1B) receptor antagonists, such as neliptan (SSR149415).

112. The method or composition of any one of claims 1-111, wherein: The cargo in the MEV contains catalase, GFP, luciferase, nerve growth factor (NGF), TrkA (tropomyosin kinase A), neurotrophic factors, including but not limited to NT-3, NT-4, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), psilocin / dephosphorylated psilocin, halamine, temozolomide, levamisole and / or rhodamine; and Optionally, the composition is formulated for use with neurons, astrocytes, oligodendrocytes, microglia, ependymal cells, and / or neural stem cells for administration via intranasal application.

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

114. The method or composition of any one of claims 1-25 and 27-112, wherein the exogenous cargo in the MEV is loaded in a purified or partially purified MEV.

115. The method or composition of any one of claims 1-114, wherein: The microalgae mentioned are species of Chlorella; The MEV in the composition contains heterologous bioactive molecular cargo that has been exogenously introduced into the isolated MEV, such that the vesicles containing the heterologous bioactive molecular cargo in the composition contain, on average, the same heterologous cargo, wherein: The goods described are heterologous to Chlorella; and The goods are biomolecules or small molecule drugs.

116. The method or composition of any one of claims 1-114, wherein: The MEV is an extracellular vesicle of Chlorella; The extracellular vesicles of *Chlorella* contain heterologous bioactive molecular cargoes introduced endogenously into the extracellular vesicles by the microalgae, wherein: The cargo molecules are heterologous to Chlorella; and The bioactive cargo is a biomolecule intended for the treatment of diseases, conditions, or illnesses of the brain or involving the brain.

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

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

119. The method or composition of any one of claims 1-116, wherein the Chlorella species is a pseudoChlorella species.

120. The method or composition of claim 118, wherein the Chlorella species is common Chlorella or is selected from the following Chlorella species: Chlorella kJ, Chlorella beyle, and Chlorella hussi.

121. The method or composition of any one of claims 1-118, wherein the bioactive molecule is any molecule that can be used to detect or diagnose the disease, symptom or condition of any one of claims 103-112.

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